CN112876414B - Polyamine-modified naphthalimide conjugate, and preparation method and application thereof - Google Patents
Polyamine-modified naphthalimide conjugate, and preparation method and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of pharmaceutical chemistry, and particularly relates to a polyamine-modified naphthalimide conjugate, and a preparation method and application thereof. The naphthalimide-polyamine conjugate has good anti-tumor activity, and has better in-vivo and in-vitro anti-liver cancer growth and metastasis activity than amonafide, and wider anti-tumor spectrum. Different from classical amonafide, the conjugate provided by the invention inhibits autophagy through targeting lysosomes, and regulates and controls polyamine metabolism and functions in a tumor cell microenvironment to play roles in resisting liver cancer growth and metastasis. 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 provides a new thought and a new research direction for treating late-stage liver cancer by finding the naphthalimide complex capable of regulating and controlling subcellular organelles and tumor microenvironment for the first time.
Description
Technical Field
The invention belongs to the technical field of pharmaceutical chemistry, and particularly relates to a polyamine-modified naphthalimide conjugate, and a preparation method and application thereof.
Background
Hepatocellular carcinoma (HCC) is a malignant tumor which is found late and has high lethality, and accounts for the sixth part of the malignant tumors, about 90 percent of liver cancer patients cannot survive for more than 5 years after operation, and although the progress is made in the combined cancer treatment technology, the current anti-cancer drugs cannot solve the problems of relapse and metastasis after liver cancer resection.
In recent years, in addition to physical properties, the biological activity of naphthalimides as a multifunctional antitumor active compound has been increasing. As the intercalation of the planar aromatic rings in the structure between DNA base pairs can distort the DNA backbone conformation and interfere the interaction of DNA-protein so as to play a role in tumor resistance, the naphthalimide represented by amonafide and the derivative thereof become a good anti-tumor DNA intercalator.
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 isoprostylamine (BENSpm), bisethyl spermine (BESpm), bisethyl homoprostylamine (BEHSpm). The compounds can be recognized and absorbed by PTS, inhibit polyamine synthetase ODC and S-AdeMetDC, and simultaneously up-regulate SSAT activity, thereby rapidly consuming intracellular polyamine to inhibit tumor proliferation and differentiation. 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. Recent researches show that the polyamine conjugate has a good effect in resisting tumor metastasis, and the targeted liver cancer and liver cancer metastasis effects of the polyamine-modified naphthalimide conjugate are not reported.
The current research shows that besides being used as a molecular probe to detect the distribution of the medicine in subcellular organelles, a plurality of naphthalimide composite Polyamine analogs can simultaneously target tumor cells through Polyamine Transporters (PTs) highly expressed by the tumor cells, and show the anti-tumor growth and transfer activities by regulating and controlling the tumor Polyamine microenvironment and subcellular organelle functions, reversing DNA damage repair and enhancing the T immunity around tumors. Therefore, the polyamine-modified naphthalimide conjugate targets a tumor high polyamine microenvironment to regulate subcellular organelles and nuclear functions, and becomes an effective means for reversing drug resistance and relieving T cell immunosuppression around tumors. Compared with a non-drug-resistant system, the cisplatin can obviously increase the contents of putrescine (Put), spermidine (Spd) and spermine (Spm) of various drug-resistant cell systems, and preliminarily shows that the tumor high 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-polyamine 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 nucleuses by regulating and controlling polyamine steady state, show good antitumor growth and transfer activity, and simultaneously enhance antitumor immune function.
Disclosure of Invention
In view of this, the present invention aims to overcome the defects in the prior art, and provide a polyamine-modified naphthalimide conjugate with high stability and high targeting property, so as to study whether the naphthalimide-polyamine conjugate has a dual inhibition effect on subcellular organelles and cell nuclei, and further study whether the naphthalimide-polyamine conjugate can synergistically inhibit invasion and migration of tumor cells after entering into a body.
The invention also provides a preparation method of the polyamine modification-based naphthalimide conjugate.
The invention further provides application of the polyamine-modified naphthalimide conjugate in preparing tumor treatment medicines.
In order to achieve the purpose, the technical scheme of the invention is realized as follows:
a polyamine-modified naphthalimide conjugate has a structural general formula shown in formula I or formula II:
wherein R is 1 And R 2 Is a single ligand or a complex ligand, and R 1 、R 2 The same or different, is a chain alkyl with the carbon number less than or equal to 20, a cyclic alkyl 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 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, p is 0, 1, 2, 3, 4 or 5, R 3 And R4 are the same or different and are respectively and independently selected from one of H, methyl, ethyl or propyl.
Specifically, the polyamine-modified-based naphthalimide conjugate is a compound with the following structure:
the preparation method of the polyamine-modified-based naphthalimide conjugate (8a-8d) comprises the following steps:
(1) reacting the compound 2a (3-NH) 2 -1,8 Naphthalenedicarboxylic acid glycoside) or 2b (4-NH) 2 -1, 8-naphthalenedicarboxylic acid glycoside) and K 2 CO 3 Is placed in CH 3 Adding polyamine compound into CN under ice bath condition
Reacting the acetonitrile solution at 80-85 ℃ for 4-5h, cooling to room temperature after the reaction is finished, and concentrating and separating to obtain an intermediate compound I;
(2) dissolving the intermediate compound I in Dichloromethane (DCM), adding TEA and chloroacetyl chloride under the ice bath condition, reacting for 1.5-2h, detecting by TLC, and separating to obtain an intermediate compound II;
(3) the separated intermediate compound II and NaN 3 Dissolving the intermediate compound in N, N-Dimethylformamide (DMF), reacting at 60-70 ℃ overnight to obtain an intermediate compound III, reducing the intermediate compound III to obtain an intermediate compound IV, and reacting the intermediate compound IV with HCl in a methanol solution for deprotection to obtain the target compound.
Wherein the content of the first and second substances,R 3 and R4 are the same or different and are respectively and independently selected from one of H, methyl, ethyl or propyl.
Specifically, in the step (1), the compound 2a or 2b is reacted with K 2 CO 3 The molar ratio of the polyamine compound is 1 (3-5): 1; in the step (2), the molar ratio of the intermediate compound I to TEA and chloroacetyl chloride is 2: 1: (2-3); the intermediate compound II and NaN in the step (3) 3 The molar ratio of (1) to (2-3).
Further, the preparation method of the compound 2a or 2b in the step (1) is as follows:
dissolving the compound 1a or 1b in concentrated sulfuric acid under an ice bath condition to obtain a concentrated sulfuric acid solution of the compound 1a or 1b, dripping mixed acid prepared from concentrated nitric acid and concentrated sulfuric acid into the concentrated sulfuric acid solution of the compound 1a or 1b 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 with water to be neutral, drying to obtain a filter cake, reacting the filter cake with stannous chloride and concentrated hydrochloric acid at the temperature of 80-85 ℃ for 1-2h, carrying out suction filtration, washing with water to be neutral, and drying to obtain the compound 2a or 2 b.
Specifically, in the preparation of the compound 2a or 2b, the solid-to-liquid ratio of the compound 1a or 1b to concentrated nitric acid is (1.6-1.8):1 g/mL.
The synthesis route of the target compound (8a-8d) is as follows:
the synthesis route of the target compound (13a-13c) is as follows:
the synthetic route of the target compounds (17 and 21) is as follows:
specifically, polyamine compounds
Reference (a) Wang, y.x.; zhang, x.; zhao, j.; xie, s.q.; wang, C.J. synthetic and biological evaluation.J. Med.chem.2012,55, 3502-3512; b) dai, f.j.; li, Q.; wang, y.x.; ge, c.c.; feng, C.; xie, s.q.; he, h.; xu, x; wang, C.J.design, synthesis, and biological evaluation of mitophornda-targeted flavone-polyamine conjugates with immunological activity.J.Med.chem.2017,60, 2071-; c) li, J.; tian, R.; ge, C.; chen, y.; liu, x.; wang, y.; yang, y.; luo, w.; dai, f.; wang, s.; chen, s.; xie, s.; wang, C.discovery of the polyamine conjugate with benzol [ cd]indole-2 (1H) -one as a lysosometargeted anti-inflammatory agent.J.Med.chem.2018,61, 6814-6829.).
The polyamine-modified naphthalimide conjugate is applied to preparing tumor treatment medicines.
Specifically, the tumor refers to human breast cancer, human liver cancer or human lung cancer.
Specifically, the medicament comprises a polyamine-modified-based naphthalimide conjugate and a pharmaceutically acceptable carrier.
The invention utilizes the special stability of the naphthalimide compound different from amonafide, simultaneously utilizes the characteristic that tumor cells are different from normal cells and have more polyamine intake and uses polyamine transport receptors on the surface of the tumor cells to solve the problems of poor solubility and the like of the conventional amonafide, and firstly utilizes an organic synthesis means to synthesize the naphthalimide conjugate based on polyamine modification.
The designed and synthesized naphthalimide conjugate can enhance the targeting property of naphthalimide medicines, and simultaneously, the regulation of lysosome and nuclear functions by targeting a tumor high polyamine microenvironment becomes an effective means for reversing drug resistance and relieving T cell immune suppression around tumors, thereby reducing the repair proportion of DNA and enhancing the effect of naphthalimide analogues on innate and acquired drug-resistant cells.
Compared with the prior art, the invention has the beneficial effects that:
1. the invention discovers the good treatment effect of the novel naphthalimide-polyamine conjugate on liver cancer and late-stage liver cancer for the first time. Unlike classical amonafide, the conjugate of the invention inhibits autophagy by targeting lysosomes, regulates polyamine metabolism and functions in a tumor cell microenvironment to play a role in resisting liver cancer growth and metastasis, and the regulation and control function is closely related to HMGB1/p62/LC3II/LC3I and p 53/SSAT/beta-catenin pathways. The double targeting effects of subcellular organelles and cell nucleuses are realized by modifying the naphthalimide structure.
2. By targeting the contents of key enzymes PAO (polyamine oxidase) SSAT (spermine-N-acetyltransferase) and three endogenous small molecules Put (putrescine), Spd (spermidine) and Spm (spermine) in a tumor high polyamine microenvironment, the functions of lysosomes and cell nuclei are regulated, the anti-tumor immune response is enhanced, and the tumor-resistant immune response has better treatment potential on late-stage metastatic tumors.
3. The upregulation of p53 and γ H2AX by naphthalimide-polyamine conjugate 13b suggests that its effect is also closely related to DNA damage.
4. The invention utilizes the polyamine transport protein highly expressed on the surface of the tumor cells, improves the targeting property of the medicament to the tumor cells, improves the bioavailability and reduces the toxic and side effect to normal cells. Meanwhile, the polyamine-modified naphthalimide heterocyclic compound plays an important role in adjusting the high polyamine microenvironment of the tumor, and plays an important role in treating late metastatic tumor and relieving the immunosuppression around the tumor.
5. The invention improves the water solubility and fat solubility of naphthalimide medicines and increases the intake of tumor cells to the medicines. The invention also improves the stability of the medicament, prolongs the half-life period, reduces the dosage and improves the maximum tolerance of organisms to the medicament by modifying the naphthalimide analogue.
Drawings
FIG. 1 shows the comparison of the inhibition rates of the positive drugs amonafide (FIG. 1A) and compound 13B (FIG. 1B) with different concentrations on liver cancer cells (HepG-2 and Huh-7) and normal liver cells (HL-7702);
FIG. 2 shows the in vivo anti-tumor activity of compound 13b and amonafide group on hepatocarcinoma;
FIG. 3 is the in vivo anti-metastatic activity of 13b and amonafide group on HCC tumors;
FIG. 4 is subcellular organelle localization of 13b in living cells;
FIG. 5 is a graph showing the change in inhibition rate of the naphthalimide-polyamine conjugate 13b in the presence or absence of Spd (an inhibitor of polyamine transport system highly expressed on the surface of tumor cell membranes);
FIG. 6 is the effect of 10 μ M concentration of the naphthalimide-polyamine conjugate 13B and amonafide on HMGB1, p62, LC3II/LC3I and p53 proteins (A), p53 gene (B), p62 gene (C), LC3II/LC3I gene (D), HMGB1 gene (E) expression in hepatoma cells after 24 hours of incubation;
FIG. 7 is the effect of 10 μ M concentration of the naphthalimide-polyamine conjugate 13b on SSAT gene and protein expression in hepatoma cells after 24 hours incubation;
FIG. 8 shows the effect of 10 μ M concentration of the naphthalimide-polyamine conjugate 13b on the expression of β -catenin gene and protein in hepatoma cells after 24 hours incubation;
FIG. 9 is a graph of the effect of a naphthalimide-polyamine conjugate 13b on PAO (polyamine oxidase) activity in hepatoma cells;
FIG. 10 is a graph showing the effect of a naphthalimide-polyamine conjugate 13b on the contents of three endogenous small molecule putrescine (Put), spermidine (Spd) and spermine (Spm) in the polyamine cycle in a polyamine microenvironment of tumor cells;
FIG. 11 is the effect of 10 μ M concentration of the naphthalimide-polyamine conjugate 13b on γ H2AX gene and protein expression in hepatoma cells after 24 hours incubation;
FIG. 12 is a representative HPLC profile of a naphthalimide-polyamine conjugate 13 b;
FIG. 13 is a stability test of the naphthalimide-polyamine conjugate 13b in water;
fig. 14 is a graph showing changes in the absorption spectrum (a) and emission spectrum (b) of the naphthalimide-polyamine conjugate 13b in water and PBS.
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, the cis. is cisplatin, the oxp. is oxaliplatin, and the AF is amonafide.
The invention realizes the connection of naphthalimide analogs with different structures and polyamine by a chemical synthesis means, thereby synthesizing a series of naphthalimide conjugates.
The test results show that the naphthalimide conjugate has better activity on tumor growth and metastasis, particularly the growth and metastasis of liver cancer.
The naphthalimide conjugate is used as an antitumor compound, and the design, synthesis and anticancer activity test of the naphthalimide conjugate aims to prepare an anticancer active molecule with broad spectrum, high efficiency and low toxicity, so that a novel candidate drug is provided for clinical cancer treatment.
On the basis, 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 conjugates based on polyamine modification for the first time, and characterizes the structure of a target compound to test the in vitro and in vivo anti-tumor activity of the naphthalimide conjugates, and the main research content is as follows:
the polyamine-modified-based naphthalimide conjugate 8a-8d,13a-13c,17 and 21 compound design idea of the invention 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 structure of the polyamine analogue is introduced into the naphthalimide conjugate, and the targeting property is enhanced, and simultaneously, the water solubility and the stability of the compound are enhanced, so that the defects of poor solubility, poor stability, low in-vivo utilization rate and the like of parent nucleus cisplatin are overcome.
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 molecular system to synthesize 8a-8d,13a-13c,17 and 21 respectively, and aims to explore the influence of different polyamine structures on the activity of the naphthalimide conjugate.
The method for synthesizing the naphthalimide parent nucleus is well known to those skilled in the art, and specifically refers to the following steps: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-Naphthalimide Conjugate as Substrates for Polyamine Transporters Preferentially Accumulates in Cancer Cells and Minimizes Side Effects in vitro and in vivo.Front Chem.2020,8,166.doi:10.3389/fchem.2020.00166.
example 1: 8a of
1 H NMR(300MHz,Deuterium Oxide)δ7.90(s,1H),7.76(s,3H),7.41(s,1H),4.02(s,2H),3.83(s, 2H),3.05(s,6H),2.90(s,2H),2.57(s,1H),2.02(d,J＝14.4Hz,4H),1.60(s,2H),0.83(s,3H). 13 C NMR(75MHz,Deuterium Oxide)δ164.61,163.98,135.35,134.59,130.93,130.41,127.68, 123.37,122.95,122.32,119.85,49.34,45.45,44.59,44.24,41.20,37.34,24.19,22.65,19.11, 10.09.ESI-MS(positive ion mode):m/z[M+H] + :calcd:426.18；obsd:426.18Calcd for C 23 H 31 N 5 O 3 : C 64.92％,H 7.34％,N 16.46％.Found:C 65.10％,H 7.31％,N 16.38％.
The preparation method comprises the following steps:
(1) adding 100mmol (19.8g) of compound 1a (1, 8-naphthalic acid glucoside) and 100mL of concentrated sulfuric acid into a 250mL volumetric flask, stirring and dissolving under 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 1a under ice bath condition, continuing to react for 2.5h after dripping, and after the reaction is finished, adding the reaction solutionPouring into ice water, standing, performing suction filtration, washing to be neutral, and drying to obtain a filter cake; placing the dried filter cake into 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 3-amino substituted compound 2a (3-NO) 2 -1,8 naphthalenedicarboxylic acid glycoside);
(2) the compounds 2a (1mmol) and K 2 CO 3 (5mmol) was placed in 10mL CH 3 Adding 5mL of acetonitrile solution of the compound 3a (1mmol) into CN under an ice bath condition, standing for 0.5h, heating the reaction solution to 85 ℃ for reaction for 5h, cooling to room temperature after the reaction is finished, concentrating and separating to obtain a compound 4a, and separating by adopting column chromatography, wherein the adopted separation solvent is DCM: MeOH: 100:1-100:3 (v/v);
(3) dissolving the compound 4a (1mmol) in Dichloromethane (DCM), adding TEA (0.5mmol) and chloroacetyl chloride (1.5mmol) under ice bath condition, reacting for 1.5-2h, detecting by TLC and separating to obtain a compound 5 a;
(4) the isolated compound 5a (1mmol) was reacted with NaN 3 (3mmol) is dissolved in DMF and reacts at 70 ℃ overnight to obtain a compound 6a, the compound 6a is reduced in methanol solution by a Pd/C catalyst to obtain a compound 7a, and the compound 7a reacts with 4M HCl in methanol solution to remove protection and obtain a compound 8 a.
Example 2: 8b
1 H NMR(300MHz,Deuterium Oxide)δ7.46(s,1H),7.11(d,J＝43.3Hz,3H),6.77(s,1H),3.76 (d,J＝33.6Hz,4H),3.08(d,J＝63.5Hz,6H),2.12(s,2H),1.62(d,J＝27.3Hz,6H). 13 C NMR(75 MHz,Deuterium Oxide)δ164.91,161.02,160.15,134.85,130.03,127.40,121.46,120.75, 118.96,118.44,47.13,46.78,40.33,38.63,25.38,23.88,22.69.ESI-MS(positive ion mode): m/z[M+H] + :calcd:412.09,obsd:412.09.Calcd for C 22 H 29 N 5 O 3 :C 64.21％,H 7.10％,N 17.02％. Found:C 64.20％,H 7.09％,N 17.00％.
The method for producing compound 8b in example 2 differs from example 1 in that, in step (2), compound 2a is reacted with compound 3b to finally obtain compound 8 b.
Example 3: 8c
1 H NMR(300MHz,Deuterium Oxide)δ7.82(d,J＝7.2Hz,1H),7.68–7.60(m,2H),7.57(d,J ＝2.1Hz,1H),7.36(t,J＝7.8Hz,1H),4.01(s,2H),3.80(t,J＝7.1Hz,2H),3.04(t,J＝7.6Hz,4H), 2.96(t,J＝7.0Hz,2H),1.91(t,J＝7.9Hz,2H),1.77–1.61(m,4H). 13 C NMR(75MHz, Deuterium Oxide)δ165.26,164.39,163.73,135.33,134.53,130.76,130.31,127.68,123.08, 122.60,121.92,120.38,47.07,45.31,41.24,38.79,37.43,30.20,23.93,22.80.ESI-MS (positive ion mode):m/z[M+H] + :calcd:398.13；obsd:398.13.Calcd for C 21 H 27 N 5 O 3 :C 63.46％,H 6.85％,N 17.62％.Found:C 63.39％,H 6.78％,N 17.58％.
The method for producing compound 8c in example 3 differs from example 1 in that, in step (2), compound 2a is reacted with compound 3c to finally obtain compound 8 c.
Example 4: 8d
1 H NMR(300MHz,Deuterium Oxide)δ7.58(d,J＝6.9Hz,1H),7.39(d,J＝7.0Hz,2H),7.30(d, J＝2.0Hz,1H),7.18(s,1H),3.95(s,2H),3.54(d,J＝8.3Hz,2H),3.03(s,4H),2.94(s,2H),1.72 –1.60(m,8H),1.47(s,3H). 13 C NMR(75MHz,Deuterium Oxide)δ165.04,163.94,163.24, 134.17,130.35,130.02,127.47,122.55,122.12,121.38,120.02,119.34,47.16,46.91,41.20, 39.72,38.79,24.11,23.93,23.24,22.81.ESI-MS(positive ion mode):m/z[M+H] + :calcd:412.03； obsd:412.03.Calcd for C 22 H 29 N 5 O 3 :C 64.21％,H 7.10％,N 17.02％.Found:C 64.20％,H 6.92％,N 16.98％.
The method for producing compound 8d in example 4 differs from example 1 in that compound 1b is used instead of compound 1a in step (1) to finally obtain compound 2b (4-NO) substituted with amino at the 4-position 2 -1,8 naphthalenedicarboxylic acid glycoside);
in step (2), the compound 2b reacts with the compound 3d to finally obtain a compound 8 d.
Example 5: 13a of
1 H NMR(300MHz,Deuterium Oxide)δ7.94(s,1H),7.84–7.70(m,2H),7.60–7.28(m,2H), 4.18–3.97(m,2H),3.87(s,2H),3.01(d,J＝47.9Hz,12H),2.07(q,J＝13.8,12.9Hz,6H),1.67 –1.48(m,2H),0.87(p,J＝7.5Hz,3H). 13 C NMR(75MHz,Deuterium Oxide)δ165.34,164.67, 164.06,135.35,134.62,130.47,127.72,123.13,122.47,119.97,49.37,45.53,44.63,44.55, 44.21,41.23,37.35,24.23,22.63,19.12,10.11,10.10.ESI-MS(positive ion mode):m/z[M]+: calcd:483.10；obsd:483.10.Calcd for C 26 H 38 N 6 O 3 :C 64.71％,H 7.94％,N 17.41％.Found:C 64.68％,H 7.84％,N 17.40％.
The method for producing compound 13a in example 5 differs from example 1 in that step (2) to step (4):
(2) the compounds 2a (1mmol) and K 2 CO 3 (3mmol) was placed in 10mL CH 3 Adding 5mL of acetonitrile solution of the compound 9a (1mmol) into CN under an ice bath condition, standing for 0.5h, heating the reaction solution to 85 ℃ for reaction for 5h, cooling to room temperature after the reaction is finished, concentrating and separating to obtain a compound 10a, and separating by adopting column chromatography, wherein the adopted separation solvent is DCM: MeOH: 100:1-100:3 (v/v);
(3) dissolving the compound 10a (1mmol) in Dichloromethane (DCM), adding TEA (0.5mmol) and chloroacetyl chloride (1.5mmol) under ice bath condition, reacting for 1.5-2h, detecting by TLC and separating to obtain a compound 11 a;
(4) the isolated compound 11a (1mmol) was reacted with NaN 3 (3mmol) is dissolved in DMF and reacts at 70 ℃ overnight to obtain a compound 12a1, the compound 12a1 is reduced in methanol solution by a Pd/C catalyst to obtain a compound 12b1, and the compound 12b1 is reacted with 4M HCl in methanol solution to remove protection to obtain a compound 13 a.
Example 6: 13b
1 H NMR(300MHz,Deuterium Oxide)δ8.05(dd,J＝7.8,3.6Hz,2H),7.98(d,J＝8.1Hz,1H), 7.76(d,J＝8.2Hz,1H),7.51(t,J＝8.0Hz,1H),4.19(s,2H),3.90(t,J＝6.9Hz,2H),3.07(ddd,J ＝15.6,11.2,7.2Hz,10H),2.93(t,J＝7.8Hz,2H),2.10–1.90(m,6H),1.59(q,J＝7.5Hz,2H), 0.87(td,J＝7.5,1.5Hz,3H). 13 C NMR(75MHz,Deuterium Oxide)δ166.49,164.94,164.32, 138.42,131.77,129.13,126.98,120.97,120.65,49.35,45.48,44.63,44.49,44.20,41.40, 37.26,24.22,24.22,22.62,19.11,10.10.ESI-MS(positive ion mode):m/z[M+H] + :calcd:483.23； obsd:483.23.Calcd for C 26 H 38 N 6 O 3 :C 64.71％,H 7.94％,N 17.41％.Found:C 64.68％,H 7.89％,N 17.35％.
The method for producing compound 13b in example 6 differs from example 5 in that compound 2a and compound 9b are reacted in step (2) to finally obtain compound 13 b.
Example 7: 13c
1 H NMR(300MHz,Deuterium Oxide)δ7.31(d,J＝6.9Hz,1H),7.08(d,J＝8.5Hz,1H),7.02– 6.90(m,3H),3.83(s,2H),3.46(d,J＝7.1Hz,2H),2.96(t,J＝6.8Hz,8H),2.88(d,J＝6.5Hz,2H), 1.64(d,J＝9.4Hz,12H),0.99(t,J＝7.1Hz,4H). 13 C NMR(75MHz,Deuterium Oxide)δ164.74, 163.35,162.58,127.35,119.35,118.78,57.34,47.11,46.88,41.13,38.75,30.23,23.87, 23.23,22.91,22.82,22.73,16.78.ESI-MS(positive ion mode):m/z[M]+:calcd:495.10；obsd: 495.10.Calcd for C 26 H 38 N 6 O 3 :C 64.71％,H 7.94％,N 17.41％.Found:C 64.70％,H 7.90％,N 17.35％.
Compound 13c of example 7 is prepared by a method different from that of example 5 in that compound 1b is used instead of compound 1a in step (1) to finally obtain compound 2b (4-NO) substituted with amino at the 4-position 2 -1,8 naphthalenedicarboxylic acid glycoside); in the step (2), the compound 2b reacts with the compound 9c to finally obtain a compound 13 c.
Example 8: 17
1 H NMR(300MHz,Deuterium Oxide)δ7.73(s,1H),7.61–7.40(m,3H),7.29(s,1H),3.98(s, 2H),3.83–3.66(m,2H),2.98(d,J＝16.1Hz,4H),1.88(s,2H),1.68–1.55(m,2H),0.95– 0.84(m,3H). 13 C NMR(75MHz,Deuterium Oxide)δ163.62,134.46,130.66,130.20,127.64, 122.95,122.40,121.76,120.25,119.56,49.31,45.05,41.24,37.46,24.17,19.19,10.25. ESI-MS(positive ion mode):m/z[M+H] + :calcd:369.11；obsd:369.11.Calcd for C 20 H 24 N 4 O 3 :C 65.20％,H 6.57％,N 15.21％.Found:C 65.18％,H 6.48％,N 15.19％.
The method for producing compound 17 in example 8 differs from example 5 in that step (2) to step (4):
(2) the compounds 2a (1mmol) and K 2 CO 3 (3mmol) was placed in 10mL CH 3 Adding 5mL of acetonitrile solution of a compound 13(1mmol) into CN under an ice bath condition, standing for 0.5h, heating the reaction solution to 85 ℃ for reaction for 5h, cooling to room temperature after the reaction is finished, concentrating and separating to obtain a compound 14, separating by adopting column chromatography,the isolation solvent used was DCM: MeOH ═ 100:1 to 100:3 (v/v);
(3) dissolving the compound 14(1mmol) in Dichloromethane (DCM), adding TEA (0.5mmol) and chloroacetyl chloride (1.5mmol) under the ice bath condition, reacting for 1.5-2h, detecting by TLC and separating to obtain a compound 15;
(4) the isolated compound 15(1mmol) was reacted with NaN 3 (3mmol) is dissolved in DMF, after reaction at 70 ℃ overnight, the compound is reduced in methanol solution by Pd/C catalyst to obtain a compound 16, and the compound 16 reacts with 4M HCl in methanol solution to remove protection to obtain a compound 17.
Example 9: 21
1 H NMR(300MHz,Chloroform-d)δ8.64–8.32(m,3H),7.99(d,J＝8.5Hz,1H),7.61(d,J＝ 7.9Hz,1H),4.29(d,J＝7.0Hz,2H),3.66(d,J＝12.3Hz,2H),2.73–2.62(m,2H),2.39(d,J＝ 11.0Hz,6H). 13 C NMR(75MHz,Chloroform-d)δ171.25,164.02,163.48,138.40,132.53, 130.91,128.48,126.42,125.76,122.89,122.40,117.33,116.06,56.95,45.73,45.59,38.03. ESI-MS(positive ion mode):m/z[M] + :calcd:340.93；obsd:340.93.Calcd for C 18 H 20 N 4 O 3 :C 63.52％,H 5.92％,N 16.46％.Found:C 63.49％,H 5.89％,N 16.38％.
The method for producing compound 21 in example 9 differs from that in example 8 in that compound 1a is replaced with compound 1b in step (1) to finally obtain compound 2b (4-NO) substituted with amino at the 4-position 2 -1,8 naphthalenedicarboxylic acid glycoside);
step (2) to step (4):
(2) reacting compound 2b (1mmol) with K 2 CO 3 (3mmol) was placed in 10mL CH 3 Adding 5mL of acetonitrile solution of compound 18(1mmol) into CN under ice bath condition, standing for 0.5h, heating the reaction solution to 85 ℃ for reaction for 5h, cooling to room temperature after the reaction is finished, concentrating and separating to obtain compound 19, separating by column chromatography, collectingThe isolation solvent used was DCM: MeOH ═ 100:1 to 100:3 (v/v);
(3) dissolving the compound 19(1mmol) in Dichloromethane (DCM), adding TEA (0.5mmol) and chloroacetyl chloride (1.5mmol) under the ice bath condition, reacting for 1.5-2h, detecting by TLC and separating to obtain a compound 20;
(4) the isolated compound 20(1mmol) was reacted with NaN 3 (3mmol) is dissolved in DMF and reacted at 70 ℃ overnight, and then reduced in methanol solution with Pd/C catalyst to obtain compound 21.
Test example 1: evaluation of target molecule biological Activity
(1) In vitro antitumor Activity test
The cancer cell lines selected in the test comprise: human breast cancer cells (MDA-MB-231 and MCF-7), human lung adenocarcinoma cells (A549), and cisplatin-resistant human lung adenocarcinoma cells (A549cisR), human hepatoma cells (HepG-2 and Huh-7).
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 3000-5000 cells/well, and the last column was left as a zero-adjustment well. 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 calculated 50 Values, 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 parent nucleus modified polyamine analogs 8a-8d,13a-13c,17 and 21 (IC) 50 Unit: μ M) in human tumor cell lines (RF) a :Resistant factor＝IC 50 (A549cisR)/IC 50 (A549))
And (3) annotation: [ a ] A]The RF(resistance factor)is defined as the IC 50 value in A549cisR cells/IC 50 value in A549 cells.[b]FI (fold increase)is defined as IC 50 (amonafide)/IC 50 (13b).[c]FI(fold increase)is defined as IC 50 (cisplatin)/IC 50 (13b). [d]An average of three measurements.ND＝not determined.
Table 1 shows that the in vitro antitumor activity test results show that the naphthalimide-polyamine conjugates 8a-8d,13a-13c,17 and 21 show significantly better antitumor activity than amonafide, the polyamine ligands with different structures have significant influence on the antitumor activity of the compounds, and show better activity on various tested tumor cells, and the naphthalimide mother nucleus modified polyamine analogue 13b shows better activity on cisplatin-resistant a549cisR cells, has no cross resistance with cisplatin, has an RF value of 0.76, and has greater advantages compared with 4.01 of cisplatin. And the lead compound 13b has better effect on HepG-2 and Huh-7 than the positive drugs of amonafide, cisplatin and oxaliplatin, the FI value is 6.74-15.33, the FI value is the highest in liver cancer cells, the contrast effect in other tumors is not obvious, the nmol level is reached, and the good targeting on liver cancer is shown in vitro.
(2) In vitro normal cytotoxicity assay
Human normal hepatocytes (HL-7702) required for the test were purchased from an outsider, and the killing ability of the lead compound 13b against normal cells was measured by the above-mentioned MTT method.
TABLE 2 Effect of 13b of naphthalimide indole core-modified polyamine analogs on the viability of cancer and comparable normal cells (IC) 50 Unit: μ M).
[a]SI(selectivity index)is defined as IC 50 in HL-7702/IC 50 in HepG-2.[b]SI(selectivity index)is defined as IC 50 in HL-7702/IC 50 in Huh-7.
From the test results in table 2, it can be seen that, in addition to the great difference in the antitumor activity, the naphthalimide mother nucleus modified polyamine analog 13b has a great difference in the normal liver cells, and has an SI value of 4.77-6.43, which is 11 times that of amonafide and the clinical common chemotherapeutic drugs cisplatin and oxaliplatin, respectively, and shows a lower toxicity to the normal cells in vitro.
As shown in FIG. 1, similar phenomena could be found from the comparison of the inhibition rates of the positive drugs amonafide (FIG. 1A) and compound 13B (FIG. 1B) on liver cancer cells (HepG-2 and Huh-7) and normal liver cells (HL-7702) at different concentrations. Test example 2: in vivo antitumor activity of different concentrations of naphthalimide compound 13b and amonafide in liver cancer;
taking HCC liver cancer cells in logarithmic growth 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 proportion 6 mu.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 grouped, including the control group, compound 13b group at 1mg/kg, 3mg/kg, 5mg/kg, and amonafide group at 5 mg/kg. The administration was administered seven times a day, seven days after the end of administration, and the tumor volume was measured by weighing each day. Seven days later the mice were sacrificed by dislocation, the orthotopic tumors and various organs were dissected out and recorded by weighing.
FIG. 2 shows the in vivo antitumor activities of compound 13b and amonafide group against liver cancer, and mice were divided into a negative control group, amonafide group (5mg/kg), 13b group (1mg/kg), 13b group (3mg/kg) and 13b group (5 mg/kg). In fig. 2, a is the relative weight of PBS, amonafide, and 13b group mice; b is the tumor weight of PBS, amonafide and group 13B at the end of the experiment; c is the tumor image of PBS, amonafide and 13b groups at the end of the experiment, first row, PBS control group; second line, amonafide group (5 mg/kg); third row, 13b (1 mg/kg); fourth line, 13b (3 mg/kg); fifth element, 13b (5 mg/kg); d is the organ weight index (organ weight/body weight) x 100% calculated for PBS, amonafide and 13b groups at the end of the experiment, including heart, liver, kidney, lung and spleen,. P < 0.001.
The experimental results in fig. 2 show that the inhibition rate of the lead compound 13b in the embodiment of the present invention at the doses of 1mg/kg (58.52%), 3mg/kg (70.14%) and 5mg/kg (76.01%) is better than that of the positive control group at 5mg/kg (46.91%), which shows better anti-liver cancer activity in vivo. And the organ index and body weight change of the liver cancer are not greatly different from those of the positive group, which indicates lower in vivo toxicity. . Test example 3: in vivo anti-tumor metastasis activity of naphthalimide compounds 13b and amonafide with different concentrations in liver cancer;
taking liver cancer cells in logarithmic growth phase, digesting, centrifuging, washing the cells by using sterile PBS buffer solution, collecting the cells after centrifuging, and resuspending by using glucose injection. After staining with trypan blue solution, the cell suspension was aspirated by 10. mu.l using 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 proportion 6 200 μ l/200 μ l, then 200 μ l of cell suspension was injected by tail vein injection. Seven days later, the tumor-inoculated mice were randomly divided into groups including the control group, compound 13b group at 1mg/kg, 3mg/kg, 5mg/kg, and amonafide group at 5mg/kg, and the administration was performed seven times per day, and seven days after the administration was completed were observed and weighed daily. Seven days later, the mice were killed by dislocation, the lungs and organs of the mice were dissected and removed and weighed and recorded, the lungs were fixed in picric acid paraformaldehyde solution, and then taken out for photography and the knot number was calculated.
FIG. 3 is the in vivo anti-metastatic activity of 13b and amonafide group on HCC tumors; in FIG. 3, A is 13b and the body weight of the mice during the amonafide treatment, and the body weight of the control group for 14 days; b is statistics of lung metastasis nodules of the mice after treatment of 13B, amonafide and a control group for 14 d; c is representative image of lung metastasis of mice intravenously injected every two days for control group, 13b (1mg/kg), 13b (3mg/kg), 13b (5mg/kg) and positive control group (5mg/kg) (n is 8/group), P < 0.01; p < 0.001. The experimental result of fig. 3 shows that the inhibition rate of lead compound 13b in the present invention at doses of 1mg/kg (58.48%), 3mg/kg (65.55%), and 5mg/kg (75.02%) is better than that of the positive control group of 5mg/kg (55.77%), which shows better activity against liver cancer metastasis in vivo. And the organ index and body weight change of the liver cancer are not greatly different from those of the positive group, which indicates lower in vivo toxicity.
Test example 4: confocal determination of the localization of the naphthalimide Compound 13b in subcellular organelles
Lysosome, endoplasmic reticulum, golgi and mitochondrial fluorochromes were examined for localization in lysosome, endoplasmic reticulum, golgi and mitochondria, respectively, using a german lycra laser confocal microscope (Leica, Wetzlar, Germany). 13b (10. mu.M) was added to the confocal laser cell culture dish and incubated with HCC cells in 5000 cells/dish for 2 hours in advance. Harvested cells were washed 3 times with PBS and stained for 30 minutes for capture using a Leika laser confocal microscope.
FIG. 4 shows subcellular organelle localization of 13b in living cells. The HepG2 cells were treated with 13b (10 μ M) for 8h, then stained with a mitochondrial tracker (mito tracker), a lysosomal tracker (Lyso tracker), an endoplasmic reticulum tracker (ER tracker), and a Golgi tracker, and images were obtained by confocal microscopy. The results of the experiment in fig. 4 show that 13b is localized in lysosomes, but the compound is not seen in other organelles, indicating that targeting liver cancer growth and metastasis of polyamine-modified naphthalimide conjugate 13b of the present invention is closely related to initiation of lysosomal function.
Test example 5: change in inhibition rate of naphthalimide conjugate 13b in the presence or absence of Spd (inhibitor of polyamine transport system highly expressed on tumor cell membrane surface);
during the culture of tumor cells, a certain dosage of PAT transporter inhibitor is added to study the cell inhibition rate and IC 50 A change in value. The specific method comprises the following steps:
the assayed HepG-2(5 x 10) in the logarithmic growth phase 5 Perwell) and Huh-7(2 x 10) 5 /well) cells were plated in six-well plates and placed at 37 ℃ in 5% CO 2 Culturing in the incubator, allowing the cells to grow in an adherent manner, adding different concentrations of amonafide and target compound 13b as positive reference drugs into a 96-well plate, and adding sub-drugsThe spermine group is prepared by adding 50 μ M spermidine at the same time and aminoguanidine (1mM) at the same time when the same dosage of spermidine is added to prevent interference of blood serum to the drug, and testing the inhibition rate and IC by MTT after the same time as the drug group 50 As a control, an equal amount of medium was added at the same time without the spermidine addition group.
The experimental results in fig. 5 show that the inhibition rate is greatly changed in the presence of the inhibitor Spd of PAT transporter, and the polyamine transporter is proved to be involved in transmembrane transport of polyamine-modified-based naphthalimide conjugate 13b and to have different selectivity for possible products of different tumor cells.
Test example 6: based on the influence of polyamine-modified naphthalimide conjugate 13b and amonafide (10 mu M, fostering for 24 hours) on the expression of genes and proteins of start lysosome autophagy phenomenon, apoptosis inhibition and migration related proteins HMGB1, p62, LC3II/LC3I, p53, SSAT, beta-catenin and gamma H2AX in liver cancer cells;
western Blotting experiment detects the influence of target compounds on the contents of HMGB1, p62, LC3II/LC3I, p53, SSAT, beta-catenin and gamma H2AX proteins.
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 × membrane transferring liquid, and placing the membrane transferring liquid into a refrigerator for precooling, wherein the preparation method of the membrane transferring liquid comprises 100ml of 10 × membrane transferring liquid, 200ml of methanol and 700ml of distilled water. Pouring part of the membrane transferring liquid into a tray, cutting the gel in the tray, cutting off the concentrated gel, activating the PVDF membrane in methanol in advance, covering the gel, placing the gel 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 a1 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 a1 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 experimental result of fig. 6 shows that, compared with the positive drug amonafide, 13b can obviously up-regulate the expression of p62, LC3I and p53 proteins and inhibit the expression of HMGB1 and LC3II proteins, which proves that 13b inhibits the growth and metastasis of liver cancer and is closely related to HMGB1/p62/LC3II/LC3I pathway.
The experimental results of fig. 7 and fig. 8 show that 13b can simultaneously up-regulate SSAT and down-regulate the expression of β -catenin, and prove that 13b inhibits the growth and metastasis of liver cancer and is closely related to the p53/SSAT/β -catenin pathway.
The results of fig. 6 and fig. 11 show that 13b can significantly up-regulate the expression of p53 and γ H2AX proteins, demonstrating that 13b inhibits the growth and metastasis of liver cancer and is also closely related to DNA damage.
Test example 7: the effect of polyamine-modified based naphthalimide conjugates 13b on PAO (polyamine oxidase) activity in hepatoma cells;
detecting the influence of the naphthalimide indole heterocyclic compound 13b on the PAO (polyamine oxidase) activity in the hepatoma cells by adopting the PAO (polyamine oxidase) activity and a 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 tumor cell polyamine microenvironment plays a very important role, and in the tumor polyamine cycle, enzymes playing a key role in polyamine synthesis and metabolism mainly comprise ODC, SSAT and PAO. Among them, PAO plays a key role in polyamine metabolism.
The experimental results in fig. 9 show that the polyamine-modified naphthalimide-based conjugate 13b can significantly up-regulate the PAO content, indicating that it plays a crucial role in interfering with the tumor microenvironment.
Test example 8: based on the influence of the polyamine-modified naphthalimide conjugate 13b on the contents of three endogenous small molecules of putrescine (Put), spermidine (Spd) and spermine (Spm) in polyamine circulation in a 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;
hexaminehydrochloride 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 μ 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 analysis conditions of polyamine high performance liquid chromatography:
(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 reacting for 30min in a dark place;
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 3.
TABLE 3 polyamine concentration in the standard solution
The results of fig. 10 show that based on polyamine-modified naphthalimide conjugate 13b, the contents of three key endogenous small molecules Put, Spd and Spm in a tumor polyamine microenvironment can be obviously reduced, which indicates that 13 b-mediated antitumor activity is closely related to the reduction of the tumor polyamine microenvironment, and the latest research indicates 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 besides the enhancement of targeting on tumor cells and the overcoming of drug resistance, the naphthalimide conjugate 13b also plays an important role in the release of tumor immune suppression.
Test example 9: based on polyamine-modified naphthalimide conjugate 13b representative HPLC profile and stability in water test;
and (3) respectively culturing the polyamine-modified naphthalimide conjugate 13b in 1mM aqueous solution in a constant-temperature shaking incubator at 37 ℃ for 24h, 48h and 72h, and then detecting the HPLC (high performance liquid chromatography) pattern and the stability of the medicament in water by using 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.
Table 4 shows the mobile phase conditions for the high performance liquid phase of polyamine-modified-based naphthalimide conjugates 13b
Table 5 shows the results of the purity test of the naphthalimide-polyamine conjugates
The mobile phase conditions for the high performance liquid phase are shown in table 4, the test results are shown in table 5, which shows that the purity of the novel naphthalimide-polyamine conjugate is greater than 95%, the representative HPLC graphs are shown in fig. 12 and 13, fig. 14 is the change of the absorption spectrum (a) and the emission spectrum (b) of the naphthalimide-polyamine conjugate 13b in water and PBS, the maximum excitation and emission wavelengths of the compound 13b can be obtained from fig. 14, and fig. 14 illustrates that the clear location of 13b in subcellular organelles can be obtained by excitation in a confocal microscope with the wavelengths. The results in fig. 12, 13 and 14 show that compound 1b has a higher stability in water.
In conclusion, the compound synthesized by the invention 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, in addition, because the polyamine-modified naphthalimide conjugate has better targeting property on liver cancer and liver cancer metastasis, the high selectivity on tumor cells is improved, in addition, the compound provided by the invention solves the problems of poor solubility and more complicated clinical compatibility of the traditional antitumor drugs, and has better water solubility. Compared with the classical naphthalimide compound amonafide, the polyamine-modified naphthalimide conjugate 13b inhibits autophagy by targeting lysosomes, regulates polyamine metabolism and functions in a tumor cell microenvironment to play the activities of resisting liver cancer growth and metastasis, and the regulation and control effect is closely related to HMGB1/p62/LC3II/LC3I and p 53/SSAT/beta-catenin pathways. By targeting the contents of key enzymes PAO (polyamine oxidase) SSAT (spermine-N-acetyltransferase) and three endogenous small molecules Put (putrescine), Spd (spermidine) and Spm (spermine) in a tumor high polyamine microenvironment, the functions of lysosomes and cell nuclei are regulated, the anti-tumor immune response is enhanced, and the tumor-resistant immune response has better treatment potential on late-stage metastatic tumors. Meanwhile, the upregulation of p53 and gamma H2AX by the naphthalimide-polyamine conjugate 13b indicates that the effect is also closely related to DNA damage. 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, improves the in vivo utilization rate, enhances the curative effect and reduces the toxic and side effects of the conventional chemotherapeutic drugs on spleen, kidney and the like. And a novel naphthalimide conjugate targeting liver cancer and late-stage liver cancer is found for the first time, and a naphthalimide complex capable of regulating subcellular organelles and a tumor microenvironment is found for the first time, so that a new thought and a new research direction are provided for treating late-stage liver cancer.
Claims (5)
1. A polyamine-modified-based naphthalimide conjugate of the formula:
2. the method for preparing a polyamine-modified-based naphthalimide conjugate according to claim 1, comprising the steps of:
(1) reacting compounds 2a and K 2 CO 3 Is placed in CH 3 Adding polyamine compound into CN under ice bath condition
Reacting the acetonitrile solution at 85 ℃ for 5 hours, cooling to room temperature after the reaction is finished, and concentrating and separating to obtain an intermediate compound I;
(2) dissolving the intermediate compound I in dichloromethane, adding TEA and chloroacetyl chloride under the ice bath condition, reacting for 1.5-2h, detecting by TLC, and separating to obtain an intermediate compound II;
(3) the separated intermediate compound II and NaN 3 Dissolving in N, N-dimethylformamide, reacting at 70 deg.C overnight to obtain intermediate compound III, and reducing to obtain intermediate compound III
Intermediate compounds
In a methanol solution, reacting with HCl for deprotection to obtain a target compound;
the compound 2a is 3-NH 2 -1,8 naphthalenedicarboxylic acid glycoside.
3. The process according to claim 2, wherein the compounds 2a and K in step (1) 2 CO 3 And the molar ratio of the polyamine compound is 1: 3: 1; in the step (2), the molar ratio of the intermediate compound I to TEA and chloroacetyl chloride is 2: 1: 3; the intermediate compound II and NaN in the step (3) 3 In a molar ratio of 1: 3.
4. The polyamine-modified naphthalimide conjugate of claim 1, wherein the tumor is human breast cancer or human liver cancer.
5. The use according to claim 4, wherein the medicament comprises a polyamine-modified based naphthalimide conjugate and a pharmaceutically acceptable carrier.
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