Anti-tumor drug compound targeting mitochondrial topoisomerase I as well as preparation method and application of anti-tumor drug compound

Abstract

The invention discloses an anti-tumor drug compound targeting mitochondrial topoisomerase I as well as a preparation method and application of the anti-tumor drug compound. The drug compound has a molecular structure as shown in a formula I, and comprises an anthracene [1, 2-d] imidazole-6, 11-diketone group and a triphenylphosphine group which are connected by an alkyl chain, the compound can target a mitochondrial cell nucleus and inhibit topoisomerase I, so that an anti-tumor effect is achieved. The reaction for preparing the compound does not need harsh reaction conditions and complex processes.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of anti-tumor drug development, and particularly relates to an anti-tumor drug compound targeting mitochondrial topoisomerase I and a preparation method and application thereof. BACKGROUND

[0002] Deoxyribonucleic acid (DNA) mainly exists in the nucleus and mitochondria, and is a carrier of genetic information. DNA is closely related to the growth, development and canceration of organisms, and thus is the most common target of anti-tumor drugs. Further twisting of double helix DNA forms a tertiary structure of DNA, and supercoiling is the main form of the tertiary structure of DNA. According to the twisting direction, supercoiled DNA can be divided into positive supercoiling and negative supercoiling. When DNA is in a "relaxed" state, the two strands of the double helix usually twist along the middle axis in a way that one turn is placed every 10.4-10.5 base pairs to produce supercoiling. If the twisting direction of DNA is consistent with the rotation direction of the double helix, it is called positive supercoiling, and vice versa.

[0003] As a carrier of genetic information, DNA mainly exists in a supercoiled state in prokaryotic or eukaryotic cells. DNA supercoiling plays a crucial role in the normal growth and proliferation of cells, and its supercoiled state needs to be regulated by topoisomerases. During the processes of transcription, replication and gene expression, the DNA double helix needs to be constantly converted between supercoiled and uncoiled states, and all these physiological processes need the regulation of DNA topoisomerases. Topoisomerases can change the topological structure of DNA by repeatedly breaking and connecting the phosphodiester bonds of DNA through two consecutive transesterification reactions. The activity of topoisomerases in normal cells is strictly controlled, but in tumor cells, the activity is in a high level of expression state, so topoisomerases are a new drug target. SUMMARY

[0004] Based on the above reasons, the first object of the present application is to provide an anti-tumor drug compound targeting mitochondrial topoisomerase I, which connects an anthra[1,2-d]imidazole-6,11-dione group and a triphenylphosphine group through an alkyl chain, so that the compound has good water solubility, cell membrane permeability and targeting property, can target mitochondria, inhibit topoisomerase I, and improve the killing of tumor cells.

[0005] The second object of the present application is to provide a preparation method of an anti-tumor drug compound targeting mitochondrial topoisomerase I. The reaction of the terminal bromine in the compound of formula II with triphenylphosphine can obtain a quaternary phosphonium salt to obtain the anti-tumor drug compound. The reaction does not require harsh reaction conditions and a complex process.

[0006] A third object of the present application is to provide an application of an anti-tumor drug compound targeting mitochondrial topoisomerase I in an anti-tumor drug, and to provide an anti-tumor drug; the drug can target mitochondria, change the topological structure of supercoiled DNA, relax supercoiled DNA, and achieve significant inhibition of topoisomerase I.

[0007] The first object of the present application can be achieved by adopting the following technical solutions:

[0008] An anti-tumor drug compound targeting mitochondrial topoisomerase I, the molecular structure formula is shown as formula I:

[0009] ;

[0010] Wherein, n is an integer from 2 to 8.

[0011] Further, n is 5; the chemical name of the compound shown in formula I is [6-(4-(6,11-dioxo-6,11-dihydro-1H-anthra[1,2-d]imidazol-2-yl)phenoxy)hexyl]triphenylphosphine.

[0012] The second object of the present application can be achieved by adopting the following technical solutions:

[0013] The preparation method of the anti-tumor drug compound targeting mitochondrial topoisomerase I, the compound shown in formula II is reacted with triphenylphosphine to prepare the anti-tumor drug compound targeting mitochondrial topoisomerase I;

[0014] .

[0015] Further, the reaction is carried out in a solvent, and the reaction solvent is DMF and / or DEF.

[0016] Further, the reaction temperature is 100-120℃; the reaction time is 12-72h.

[0017] Further, the molar ratio of the compound shown in formula II to triphenylphosphine is 1: (1-1.2).

[0018] Further, the preparation path of the compound of formula II is as follows:

[0019] ;

[0020] The reaction is carried out in a potassium carbonate-containing acetone solution; the molar ratio of the compound of formula V, potassium carbonate and the compound of formula IV is (1.1-1): (0.6-0.8): 1.

[0021] Further, the reaction temperature is 110-118℃, and the reaction time is 6-36h.

[0022] Further, the preparation path of the compound of formula IV is as follows:

[0023] ;

[0024] The molar ratio of the compound of formula III to 4-hydroxybenzaldehyde is (1-1.1): 1.

[0025] Further, the reaction is carried out in acetic acid solution, the reaction temperature is 110-118℃, and the reaction time is 6-36h.

[0026] The third object of the present application can be achieved by adopting the following technical solution:

[0027] The application of the anti-tumor drug compound targeting mitochondrial topoisomerase I in preparing an anti-tumor drug targeting mitochondrial topoisomerase I.

[0028] The fourth object of the present application can be achieved by adopting the following technical solution:

[0029] An anti-tumor drug targeting mitochondrial topoisomerase I, comprising the anti-tumor drug compound targeting mitochondrial topoisomerase I described above.

[0030] Further, the tumor is any one of colorectal adenocarcinoma, lung cancer, cervical cancer, breast cancer or malignant melanoma.

[0031] Compared with the prior art, the present application has the following beneficial effects:

[0032] 1. The anti-tumor drug compound targeting mitochondrial topoisomerase I of the present application comprises an anthra[1,2-d]imidazole-6,11-dione group and a triphenylphosphine group, which are connected by an alkyl chain; the positive charge property of the triphenylphosphine group enables the compound to target the mitochondrial nucleus and aggregate in the cell; the compound enables topoisomerase I to break and reconnect DNA single strands, changes the topological structure of supercoiled DNA, and relaxes the supercoiled DNA; thereby achieving inhibition of topoisomerase I and achieving an anti-tumor effect.

[0033] 2. The preparation method of the present application enables the alkyl group at the end of the anthra[1,2-d]imidazole-6,11-dione group to react with triphenylphosphine to obtain a quaternary phosphonium salt, forming a stable compound, achieving water solubility, cell membrane permeability, targeting and topoisomerase I inhibition of the compound. The reaction does not require harsh reaction conditions and a complex process.

[0034] 3. The application of the anti-tumor drug compound targeting mitochondrial topoisomerase I in the anti-tumor drug, and the anti-tumor drug can effectively inhibit topoisomerase I, and has excellent killing effect on tumors including colorectal adenocarcinoma, lung cancer, cervical cancer, breast cancer or malignant melanoma. BRIEF DESCRIPTION OF DRAWINGS

[0035] Figure 1 NMR spectrum of drug MitoA;

[0036] Figure 2 UV-visible light absorption spectrum of drug MitoA;

[0037] Figure 3 Stability-UV-visible light absorption spectrum of drug MitoA;

[0038] Figure 4 Stability-high pressure liquid chromatogram of drug MitoA;

[0039] Figure 5 a is the UV titration diagram of pBR322 DNA, and b is the UV titration detection line test of pBR322 DNA;

[0040] Figure 6 pBR322 DNA binding constant diagram;

[0041] Figure 7 pBR322 DNA fluorescence competitive test of drug MitoA;

[0042] Figure 8 Viscosity response experiment of drug MitoA;

[0043] Figure 9 Fluorescence anti-interference test of drug MitoA;

[0044] Figure 10 Topoisomerase I inhibition test of drug MitoA;

[0045] Figure 11 Localization experiment of drug MitoA in mitochondria;

[0046] Figure 12 Cytotoxicity test of drug MitoA (A) / (B);

[0047] Figure 13 Chemical structural formula of drug MitoA. DETAILED DESCRIPTION

[0048] The technical solutions of the present application will be clearly and completely described below in combination with specific embodiments. Apparently, the described embodiments are only some of the embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work fall within the scope of protection of the present application.

[0049] An anti-tumor drug compound targeting mitochondrial topoisomerase I, the molecular structure formula is shown as formula I:

[0050] ;

[0051] Wherein, n is an integer of 2-8.

[0052] In the compound of formula I, anthra[1,2-d]imidazole-6,11-dione group and triphenylphosphine group and triphenylphosphine group are contained, and the two groups are connected by an alkyl chain. In the structure of the compound, the positive charge property of triphenylphosphine can make the compound target the mitochondrial nucleus and aggregate in the cell; and the anthra[1,2-d]imidazole-6,11-dione group acts on topoisomerase I, so that topoisomerase I breaks and reconnects the single strand of DNA, changes the topological structure of supercoiled DNA, and relaxes the supercoiled DNA; thereby realizing the inhibition of topoisomerase I and realizing the anti-tumor effect.

[0053] In the present embodiment, n can be 2, 3, 4, 5, 6, 7, 8, and the length of the alkyl chain is C3, C4, C5, C6, C7, C8 and C9 alkyl chain.

[0054] As one of the embodiments, when n is 5, the chemical name of the compound of formula I is [6-(4-(6,11-dioxo-6,11-dihydro-1H-anthra[1,2-d]imidazol-2-yl)phenoxy)hexyl]triphenylphosphine; the structure formula is as follows:

[0055] .

[0056] The present application also provides a preparation method of the anti-tumor drug compound targeting mitochondrial topoisomerase I, the compound of formula II is reacted with triphenylphosphine to prepare the anti-tumor drug compound targeting mitochondrial topoisomerase I;

[0057] .

[0058] The alkyl chain end contained in the anthra[1,2-d]imidazole-6,11-dione group has a terminal bromine, which can react with triphenylphosphine to obtain a quaternary phosphonium salt, forming a stable compound, which is an antitumor drug compound targeting mitochondrial topoisomerase I. The water solubility, cell membrane permeability, targeting and topoisomerase I inhibitory effect of the compound are achieved. The reaction does not require harsh reaction conditions and complex process.

[0059] As one of the embodiments, the reaction is carried out in a solvent, and the solvent of the reaction is DMF and / or DEF. DMF and DEF have good solubility, and can realize a reaction environment at a high temperature, which is beneficial to the reaction.

[0060] As one of the embodiments, the temperature of the reaction is 100-120℃; and the reaction time is 12-72h.

[0061] As one of the embodiments, the molar ratio of the compound of formula II to triphenylphosphine is 1: (1-1.2).

[0062] As one of the embodiments, the reaction further includes a post-treatment step. The post-treatment step is: cooling the reaction solution, adding toluene and standing, after solid-liquid separation, the solid phase is purified by column chromatography to obtain the antitumor drug compound targeting mitochondrial topoisomerase I of formula I.

[0063] In the embodiment, any one of centrifugation, filtration or suction filtration is used for solid-liquid separation. The mobile phase of column chromatography is methanol and dichloromethane with a volume ratio of 1: 50.

[0064] As one of the embodiments, the preparation path of the compound of formula II is as follows:

[0065] ;

[0066] The reaction is carried out in an acetone solution containing potassium carbonate; and the molar ratio of the compound of formula V, potassium carbonate and the compound of formula IV is (1.1-1): (0.6-0.8): 1.

[0067] As one of the embodiments, the temperature of the reaction is 110-118℃, and the reaction time is 6-36h.

[0068] As one of the embodiments, the reaction further includes a post-treatment step; the post-treatment step is: cooling the reaction solution, suction filtration to obtain the filtrate and evaporate to dryness; adding deionized water and standing, after solid-liquid separation, the liquid phase is spin dried, and the solid phase is purified by column chromatography to obtain the compound of formula II.

[0069] In the present embodiment, the solid-liquid separation is any one of centrifugation, filtration or suction filtration. The column chromatography uses methanol and dichloromethane in a volume ratio of 1: 100 as the mobile phase.

[0070] As one of the embodiments, the preparation route of the compound of formula IV is as follows:

[0071] ;

[0072] The molar ratio of the compound of formula III to 4-hydroxybenzaldehyde is (1-1.1): 1.

[0073] As one of the embodiments, the reaction is carried out in acetic acid solution, the reaction temperature is 110-118℃, and the reaction time is 6-36h.

[0074] As one of the embodiments, the reaction further includes a post-treatment step after the reaction; the post-treatment step is: cooling the reaction solution, neutralizing by adding saturated sodium hydroxide aqueous solution, and then purifying the compound of formula IV by column chromatography after solid-liquid separation.

[0075] In the present embodiment, the solid-liquid separation is any one of centrifugation, filtration or suction filtration. The column chromatography uses methanol and dichloromethane in a volume ratio of 1: 100 as the mobile phase.

[0076] The present application also provides the use of the anti-tumor drug compound targeting mitochondrial topoisomerase I in the preparation of an anti-tumor drug targeting mitochondrial topoisomerase I.

[0077] The present application also provides an anti-tumor drug targeting mitochondrial topoisomerase I, comprising the anti-tumor drug compound targeting mitochondrial topoisomerase I described above.

[0078] As one of the embodiments, the tumor is any one of colorectal adenocarcinoma, lung cancer, cervical cancer, breast cancer or malignant melanoma.

[0079] The following is further illustrated with specific examples.

[0080] Example 1: Synthesis of drug MitoA

[0081] S1: Synthesis of compound 2-(4-hydroxyphenyl)-3H-anthra[1,2-d]imidazol-6,11-dione of formula IV

[0082] Take 1 mmol of 1,2-diaminoanthracene-9,10-dione and 1.1 mmol of 4-hydroxybenzaldehyde in acetic acid solvent, heated to 120°C reflux; after 12h reaction, the reaction solution was quenched with cold deionized water, then neutralized with saturated aqueous sodium hydroxide solution, and the solid was precipitated to obtain the crude product; column chromatography on silica gel was used to separate the purified product 2-(4-hydroxyphenyl)-3H-anthra[1,2-d]imidazole-6,11-dione; the mobile phase of the column chromatography separation was methanol and dichloromethane at a volume ratio of 1:100.

[0083] S2: Compound of formula II 2-(4-((6-bromohexyl)oxy)phenyl)-4,5-dihydro-3H-3λ 4 -anthra[1,2-d]imidazole-6,11-dione synthesis:

[0084] Take 1.1 mmol of 1,6-dibromohexane, 0.7 mmol of potassium carbonate and 1 mmol of 2-(4-hydroxyphenyl)-3H-anthra[1,2-d]imidazole-6,11-dione in acetone solvent, heated to 65°C reflux, after 8h reaction, the reaction solution was cooled to room temperature, then filtered to obtain the filtrate, rotary evaporation, then added cold deionized water and stood for 30 min, then filtered to obtain the solid, to obtain the crude product, column chromatography on silica gel was used to separate the purified product 2-(4-((6-bromohexyl)oxy)phenyl)-4,5-dihydro-3H-3λ 4 -anthra[1,2-d]imidazole-6,11-dione; the mobile phase of the column chromatography separation is preferably methanol and dichloromethane at a volume ratio of 1:200.

[0085] S3: Compound of formula I (6-(4-(6,11-dioxo-6,11-dihydro-1H-anthra[1,2-d]imidazol-2-yl)phenoxy)hexyl)triphenylphosphonium synthesis:

[0086] Take 1.2 mmol of triphenylphosphine, 1.0 mmol of 2-(4-((6-bromohexyl)oxy)phenyl)-4,5-dihydro-3H-3λ 4 -anthra[1,2-d]imidazole-6,11-dione, in N,N-dimethylformamide solvent, heated to 110°C reflux, after 24h reaction, the reaction solution was cooled to room temperature, then added cold toluene and stood for 30 min, then filtered to obtain the solid, to obtain the crude product, column chromatography on silica gel was used to separate the purified product (6-(4-(6,11-dioxo-6,11-dihydro-1H-anthra[1,2-d]imidazol-2-yl)phenoxy)hexyl)triphenylphosphonium (named MitoA); the mobile phase of the column chromatography separation is preferably methanol and dichloromethane at a volume ratio of 1:50.

[0087] The compound of formula I (6-(4-(6,11-dioxo-6,11-dihydro-1H-antheno[1,2-d]imidazol-2-yl)phenoxy)hexyl)triphenylphosphonium 1 The H NMR spectrum is as shown in Figure 1 The data are as follows:

[0088] 1 H NMR (400 MHz, DMSO-d6) δ 13.06 (s, 1H), 8.40 (d, J = 8.4 Hz, 2H), 8.27-8.23 (m, 2H), 8.10 (s, 2H), 7.97-7.95 (m, 2H), 7.93-7.88 (m, 3H), 7.84-7.75 (m, 12H), 7.10 (d, J = 8.9 Hz, 2H), 4.06 (t, J = 6.4 Hz, 2H), 3.63-3.56 (m, 2H), 1.75-1.68 (m, 2H), 1.57-1.45 (m, 6H).

[0089] The compound was tested for pharmaceutical properties:

[0090] (1) UV-visible absorption spectrum of the drug MitoA and its stability

[0091] A test mother liquor of the drug obtained in Example 1 at a concentration of 1 mmol / L in dimethyl sulfoxide (DMSO) was prepared for use.

[0092] It was added to a deionized water solution, shaken well, diluted to 10 μmol / L of the drug working solution, and the solution was transferred to a cuvette using ultrasonic equipment to remove air bubbles. The UV-visible absorption spectrum of the solution was determined, and the UV-visible absorption spectrum is shown in Figure 2 .

[0093] As shown in Figure 2 , it can be seen that the UV absorption intensity of the drug MitoA at 425 nm is relatively strong.

[0094] The working solution was placed for 48 h, and then the UV-visible absorption spectrum of the solution was determined again. The UV-visible absorption spectrum is shown in Figure 3 .

[0095] As shown in Figure 3 , the UV absorption intensity between 250-650 nm is basically unchanged, indicating that the drug MitoA has good stability within 48 h.

[0096] (2) Stability of the drug MitoA - high pressure liquid chromatogram

[0097] A test mother liquor of the drug obtained in Example 1 at a concentration of 1 mmol / L in dimethyl sulfoxide (DMSO) was prepared for use.

[0098] Add it to a deionized water solution and shake well to dilute to a 10 μmol / L working solution. Remove air bubbles using an ultrasonic instrument, transfer the solution to a sample vial, and perform high-performance liquid chromatography (HPLC) analysis. Let the working solution stand for 48 hours, then perform HPLC analysis again. The chromatograms from both tests are shown below. Figure 4 As shown.

[0099] Figure 4 In the test, as time changed, the peak position of the test sample was at t=9.25min after 48h, indicating that the drug MitoA did not decompose and had good stability within 48h.

[0100] (3) Test of the binding ability of drug MitoA to pBR322 DNA

[0101] pBR322 DNA UV titration diagram of drug MitoA ( Figure 5 Figure a); pBR322 DNA UV titration detection line test ( Figure 5 (Figure b); pBR322 DNA binding constant diagram ( Figure 6 ).

[0102] Prepare a test stock solution of dimethyl sulfoxide (DMSO) of the drug obtained in Example 1 at a concentration of 1 mmol / L for later use; add 4 mL of deionized water to the reference cell of the UV-Vis spectrophotometer, and add 10 μM of the compound aqueous solution to the sample cell. Using a pipette, add pBR322 DNA stock solution (0.00 μM, 0.50 μM, 0.10 μM, 0.15 μM, 0.20 μM, 0.25 μM, 0.30 μM, 0.35 μM, 0.40 μM, 0.45 μM, 0.50 μM, 0.55 μM, 0.60 μM) to the sample cell each time, so that the concentration ratio of DNA to drug (C) is... DNA / C MitoA Increase the concentration until the absorption peak at 425 nm no longer decreases. After each thorough mixing for approximately 2 minutes, monitor the changes in the UV absorption spectrum of the drug within the 250-650 nm range. The results are as follows: Figure 5 As shown.

[0103] Figure 5 Figure a shows that the UV absorption intensity at 425 nm gradually decreases with the addition of pBR322 DNA, indicating that pBR322 DNA can bind to the drug MitoA. Figures a and b quantitatively compare the binding strength between the drug and pBR322 DNA using the formula D / Δε. ap =D / Δε+1 / [Δε×K a In the formula, D is the concentration of DNA; Δε ap =|εa - ε f | ; ε a is the apparent molar extinction coefficient of the compound a = A obs / [compound], i.e. the absorbance value of the compound after adding DNA of different concentrations obs divided by the concentration of the compound; ε f is the molar extinction coefficient of the compound without adding DNA; K a is the binding constant of the compound with DNA. D / Δε ap is plotted against D, and the slope divided by the corresponding intercept is obtained by linear fitting, and then K is obtained a = 1.0 x 10 6 M -1 .

[0104] (4) Fluorescence competitive test of drug MitoA with pBR322 DNA

[0105] Prepare a test mother liquor of dimethyl sulfoxide (DMSO) of the drug obtained in Example 1 at a concentration of 1 mmol / L for standby use.

[0106] In the test solution, 3.3 ng / ml of pBR322 DNA and 3.3 ng / ml of Hoechst stock solution are added to 4 mL of deionized water solution and shaken well, and the solution is transferred to a cuvette, and different concentrations of the drug MitoA (0 μM, 0.25 μM, 0.5 μM, 0.75 μM, 1.00 μM, 1.25 μM, 1.50 μM, 1.75 μM, 2.00 μM, 2.25 μM, 2.50 μM, 2.75 μM) are added to the cuvette for testing, and the fluorescence spectrum is shown in Figure 7 .

[0107] As shown in Figure 7 , with the increase of the concentration of the drug, the fluorescence intensity of Hochst dye is gradually decreasing, which indicates that the drug gradually displaces the Hochst dye, proving that the binding mode of the drug with DNA is most likely groove binding.

[0108] (5) Viscosity response experiment of drug MitoA

[0109] Prepare a test mother liquor of dimethyl sulfoxide (DMSO) of the drug obtained in Example 1 at a concentration of 1 mmol / L for standby use.

[0110] In the test solution, 4 ml of mixed solvent of glycerol and deionized water in different proportions (glycerol: deionized water = 0:10, 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, 9:1, 10:0) were taken respectively, then the drug MitoA stock solution (final concentration of 10 μM) was added, fluorescence scanning (excitation wavelength 425 nm, detection band 440-800 nm) was carried out, the relative fluorescence intensity in each system was measured, and the results are shown in Table 1. Figure 8

[0111] It can be seen from Table 1 that with the increase of solvent viscosity, the relative fluorescence intensity becomes stronger. Figure 8

[0112] (6) Fluorescence specificity test of drug MitoA

[0113] A test stock solution of 1 mmol / L drug prepared in Example 1 in dimethyl sulfoxide (DMSO) was prepared for use.

[0114] 10 μM of drug MitoA was mixed with ions and substances (1-23): K + (10 μM) / Co 2+ (10 μM) / Ca 2+ (10 μM) / Cu 2+ (10 μM) / Mn 2+ (10 μM) / ClO 3- (10 μM) / SO3 2- (10 μM) / HCO 3- (10 μM) / SO4 2- (10 μM) / NO 3- (10 μM) / Cl - (10 μM) / di-H2O (10 μM) / Glucose (10 μM) / L-Isoleucine (10 μM) / L-Threonine (10 μM) / L-Arginine (10 μM) / L-Leucine (10 μM) / Ascorbic acid (10 μM) / BSA (0.25 μM) / RNA (0.25 μM) / DNA (0.25 μM) / S2O8 2- (10 μM) / Gly were mixed into the same cuvette for fluorescence test. The test results are shown in Table 2. Figure 9

[0115] It can be seen from Table 2 that the fluorescence response of MitoA to high viscosity is significantly higher than that to other potential factors. Figure 9

[0116] (7) Topoisomerase I inhibition test of drug MitoA ​​​​

[0117] Prepare the test stock solution of the drug MitoA obtained in Example 1 in dimethyl sulfoxide (DMSO) at a concentration of 1 mmol / L for use.

[0118] Prepare the test stock solution of the drug CPT in dimethyl sulfoxide (DMSO) at a concentration of 1 mmol / L for use.

[0119] Topo I is used directly in the experiment without further processing, and the specific experimental steps are as follows:

[0120] S1: Mix 35 mM Tris-HCl, 72 mM KCl, 2.5 mM DTT, 5 mM MgCl2, 2 mM spermidine, 0.1 μg / mL BSA, 0.25 μg pBR322 DNA, 2 Unit Topo I, a certain concentration range of drug MitoA or CPT, and a total volume of 20 μL.

[0121] S2: After incubating the prepared reaction mixture at 37°C for 30 min, add 4 μL of 5×Loading Buffer reaction termination solution to terminate the reaction.

[0122] S3: Add the reaction mixture to a 0.9% agarose gel in TBE buffer and electrophorese at a constant voltage of 80 V.

[0123] S4: Stain the gel with 1 μg / mL ethidium bromide solution, collect the picture and analyze it using a Tanon-3500 gel imaging analysis system. The specific results are shown in Figure 10

[0124] Topoisomerase I (Topo I) can break and reconnect pBR322 DNA single strands, thereby changing the topological structure of supercoiled DNA, relaxing the supercoiled DNA, and showing a slower migration rate in gel electrophoresis experiments. By using Topo I unwinding inhibition experiments, the Topo I inhibition effect of the drug can be clearly observed.

[0125] (8) Mitochondrial localization experiment of drug MitoA

[0126] Prepare the test stock solution of the drug MitoA obtained in Example 1 in dimethyl sulfoxide (DMSO) at a concentration of 1 mmol / L for use.

[0127] ​A549R cells of appropriate density were inoculated into sterilized 35mm imaging culture dishes and cultured in a CO2 incubator (temperature 37℃, 5% CO2), after the cells adhered, drug MitoA and commercial dye MitoTracker Deep Red FM were added to the cells, the final concentration of drug MitoA was 5μM, and the concentration of MitoTracker Deep Red FM dye was according to the requirements of the kit working solution, half an hour later, the culture medium was discarded, the cells were washed with PBS buffer for 3 times, and then fluorescence imaging was carried out (MitoA channel: excitation wavelength: 450nm, emission wavelength: 570-590nm; MitoTracker Deep Red FM channel: 633nm, excitation wavelength: 650-670nm), and the results are shown in Figure 11 .

[0128] Specifically, the co-localization of the mitochondrial channel luminescence mode was studied using MitoTracker Deep Red FM and MitoA, and a high co-localization coefficient (Pearson coefficient: 0.71) was observed, indicating effective mitochondrial targeting accumulation, and the co-localization coefficient of MitoA and MitoTracker Deep Red FM is shown in Figure 11 , the results show that drug MitoA specifically targets mitochondria.

[0129] (9) Cell toxicity test of drug MitoA

[0130] Prepare a test stock solution of dimethyl sulfoxide (DMSO) containing the drug obtained in Example 1 at a concentration of 1mmol / L for later use.

[0131] Prepare a test stock solution of 0.9% physiological saline containing drug CDDP at a concentration of 2mmol / L for later use.

[0132] Cell toxicity was determined by MTT method, and the specific steps are as follows:

[0133] S1: The experimental cells were placed in a culture incubator at 37℃, 5% CO2 saturated humidity, and grown to the logarithmic phase, 0.25% trypsin was used to collect cells, the concentration of cell suspension was adjusted, and the cell density was about 1.0×10 4 / mL, 100mL per well was inoculated into a 96-well plate, and the cell density was about 3-5×10 3 / well. Incubate in a 37℃, 5% CO2 saturated humidity incubator for 24h.

[0134] S2: Add drugs. Add drugs at different concentration gradients. Perform three parallel samples for each concentration. The positive control group (culture medium, cells, different concentrations of CDDP, MTT, DMSO) is incubated at 37℃ and 5% CO2 saturated humidity for 44 hours. The experimental group (culture medium, cells, different concentrations of MitoA, MTT, DMSO) is incubated at 37℃ and 5% CO2 saturated humidity for 44 hours. The blank group (culture medium, cells, MTT, DMSO) is incubated at 37℃ and 5% CO2 saturated humidity for 44 hours.

[0135] S3: Add 20 μL of MTT solution (5 mg / mL, i.e., 0.5% MTT) to each well and continue culturing for 4 h.

[0136] S4: Terminate the culture, discard the culture medium in the wells, add 150 mL of DMSO to each well, and place on a shaker at low speed for 30 min to fully dissolve the crystals. Detect the OD value of each well at 595 nm wavelength using an ELISA reader, and determine the related cell proliferation inhibition rate and half-maximal inhibitory concentration (IC50). 50 The growth inhibition rate is calculated using the following formula: Growth inhibition rate = 1 - [OD] 实验 / OD 空白 By plotting the inhibition rate against drug concentration, a dose-response curve can be obtained, such as... Figure 12 As shown; calculate IC from it. 50 The values ​​are shown in Table 1.

[0137] Table 1.

[0138]

[0139] The results are as follows Figure 12 As shown in Table 1, the IC50 values ​​of drugs MitoA and CDDP in A549R cells... 50 In comparison, the IC50 of drug MitoA 50 Much smaller than CDDP.

[0140] In summary, the antitumor drug compound targeting mitochondrial topoisomerase I of this application has good water solubility, cell membrane permeability, and targeting properties; it can kill tumor cells by targeting mitochondria and inhibiting topoisomerase I; and it has excellent effects, especially on A549 cisplatin-resistant cells.

[0141] The above embodiments are merely preferred embodiments of the present invention and should not be construed as limiting the scope of protection of the present invention. Any non-substantial changes and substitutions made by those skilled in the art based on the present invention shall fall within the scope of protection claimed by the present invention.

Claims

1. An antitumor drug compound targeting mitochondrial topoisomerase I, characterized in that, The molecular structure is shown in Formula I: ； Where n is an integer between 2 and 8.

2. The antitumor drug compound targeting mitochondrial topoisomerase I according to claim 1, characterized in that, n is 5; the chemical name of the compound with the structure shown in Formula I is [6-(4-(6,11-dioxo-6,11-dihydro-1H-anthra[1,2-d]imidazol-2-yl)phenoxy)hexyl]triphenylphosphine.

3. The method for preparing the antitumor drug compound targeting mitochondrial topoisomerase I according to claim 1 or 2, characterized in that, The compound with the structure shown in Formula II was reacted with triphenylphosphine to prepare the antitumor drug compound that targets mitochondrial topoisomerase I. 。 4. The method for preparing the antitumor drug compound targeting mitochondrial topoisomerase I according to claim 3, characterized in that, The reaction is carried out in a solvent, which is DMF and / or DEF; the reaction temperature is 100-120℃; and the reaction time is 12-72h.

5. The method for preparing the antitumor drug compound targeting mitochondrial topoisomerase I according to claim 3, characterized in that, The molar ratio of the compound with the structure shown in Formula II to triphenylphosphine is 1:(1-1.2).

6. The method for preparing the antitumor drug compound targeting mitochondrial topoisomerase I according to claim 3, characterized in that, The preparation route of compound II is as follows: ； The reaction is carried out in an acetone solution containing potassium carbonate; the molar ratio of compound V, potassium carbonate, and compound IV is (1.1-1): (0.6-0.8):

1. The reaction temperature is 110-118℃, and the reaction time is 6-36h.

7. The method for preparing the antitumor drug compound targeting mitochondrial topoisomerase I according to claim 6, characterized in that, The preparation route of compound IV is as follows: ； The molar ratio of the compound with the structure shown in Formula III to 4-hydroxybenzaldehyde is (1-1.1):1; the reaction is carried out in acetic acid solution; the reaction temperature is 110-118℃, and the reaction time is 6-36h.

8. The use of the antitumor drug compound targeting mitochondrial topoisomerase I as described in claim 1 or 2 in the preparation of an antitumor drug targeting mitochondrial topoisomerase I.

9. An antitumor drug targeting mitochondrial topoisomerase I, characterized in that, An antitumor drug compound comprising the target mitochondrial topoisomerase I as described in claim 1 or 2.

10. The antitumor drug targeting mitochondrial topoisomerase I according to claim 9, characterized in that, The tumor is any one of colorectal adenocarcinoma, lung cancer, cervical cancer, breast cancer, or malignant melanoma.

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