Multifunctional skeleton molecule for controllably and visually monitoring drug release as well as preparation method and application of multifunctional skeleton molecule

Abstract

The invention discloses a multifunctional skeleton molecule for controllably and visually monitoring drug release and a preparation method and application thereof, the multifunctional skeleton molecule has a structure as shown in formula I, and comprises a tetrazine structural unit and a phenanthrene dioxin skeleton, fluorescence of phenanthrene dioxin is quenched through a fluorescence resonance energy transfer effect between tetrazine and a phenanthrene dioxin mother nucleus. The multifunctional skeleton molecule provided by the invention can be subjected to a Diels-Alder biological orthogonal bond cleavage reaction with reverse electron requirements through a tetrazine group and a trans-cyclooctene, 3-isocyanopropyl or 3-isocyanopropyl-1-carbamoyl modified prodrug, so that a quenching effect is destroyed, fluorescence is recovered, meanwhile, the prodrug is activated, and the fluorescence intensity is improved. The controllable release and visual monitoring of the medicine are realized. In addition, according to the multifunctional skeleton molecule provided by the invention, a targeting group can be connected to a phenanthrene dioxin skeleton, so that controllable release of drugs at a targeting part and real-time visual monitoring of drug release are realized. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to the field of organic synthesis and biomedicine technology, and in particular to a multifunctional skeleton molecule for controllable and visual monitoring of drug release, and a preparation method and application thereof. Background Art

[0002] Bioorthogonal reactions refer to a type of chemical reaction that can be carried out in living cells. By modifying the substrate and optimizing the reaction conditions, the reaction is transferred from the round-bottom flask to the biological system. This type of reaction can occur under physiological conditions in the body, will not interfere with other biochemical reactions occurring simultaneously in the body, and will not damage the organism and the target biological molecules. It has the advantages of mild conditions, high specificity, and biocompatibility. With the emergence and development of bioorthogonal technology as a tool for chemical biology research, it is applied to the selective modification of proteins in organisms, providing a powerful means to clarify the structure and function of organisms and the occurrence, development and intervention of diseases.

[0003] So far, scientists have developed and utilized various bioorthogonal reactions to achieve various functional modifications of proteins, including fluorescence imaging, activity regulation, dynamic tracking, etc. However, most of the interventions on targets are currently single-site, single-function modifications, and there are few reports on dual or even multi-functional target interventions. Therefore, achieving multifunctional intervention of targets can expand their applications in disease diagnosis and treatment. Summary of the invention

[0004] To solve the above problems, the present invention provides a multifunctional skeleton molecule for controllable and visual monitoring of drug release, and a preparation method and application thereof. The multifunctional skeleton molecule can undergo a reverse electron demand Diels-Alder bioorthogonal bond cleavage reaction with a prodrug modified with trans-cyclooctene, 3-isocyanopropyl or 3-isocyanopropyl-1-carbamoyl to release the drug and restore fluorescence at the same time; and the multifunctional skeleton molecule can achieve controllable release of drugs at targeted sites and real-time visual monitoring of drug release by connecting targeting groups to the phenanthrene dioxin skeleton, and has good application prospects in tumor imaging, tumor treatment and the like.

[0005] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0006] The first aspect of the present invention provides a multifunctional backbone molecule, wherein the multifunctional backbone molecule has the following general structural formula:

[0007]

[0008] Wherein R is H or methyl phenylboronate.

[0009] The second aspect of the present invention provides a method for preparing the multifunctional backbone molecule according to the first aspect, comprising the following steps:

[0010] S1, reacting (4-(6-methyl-1,2,4,5-tetrazine-3-yl)phenyl)methanol shown in formula 2 with 9,10-phenanthrenequinone-3-carboxylic acid shown in formula 4 in the presence of a condensing agent, an alkali reagent and a solvent to obtain an intermediate PQ-Tz;

[0011] S2, treating the intermediate PQ-Tz and (4-(2-(vinyloxy)ethoxy)carbonyl)phenylboronic acid shown in Formula 5 or ethyleneoxyethanol shown in Formula 6 with light in the presence of a solvent to obtain the multifunctional skeleton molecule;

[0012] The structures of Formula 2, Formula 3, intermediate PQ-Tz, Formula 5 and Formula 6 are shown below:

[0013]

[0014]

[0015] Furthermore, in S1, the condensing agent is selected from one or more of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N,N'-diisopropylcarbodiimide, and N,N'-dicyclohexylcarbodiimide.

[0016] Furthermore, in S1, the alkaline reagent is selected from one or more of 4-dimethylaminopyridine, 1,8-diazabicyclo[5.4.0]undec-7-ene, and dimethylacetamide.

[0017] Further, in S1, the solvent is preferably dichloromethane and / or N,N-dimethylformamide,

[0018] Furthermore, in S1, the molar ratio of the 9,10-phenanthrenequinone-3-carboxylic acid to the condensation agent and the alkaline reagent is 1:(1-2):(04-0.6), for example, 1:1.5:0.5.

[0019] Furthermore, in S1, the reaction temperature is 20-40°C, and the reaction time is 1-3h.

[0020] In some preferred embodiments, (4-(6-methyl-1,2,4,5-tetrazine-3-yl)phenyl)methanol shown in Formula 2 and 9,10-phenanthrenequinone-3-carboxylic acid shown in Formula 4 are first dissolved in a solvent at 0°C, and then a condensing agent and an alkali reagent are added and the temperature is raised to 20-40°C for reaction, and the intermediate PQ-Tz is obtained by extraction and separation.

[0021] In some preferred embodiments, (4-(6-methyl-1,2,4,5-tetrazine-3-yl)phenyl)methanol in S1 can be prepared by the following method: 4-(hydroxymethyl)benzonitrile, acetonitrile, and nickel trifluoromethanesulfonate are placed in a sealed tube, hydrazine hydrate is gradually added, and the mixture is reacted at 60°C for 24 hours, and then water is added, and the mixture is extracted with dichloromethane four times until the purple-red color of the organic phase becomes lighter, the organic phases are combined, spin-dried, dichloromethane and an oxidant are added, and the mixture is reacted at room temperature for 2 hours, and separated on a thick preparation plate to obtain purple-red crystals, i.e. (4-(6-methyl-1,2,4,5-tetrazine-3-yl)phenyl)methanol; the oxidant includes but is not limited to iodophenyl diacetate.

[0022] In some preferred embodiments, 9,10-phenanthrenequinone-3-carboxylic acid in S1 can be prepared by the following method:

[0023] Dissolve 3-acetylphenanthrene in 1,4-dioxane, add sodium hypochlorite (available chlorine>7.5%) and NaOH, stir at 65°C for 6h, cool to room temperature, add sodium thiosulfate, adjust pH to 1-2 with 2M hydrochloric acid, extract twice with ethyl acetate, dry over anhydrous sodium sulfate, remove solvent by rotary evaporation, and dry in vacuo to obtain a white solid;

[0024] Dissolve 18-crown ether-6 and chromium trioxide in acetic acid, add distilled water, and then dissolve the white solid prepared above in acetic acid, and react at 60°C for 10 hours. Then add a large amount of distilled water, and a large amount of orange-yellow precipitate is generated, which is filtered, and the filter cake is washed twice with a mixed solution of acetic acid: water (1:1, v / v), washed twice with dichloromethane, and dried to obtain an orange-yellow solid, namely 9,10-phenanthrenequinone-3-carboxylic acid.

[0025] In some preferred embodiments, (4-(2-(vinyloxy)ethoxy)carbonyl)phenylboronic acid in S2 can be prepared by the following method:

[0026] 2-(vinyloxy)ethane-1-ol and p-carboxyphenylboronic acid are reacted in the presence of a condensing agent, an alkaline reagent and a solvent, and the (4-(2-(vinyloxy)ethoxy)carbonyl)phenylboronic acid is obtained through extraction and separation; the condensing agent is selected from one or more of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N,N'-diisopropylcarbodiimide, and N,N'-dicyclohexylcarbodiimide; the alkaline reagent is selected from one or more of 4-dimethylaminopyridine, 1,8-diazabicyclo[5.4.0]undec-7-ene, and dimethylacetamide; and the solvent is selected from one or more of dichloromethane and N,N-dimethylformamide.

[0027] Furthermore, in S2, the solvent includes but is not limited to acetonitrile.

[0028] Further, in S2, in the step of illumination treatment: the light source is white light, the energy of the light source is 10-20W, and the illumination treatment time is 0.5-2h; for example, a 15W strong white light flashlight is used for illumination for 1h.

[0029] The third aspect of the present invention provides a use of the multifunctional skeleton molecule described in the first aspect in the preparation of a drug with controlled release and visual monitoring.

[0030] Furthermore, the drug also contains a prodrug modified with trans-cyclooctene, 3-isocyanopropyl or 3-isocyanopropyl-1-carbamoyl; the tetrazine group in the multifunctional skeleton molecule in the drug undergoes a reverse electron demand Diels-Alder bioorthogonal bond cleavage reaction with the trans-cyclooctene, 3-isocyanopropyl or 3-isocyanopropyl-1-carbamoyl in the prodrug to restore the fluorescence properties of the multifunctional skeleton molecule and activate the prodrug at the same time.

[0031] Furthermore, the prodrug includes but is not limited to one of the following structures:

[0032]

[0033] Furthermore, the drug is used for tumor imaging and / or tumor treatment; the tumor includes but is not limited to human non-small cell lung cancer and cervical cancer.

[0034] Compared with the prior art, the present invention has the following beneficial effects:

[0035] The present invention provides a multifunctional skeleton molecule, which includes a tetrazine structural unit and a phenanthrene dioxin skeleton, and the fluorescence of phenanthrene dioxin can be quenched by the fluorescence resonance energy transfer effect between tetrazine and phenanthrene dioxin parent nucleus. The multifunctional skeleton molecule has good biocompatibility, and can achieve in vivo targeting of specific parts (such as tumor cells) by connecting a targeting group (such as methyl phenylboronic acid) to the phenanthrene dioxin skeleton, and undergo a reverse electron demand Diels-Alder bioorthogonal bond cleavage reaction with a prodrug modified with trans-cyclooctene, 3-isocyanopropyl or 3-isocyanopropyl-1-carbamoyl, and click release of drugs while restoring fluorescence, thereby achieving controlled release and visual monitoring of drugs at the target site, and has good application prospects in the diagnosis and treatment of tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 :A is the fluorescence spectra of PDO-Tz and PDO-Pz at different concentrations (10, 25, 50 μM); B is the fluorescence spectra of PDO-Tz and PDO-Pd at different concentrations (10, 25, 50 μM);

[0037] Figure 2:A is the fluorescence spectra of PDO-Pz at different concentrations (10, 25, 50, 125 μM); B is the linear relationship between the fluorescence intensity and concentration of PDO-Pz;

[0038] Figure 3 :A is the fluorescence spectra of PDO-Pd at different concentrations (1, 10, 25, 50, 125 μM); B is the linear relationship between the fluorescence intensity and concentration of PDO-Pd;

[0039] Figure 4 : A is the HPLC graph of the click release reaction of the prodrug ICPr-mp and PDO-Tz; B is the HPLC graph of the click release reaction of the prodrug ICPrc-mmc and PDO-Tz; C is the quantitative analysis of the release rate of the prodrugs ICPr-mp and ICPrc-mmc (HPLC method);

[0040] Figure 5 : A is the HPLC graph of the click release reaction of prodrug TCO-Dox and PDO-Tz; B is the quantitative analysis of the conversion rate and release rate of prodrug TCO-Dox (HPLC method);

[0041] Figure 6 :A is the HPLC graph of the reaction of 1200μM PDO-Tz and 200μM ICPr-mp at different time points (5min, 30min, 60min, 120min, 240min); B is the linear relationship graph between the peak area of ​​PDO-Pz and the peak area of ​​active drug mp;

[0042] Figure 7 :A is the HPLC graph of the reaction of different concentrations of PDO-Tz (100μM, 200μM, 400μM) with different concentrations of TCO-Dox (200μM, 200μM, 300μM); B is the linear relationship graph between the peak area of ​​PDO-Pd and the peak area of ​​the active drug Dox;

[0043] Figure 8 : Cell imaging results after 1% DMSO, trans-cyclooctene conjugated prodrug, PDO-Tz, trans-cyclooctene conjugated prodrug and PDO-Tz were incubated in A549 cells for 1 hour and 2 hours;

[0044] Fig. 9 : Cell imaging results of 1% DMSO, trans-cyclooctene conjugated prodrug, PBA-PDO-Tz, trans-cyclooctene conjugated prodrug and PBA-PDO-Tz incubated in 293T normal cells and HeLa tumor cells for 1 hour;

[0045] Fig.10: A is the survival rate of A549 cells treated with different concentrations of PDO-Tz; B is the inhibition rate of TCO-Dox prodrug system or related compounds on A549 cell proliferation; Among them, the P value of the drug treatment group was compared with the DMSO group, and the two-tailed unpaired t-test was used to calculate, ns: no significant difference, *P<0.05, ***P<0.001, ****P<0.0001;

[0046] Fig.11 : A is a schematic diagram of the construction of the tumor-bearing mouse model and the method of drug administration; B is the imaging of tumor tissue sections of A549 tumor-bearing mice treated with PBS, PBA-PDO-Tz (5 mg / kg), TCO-Dox (5 mg / kg), and PBA-PDO-Tz (5 mg / kg) + TCO-Dox (5 mg / kg), the cell nuclei were stained with propidium iodide (PI), and the rhodamine (RB) and DAPI channels were used for imaging, scale bar: 100 μm. DETAILED DESCRIPTION

[0047] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more related listed items.

[0048] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0049] Example 1

[0050] This example relates to the preparation of a multifunctional skeleton molecule PDO-Tz, and the specific operations are as follows:

[0051] (1) Synthesis of intermediate 2(4-(6-methyl-1,2,4,5-tetrazine-3-yl)phenyl)methanol

[0052]

[0053] 4-(Hydroxymethyl)benzonitrile (400 mg, 3.0 mmol), nickel trifluoromethanesulfonate (336 mg, 1.5 mmol), acetonitrile (1.25 mL, 30.0 mmol) and hydrazine monohydrate (1.74 mL, 150.0 mmol) were combined in a sealed tube. The mixture was stirred at 60 ° C for 24 hours and cooled to 0 ° C. Subsequently, water was added and the reaction mixture was extracted with dichloromethane. The combined organic layer was washed with brine and dried over Na2SO4. After removing the solvent, the residue was dissolved in dichloromethane (10 mL) and PhI(OAc)2 (1.45 g, 4.5 mmol) was added. The solution was stirred at room temperature for 2 hours and concentrated under reduced pressure. The residue was purified by column chromatography to obtain 219 mg of (4-(6-methyl-1,2,4,5-tetrazine-3-yl)phenyl)methanol as a purple solid with a two-step yield of 36%.

[0054] 1 HNMR (400MHz, CDCl3) δ8.59(d,J=8.6Hz,2H),7.59(d,J=8.6Hz,2H),4.84(s,2H),3.10(s,3H).

[0055] (2) Synthesis of intermediate 4,9,10-phenanthrenequinone-3-carboxylic acid

[0056]

[0057] 3-acetylphenanthrene (300mg, 1.36mmol) is dissolved in 1,4-dioxane (6mL). NaClO solution (10mL, available chlorine 7.5%) and 1M NaOH aqueous solution (25mL) are added to the solution and stirred at 65 ℃ for 6 hours. After cooling to room temperature, sodium thiosulfate (6g, 38.0mmol) is added and stirred for neutralization. 2M hydrochloric acid is used to adjust pH to 1-2, ethyl acetate is extracted, and the organic phase is washed with saturated brine, and then vacuum dried to obtain the phenanthrene-3-carboxylic acid of white solid. The solid is directly used in the next step without further purification.

[0058] 18-crown-6 (60 mg, 0.23 mmol) and chromium trioxide (68 mg, 0.68 mmol) were added to an acetic acid / water (v / v=10 / 1) (5.5 mL) solution, and phenanthrene-3-carboxylic acid (100 mg, 0.45 mmol) was added under stirring. The reaction mixture was stirred at 60°C for 10 hours. Then water was added to the reaction mixture to precipitate, and the precipitate was washed with acetic acid / water (v / v=1 / 1) and ether to obtain 45 mg of yellow solid 9,10-phenanthrenequinone-3-carboxylic acid, with a two-step yield of 50%.

[0059] 1H NMR (400MHz, DMSO-d6) δ8.71 (s, 1H), 8.35 (d, J = 8.0Hz, 1H), 8.14-8.02 (m, 3H), 7.83-7.79 (m, 1H), 7.60-7.56 (m, 1H).

[0060] (3) Synthesis of the framework PQ-Tz

[0061]

[0062] 9,10-Phenanthrenequinone-3-carboxylic acid (420 mg, 1.67 mmol) and (4-(6-methyl-1,2,4,5-tetrazine-3-yl)phenyl)methanol (281 mg, 1.39 mmol) were added to dichloromethane (50 mL) at 0°C, and N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDCI) (840 mg, 4.4 mmol) and 4-dimethylaminopyridine (DMAP) (60 mg, 0.6 mmol) were added under stirring. The reaction mixture was allowed to warm to room temperature and maintained for 2 hours. It was then diluted with water (50 mL) and extracted with dichloromethane. The combined organic phases were washed with brine, dried over Na2SO4, and concentrated under reduced pressure. The residue was purified by column chromatography to give 334 mg of red solid PQ-Tz with a yield of 55%.

[0063] 1 H NMR (400MHz, CDCl3) δ8.76 (s, 1H), 8.66 (d, J = 8.0Hz, 2H), 8.29-8.22 (m, 2H), 8.17-8.13 (m ,2H),7.80-7.76(m,1H),7.72-7.70(m,2H),7.55-7.51(m,1H),5.56(s,2H),3.12(s,3H).

[0064] 13 C NMR (100MHz, CDCl3) δ180.1,179.6,167.5,165.0,163.8,139.9,136.3,136.2,136.1,135.0 ,133.6,132.0,131.1,130.8,130.6,130.2,130.0,128.9,128.3,125.6,124.4,66.9,21.2.

[0065] (4) Synthesis of multifunctional backbone molecule PDO-Tz

[0066]

[0067] PQ-Tz (220 mg, 0.5 mmol) and ethyleneoxyethanol (440 μL, 5.0 mmol) were dissolved in acetonitrile (250 mL). The mixture was then stirred and irradiated with a handheld white LED lamp for 1 hour. The reaction mixture was then extracted with ethyl acetate. The combined organic layers were washed with brine, dried over Na2SO4, and concentrated under reduced pressure. The residue was purified by preparative thin layer chromatography to give a light red solid PDO-Tz 168 mg with a yield of 64%.

[0068] 1 H NMR(400MHz, DMSO-d6)δ9.33(s,1H),8.78–8.76(m,1H),8.51-8.49(m,2H),8.22–8.10(m,3H),7.82-7.80(m,2H),7.73–7.65(m,2H),5. 77–5.76(m,1H),5.58(s,2H),4.53-4.48(m,1H),4.42-4.37(m,1H),4.28(brs,1H),3.83–3.80(m,2H),3.57-3.47(m,2H),2.99(s,3H).

[0069] 13 C NMR(100MHz,DMSO-d6)δ167.1,165.7,163.0,140.8,140.7,135.3,132.6,132.5,131.5 2,131.50,129.7,128.9,128.7,128.6,128.4,127.7,127.6,126.7,126.6,126.2,126. 17,126.12,126.0,125.96,125.8,125.6,125.5,125.4,125.36,124.6,122.9,122.88,120.9,120.6,120.5,120.3,94.3,94.0,70.2,70.1,66.2,66.0,65.8,65.7,60.0,20.8.

[0070] MS (ESI) m / z: [M+H] + Calculated value C 29 H 24 N4O6,525.45; test value 524.53.

[0071] Example 2

[0072] This example relates to the preparation of a multifunctional backbone molecule PBA-PDO-Tz, and the specific operations are as follows:

[0073] (1) Synthesis of intermediate 5(4-(2-(vinyloxy)ethoxy)carbonyl)phenylboronic acid

[0074]

[0075] 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoic acid (500 mg, 2.0 mmol) and ethyleneoxyethanol (360 μL, 2.0 mmol) were added to dichloromethane (50 mL) at 0°C, and N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDCI) (768 mg, 4.0 mmol) and 4-dimethylaminopyridine (DMAP) (100 mg, 0.8 mmol) were added under stirring. The reaction mixture was allowed to warm to room temperature and maintained for 2 hours. It was then diluted with water (50 mL) and extracted with dichloromethane. The combined organic phases were washed with brine, dried over Na2SO4, and concentrated under reduced pressure. The residue was purified by column chromatography to obtain 450 mg of 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoate with a yield of 70%.

[0076] 1 H NMR (400MHz, CDCl3) δ8.03(d,J=7.8Hz,2H),7.87(d,J=7.9Hz,2H),6.51(dd,J=14.3,6.8Hz,1H),4.60–4 .51(m,2H),4.24(dd,J=14.4,2.3Hz,1H),4.07(dd,J=6.9,2.3Hz,1H),4.05–4.01(m,2H),1.36(s,12H).

[0077] (2) Synthesis of multifunctional backbone molecule PBA-PDO-Tz

[0078]

[0079] PQ-Tz (54 mg, 0.12 mmol) and 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoate (202 mg, 0.62 mmol) were dissolved in acetonitrile (32 mL). The mixture was then stirred and irradiated with a handheld white LED lamp for 2 hours. The reaction mixture was then extracted with ethyl acetate. The combined organic layers were washed with brine, dried over Na2SO4, and concentrated under reduced pressure. The residue was purified by preparative thin layer chromatography to give a light red solid PBA-PDO-Tz 55 mg with a yield of 68%.

[0080] 1H NMR(400MHz, DMSO-d6)δ9.33(d,J=11.0Hz,1H),8.81–8.72(m,1H),8.53(d,J=8.0Hz ,2H),8.22(s,2H),8.21–8.11(m,3H),7.84(dd,J=8.4,3.0Hz,2H),7.68(dd,J=8.3, 4.1Hz,4H),7.57(dd,J=16.9,7.8Hz,2H),5.88(d,J=6.0Hz,1H),5.60(s,2H),4.55( t,J=12.3Hz,1H),4.42(dt,J=19.1,9.1Hz,3H),4.16(d,J=5.0Hz,2H),3.01(s,3H).

[0081] 13 C NMR (100MHz, DMSO-d6) δ167.64,166.20,166.14,163.58,141.31,141.27,135. 84,134.44,134.41,133.08,132.83,130.89,130.84,129.19,129.10,128.13, 128.08,127.14,127.06,126.74,126.62,125.95,125.89,125.15,123.42,121.39,120.99,120.72,94.24,93.90,66.55,66.41,66.29,66.24,64.04,21.31.

[0082] MS (ESI) m / z: [M+H] + Calculated value C 36 H 29 BN4O9, 673.21; test value 672.46.

[0083] Example 3

[0084] This embodiment relates to the preparation of a fluorescent molecule PDO-Pz, and the specific operations are as follows:

[0085]

[0086] PDO-Tz (10 mg, 0.02 mmol) was dissolved in 0.2 mL of dry DMSO, and ICPr-mp (10 mg, 0.04 mmol) was added to the reaction system and stirred at 37°C for 6 h. The reaction was monitored by thin layer chromatography. After the reaction was complete, the solvent was removed by freeze drying, and the residue was separated by preparative thin layer chromatography (eluent polarity PE: EA = 1:1) to obtain a light yellow crystalline solid (8 mg, 76%).

[0087] 1 H NMR(400MHz, DMSO-d6)δ9.32(d,J=13.4Hz,1H),8.81(t,J=7.0Hz,1H),8.29–8 .12(m,3H),7.81–7.67(m,4H),7.57(d,J=7.2Hz,1H),7.32(d,J=8.0Hz,1H),5 .77(d,J=5.2Hz,1H),5.46(s,1H),4.53–4.49(m,2H),4.42-4.37(m,1H),3.96 (s,1H),3.83-3.79(m,2H),3.72(s,1H),3.64–3.47(m,4H),2.11–2.10(m,3H).

[0088] 13 C NMR(100MHz,DMSO-d6)δ166.9,166.3,155.7,147.7,140.7,135.7,135.65,134.3,134.2,133.0,132 .9,130.2,129.3,129.2,129.0,128.9,128.85,128.8,128.3,128.2,127.2,127.1,127.08,127.03, 126.7,126.67,126.64,126.5,126.46,126.43,126.1,125.9,125.5,125.1,125.0,124.7,123.4,121.4,121.36,121.1,121.0,120.8,94.8,94.5,70.7,70.6,66.9,66.7,66.5,63.2,60.4,52.8,24.9.

[0089] MS (ESI) m / z: [M+H] + Calculated value C 30 H 28 N3O6, 526.20; test value 526.17.

[0090] Example 4

[0091] This embodiment relates to the preparation of a fluorescent molecule PDO-Pd, and the specific operations are as follows:

[0092]

[0093] PDO-Tz (10 mg, 0.02 mmol) was dissolved in 1 mL of dry DMSO, cis-cyclooctene (68 μL, 0.5 mmol) was added to the reaction system, and stirred at 110°C for 12 h. The reaction was monitored by thin layer chromatography, and after the reaction was complete, the solvent was removed by freeze drying, and the residue was separated by preparative thin layer chromatography (eluent polarity PE: EA = 1.5:1) to obtain a white crystalline solid (7 mg, 58%).

[0094] 1 H NMR(400MHz, DMSO-d6)δ9.37(s,1H),8.81(dd,J=7.8,1.0Hz,1H),8.29–8.11(m,3H),7.78–7.65(m,4H),7. 52–7.46(m,2H),5.78(dt,J=4.0,1.9Hz,1H),5.56(d,J=2.6Hz,2H),4.70(q,J=5.6Hz,1H),4.52(td,J=11.2 ,2.2Hz,1H),4.40(ddd,J=11.4,7.5,1.7Hz,1H),3.87–3.77(m,2H),3.53(ddq,J=17.5,11.7,5.9,5.4Hz,2 H),2.84(dd,J=7.6,5.0Hz,2H),2.69(d,J=6.9Hz,2H),2.67(s,3H),1.71(s,2H),1.47(s,2H),1.33(s,4H).

[0095] 13 C NMR(100MHz,DMSO-d6)δ166.3,160.4,157.7,139.3,138.57,138.55,138.2,136.78,136.73,135.7, 133.0,131.0,129.6,129.4,128.9,128.3,128.2,127.2,127.1,126.71,126.68,126.64,126.52,12 6.46,126.4,126.3,126.1,125.91,125.88,125.14,125.11,123.44,123.39,121.4,121.1,121.0,120.8,94.8,94.5,70.7,70.6,66.7,66.6,66.5,60.4,30.5,29.5,28.5,26.8,26.5,26.0,25.8,20.3.

[0096] MS (ESI) m / z: [M+H] + Calculated value C 37 H37 N2O6,605.27; test value 605.36.

[0097] Example 5

[0098] The multifunctional skeleton molecule PDO-Tz was used as a control sample to study the fluorescence properties of the fluorescent molecules PDO-Pz and PDO-Pd prepared in Examples 3 and 4. The specific operation was as follows:

[0099] PDO-Tz, PDO-Pz and PDO-Pd were prepared into solutions with concentrations of 10, 25 and 50 respectively using DMSO:PBS (1:9, v / v), and their fluorescence emission spectra were tested at an excitation wavelength of 365 nm.

[0100] Test results such as Figure 1 As shown in the figure, compared with PDO-Pz and PDO-Pd, the fluorescence of PDO-Tz is significantly weaker. After reacting with ICPr-mp or cis-cyclooctene, it has a better fluorescence enhancement effect, with the maximum enhancement times being 15 times and 44 times, respectively; and Figure 2 , 3 It can be seen that the concentration of PDO-Pz is positively correlated with the fluorescence intensity within 10-125 μM (R 2 =0.9874), and the concentration of PDO-Pd was positively correlated with the fluorescence intensity in the range of 1-125 μM (R 2 =0.9517).

[0101] Example 6

[0102] Taking the multifunctional skeleton molecule PDO-Tz as an example, the drug release after the multifunctional skeleton molecule reacts with trans-cyclooctene, 3-isocyanopropyl or 3-isocyanopropyl-1-carbamoyl modified prodrugs is studied as follows:

[0103] (1) The prodrugs ICPrc-mmc and ICPr-mp were prepared into 4 mM solutions with DMSO:PBS (1:9, v / v), and PDO-Tz was prepared into 24 mM solutions with DMSO:PBS (1:9, v / v). 250 μL of glutathione (c=8 mM) as a reducing agent and 250 μL of 7-hydroxycoumarin (c=2.0 mM) as an internal standard and 250 μL of ICPrc-mmc (c=4.0 mM) or 250 μL of ICPr-mp (c=4.0 mM) and 250 μL of PDO-Tz (c=24 mM) were mixed and incubated at 37°C. HPLC traces were recorded at 5 and 240 minutes at the specified wavelength (10 μL injection).

[0104] The results are as follows Figure 4As shown, the absorption peaks of the prodrug molecules ICPr-mp and ICPrc-mmc disappeared at 240 min, and mp and mmc were successfully released. At the same time, the fluorescent molecule PDO-Pz was also generated. Through quantitative analysis, it was further concluded that the release rate of the prodrug ICPr-mp was 94.8%; the release rate of ICPrc-mmc was 96.1%.

[0105] (2) The prodrug TCO-DOX was prepared into a solution with a concentration of 3 mM using a solution of DMSO:PBS (1:9, v / v), and the PDO-Tz was prepared into a solution with a concentration of 3 mM using a solution of DMSO:PBS (1:9, v / v). 250 μL of 7-hydroxycoumarin (c=1.0 mM) as an internal standard, 250 μL of TCO-DOX (c=3 mM), and 250 μL of PDO-Tz (c=3 mM) were mixed and incubated at 37°C. HPLC traces were recorded at the specified wavelength at 5 minutes (10 μL injection).

[0106] The results are as follows Figure 5 As shown: the absorption peak of the prodrug molecule TCO-Dox disappears at 5 minutes, successfully releasing the Dox original drug, and also generating the fluorescent molecule PDO-Pd. The conversion rate of the prodrug TCO-Dox reaches 99%, but the release rate of the Dox original drug is only 53%, the reason is that some compounds form cyclization products without further releasing the original drug.

[0107] (3) Linear relationship analysis test of prodrug release

[0108] Effect of reaction time on prodrug release: The prodrug ICPr-mp was prepared into a solution with a concentration of 200 μM using DMSO:PBS (1:9, v / v), and PDO-Tz was prepared into a solution with a concentration of 1200 μM using DMSO:PBS (1:9, v / v). 250 μL of glutathione (c=8 mM) as a reducing agent and 250 μL of 7-hydroxycoumarin (c=2.0 mM) as an internal standard and 250 μL of ICPrc-mmc (c=2.0 mM) or 250 μL of ICPr-mp (c=200 μM) and 250 μL of PDO-Tz (c=1200 μM) were mixed and incubated at 37°C. HPLC traces (10 μL injection) were recorded at 5, 30, 60, 120 and 240 minutes at the specified wavelength.

[0109] The results are as follows Figure 6 As shown in the figure, the amount of PDO-Pz generated during the reaction of PDO-Tz and prodrug ICPr-mp is also linearly correlated with the amount of original drug mp generated, and the peak area of ​​PDO-Pz is positively correlated with the peak area of ​​original drug mp (R 2=0.9917). The fluorescence intensity of PDO-Pz is also linearly correlated with its concentration. Therefore, the fluorescence intensity of the fluorescent molecule (PDO-Pz) is positively correlated with the concentration of the original drug (mp). The concentration of the released drug can be measured by measuring the fluorescence intensity, thus achieving real-time quantitative visual monitoring of drug release.

[0110] Effect of the concentration ratio of the multifunctional backbone molecule PDO-Tz and the prodrug on the release of the prodrug: The prodrug TCO-Dox was prepared into solutions with concentrations of 200 μM and 300 μM using DMSO:PBS (1:9, v / v), and the PDO-Tz was prepared into solutions with concentrations of 100 μM, 200 μM, and 400 μM using DMSO:PBS (1:9, v / v); 250 μL of 7-hydroxycoumarin (c=200 μM) as an internal standard, 250 μL of TCO-Dox (c=200 μM or 300 μM), and 250 μL of PDO-Tz (c=100 μM, 200 μM, or 400 μM) were mixed to obtain a concentration ratio (P:T) of PDO-Tz to TCO-Dox in the solution of 1:2, 1:1, and 2:1.5, and incubated at 37°C. HPLC traces were recorded at 5 minutes at the specified wavelength (10 μL injection).

[0111] The results are as follows Figure 7 As shown in Figure 2, the amount of PDO-Pd generated during the reaction of PDO-Tz and prodrug TCO-Dox is also linearly correlated with the amount of original drug Dox generated, and the peak area of ​​PDO-Pd is positively correlated with the peak area of ​​original drug Dox (R 2 =0.9964). The fluorescence intensity of PDO-Pd is also linearly correlated with its concentration. Therefore, the fluorescence intensity of the fluorescent molecule (PDO-Pd) is positively correlated with the concentration of the original drug (Dox). The concentration of the released drug can be measured by measuring the fluorescence intensity, thus achieving real-time quantitative visual monitoring of drug release.

[0112] Example 7

[0113] This embodiment involves the application of multifunctional skeleton molecules PDO-Tz and PBA-PDO-Tz in cell imaging, and the specific operations are as follows:

[0114] Take the tumor cells (A549 cells, HEK293T cells or HeLa cells) in the logarithmic growth phase and add 1×10 5 Each well was inoculated with 1 mL of culture medium and cultured at 37°C and 5% CO2 for 12 h. After the cells adhered to the wall, different concentrations of compounds were added for a certain period of time (if light was required, a cell phototoxicity irradiator was used at 12.5 mW / cm 2The cells were incubated in an incubator for the corresponding time, the medium was removed, and the cells were washed three times with 200 μL of PBS solution. Then, 200 μL of 4% paraformaldehyde was added to each well, and the cells were fixed in the dark at room temperature for 15 min. The fixative was removed, and the cells were washed three times with 200 μL of PBS solution. Finally, 200 μL of anti-fluorescence quencher was added to each well, and images were taken using a laser confocal microscope. The images were analyzed and processed using ZEN 2011 microscope imaging software.

[0115] Figure 8 The confocal fluorescence imaging diagrams of different compounds in A549 cells over time and concentration change, in which: Blank is the blank control group, PDO-Tz is the test group incubated for 2 hours with 50μM PDO-Tz, TCO-Dox is the test group incubated for 2 hours with 100μM prodrug TCO-Dox, 1 is the test group incubated for 2 hours with 50μM PDO-Tz and then 100μM prodrug TCO-Dox for 1 hour, 2 is the test group incubated for 2 hours with 50μM PDO-Tz and then 100μM prodrug TCO-Dox for 2 hours. Figure 8 It can be seen that the backbone molecule PDO-Tz and the prodrug TCO-Dox can be rapidly taken up by A549 cells, and undergo a bioorthogonal shear reaction inside the cells to release PDO-Pd with a fluorescent signal.

[0116] Fig. 9 Confocal fluorescence imaging of the selective uptake of the prodrug system in HEK293T cells (A) and HeLa cells (B). Fig. 9 In A: Blank is the blank control group, TCO-Dox is the test group incubated for 1 hour with the addition of prodrug TCO-Dox, PDO-Tz is the test group incubated for 1 hour with the addition of 50μM PDO-Tz, 1 and 3 are the test groups incubated for 1 hour with the addition of 50μM PDO-Tz and then 100μM prodrug TCO-Dox, PBA-PDO-Tz is the test group incubated for 1 hour with the addition of 50μM PBA-PDO-Tz, 2 and 4 are the test groups incubated for 1 hour with the addition of 50μM PBA-PDO-Tz and then 100μM prodrug TCO-Dox. Fig. 9 It can be seen that the PBA-PDO-Tz group in HeLa cells showed obvious blue fluorescence of PDO-Pd, while the blue fluorescence of the control group was weak. The blue fluorescence of both groups in 293T cells was relatively weak. This result shows that the backbone molecule PBA-PDO-Tz connected to the target head can be selectively taken up by tumor cells, and undergo a bioorthogonal shear reaction with TCO-Dox in the cell to release PBA-PDO-Pd molecules with fluorescent signals. This targeting group-modified PDO-Tz has molecular targeting and can accumulate in tumor cells.

[0117] Example 8

[0118] This example takes the prodrug system composed of PDO-Tz and TCO-Dox as an example to study the inhibitory effect of the prodrug system on A549 cell proliferation. The specific operation is as follows:

[0119] Take the tumor cells to be tested in the logarithmic growth phase, digest them with trypsin, add culture medium to dilute them into single cell suspension for counting. Then inoculate them into 96-well culture plates at a density of 3000 cells per well, and the volume of cell suspension in each well is 100μL. Culture at 37℃ and 5% CO2 for 12h. After the cells adhere to the wall, remove the culture medium, add 200μL of compound culture medium solution of different concentrations to each well, and set three replicate wells for each concentration (use cell phototoxicity irradiator at 12.5mW / cm if light is needed) 2 After 72 hours of exposure, the culture medium was removed and 100 μL of CCK-8 culture medium solution (600 μL of CCK-8 dissolved in 6 mL of culture medium) was added. After incubation at 37°C for 0.5-1 hour, the OD value at a wavelength of 450 nm was read using a multifunctional microplate reader (the OD value of the control group was about 1.0). The inhibition rate of different concentrations of compounds on tumor cells was calculated according to the formula of inhibition rate = [1-(OD value of the experimental group-OD value of the blank group) / (OD value of the control group-OD value of the blank group)] × 100%. The S-shaped dose-inhibition rate curve was drawn using the nonlinear regression model using GraphPad Prism software, and the IC was fitted and calculated. 50 value.

[0120] Depend on Fig.10 It can be seen that PDO-Tz showed lower toxicity EC to A549 cells 50 Greater than 50μM. After adding the prodrug TCO-Dox, the inhibitory activity on A549 cell proliferation was basically consistent with that of the positive control compound Dox. After the backbone molecule PDO-Tz was taken up by A549 cells, it could quickly undergo a bioorthogonal shear reaction with the prodrug TCO-Dox to release the drug molecule Dox.

[0121] Example 9

[0122] This example takes the prodrug system composed of PBA-PDO-Tz and TCO-Dox as an example, and studies the feasibility of the prodrug system in vivo through a mouse xenograft tumor slice imaging experiment. The specific operation is as follows:

[0123] like Fig.11 As shown in A, 6-8 week old female nude mice were subcutaneously inoculated with A549 cells (1×10 7 Cells / mouse) were used to establish a nude mouse model of human non-small cell lung cancer xenografts. When the tumor tissue grew to 75 mm 3The mice were randomly divided into 4 groups, 3 mice in each group, including control group, compound PBA-PDO-Tz (5 mg / kg), compound TCO-Dox (5 mg / kg) and compound PBA-PDO-Tz (5 mg / kg) and compound TCO-Dox (5 mg / kg) combination group. Compound PBA-PDO-Tz (5 mg / kg) was injected into the mice through the tail vein for 1 hour, and then compound TCO-Dox (5 mg / kg) was injected into the mice through the tumor. After 1 hour, the mice were euthanized. The tumor was then removed and cut into 0.5 μm tissue sections. The cell nuclei were stained with propidium iodide and washed with PBS, and finally imaged with a laser confocal microscope. The images were analyzed and processed by ZEN 2011 microscope imaging software.

[0124] The results are as follows Fig.11 As shown in B: Only the tumor tissue sections of the PBA-PDO-Tz / TCO-Dox treatment group showed obvious blue fluorescence, indicating that the PBA-PDO-Tz / TCO-Dox prodrug system can release fluorescent molecules PBA-PDO-Pd and anti-tumor drugs Dox by bioorthogonal shear reaction at the tumor site in mice. The prodrug system can simultaneously achieve targeted controllable drug release, visual monitoring of drug release, and imaging of target sites.

[0125] In summary, the multifunctional skeleton molecule prepared by the present invention has almost no photoactivity before activation. After reacting with a prodrug modified with trans-cyclooctene, 3-isocyanopropyl or 3-isocyanopropyl-1-carbamoyl, the fluorescence can be restored and the drug can be released, thereby realizing controlled release and visual monitoring of the drug. Moreover, the multifunctional skeleton molecule can realize controlled release of the drug in tumor cells and real-time visual monitoring of drug release by connecting a targeting group to the phenanthrene dioxin skeleton, and has good application prospects in tumor imaging and treatment.

[0126] The above-described embodiments are only preferred embodiments for fully illustrating the present invention, and the protection scope of the present invention is not limited thereto. Equivalent substitutions or changes made by those skilled in the art based on the present invention are within the protection scope of the present invention. The protection scope of the present invention shall be subject to the claims.

Claims

1. A multifunctional backbone molecule, characterized in that: The multifunctional skeleton molecule has the following general structural formula: Wherein, R is H or methyl phenylborate.

2. A method for preparing the multifunctional backbone molecule according to claim 1, characterized in that: The following steps are involved: S1, reacting (4-(6-methyl-1,2,4,5-tetrazine-3-yl)phenyl)methanol shown in formula 2 with 9,10-phenanthrenequinone-3-carboxylic acid shown in formula 4 in the presence of a condensing agent, an alkali reagent and a solvent to obtain an intermediate PQ-Tz; S2, treating the intermediate PQ-Tz and (4-(2-(vinyloxy)ethoxy)carbonyl)phenylboronic acid shown in Formula 5 or ethyleneoxyethanol shown in Formula 6 with light in the presence of a solvent to obtain the multifunctional skeleton molecule; The structures of Formula 2, Formula 3, intermediate PQ-Tz, Formula 5 and Formula 6 are shown below:

3. The preparation method according to claim 2, characterized in that: In S1, The condensing agent is selected from one or more of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N,N'-diisopropylcarbodiimide, and N,N'-dicyclohexylcarbodiimide; The alkaline reagent is selected from one or more of 4-dimethylaminopyridine, 1,8-diazabicyclo[5.4.0]undec-7-ene, and dimethylacetamide; The solvent is dichloromethane and / or N,N-dimethylformamide.

4. The preparation method according to claim 2 or 3, characterized in that: The molar ratio of the 9,10-phenanthrenequinone-3-carboxylic acid to the condensation agent and the alkaline reagent is 1:(1-2):(0.4-0.6).

5. The preparation method according to claim 2, characterized in that: In S1, the reaction temperature is 20-40°C and the reaction time is 1-3h; First, (4-(6-methyl-1,2,4,5-tetrazine-3-yl)phenyl)methanol shown in formula 2 and 9,10-phenanthrenequinone-3-carboxylic acid shown in formula 4 are dissolved in a solvent at 0°C, and then a condensation agent and an alkali reagent are added and the temperature is raised to 20-40°C for reaction, and the intermediate PQ-Tz is obtained through extraction and separation.

6. The preparation method according to claim 2, characterized in that: In S2, in the step of light treatment: the light source is white light, the energy of the light source is 10-20W, and the light treatment time is 0.5-2h.

7. Use of the multifunctional skeleton molecule according to claim 1 in the preparation of a drug with controlled release and visual monitoring.

8. The use according to claim 7, characterized in that: The drug also includes a trans-cyclooctene, 3-isocyanopropyl or 3-isocyanopropyl-1-carbamoyl modified prodrug; The tetrazine group in the multifunctional skeleton molecule undergoes a reverse electron demand Diels-Alder bioorthogonal bond cleavage reaction with the trans-cyclooctene, 3-isocyanopropyl or 3-isocyanopropyl-1-carbamoyl group in the prodrug to restore the fluorescence properties of the multifunctional skeleton molecule and activate the prodrug at the same time.

9. The use according to claim 8, characterized in that: The prodrug is one of the following structures:

10. The use according to claim 7, characterized in that: The drug is used for tumor imaging and / or tumor treatment; the tumors include human non-small cell lung cancer and cervical cancer.

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