CN112898301B

CN112898301B - Naphthalimide indole heterocyclic compound, and preparation method and application thereof

Info

Publication number: CN112898301B
Application number: CN202110125413.2A
Authority: CN
Inventors: 马静; 王佳佳; 田智勇; 方东; 孙华; 谢松强
Original assignee: Henan University
Current assignee: Henan University
Priority date: 2021-01-29
Filing date: 2021-01-29
Publication date: 2022-03-08
Anticipated expiration: 2041-01-29
Also published as: CN112898301A

Abstract

The invention belongs to the technical field of pharmaceutical chemistry, and particularly relates to a naphthalimide indole heterocyclic compound, and a preparation method and application thereof. The naphthalimide indole heterocyclic compound has good antitumor activity, the in vivo and in vitro antitumor activity of the naphthalimide indole heterocyclic compound is better than that of amonafide, and the antitumor spectrum is wider. Different from classical amonafide, the compound provided by the invention regulates and controls subcellular organelle and nuclear function reversal drug resistance by targeting contents of key enzymes PAO (polyamine oxidase) and three endogenous micromolecules Put (putrescine), Spd (spermidine) and Spm (spermine) in a tumor polyamine microenvironment, simultaneously enhances anti-tumor immune response, and has better treatment potential on late metastatic tumors. The complex also solves the problems of poor solubility, complex clinical compatibility, poor immunity of patients in clinical application of chemotherapeutic drugs and the like of the conventional naphthalimide analogs represented by amonafide, and has good water solubility.

Description

Naphthalimide indole heterocyclic compound, and preparation method and application thereof

Technical Field

The invention belongs to the technical field of pharmaceutical chemistry, and particularly relates to a naphthalimide indole heterocyclic compound, and a preparation method and application thereof.

Background

The main reasons for the failure of the current chemotherapeutic drugs in treating cancers, particularly metastatic cancers, are lack of targeting property, drug resistance, weak anti-tumor metastasis activity, immunosuppression and the like. Naphthalimide and derivatives thereof represented by amonafide are DNA intercalators with good anti-tumor effect, and the planar aromatic ring in the structure is inserted between DNA base pairs to distort the conformation of a DNA backbone and interfere the interaction of DNA-protein, thereby playing the anti-tumor effect. As early as 1973, the Brana topic is combined into a series of naphthalimide-polyamine derivatives with antitumor activity, wherein two compounds, namely ananafide (Amonafide) and Mitonafide (Mitonafide), enter a clinical second-stage test, Amonafide has a good inhibitory effect on leukemia cells (P388) and leukemia cells (L1210), Mitonafide has a good antitumor activity on human oral epithelial cancer cells (KB) and cervical cancer (Hela), but Amonafide can cause different degrees of influences on human bone marrow inhibition, vomiting, phlebitis and the like; however, Mitonafide causes serious toxicity to the human body, which limits the clinical application of Mitonafide, so that the improvement of the targeting property and the solution of the immunosuppression problem of the Mitonafide by modifying the naphthalimide structure become the key points of the research and development of the current chemotherapeutic drugs.

Polyamine analogs are compounds similar to polyamine structures, can be recognized and taken up by Polyamine Transporters (PTS), cannot replace some physiological functions of polyamines per se, but can inhibit the utilization of polyamines by tumor cells in various ways, thereby inducing apoptosis of cells. The first generation of polyamine analogs (end group symmetry) were synthesized by Wallace et al and included bisethyl isospermine (BENSpm), bisethyl spermine (BESpm), and bisethyl homopspermine (BEHSpm). The compounds can be recognized and absorbed by PTS, inhibit polyamine synthase ODC and S-AdeMetDC, and simultaneously up-regulate the activity of SSAT, thereby rapidly consuming polyamine in cells to inhibit the proliferation and differentiation of tumors. In addition, BENSpm has a certain synergistic effect when being combined with antitumor drugs such as taxol, cisplatin and 5-fluorouracil. The second generation polyamine analogs were first synthesized by Woster et al, modifying BENSpm mainly to N1-propargyl-N11-ethyl isospermine (PENSpm) or N1-cyclopropyl-N11-ethyl isospermine (CPENSpm), and compared with the first generation polyamine analogs, were able to express higher activity of SSAT, thereby depleting the polyamines in tumor cells more rapidly and further inducing apoptosis. At present, the main key point of modifying the structure of the naphthalimide is targeted modification of the naphthalimide.

Fig. 1 shows structural formulas of amonafide, mitonaphthylamine, cisplatin and oxaliplatin, and the current research shows that besides being used as a molecular probe to detect the distribution of drugs in subcellular organelles, various naphthalimide composite Polyamine analogs can simultaneously target tumor cells through Polyamine Transporters (PTs) highly expressed by the tumor cells, and finally show the anti-tumor growth and transfer activities by regulating and controlling the tumor Polyamine microenvironment and the subcellular organelle functions, reversing DNA damage repair and enhancing the T immunity around tumors.

The inventor finds that the targeted tumor polyamine microenvironment regulation subcellular organelles and nuclear functions through various naphthalimide indole heterocyclic compounds become an effective means for reversing drug resistance and relieving T cell immunosuppression around tumors through tests. Compared with a non-drug-resistant system, the cisplatin can obviously increase the contents of putrescine (Put), spermidine (Spd) and spermine (Spm) in various drug-resistant cell systems, and preliminarily shows that the tumor polyamine microenvironment is closely related to drug resistance. In clinical experiments, it is found that amonafide can show good antitumor activity by targeting DNA damage, one of the reasons of cisplatin resistance is the repair effect after DNA damage, therefore, by designing and synthesizing a naphthalimide indole heterocyclic compound, DNA damage can be enhanced, DNA damage repair reversal drug resistance can be improved, and the synthesized part of the naphthalimide analogue modified by polyamine can target subcellular organelles and cell nucleus by regulating and controlling polyamine steady state, and shows good antitumor growth and transfer activity.

Disclosure of Invention

In view of the above, the present invention aims to overcome the defects in the prior art, and provide a high-stability and high-targeting naphthalimide indole heterocyclic compound, study whether the naphthalimide indole heterocyclic compound has dual inhibitory effects on subcellular organelles and nuclei, and study whether the naphthalimide indole heterocyclic compound can synergistically inhibit the invasion and migration of tumor cells after entering the body.

The invention also provides a preparation method of the naphthalimide indole heterocyclic compound.

The invention further provides application of the naphthalimide indole heterocyclic compound in preparing a tumor treatment medicament.

In order to achieve the purpose, the technical scheme of the invention is realized as follows:

a naphthalimide indole heterocyclic compound has a structural general formula shown in formula I:

x is 1, 2 or 3, R is a chain alkyl group with the carbon number of less than or equal to 20, a cyclic alkyl group with the carbon number of 4-8, a heteroaromatic ring with the carbon number of 5-8, a heteroaromatic ring substituted by the chain alkyl group with the carbon number of 1-4, a non-heteroaromatic ring, a polyamine analogue PA or a hydrogen atom.

Specifically, the Polyamine Analog (PA) is:

wherein m is 0, 1, 2, 3, 4 or 5, n is 0, 1, 2, 3, 4 or 5, and p is 0, 1, 2, 3, 4 or 5.

Specifically, the naphthalimide indole heterocyclic compound is a compound 1b-6b with the following structure:

the preparation method of the naphthalimide indole heterocyclic compound comprises the following steps:

(1) dissolving a compound 8(1, 8-naphthalic acid glucoside) in concentrated sulfuric acid under an ice bath condition to obtain a concentrated sulfuric acid solution of the compound 8, dripping mixed acid prepared from the concentrated nitric acid and the concentrated sulfuric acid into the concentrated sulfuric acid solution of the compound 8 under the ice bath condition, continuously reacting for 2-3h after dripping is finished, pouring reaction liquid into ice water after the reaction is finished, standing, carrying out suction filtration, washing to be neutral, and drying to obtain a filter cake, namely the compound 9 (3-NO) to obtain a filter cake₂-1,8 naphthalenedicarboxylic acid glycoside);

(2) reacting the compound 9 in the step (1) with stannous chloride and concentrated hydrochloric acid at 80-90 ℃ for 1-2h, carrying out suction filtration, washing with water to be neutral, and drying to obtain a compound 10;

(3) mixing the compound 10 and anhydrous acetonitrile under an ice bath condition, dripping mixed solution of chloroacetyl chloride and acetonitrile, reacting completely at room temperature, performing suction filtration and drying, adding chlorocyclohexane into a dry filter cake for dissolving, then quickly adding anhydrous aluminum chloride, reacting for 1-2h at the temperature of 120-; and (4) carrying out condensation reaction and the like on the compound 12a, a polyamine chain and acid anhydride for 24-48 h to obtain a compound 13 with or without a Boc protective group, and removing the Boc protective group with the protective group to obtain a compound 14.

Wherein the structural formula of the compound 10 is

The structural formula of the compound 12a is

The structural formula of the compound 13 is

Compound 14 has the structural formula

Specifically, the solid-to-liquid ratio of the compound 8 to the concentrated nitric acid in the step (1) is (1.6-1.8) to 1 g/mL; the molar ratio of the compound 10 to the chloroacetyl chloride in the step (3) is 1 (1-1.5), and the mass ratio of the compound 10 to the anhydrous aluminum chloride in the step (3) is 1 (2.4-2.6); in the step (4), the molar ratio of the compound 12a to the polyamine chain and the anhydride is 1: (1-2): (2-3).

Specifically, the polyamine chain in the step (4) is the following compound:

the synthetic route of the naphthalimide indole heterocyclic compound is as follows:

wherein R is₄And R₅Respectively correspond to R₁And R₂Form bearing Boc protecting group.

The naphthalimide indole heterocyclic compound is applied to preparing medicaments for treating tumors.

Specifically, the tumor refers to human cervical cancer, human breast cancer, human lung adenocarcinoma, human liver cancer, human colon cancer cell, cisplatin-resistant human lung adenocarcinoma or human prostate cancer.

Specifically, the medicament comprises a naphthalimide indole heterocyclic compound and a pharmaceutically acceptable carrier.

According to the invention, the special stability of the naphthalimide indole heterocyclic compound different from amonafide is utilized, the characteristic that the tumor cells are different from normal cells and the polyamine intake is more is utilized, and the polyamine transport receptors on the surface of the tumor cells are more, so that the problems of poor solubility and the like of the conventional amonafide are solved, and the naphthalimide indole heterocyclic compound is synthesized for the first time by utilizing an organic synthesis means.

The designed and synthesized naphthalimide indole heterocyclic compound can enhance the targeting property of naphthalimide medicines, and simultaneously, the regulation and control of subcellular organelles and nuclear functions by targeting a tumor polyamine microenvironment becomes an effective means for reversing drug resistance and relieving T cell immune suppression around tumors, so that the poisoning effect of platinum medicines by sulfur-containing protein GSH is reduced, the effective platinum content in tumor cells is further improved, the DNA repair proportion is reduced, and the effect of naphthalimide analogues on innate and acquired drug-resistant cells is enhanced.

Compared with the prior art, the invention has the beneficial effects that:

1. the polyamine transport protein with high expression on the surface of the tumor cells is utilized, so that the targeting property of the medicament to the tumor cells is improved, the bioavailability is improved, and the toxic and side effects to normal cells are reduced.

2. The double targeting effects of subcellular organelles and cell nucleuses are realized by modifying the naphthalimide structure.

3. The polyamine-modified naphthalimide heterocyclic compound plays an important role in adjusting the microenvironment of tumor polyamines and playing an important role in treating late metastatic tumors and relieving the immunosuppression around the tumors.

4. Improves the water solubility and fat solubility of naphthalimide medicines and increases the intake of tumor cells to medicines.

5. Through modification of the naphthalimide indole analogue, the stability of the medicament is improved, the half-life period is prolonged, the dosage is reduced, and the maximum tolerance of an organism to the medicament is improved.

6. The effective combination of the medicament and the DNA of tumor cells is improved, and the orally taken antitumor medicament is obtained by utilizing the special stability of the naphthalimide indole analogue superior to the amonafide analogue.

Drawings

FIG. 1 shows the structural formulas of clinical naphthalimide (amonafide, mitonaphthylamine) and platinum drugs (cisplatin, oxaliplatin);

FIG. 2 shows the in vivo antitumor activities of naphthalimide indole heterocyclic compounds 1b and amonafide at different concentrations in liver cancer;

FIG. 3 is the effect of 10 μ M concentration of naphthalimide indole heterocyclic compound 1b and amonafide on the expression of p53 gene and protein in A549cisR cells after 24h incubation;

fig. 4 is a representative HPLC profile and stability test in water of the naphthalimide indole heterocyclic compound 1B, wherein fig. 4A is a representative HPLC profile of the naphthalimide indole heterocyclic compound 1B, and fig. 4B is a stability test in water of the naphthalimide indole heterocyclic compound 1B;

FIG. 5 shows the change of inhibition rate of the naphthalimide indole heterocyclic compound 1B in the presence or absence of Spd (an inhibitor of polyamine transport system highly expressed on the surface of tumor cell membrane) in SMMC-7721 cells (A) and Snu-739 cells (B);

FIG. 6 shows the effect of naphthalimide indole heterocyclic compound 1B on PAO (polyamine oxidase) activity in SMMC-7721 cells (A) and Snu-739 cells (B);

FIG. 7 shows the effect of the heterocyclic compound 1B of naphthalimide indole on the contents of three endogenous small molecules of putrescine (Put), spermidine (Spd) and spermine (Spm) in polyamine cycle in SMMC-7721 cells (A) and Snu-739 cells (B) in a high polyamine microenvironment.

Detailed Description

The technical solution of the present invention is further described in detail by the following specific examples, but the scope of the present invention is not limited thereto.

Unless otherwise specified, each reagent referred to herein is derived from commercially available high purity reagents meeting experimental requirements; in the examples, cis. is cisplatin, oxp. is oxaliplatin and AF is amonafide.

The polyamine analogue modified by the naphthalimide indole parent nucleus is used as an anti-tumor compound, and the design, synthesis and anti-cancer activity test aim at preparing anti-cancer active molecules with broad spectrum, high efficiency and low toxicity, and provides a novel candidate drug for clinical cancer treatment. The test results of the invention show that the naphthalimide analogue has better activity on tumor growth and metastasis.

The invention introduces classic polyamine analogue fragments such as DENSpm and the like entering into clinical research stage into the existing classic naphthalimide mother nucleus, designs and synthesizes a series of naphthalimide indole analogues for the first time, characterizes the structure of a target compound, tests the in vitro and in vivo anti-tumor activity of the target compound, and has the following main research contents:

the design idea of the monofunctional naphthalimide indole nucleus modified polyamine analogue 1b-6b is as follows:

1) the polyamine transport Protein (PAT) is highly expressed on the surface of the tumor cell membrane, and the structure of polyamine analogues is introduced into target molecules, so that the targeting property can be enhanced. The polyamine analogue modified by the naphthalimide indole parent nucleus is introduced into the structure of the polyamine analogue, so that the targeting property is enhanced, and simultaneously, the water solubility and the stability of the compound are enhanced, thereby overcoming the defects of poor solubility, poor stability, low in-vivo utilization rate and the like of parent nucleus cisplatin.

2) Clinical experimental studies have shown that small differences in polyamine structures of different chain lengths can have a significant impact on their biological function. Therefore, the invention introduces different polyamine analogue star molecules which enter clinical stages into a target molecule system to synthesize 1b-6b respectively, and aims to explore the influence of different polyamine structures on the activity of polyamine analogues modified by a naphthalimide indole parent nucleus.

The method for synthesizing the naphthalimide parent nucleus is well known to those skilled in the art, and the specific synthetic method is shown in the literature: ma J, Li Y, Li L, Yue K, Liu H, Wang J, Xi Z, Shi M, ZHao S, Ma Q, Liu S, Guo S, Liu J, Hou L, Wang C, Wang PG, Tian Z, Xie S.A Polyamine-Based Dinitro-naphthalene polymers as subsystems for Polyamine Transporters Preferentially accuumulates in Cancer Cells and minimetries silicon Effects in vitro and in vivo.front chem.2020,8,166.doi: 10.3389/fchem.2020.2020.00166.

Example 1: 1b

¹H NMR(300MHz,DMSO-d6)δ8.71(s,1H),8.24(s,1H),8.06(s,1H),7.75(s,1H),4.26(d,4H), 2.94(t,J＝27.2Hz,6H),2.01(d,J＝24.3Hz,4H).¹³C NMR(75MHz,D₂O)δ167.17,164.14, 163.39,135.27,134.36,134.00,131.51,130.29,127.77,123.66,121.97,120.24,45.36,44.65,44.55, 43.07,36.51,24.13,23.76.ESI-MS(positive ion mode)m/z[M-H]⁺calcd 365.19, obsd 365.19, the preparation method is as follows:

(1) adding 100mmol (19.8g) of compound 8(1, 8-naphthalenedicarboxylic acid glucoside) and 100mL of concentrated sulfuric acid into a 250mL volumetric flask, stirring and dissolving under an ice bath condition, measuring 11.2mL of concentrated nitric acid and 50mL of concentrated sulfuric acid to prepare mixed acid, dripping into the concentrated sulfuric acid solution of the compound 8 under the ice bath condition, continuing to react for 2.5 hours after dripping, pouring the reaction solution into ice water after the reaction is finished, standing, filtering, washing to be neutral, and drying to obtain a filter cake, namely the compound 9 (3-NO)₂-1,8 naphthalenedicarboxylic acid glycoside);

(2) placing the dried compound 9 in the step (1) in a 500mL flask, adding 110g of stannous chloride and 150mL of concentrated hydrochloric acid, reacting at 85 ℃ for 2h, performing suction filtration, cleaning with 5% hydrochloric acid by mass fraction, washing with water to neutrality, and drying to obtain a compound 10a (3-NH)₂-1,8 naphthalenedicarboxylic acid glycoside).

(3) Placing 10mmol (2.14g) of compound 10a in a 250mL volumetric flask, adding 100mL of anhydrous acetonitrile, dropwise adding a mixed solution of chloroacetyl chloride 15mmol (1.19mL) and acetonitrile (about 5mL) under ice bath conditions, reacting at room temperature overnight, performing suction filtration and drying, placing a dried filter cake in a 500mL volumetric flask, adding 150mL of chlorocyclohexane to dissolve the chlorocyclohexane, then quickly adding 5.34g of anhydrous aluminum chloride, reacting at 125 ℃ for 2h, pouring a reaction solution into ice water containing hydrochloric acid (about 250mL of water is added with 5mL of hydrochloric acid) after the reaction is finished, performing suction filtration and drying to obtain a compound 12 a.

(4) Reaction of Compound 12a (1mmol) with a polyamine chain

And (2mmol) and acid anhydride (3mmol) are subjected to condensation reaction for 24-48 h to obtain a compound 1b-1 with or without a Boc protective group, and the Boc protective group is removed to obtain a compound 1 b.

The reaction formula of the synthetic route of the compound 1b is as follows:

example 2: 2b

¹H NMR(300MHz,DMSO-d6)δ8.69(d,J＝14.3Hz,2H),8.36(s,1H),7.82(s,1H),4.42(s,2H), 4.11(s,2H),3.11–2.70(m,6H),2.05(s,2H),1.79–1.41(s,4H).¹³C NMR(75MHz,D₂O)δ 167.33,164.43,163.73,135.40,134.49,130.70,130.32,127.63,123.14,122.53,121.86,120.31, 119.78,46.99,45.24,43.07,38.74,37.35,24.18,23.90,22.77.ESI-MS(positive ion mode)m/z [M+HCl]⁺calcd 417.10,obsd 417.10.

The method for preparing compound 2b in example 2 differs from example 1 in that compound 12a (1mmol) is reacted with a polyamine chain in step (4)

And (2mmol) and acid anhydride (3mmol) are subjected to condensation reaction for 24-48 h to obtain a compound with or without a Boc protective group, and the Boc protective group is removed to obtain a compound 2 b.

The reaction formula of the 2b synthetic route of the polyamine analogue modified by the naphthalimide indole parent nucleus is as follows:

example 3: 3b

¹H NMR(300MHz,DMSO-d6)δ10.66(s,J＝14.3Hz,1H),8.36(s,1H),8.81(s,1H),7.97(s,1H), 7.78(s,1H),4.10(s,2H),4.07(s,2H),2.68(s,2H),2.53(m,8H),1.78(s,2H),1.74(s,2H),1.5(s, 3H),1.38(s,4H).¹³C NMR(75MHz,D₂O)δ176.6,159.3,159.3,145.2,132.0,131.6,129.5,128.1, 124.3,123.6,123.0,117.1,116.4,56.1,49.6,49.6,46.0,46.0,39.4,34.6,31.9,28.6,25.3,25.3. ESI-MS(positive ion mode)m/z[M]⁺calcd 437.24,obsd 437.24.

The preparation of compound 3b in example 3 differs from that of example 1 in that compound 12a (1mmol) is reacted with a polyamine chain in step (4)

And (2mmol) and acid anhydride (3mmol) are subjected to condensation reaction for 24-48 h to obtain a compound with or without a Boc protective group, and the Boc protective group is removed to obtain a compound 3 b.

The reaction formula of the 3b synthetic route of the polyamine analogue modified by the naphthalimide indole parent nucleus is as follows:

example 4: 4b

¹H NMR(300MHz,DMSO-d₆)δ9.01(s,1H),8.64(s,1H),8.19(s,1H),7.82(s,1H),4.01(s,2H), 2.88(s,8H),1.67(s,8H),1.25(d,J＝46.0Hz,3H).¹³C NMR(75MHz,D₂O)δ164.12,163.59, 134.42,131.75,130.95,129.60,128.29,125.55,124.78,122.72,120.38,47.11,46.86,39.64,38.73, 29.49,24.05,23.84,23.08,22.68.ESI-MS(positive ion mode)m/z[M-H]⁺calcd 393.19,obsd 393.19.

The method for preparing compound 4b in example 4 differs from example 1 in that compound 12a (1mmol) and the polyamine chain are reacted in step (4)

And (2mmol) and acid anhydride (3mmol) are subjected to condensation reaction for 24-48 h to obtain a compound with or without a Boc protective group, and the Boc protective group is removed to obtain a compound 4 b.

The reaction formula of the 4b synthetic route of the polyamine analogue modified by the naphthalimide indole parent nucleus is as follows:

example 5: 5b

¹H NMR(300MHz,Deuterium Oxide)δ7.86(s,1H),7.75(s,1H),7.68(s,1H),7.46(d,J＝0.9Hz, 1H),4.27(s,2H),3.85(m,J＝6.5Hz,2H),3.62(d,J＝7.1Hz,10H),2.15–1.94(m,6H),1.14(t,J ＝7.1Hz,4H).¹³C NMR(75MHz,D₂O)δ168.78,166.10,165.54,135.78,132.97,132.31,126.04, 125.91,125.57,124.26,123.69,121.81,59.01,46.30,38.14,38.10,25.80,25.38,25.33,24.38,24.29, 18.38.ESI-MS(positive ion mode)m/z[M+HCl]⁺calcd 460.12,obsd 460.12.

The compound 5b of example 5 was prepared by a method different from that of example 1 in that, in the step (4), the compound 12a (1mmol) and the polyamine chain were bonded

And (2mmol) and acid anhydride (3mmol) are subjected to condensation reaction for 24-48 h to obtain a compound with or without a Boc protective group, and the Boc protective group is removed to obtain a compound 5 b.

The reaction formula of the 5b synthetic route of the polyamine analogue modified by the naphthalimide indole parent nucleus is as follows:

example 6: 6b

¹H NMR(300MHz,Deuterium Oxide)δ7.72(s,1H),7.43(s,1H),7.35(s,1H),7.24(s,1H),4.26 (s,2H),3.69(d,2H),3.13(m,J＝7.7Hz,4H),2.09(d,J＝7.8Hz,2H),1.64(m,J＝36.0Hz,6H).¹³C NMR(75MHz,D₂O)δ169.38,166.15,165.42,133.65,132.26,129.95,125.71,124.77,124.08, 122.36,122.10,121.66,49.61,46.87,46.77,41.89,38.89,38.83,26.05,25.57,25.44.ESI-MS (positive ion mode)m/z[M+HCl]⁺calcd 417.08,obsd 417.08.

The preparation of compound 6b in example 6 differs from that of example 1 in that in step (4), compound 12a (1mmol) is reacted with a polyamine chain

And (2mmol) and acid anhydride (3mmol) are subjected to condensation reaction for 24-48 h to obtain a compound with or without a Boc protective group, and the Boc protective group is removed to obtain a compound 6 b. The 6b synthetic route of the polyamine analogue modified by the naphthalimide indole parent nucleus has the reaction formula:

test example 1: evaluation of target molecule biological Activity

(1) In vitro antitumor Activity test

The cancer cell lines selected for this test included: human cervical cancer cells (Hela), human breast cancer cells (MDA-MB-231), human breast cancer cells (MCF-7), human lung adenocarcinoma cells (A549), human liver cancer cells (SMMC7721), human colon cancer cells (HCT-116), cisplatin-resistant human lung adenocarcinoma cells (A549cisR), human prostate cancer cells (PC3), human prostate cancer cells (DU 145).

The test method comprises the following steps: 100 μ L of cell suspension was added to a 96-well plate, the cell density was controlled at 3000-. After 24 hours of incubation in the 37 ℃ cell incubator, 100. mu.L of a compound medium solution having a gradient concentration (0.2, 0.6, 1.3, 3.2, 7.5, 17.8, 42.2, 100, unit. mu.M) was added to the 96-well plate, and the cell incubator was further incubated at 37 ℃ for 48 hours. 20. mu.L of 5mg/mL MTT solution was added to each well of a 96-well plate, and the plate was taken out after culturing in a 37 ℃ cell incubator for 4 hours, the medium was aspirated, and 150. mu.L DMSO was added thereto, and the plate was shaken in a shaker at 37 ℃ for 20min in the dark. The absorbance of each well was measured at 470nm with a microplate reader, and the IC thereof was calculated₅₀Values, which were repeated at least three times for each set of experiments, were measured as shown in table 1. Three positive controls, Amonafide (AF), cisplatin (Cis), and oxaliplatin (Oxp.) were added to the cells.

TABLE 1 naphthalimide indole parent nucleus modified polyamine analogs 1b-6b (IC)₅₀Unit: μ M) cytotoxicity profile (RF) in human tumor cell lines^a:Resistant factor＝IC₅₀(A549cisR)/IC₅₀(A549))

The in vitro anti-tumor activity test result of table 1 shows that 1b-6b of the polyamine analogue modified by the naphthalimide indole nucleus shows obviously better anti-tumor activity than amonafide, the polyamine ligands with different structures have obvious influence on the anti-tumor activity of the compound, the polyamine analogue shows better activity on various tested tumor cells, and the polyamine analogue modified by the naphthalimide indole nucleus shows better activity on cis-platinum-resistant A549cisR cells and has no cross resistance with cis-platinum, the RF value is between 0.70 and 0.93, and the polyamine analogue has greater advantages compared with 3.02 of the cis-platinum. Wherein, the compound 1b,5b has better effect on SMMC7721, Hela, MDA-MB-231, MCF-7, HCT-116, A549cisR than positive drugs of amonafide, cisplatin and oxaliplatin. Particularly, the lead compound 1b achieves the nmol level on MCF-7 and HCT-116, and shows obviously better positive activity.

(2) In vitro normal cytotoxicity assay

Mouse embryonic fibroblasts (3T3) and human normal hepatocytes (HL-7702) required for the test were purchased externally, and the killing ability of lead compound 1b against normal cells was measured by the above MTT method.

TABLE 2 Effect of 1b of naphthalimide indole core-modified polyamine analogs on the viability of cancer cells and normal cells comparable thereto (IC)₅₀Unit: μ M).

SI(selectivity index)is defined as IC₅₀ in HL-7702/IC₅₀ in SMMC-7721.

From the test results in table 2, it can be seen that, in addition to the great difference in the antitumor activity of the naphthalimide indole nucleus-modified polyamine analog 1b, the difference between the naphthalimide complexes with different structures and normal hepatocytes and 3T3 cells is also great, the SI value of the compound 1b is 6.39, which is 3-27 times that of amonafide and the clinical common chemotherapeutic drugs cisplatin and oxaliplatin, respectively, and the compound shows lower toxicity to normal cells in vitro.

Test example 2: in vivo antitumor activity of naphthalimide indole heterocyclic compound 1b and amonafide with different concentrations in liver cancer

Taking mouse HCC liver cancer cells in logarithmic phase, digesting, centrifuging, washing the cells with sterile PBS buffer solution, collecting the cells after centrifuging, and resuspending with glucose injection. After staining with trypan blue solution, 10. mu.L of the cell suspension was aspirated by a pipette gun and counted in a cell counting plate to calculate the number of viable cells. Diluting the cell concentration to 10 according to a certain proportion⁶mu.L, the right underarm skin of the experimental mice was disinfected with medical alcohol, and 200. mu.L of cell suspension was injected. Seven days later, tumor-inoculated mice were randomly divided into groups including a positive control group, a 3mg/kg compound 1b group, a 5mg/kg compound 1b group, and a 5mg/kg amonafide group. The drug was administered seven times a day, and seven days after the end of the administration, the drug was weighed daily, and the tumor volume was measured. Seven days later the mice were sacrificed by dislocation, the orthotopic tumors and various organs were dissected out and recorded by weighing.

In fig. 2, a) relative weights of PBS, amonafide, and group 1b mice; B) tumor weights in PBS, amonafide and group 1b at the end of the experiment; C) tumor images of PBS, amonafide, and 1b groups at the end of the experiment, first row, PBS control group; second line, amonafide group (5 mg/kg); third row, 1b (3 mg/kg); fourth line, 1b (5 mg/kg); D) at the end of the experiment, organ weight indices (organ weight/body weight) x 100% were calculated for PBS, amonafide group, and group 1b, including heart, liver, kidney, lung, and spleen. P < 0.001.

The experimental result of fig. 2 shows that the inhibition rate (57.97%) of the lead compound 1b on the hepatoma carcinoma cells under the dosage of 3mg/kg is better than that of the positive control group by 5mg/kg (53.27%), while the inhibition rate (65.90%) of the lead compound under the dosage of 5mg/kg is obviously better than that of the positive drug amonafide, and the lead compound shows better anti-tumor activity in vivo.

Test example 3: influence of naphthalimide indole heterocyclic compound 1b and amonafide (15 mu M, fostering for 24 hours) on expression of p53 gene and protein in A549cisR cell

Western Blotting experiment for detecting influence of target compound on p53 protein content

1) Selecting proper separation and concentration gel according to the molecular weight of the antibody protein;

2) pouring the prepared 1X electrophoresis solution into a groove of a glass plate, and pulling out a sample comb;

3) loading is carried out according to the calculation of the previous protein loading volume, and 3 μ l of Marker is added to each lane after the first sample and the last sample;

4) after the sample loading is finished, putting the glass plate into an electrophoresis tank, covering a cover, and adjusting the voltage to 80V;

5) when the marker runs to the bottom of the separation gel, the electrophoresis apparatus is switched off;

6) preparing 1 multiplied by membrane transferring liquid, placing the membrane transferring liquid in a refrigerator for precooling, wherein the preparation method of the membrane transferring liquid comprises 100ml of 10 multiplied by membrane transferring liquid, 200ml of methanol and 700ml of distilled water, pouring part of the membrane transferring liquid into a tray, cutting glue in the tray, cutting off concentrated glue, activating a PVDF membrane in the methanol in advance, covering the PVDF membrane on the glue, placing the glue and the PVDF membrane on a membrane transferring clamp, and placing three layers of filter paper up and down;

7) pouring a film transferring liquid into the film transferring groove, placing a film transferring clamp into the film transferring groove, covering a cover, adjusting the current to be 200mA, and adjusting the film transferring time to be two hours;

8) after the membrane transfer is finished, preparing a 1 xTBST solution, separating the membrane from the gel, washing the membrane in the 1 xTBST solution once, transferring the membrane into a skimmed milk powder solution, and placing the membrane on a shaking bed to seal the membrane for one hour at a constant speed;

9) preparing an anti-solution, according to the antibody: preparing an anti-solution by the ratio of the milk powder solution to 1:1000, and dripping the solution into an antibody box;

10) attaching a PVDF membrane to the antibody solution, removing bubbles, placing in a refrigerator at 4 ℃ and standing overnight;

11) the next day, recovering the primary antibody solution, preparing a 1 xTBST solution, washing the PVDF membrane with the 1 xTBST solution for three times, ten minutes each time, selecting a proper secondary antibody solution for preparation, and incubating the PVDF membrane at room temperature for about 2 hours;

12) after the time is over, washing the PVDF membrane with 1 xTBST solution for three times, mixing the PVDF membrane with a Beyo ECL developing solution according to the proportion of 1:1, and spraying the PVDF membrane with the developing solution;

13) and (5) exposing and developing.

The beta-actin internal reference used in the test is a protein standard value, and the specific protein content is obtained by comparing the protein standard value with the specific protein content.

FIG. 3, A) Effect of 1b at a concentration of 10. mu.M on the expression of p53 protein in hepatoma cells; B) effect of 1b at a concentration of 10 μ M on p53 gene expression in hepatoma cells.

From fig. 3, it can be seen that compared with the positive drug amonafide, 1b can significantly up-regulate the content of p53 protein, and has concentration dependence, indicating that the apoptosis pathway of cell nucleus plays an important role in 1b anti-tumor mechanism.

Test example 4: naphthalimide indole heterocyclic compound 1b representative HPLC pattern and stability test in water

And (3) respectively culturing the aqueous solution of the naphthalimide indole heterocyclic compound 1b in a constant-temperature shaking incubator at 37 ℃ for 24h, 48h and 72h, and detecting the HPLC (high performance liquid chromatography) spectrum and the stability of the medicament in water by adopting HPLC. Wherein HPLC adopts the model of Waters E2695-2998, Venusil MP C18 col mu Mn chromatographic column, HRMS detects the product peak therein, and ESI ion source. The mobile phase conditions for the high performance liquid phase are shown in table 3.

Table 3 mobile phase conditions for high performance liquid phase of naphthalimide indole heterocyclic compound 1 b.

Fig. 4A is a representative HPLC profile of a naphthalimide indole heterocyclic compound 1B, fig. 4B is a stability test of the naphthalimide indole heterocyclic compound 1B in water, and the results of fig. 4B show that compound 1B has higher stability in water.

Test example 5: change in inhibition Rate of Naphthylimidinoindole heterocyclic Compound 1b in the Presence of Spd (inhibitor of polyamine transport System highly expressed on tumor cell Membrane surface)

In tumorsDuring cell culture, a certain dosage of PAT transporter inhibitor is added to study the cell inhibition rate and IC₅₀A change in value. The specific method comprises the following steps:

human hepatoma cells SMMC-7721(5 x 10) to be assayed in logarithmic growth phase⁵Per well) and human hepatoma cells Snu-739 (2 x 10)⁵/well) cells were plated in six-well plates and placed at 37 ℃ with 5% CO₂The culture box is used for culturing, cells grow in an adherent manner, then positive reference drugs of amonafide and a target compound 1b with different concentrations are respectively added into a 96-well plate, meanwhile, when a spermidine group is added, the same dose of 50 mu M spermidine is simultaneously added, aminoguanidine (1mM) is added at the same time of adding spermidine to prevent interference of serum to drugs, and after the time of acting the same as the drug group, MTT is adopted to test the inhibition rate and IC of the spermidine group₅₀As a control, an equal amount of medium was added at the same time without the spermidine addition group.

As shown in figure 5, in the presence of an inhibitor Spd of PAT transporter, the inhibition rate is greatly changed, which proves that polyamine transporter is involved in transmembrane transport of naphthalimide indole heterocyclic compound 1b and has different selectivity for possible products of different tumor cells.

Test example 6: effect of Naphthylimidinoindole heterocyclic Compound 1b on PAO (polyamine oxidase) Activity in liver cancer cells

Detecting the influence of the naphthalimide indole heterocyclic compound 1b on the PAO (polyamine oxidase) activity in the liver cancer cells by adopting the PAO (polyamine oxidase) activity and detection kit;

the tumor microenvironment plays a very important role in the development of tumors, and the research on the tumor microenvironment has become a part of important attention in the research and development of anti-tumor drugs at present, wherein the polyamine microenvironment of tumor cells plays a very important role, and in the tumor polyamine cycle, the enzymes playing a key role in the synthesis and metabolism of polyamines are mainly ODC, SSAT and PAO. Wherein PAO plays a key role in polyamine metabolism, and the experimental result of figure 6 shows that the naphthalimide indole heterocyclic compound 1b can obviously up-regulate the PAO content, indicating that the naphthalimide indole heterocyclic compound plays a crucial role in interfering the tumor microenvironment.

Test example 7: influence of naphthalimide indole heterocyclic compound 1b on contents of three endogenous small molecules of putrescine (Put), spermidine (Spd) and spermine (Spm) in polyamine circulation in high polyamine microenvironment of tumor cells

(1) Reagent for HPLC and method for preparing solution

Hydrochloric acid solution (final concentration 0.1 mol/L): sucking 9ml of concentrated hydrochloric acid, slowly dripping the concentrated hydrochloric acid into 900ml of triple distilled water, and uniformly mixing for later use;

hexamethylenediamine hydrochloride standard solution (final concentration 17.76. mu.g/ml): weighing 1.776mg of hexamethylenediamine by a precision balance, adding into a 100ml volumetric flask for later use, adding the prepared hydrochloric acid solution, uniformly mixing, and storing in dark place;

putrescine hydrochloride standard solution (final concentration 121. mu.g/ml): weighing 12.1mg of putrescine by using a precision balance, adding the putrescine into a volumetric flask of 100ml for later use, adding the prepared hydrochloric acid solution, uniformly mixing, and storing in a dark place;

spermine hydrochloric acid standard solution (final concentration 152. mu.g/ml): weighing 15.2mg of spermine by using a precision balance, adding into a 100ml volumetric flask for later use, adding the prepared hydrochloric acid solution, uniformly mixing, and storing in a dark place;

spermidine hydrochloride standard solution (final concentration 116. mu.g/ml): weighing 11.6mg of spermidine by using a precision balance, adding into a 100ml volumetric flask for later use, adding the prepared hydrochloric acid solution, uniformly mixing, and storing in a dark place;

dansyl chloride-acetone solution (final concentration 2 mg/ml): weighing dansyl chloride 12mg by a precision balance, and adding into 6ml of acetone solution for later use. The mixture is uniformly mixed and stored in dark, and the best preparation is realized when the mixture is used;

perchloric acid solution (final concentration 20%): weighing 40ml of perchloric acid solution, slowly dropwise adding the perchloric acid solution into a 200ml volumetric flask containing 160ml of triple distilled water, and mixing for later use;

saturated sodium carbonate solution: weighing excessive anhydrous sodium carbonate powder in a 50ml volumetric flask, adding 50ml distilled water, placing in a water bath at 50 ℃ for heating until the anhydrous sodium carbonate powder is completely dissolved, and placing at room temperature for cooling until crystals are separated out for later use.

(2) Selection of polyamine high performance liquid chromatography analysis conditions:

(3) sample treatment and preparation:

a) taking out cells, digesting and centrifuging, collecting the cells after 3000 revolutions for 10 minutes, pouring out the culture medium, washing the cells by using a PBS solution, centrifuging to collect the cells, centrifuging, and adding 1ml of the PBS solution;

b) to the EP tube, 200. mu.l of 20% perchloric acid was added;

c) after the protein is denatured, putting the protein into a centrifugal machine for centrifugation, rotating the protein for 10 minutes at 3000, and taking supernatant;

d) taking 200 mul of polyamine standard mixed solution into an EP tube, and adding 200 mul of internal standard hexamethylene diamine;

e) weighing 200 mu l of dansyl chloropropone solution, adding and mixing, and accurately adjusting the pH value to 9.5 by using a saturated sodium carbonate solution;

f) and (3) derivatization reaction: carrying out constant-temperature water bath at 50 ℃ and carrying out light-resistant reaction for 30 min;

g) cooling the sample to room temperature;

h) adding 200 μ l of chromatographic grade ethyl acetate, mixing, extracting, and collecting supernatant;

i) the samples were filtered through a microfiltration membrane, and 20. mu.l of each sample was injected and the chromatogram recorded.

(4) Determination of polyamine standard curve:

preparation of mixed polyamine standard solution: preparing polyamine standard solution according to the method, wherein the polyamine standard solution comprises putrescine standard solution, spermine standard solution and spermidine standard solution, and the concentrations of the putrescine standard solution, the spermine standard solution and the spermidine standard solution are respectively 121 mu g/mL, 152 mu g/mL and 116 mu g/mL; respectively weighing 200 mul of spermine standard solution, 400 mul of putrescine standard solution and 200 mul of spermidine standard solution, mixing to obtain No. 1 polyamine mixed standard solution, and diluting the No. 1 mixed polyamine standard solution by using the prepared hydrochloric acid standard solution in a gradient manner by 2 times, 4 times, 8 times, 16 times, 32 times and 64 times respectively to obtain No. 2, No. 3, No. 4, No. 5, No. 6 and No. 7 mixed polyamine standard solution. The concentrations are shown in Table 4.

TABLE 4 polyamine concentrations in the Standard solutions

The results of fig. 7 show that the naphthalimide indole heterocyclic compound 1b can obviously reduce the content of three key endogenous small molecules Put, Spd and Spm in a tumor polyamine microenvironment, which indicates that the antitumor activity mediated by the compound 1b is closely related to the reduction of the tumor polyamine microenvironment, and the latest research shows that the tumor polyamine microenvironment is closely related to tumor immune escape and the expression of a tumor important immune checkpoint PD-1, so that the naphthalimide indole heterocyclic compound 1b plays an important role in relieving tumor immune suppression besides enhancing the targeting property to tumor cells and overcoming drug resistance.

In conclusion, the synthesized compound has good antitumor activity, the in vivo and in vitro antitumor activity of the compound is better than that of amonafide, cisplatin and oxaliplatin, and the stability is better. Compared with the classical naphthalimide compound amonafide, the polyamine-modified naphthalimide indole heterocyclic compound has important regulation effects on the tumor high polyamine microenvironment key enzyme PAO and the polyamine content Put, Spd and Spm, so that the tumor and the surrounding microenvironment participate in the antitumor effect of the naphthalimide indole heterocyclic compound, the regulation effects on metastatic tumors and the release of tumor surrounding immunosuppression are realized, the in vivo utilization degree is improved, the curative effect is enhanced, and the toxic and side effects of the traditional chemotherapeutic drugs on spleen, kidney and the like are reduced.

Claims

1. A naphthalimide indole heterocyclic compound, wherein the structural formula of the naphthalimide indole heterocyclic compound is as follows:

2. the use of the naphthalimide indole heterocyclic compound of claim 1 in the preparation of a medicament for the treatment of tumors.

3. The use of claim 2, wherein said tumor is human cervical cancer, human breast cancer, human lung adenocarcinoma, human liver cancer, human colon cancer cells, cisplatin-resistant human lung adenocarcinoma, or human prostate cancer.

4. The use according to claim 2, characterized in that the medicament comprises a naphthalimide indole heterocyclic compound and a pharmaceutically acceptable carrier.