Multifunctional scaffolds for controllable and visualized monitoring of drug release and preparation method and application thereof

Abstract

The application discloses a multifunctional backbone molecule for controllable and visual monitoring of drug release and a preparation method and application thereof, the multifunctional backbone molecule has a structure shown in formula I, includes a tetrazine structural unit and a phenanthrene dioxin skeleton, and fluorescence of the phenanthrene dioxin is quenched through fluorescence resonance energy transfer effect between the tetrazine and the phenanthrene dioxin parent nucleus. The multifunctional backbone molecule provided by the application can be subjected to a reverse electron demand Diels-Alder bioorthogonal bond cleavage reaction with a prodrug modified by a trans-cyclooctene, 3-isocyanopropyl or 3-isocyanopropyl-1-carbamoyl group, so that the quenching effect is destroyed, fluorescence is recovered, the prodrug is activated, controllable release and visual monitoring of the drug are realized. In addition, the multifunctional backbone molecule provided by the application can be connected with a targeting group on the phenanthrene dioxin skeleton, so that controllable release of the drug at a target site and real-time visual monitoring of the drug release are realized.

Description

TECHNICAL FIELD

[0001] The present application relates to the fields of organic synthesis and biological medicine, and in particular to a multifunctional backbone molecule for controllable and visual monitoring of drug release and a preparation method and application thereof. BACKGROUND

[0002] Bioorthogonal reaction refers to a kind 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 a round-bottom flask to a biological system. Such reactions can occur under physiological conditions in the body, do not interfere with other biochemical reactions occurring simultaneously in the body, and do not damage the organism and the target biological molecule. Bioorthogonal reactions have the advantages of mild conditions, high specificity, and biocompatibility. With the emergence and development of bioorthogonal technology as a tool for chemical biology research, the application of bioorthogonal reactions to the selective modification of proteins in organisms provides a powerful means for elucidating the structure and function of organisms and the occurrence, development, and intervention of diseases.

[0003] To date, scientists have developed various bioorthogonal reactions to achieve various functional modifications of proteins, including fluorescence imaging, activity regulation, and dynamic tracking. However, most current interventions on targets are single-site, single-function modifications, and there are few reports on dual or multiple functional target interventions. Therefore, achieving multifunctional intervention of targets can expand their applications in disease diagnosis and treatment. SUMMARY

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

[0005] To solve the above technical problems, the present application provides the following technical solutions:

[0006] The present application provides a multifunctional backbone molecule in a first aspect, which has the following structural general formula:

[0007]

[0008] wherein R is H or phenylboric acid methyl ester.

[0009] The second aspect of the present application provides a preparation method of the multifunctional backbone molecule of the first aspect, comprising the following steps:

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

[0011] S2, the intermediate PQ-Tz is subjected to light treatment with (4-(2-(ethylenoxy) ethoxy) carbonyl) phenylboronic acid shown in formula 5 or ethylenoxy ethanol shown in formula 6 in the presence of a solvent to obtain the multifunctional backbone molecule;

[0012] The structures of formula 2, formula 3, intermediate PQ-Tz, formula 5 and formula 6 are as follows:

[0013]

[0014]

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

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

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

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

[0019] Further, in S1, the reaction temperature is 20-40℃, and the reaction time is 1-3h.

[0020] In some preferred embodiments, (4-(6-methyl-1, 2, 4, 5-tetrazin-3-yl) phenyl) methanol shown in formula 2 is dissolved in a solvent at 0℃ with 9, 10-phenanthrenequinone-3-carboxylic acid shown in formula 4, then a condensing agent and a basic reagent are added and the temperature is raised to 20-40℃ for reaction, and the obtained intermediate PQ-Tz is extracted and separated.

[0021] In some preferred embodiments, (4-(6-methyl-1,2,4,5-tetrazin-3-yl)phenyl)methanol in S1 can be prepared by the following method: 4-(hydroxymethyl)benzonitrile, acetonitrile, nickel trifluoromethanesulfonate, were placed in a sealed tube, hydrazine hydrate was gradually added, and the mixture was reacted at 60 °C for 24 h. Then water was added, and the mixture was extracted with dichloromethane for 4 times until the organic phase became light purple. The organic phase was combined and rotary evaporated. Dichloromethane and an oxidant were added, and the mixture was reacted at room temperature for 2 h. The product was separated by thick preparation plate to obtain (4-(6-methyl-1,2,4,5-tetrazin-3-yl)phenyl)methanol in the form of light purple crystals; the oxidant includes but is not limited to phenyliodine diacetate.

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

[0023] 3-acetylphenanthrene was dissolved in 1,4-dioxane, sodium hypochlorite (available chlorine > 7.5%) and NaOH were added, and the mixture was stirred at 65 °C for 6 h. After cooling to room temperature, sodium thiosulfate was added, and then the pH was adjusted to 1-2 with 2M hydrochloric acid. The mixture was extracted with ethyl acetate twice, dried over anhydrous sodium sulfate, rotary evaporated to remove the solvent, and vacuum dried to obtain white solid;

[0024] 18-crown-6 ether, chromium trioxide were dissolved in acetic acid, distilled water was added, and the above-prepared white solid was dissolved in acetic acid. The mixture was reacted at 60 °C for 10 h. Then a large amount of distilled water was added, and a large amount of orange precipitate was generated. The mixture was filtered, the filter cake was washed twice with a mixture of acetic acid and water (1:1, v / v) and twice with dichloromethane, and dried to obtain 9,10-phenanthrenequinone-3-carboxylic acid in the form of orange solid.

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

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

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

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

[0029] The third aspect of the present application provides a use of the multifunctional backbone molecule of the first aspect in the preparation of a drug that can be released in a controllable manner and visualized.

[0030] Further, the drug further comprises a trans-cyclooctene, 3-isocyano propyl or 3-isocyano propyl-1-carbamoyl modified prodrug; the tetrazine group in the multifunctional backbone molecule and the trans-cyclooctene, 3-isocyano propyl or 3-isocyano propyl-1-carbamoyl in the prodrug perform a reverse electron demand Diels-Alder bio-orthogonal bond cleavage reaction, restoring the fluorescence properties of the multifunctional backbone molecule, and simultaneously activating the prodrug.

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

[0032]

[0033] Further, 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 application has the following beneficial effects:

[0035] The present application provides a multifunctional backbone molecule, which includes a tetrazine structural unit and a phenanthrene dioxin skeleton, and the fluorescence of the phenanthrene dioxin can be quenched through the fluorescence resonance energy transfer effect between the tetrazine and the phenanthrene dioxin nucleus. The above-mentioned multifunctional backbone molecule has good biocompatibility, can be connected with a targeting group (such as methyl phenylboronic acid) on the phenanthrene dioxin skeleton, realizes targeting a specific part (such as a tumor cell) in vivo, and performs a reverse electron demand Diels-Alder bio-orthogonal bond cleavage reaction with a trans-cyclooctene, 3-isocyano propyl or 3-isocyano propyl-1-carbamoyl modified prodrug, restores fluorescence while click releases drugs, realizes controllable release and visualized monitoring of drugs at the targeted site, and has a good application prospect in the diagnosis and treatment of tumors. BRIEF DESCRIPTION OF DRAWINGS

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

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

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

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

[0040] Figure 5 : A is the HPLC chart 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 chart of the reaction of 1200 μM PDO-Tz and 200 μM ICPr-mp at different time points (5 min, 30 min, 60 min, 120 min, 240 min); B is the linear relationship diagram of the peak area of PDO-Pz and the peak area of active drug mp;

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

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

[0044] Figure 9 : 1% DMSO, trans-cyclooctene conjugated prodrug, PBA-PDO-Tz, trans-cyclooctene conjugated prodrug and PBA-PDO-Tz after incubation in 293T normal cells, HeLa tumor cells for 1 hour, cell imaging results;

[0045] Figure 10: A is the survival rate of A549 cells after treatment with different concentrations of PDO-Tz; B is the inhibition rate of TCO-Dox prodrug system or related compounds on the proliferation of A549 cells; wherein, P value is calculated by two-tailed unpaired t test for comparison between drug treatment group and DMSO group, ns: no significant difference, *P < 0.05, ***P < 0.001, ****P < 0.0001;

[0046] Figure 11 : A is the schematic diagram of tumor-bearing mouse model construction and drug administration mode; 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), PBA-PDO-Tz (5 mg / kg) + TCO-Dox (5 mg / kg), the cell nucleus is stained with propidium iodide (PI), and the imaging is performed with rhodamine (RB) and DAPI channels, scale bar: 100 μm. DETAILED DESCRIPTION

[0047] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the description herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items.

[0048] The application will be further described with reference to the drawings and specific examples in order to better enable those skilled in the art to understand and practice the application, but the examples are not intended to limit the application.

[0049] Example 1

[0050] This example relates to the preparation of a multifunctional backbone molecule PDO-Tz, the specific operation is as follows:

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

[0052]

[0053] A mixture of 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) was combined in a sealed tube. The mixture was stirred at 60 °C for 24 h and cooled to 0 °C. Subsequently, water was added and the reaction mixture was extracted with dichloromethane. The combined organic layers were washed with brine and dried over Na2SO4. After removal of 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 h and concentrated under reduced pressure. The residue was purified by column chromatography to give (4-(6-methyl-1,2,4,5-tetrazin-3-yl)phenyl)methanol 219 mg in 36% yield over two steps as a purple solid.

[0054] 1 HNMR (400 MHz, CDC13) δ 8.59 (d, J = 8.6 Hz, 2H), 7.59 (d, J = 8.6 Hz, 2H), 4.84 (s, 2H), 3.10 (s, 3H).

[0055] (2) Synthesis of intermediate 49, 10-phenanthrenequinone-3-carboxylic acid

[0056]

[0057] A mixture of 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) was combined in a sealed tube. The mixture was stirred at 60 °C for 24 h and cooled to 0 °C. Subsequently, water was added and the reaction mixture was extracted with dichloromethane. The combined organic layers were washed with brine and dried over Na2SO4. After removal of 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 h and concentrated under reduced pressure. The residue was purified by column chromatography to give (4-(6-methyl-1,2,4,5-tetrazin-3-yl)phenyl)methanol 219 mg in 36% yield over two steps as a purple solid.

[0058] A mixture of 18-crown-6 (60 mg, 0.23 mmol), chromium trioxide (68 mg, 0.68 mmol) was added to a solution of acetic acid / water (v / v = 10 / 1) (5.5 mL) and phenanthrene-3-carboxylic acid (100 mg, 0.45 mmol) was added with stirring. The reaction mixture was stirred at 60 °C for 10 h. Water was then added to the reaction mixture and a precipitate was formed. The precipitate was washed with acetic acid / water (v / v = 1 / 1) and diethyl ether to give 9,10-phenanthrenequinone-3-carboxylic acid 45 mg in 50% yield over two steps as a yellow solid.

[0059] 1H NMR (400 MHz, DMSO-d6) δ 8.71 (s, 1H), 8.35 (d, J = 8.0 Hz, 1H), 8.14 - 8.02 (m, 3H), 7.83 - 7.79 (m, 1H), 7.60 - 7.56 (m, 1H).

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

[0061]

[0062] 9,10-phenanthrenequinone-3-carboxylic acid (420 mg, 1.67 mmol), (4-(6-methyl-1,2,4,5-tetrazin-3-yl)phenyl)methanol (281 mg, 1.39 mmol) were added to dichloromethane (50 mL) at 0 °C, N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDCI) (840 mg, 4.4 mmol), 4-dimethylaminopyridine (DMAP) (60 mg, 0.6 mmol) were added with stirring. The reaction mixture was allowed to warm to room temperature and kept for 2 hours. Then diluted with water (50 mL) and extracted with dichloromethane. The combined organic phase, washed with brine, dried over Na2S04and concentrated under reduced pressure. The residue was purified by column chromatography to give red solid PQ-Tz 334 mg, yield 55%.

[0063] 1 H NMR (400 MHz, CDCl3) δ 8.76 (s, 1H), 8.66 (d, J = 8.0 Hz, 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 (100 MHz, CDCl3) δ 180.1, 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 the multifunctional scaffold 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 hand-held white light LED lamp for 1 hour. The reaction mixture was then extracted with ethyl acetate. The combined organic layers were washed with brine, dried over Na2S04, and concentrated under reduced pressure. The residue was purified by preparative thin layer chromatography to give a light red solid PDO-Tz 168 mg, yield 64%.

[0068] 1 H NMR (400 MHz, DMSO-d6) δ 9.33 (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 (100 MHz, DMSO-d6) δ 167.1, 165.7, 163.0, 140.8, 140.7, 135.3, 132.6, 132.5, 131.52, 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 C 29 H 24 N4O6, 525.45; found 524.53.

[0071] Example 2

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

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

[0074]

[0075] (360 μL, 2.0 mmol) in dichloromethane (50 mL) at 0 °C, N-(3-dimethylaminopropyl)-N'- ethylcarbodiimide hydrochloride (EDCI) (768 mg, 4.0 mmol), 4-dimethylaminopyridine (DMAP) (100 mg, 0.8 mmol) were added with stirring. The reaction mixture was allowed to warm to room temperature and left 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 Na2S04and concentrated under reduced pressure. The residue was purified by column chromatography to give 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoate 450 mg, yield 70%.

[0076] 1 H NMR (400 MHz, CDC13) δ 8.03 (d, J = 7.8 Hz, 2H), 7.87 (d, J = 7.9 Hz, 2H), 6.51 (dd, J = 14.3, 6.8 Hz, 1H), 4.60 - 4.51 (m, 2H), 4.24 (dd, J = 14.4, 2.3 Hz, 1H), 4.07 (dd, J = 6.9, 2.3 Hz, 1H), 4.05 - 4.01 (m, 2H), 1.36 (s, 12H).

[0077] (2) Synthesis of multifunctional scaffold 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 hand-held white light LED lamp for 2 hours. The reaction mixture was then extracted with ethyl acetate. The combined organic layers were washed with brine, dried over Na2S04and concentrated under reduced pressure. The residue was purified by preparative thin layer chromatography to give PBA-PDO-Tz as a pink solid 55 mg, yield 68%.

[0080] 1H NMR (400 MHz, DMSO-d6) δ 9.33 (d, J = 11.0 Hz, 1H), 8.81 - 8.72 (m, 1H), 8.53 (d, J = 8.0 Hz, 2H), 8.22 (s, 2H), 8.21 - 8.11 (m, 3H), 7.84 (dd, J = 8.4, 3.0 Hz, 2H), 7.68 (dd, J = 8.3, 4.1 Hz, 4H), 7.57 (dd, J = 16.9, 7.8 Hz, 2H), 5.88 (d, J = 6.0 Hz, 1H), 5.60 (s, 2H), 4.55 (t, J = 12.3 Hz, 1H), 4.42 (dt, J = 19.1, 9.1 Hz, 3H), 4.16 (d, J = 5.0 Hz, 2H), 3.01 (s, 3H).

[0081] 13 C NMR (100 MHz, DMSO-d6) δ 167.64, 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 C 36 H 29 BN4O9, 673.21; Found 672.46.

[0083] Example 3

[0084] This example relates to the preparation of fluorescent molecule PDO-Pz, the specific operation as follows:

[0085]

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

[0087] 1 H NMR (400 MHz, DMSO-d6) δ 9.32 (d, J = 13.4 Hz, 1H), 8.81 (t, J = 7.0 Hz, 1H), 8.29 - 8.12 (m, 3H), 7.81 - 7.67 (m, 4H), 7.57 (d, J = 7.2 Hz, 1H), 7.32 (d, J = 8.0 Hz, 1H), 5.77 (d, J = 5.2 Hz, 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 (100 MHz, DMSO-d6) δ 166.9, 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 C 30 H 28 N3O6, 526.20; Found 526.17.

[0090] Example 4

[0091] This example relates to the preparation of fluorescent molecule PDO-Pd, which is prepared as follows:

[0092]

[0093] PDO-Tz (10 mg, 0.02 mmol) was dissolved in 1 mL dry DMSO and cis-cyclooctene (68 μL, 0.5 mmol) was added to the reaction and stirred at 110 °C for 12 h. The reaction was monitored by thin layer chromatography and upon completion, 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 give a white crystalline solid (7 mg, 58%).

[0094] 1 H NMR (400 MHz, DMSO-d6) δ 9.37 (s, 1H), 8.81 (dd, J = 7.8, 1.0 Hz, 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, 1.9 Hz, 1H), 5.56 (d, J = 2.6 Hz, 2H), 4.70 (q, J = 5.6 Hz, 1H), 4.52 (td, J = 11.2, 2.2 Hz, 1H), 4.40 (ddd, J = 11.4, 7.5, 1.7 Hz, 1H), 3.87 - 3.77 (m, 2H), 3.53 (ddq, J = 17.5, 11.7, 5.9, 5.4 Hz, 2H), 2.84 (dd, J = 7.6, 5.0 Hz, 2H), 2.69 (d, J = 6.9 Hz, 2H), 2.67 (s, 3H), 1.71 (s, 2H), 1.47 (s, 2H), 1.33 (s, 4H).

[0095] 13 C NMR (100 MHz, DMSO-d6) δ 166.3, 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, 126.46, 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 C 37 H37 N2O6, 605.27; test value 605.36.

[0097] Example 5

[0098] Using the multifunctional backbone molecule PDO-Tz as a control, the fluorescence properties of the fluorescent molecules PDO-Pz and PDO-Pd prepared in Examples 3 and 4 were studied. The specific procedures are as follows:

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

[0100] Test results are as follows Figure 1 As shown, compared with PDO-Pz and PDO-Pd, PDO-Tz exhibits significantly weaker fluorescence, but shows better fluorescence enhancement after reaction with ICPr-mp or cis-cyclooctene, with maximum enhancement factors of 15-fold and 44-fold, respectively; and due to Figure 2 , 3 It can be seen that the concentration of PDO-Pz is positively correlated with fluorescence intensity within the range of 10-125 μM (R0). 2 =0.9874), and the concentration of PDO-Pd was positively correlated with fluorescence intensity within the range of 1-125 μM (R = 0.9874). 2 =0.9517).

[0101] Example 6

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

[0103] (1) Prepare 4 mM solutions of the prodrugs ICPrc-mmc and ICPr-mp using DMSO:PBS (1:9, v / v), and prepare 24 mM solutions of PDO-Tz using DMSO:PBS (1:9, v / v). Mix 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 with 250 μL of ICPrc-mmc (c = 4.0 mM) or 250 μL of ICPr-mp (c = 4.0 mM) with 250 μL of PDO-Tz (c = 24 mM), and incubate at 37 °C. Record HPLC traces (10 μL injection) at the specified wavelength at 5 and 240 minutes.

[0104] The results are as follows Figure 4As shown, the absorption peak of the prodrug molecule ICPr-mp, ICPrc-mmc disappeared at 240 min, successfully releasing mp, mmc, and also generating the fluorescent molecule PDO-Pz. Through quantitative analysis, it is further concluded that the release rate of the prodrug ICPr-mp is 94.8%; the release rate of ICPrc-mmc is 96.1%.

[0105] (2) The prodrug TCO-DOX was prepared into a solution with a concentration of 3 mM using a DMSO:PBS (1:9, v / v) solution, and PDO-Tz was prepared into a solution with a concentration of 3 mM using a DMSO:PBS (1:9, v / v) solution. 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. The HPLC trace (10 μL injection) was recorded at a specified wavelength for 5 minutes.

[0106] The results are shown in Figure 5 : The absorption peak of the prodrug molecule TCO-Dox disappeared at 5 min, successfully releasing the Dox original drug, and also generating the fluorescent molecule PDO-Pd. The conversion rate of the prodrug TCO-Dox reached 99%, but the release rate of the Dox original drug was only 53%, because part of the compound formed a cyclization product 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 a DMSO:PBS (1:9, v / v) solution, and PDO-Tz was prepared into a solution with a concentration of 1200 μM using a DMSO:PBS (1:9, v / v) solution. 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, 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. The HPLC trace (10 μL injection) was recorded at a specified wavelength for 5, 30, 60, 120, and 240 minutes.

[0109] The results are shown in Figure 6 : The amount of PDO-Pz generated during the reaction of PDO-Tz and the prodrug ICPr-mp also showed a linear correlation with the amount of the original drug mp generated, and the peak area of PDO-Pz was positively correlated with the peak area of the original drug mp (R 2= 0.9917). The fluorescence intensity of PDO-Pz is also linearly related to its concentration, so the fluorescence intensity of the fluorescent molecule (PDO-Pz) is positively correlated with the concentration of the parent drug (mp), and the concentration of the released drug can be measured by measuring the fluorescence intensity, realizing real-time quantitative visualization monitoring of drug release.

[0110] Effect of the concentration ratio of multifunctional scaffold molecule PDO-Tz and prodrug on prodrug release: The prodrug TCO-Dox was prepared into a solution with a concentration of 200 μM and 300 μM using DMSO:PBS (1:9, v / v), and PDO-Tz was prepared into a solution with a concentration 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 and 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 solution with a concentration ratio (P:T) of PDO-Tz to TCO-Dox of 1:2, 1:1 and 2:1.5, and incubated at 37°C. The HPLC trace (10 μL injection) was recorded at a specified wavelength for 5 minutes.

[0111] The results are shown in Table 1: Figure 7 The amount of PDO-Pd generated during the reaction of PDO-Tz and prodrug TCO-Dox is also linearly related to the amount of parent drug Dox generated, and the peak area of PDO-Pd is positively correlated with the peak area of parent drug Dox (R 2 = 0.9964). The fluorescence intensity of PDO-Pd is also linearly related to its concentration, so the fluorescence intensity of the fluorescent molecule (PDO-Pd) is positively correlated with the concentration of the parent drug (Dox), and the concentration of the released drug can be measured by measuring the fluorescence intensity, realizing real-time quantitative visualization monitoring of drug release.

[0112] Example 7

[0113] This example relates to the application of multifunctional scaffold molecules PDO-Tz and PBA-PDO-Tz in cell imaging, and the specific operation is as follows:

[0114] Take the tumor cells (A549 cells, HEK293T cells or HeLa cells) to be tested in the logarithmic growth phase, inoculate 1×10 5 cells / well in a 24-well plate, add 1 mL of culture medium to each well, and incubate at 37°C with a 5% CO2concentration for 12 h. After the cells adhere, add different concentrations of compounds for a certain period of time (use a cell phototoxicity illuminator to irradiate at 12.5 mW / cm 2Light intensity (light for 3 min). After incubation in the incubator for the corresponding time, the medium was aspirated, washed three times with 200 μL of PBS solution, and then 200 μL of 4% paraformaldehyde was added to each well for fixation at room temperature in the dark for 15 min. The fixing solution was aspirated, washed three times with 200 μL of PBS solution, and finally 200 μL of anti-fluorescence quencher was added to each well for imaging with a laser confocal microscope. The images were analyzed and processed by ZEN 2011 microscope imaging software.

[0115] Figure 8 For the confocal fluorescence imaging graphs of different compounds in A549 cells over time and concentration, the graphs are as follows: Blank is the blank control group, PDO-Tz is the test group with 50 μM PDO-Tz added and incubated for 2 h, TCO-Dox is the test group with 100 μM prodrug TCO-Dox added and incubated for 2 h, 1 is the test group with 50 μM PDO-Tz added and incubated for 2 h, and then 100 μM prodrug TCO-Dox added and incubated for 1 h, and 2 is the test group with 50 μM PDO-Tz added and incubated for 2 h, and then 100 μM prodrug TCO-Dox added and incubated for 2 h. It can be seen that the scaffold molecule PDO-Tz and the prodrug TCO-Dox can be rapidly taken up by A549 cells, and a biological orthogonal cleavage reaction occurs in the cells to release PDO-Pd with a fluorescent signal. Figure 8

[0116] Figure 9 For the confocal fluorescence imaging graphs of the selective uptake of the prodrug system in HEK293T cells (A) and HeLa cells (B), Figure 9 In A, Blank is the blank control group, TCO-Dox is the test group with the prodrug TCO-Dox added and incubated for 1 h, PDO-Tz is the test group with 50 μM PDO-Tz added and incubated for 1 h, 1 and 3 are the test groups with 50 μM PDO-Tz added and incubated for 1 h, and then 100 μM prodrug TCO-Dox added and incubated for 1 h, PBA-PDO-Tz is the test group with 50 μM PBA-PDO-Tz added and incubated for 1 h, and 2 and 4 are the test groups with 50 μM PBA-PDO-Tz added and incubated for 1 h, and then 100 μM prodrug TCO-Dox added and incubated for 1 h. It can be seen that in HeLa cells, the PBA-PDO-Tz group shows obvious blue fluorescence of PDO-Pd, while the blue fluorescence of the control group is weak. In 293T cells, the blue fluorescence of the two groups is relatively weak. This result shows that the scaffold molecule PBA-PDO-Tz connected with a targeting head can be selectively taken up by tumor cells, and a biological orthogonal cleavage reaction occurs in the cells with TCO-Dox to release the PBA-PDO-Pd molecule with a fluorescent signal. This PDO-Tz modified with a targeting group has molecular targeting and can accumulate in tumor cells. Figure 9 ​​

[0117] Example 8

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

[0119] Tumor cells in the logarithmic growth phase were digested with trypsin and diluted with culture medium to form a single-cell suspension for counting. Cells were then seeded at a density of 3000 cells per well in 96-well plates, with a cell suspension volume of 100 μL per well. Cells were cultured at 37°C and 5% CO2 for 12 h. After cell attachment, the culture medium was removed, and 200 μL of different concentrations of compound culture medium solution was added to each well, with three replicates for each concentration. (For cells requiring light irradiation, a cell phototoxicity irradiation device was used at 12.5 mW / cm².) 2 Light intensity: 3 min. After 72 h of treatment, the culture medium was removed, and 100 μL of CCK-8 culture medium solution was added (600 μL of CCK-8 dissolved in 6 mL of culture medium). The mixture was incubated at 37℃ for 0.5-1 h, and the OD value at 450 nm was read using a multi-functional microplate reader (the OD value of the control group was around 1.0). The inhibition rate of different concentrations of the compound on tumor cells was calculated using the formula: [1 - (OD value of experimental group - OD value of blank group) / (OD value of control group - OD value of blank group)] × 100%. An S-shaped dose-inhibition rate curve was plotted using a nonlinear regression model in GraphPad Prism software, and the IC50 was fitted and calculated. 50 value.

[0120] Depend on Figure 10 It can be seen that PDO-Tz exhibits low toxicity to A549 cells. 50 The concentration was greater than 50 μM. After the addition of the prodrug TCO-Dox, the inhibitory activity against A549 cell proliferation was essentially the same as that of the positive control compound Dox. After the backbone molecule PDO-Tz was taken up by A549 cells, it rapidly underwent a bioorthogonal cleavage reaction with the prodrug TCO-Dox, releasing the drug molecule Dox.

[0121] Example 9

[0122] This embodiment uses a prodrug system composed of PBA-PDO-Tz and TCO-Dox as an example to study the feasibility of the prodrug system's in vivo response through mouse xenograft tumor section imaging experiments. The specific operation is as follows:

[0123] like Figure 11 As shown in Figure A, female nude mice aged 6-8 weeks were subcutaneously inoculated with A549 cells (1×10⁻⁶). 7 A nude mouse model of human non-small cell lung cancer xenograft tumor was established using 10 cells / mouse. The tumor tissue was allowed to grow to 75 mm. 3The mice were randomly divided into 4 groups, 3 mice in each group, which were 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 mouse body through the tail vein for 1 h, and then compound TCO-Dox (5 mg / kg) was injected into the mouse body through intratumoral injection, and after 1 h, the mice were euthanized. Then the tumor was taken out, cut into 0.5 μm tissue sections, stained with iodinated propylamine, 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 shown in Figure 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 undergo a bio-orthogonal cleavage reaction in the tumor site of the mouse body to release the fluorescent molecule PBA-PDO-Pd and the antitumor drug Dox. The prodrug system can simultaneously achieve targeted and controllable drug release, visual monitoring of drug release, and target site imaging. Figure 11 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 undergo a bio-orthogonal cleavage reaction in the tumor site of the mouse body to release the fluorescent molecule PBA-PDO-Pd and the antitumor drug Dox. The prodrug system can simultaneously achieve targeted and controllable drug release, visual monitoring of drug release, and target site imaging.

[0125] In summary, the multifunctional backbone molecule prepared by the present application has almost no photoactivity before activation, and after reaction with the prodrug modified by trans-cyclooctene, 3-isocyanopropyl or 3-isocyanopropyl-1-carbamoyl, the fluorescence can be restored and the drug can be released, achieving controllable release and visual monitoring of the drug; and the multifunctional backbone molecule can be connected with a targeting group on the phenanthrene dioxane skeleton to achieve controllable release of the drug in tumor cells and real-time visual monitoring of drug release, which has good application prospect in tumor imaging and treatment.

[0126] The above-described embodiments are only preferred embodiments of the present application, and the protection scope of the present application is not limited thereto. Any equivalent replacement or transformation made by those skilled in the art based on the present application is within the protection scope of the present application. The protection scope of the present application is subject to the claims.

Claims

1. A multifunctional skeletal molecule, characterized in that, The multifunctional skeletal molecule has the following general structural formula: ， Wherein, R is H or methyl phenylboronic acid.

2. A method for preparing the multifunctional framework molecule according to claim 1, characterized in that, Includes the following steps: S1. The (4-(6-methyl-1,2,4,5-tetraazine-3-yl)phenyl)methanol of Formula 2 and the 9,10-phenanthrenequinone-3-carboxylic acid of Formula 4 are reacted in the presence of a condensing agent, a base reagent and a solvent to obtain intermediate PQ-Tz. S2. The intermediate PQ-Tz is subjected to phototreatment in the presence of a solvent with (4-(2-(ethoxy)ethoxy)carbonyl)phenylboronic acid as shown in Formula 5 or ethoxyethanol as shown in Formula 6 to obtain the multifunctional framework molecule. The structures of Formulas 2, 4, intermediate PQ-Tz, 5, and 6 are shown below: ， 。 3. The preparation method according to claim 2, characterized in that, In S1, The condensing agent is selected from 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N , N' -Diisopropylcarbodiimide, N , N' -One or more of 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-phenanthroquinone-3-carboxylic acid to the condensing agent and the base 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 ℃, and the reaction time is 1-3 h; First, (4-(6-methyl-1,2,4,5-tetraazine-3-yl)phenyl)methanol (as shown in Formula 2) and 9,10-phenanthroquinone-3-carboxylic acid (as shown in Formula 4) are dissolved in a solvent at 0 °C. Then, a condensing agent and a base reagent are added, and the temperature is raised to 20-40 °C for reaction. After extraction and separation, the intermediate PQ-Tz is obtained.

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

7. The use of the multifunctional scaffold molecule of claim 1 in the preparation of a drug with controlled release and visual monitoring.

8. The application according to claim 7, characterized in that, The drug also contains a prodrug modified with trans-cyclooctene, 3-isocyanopropyl or 3-isocyanopropyl-1-carbamoyl; The tetrazine group in the multifunctional backbone molecule undergoes an electron-demanding Diels-Alder bioorthogonal bond cleavage reaction with the trans-cyclooctene, 3-isocyanopropyl, or 3-isocyanopropyl-1-carbamoyl group in the prodrug, restoring the fluorescence properties of the multifunctional backbone molecule and activating the prodrug.

9. The application according to claim 8, characterized in that, The prodrug has one of the following structures: 。 10. The application 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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