A bioorthogonal bond-breaking reaction system, prodrug, and applications thereof

Abstract

The present invention discloses a bioorthogonal bond-breaking reaction system, prodrugs and applications thereof. Among the existing types of bioorthogonal reactions, the tetrazine and trans-ene are currently the best systems applied to drug delivery, but they have the defect of instability; the present invention discloses a new type of bioorthogonal bond-breaking reaction, in which N-oxides can rapidly react with organosilicon borate derivatives under physiological-like conditions to obtain corresponding tertiary amines and silanols. This reaction has characteristics such as fast reaction kinetics and simple availability of reaction components, and its second-order reaction kinetic constant k2 can reach 10 3 M ‑1 s ‑1 . By using the bioorthogonal bond-breaking reaction of the present invention, corresponding intelligent fluorescent probes or prodrugs containing tertiary amine functional groups are designed, which are very stable under physiological conditions. After undergoing a click reaction with organosilicon boron reagents, corresponding tertiary amines are generated, thereby turning on fluorescence or producing corresponding pharmacological activities.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, relates to a prodrug technology, and particularly relates to a bioorthogonal bond-breaking reaction system, prodrug and application thereof. Background Art

[0002] The design of traditional prodrugs mainly relies on enzymes or endogenous small molecules (such as GSH, ROS, etc.) to activate the prodrug and release the parent drug, achieving great success in improving the druggability of candidate drugs. In recent years, medicinal chemists have proposed a new concept of bioorthogonal prodrugs. Different from traditional prodrugs, these prodrugs do not rely on endogenous stimuli, but release the parent drug through a "click" reaction with an exogenous compound under physiological conditions to exert a therapeutic effect. Compared with traditional prodrugs, bioorthogonal prodrugs have obvious advantages in terms of the specificity of drug release and spatiotemporal controllability. In the past 10 years, great progress has been made in the field of bioorthogonal prodrugs, and many types of bioorthogonal prodrugs have been designed. However, most bioorthogonal prodrugs have the problem of slow reaction kinetics, such as the second-order reaction kinetic constant being less than 10 2 M -1 s -1 , which means that a higher reactant concentration is required to ensure a reasonable drug release half-life. And a high reactant concentration will lead to potential cytotoxicity problems. Among many bioorthogonal reaction types, the kinetic constant of the bioorthogonal reaction between tetrazine and trans-ene is in the range of 10 2 ~10 3 M -1 s -1 , and this type of structure is currently the best system applied to drug delivery. Although the bioorthogonal reaction between tetrazine and trans-ene has high activity, it has defects. For example, tetrazine compounds are easily attacked by affinity reagents (such as GSH biothiol) in the body, resulting in a shortened half-life in the body. Trans-ene is easily isomerized by metalloproteins in the body to generate cis products, thus losing the ability to react with tetrazine. Therefore, it is very necessary to develop new bioorthogonal bond-breaking reactions with fast reaction kinetics and introduce them into drug delivery. Summary of the Invention

[0003] Aiming at the defects existing in the existing bioorthogonal prodrug delivery methods, the present invention discloses a bioorthogonal bond-breaking reaction system, prodrug and application thereof. N-oxide can rapidly react with organosilicon borate derivatives under quasi-physiological conditions to obtain corresponding tertiary amines and silanols, solving the following two key problems: 1. Low reaction kinetics (k < 10 2 M -1 s -1 ); 2. Poor stability of prodrugs and activators.

[0004] The present invention adopts the following technical solutions:

[0005] A bioorthogonal bond-breaking reaction system includes an N-oxide and an organosilicon boric acid derivative; the N-oxide contains a nitrogen-oxygen dipole and can rapidly react with the organosilicon boric acid derivative under quasi-physiological conditions to obtain the corresponding tertiary amine and silanol. Using this bioorthogonal bond-breaking reaction, corresponding intelligent fluorescent probes or prodrugs containing a tertiary amine functional group are designed. These probes and prodrugs are very stable under physiological conditions and generate the corresponding tertiary amine after a click reaction with an organosilicon boron reagent, thereby turning on fluorescence or generating the corresponding pharmacological activity.

[0006] In the present invention, the N-oxide is R1R2R3N + O - and the organosilicon boric acid derivative is R8R9R 10 SiB(OR 11 )2. The substituents involved are conventional substituents. The present invention utilizes the click reaction between the nitrogen oxide and the silicon boron. This reaction has characteristics such as fast reaction kinetics and simple availability of reaction components. Its second-order reaction kinetic constant k2 can reach 10 3 M -1 s -1 . Using this bioorthogonal bond-breaking reaction, corresponding intelligent fluorescent probes or prodrugs containing a tertiary amine functional group can be designed. These probes and prodrugs are very stable under physiological conditions and generate the corresponding tertiary amine after a click reaction with an organosilicon boron reagent, thereby turning on fluorescence or generating the corresponding pharmacological activity.

[0007] The present invention discloses an N-oxide prodrug with the following chemical structural formula:

[0008] The substituents involved are conventional substituents. Payload-X is the part of the parent drug with one H omitted, that is, Payload-XH is the parent drug. The present invention designs a series of self-eliminating linker structures based on the orthogonal bond-breaking reaction and uses them in the design of prodrugs of parent drugs containing various functional groups, expanding the scope of application of this bioorthogonal bond-breaking reaction for activating prodrugs; the self-eliminating linker structure is an N-oxide based on an ethylenediamine backbone, and the nitrogen-oxygen functional group can selectively respond to the silicon boron reagent. When the prodrug reacts with the silicon boron reagent, the nitrogen oxide is reduced to a tertiary amine intermediate. Utilizing the nucleophilicity of the tertiary amine nitrogen atom, this intermediate undergoes intramolecular cyclization under physiological conditions to release the parent drug (Payload-XH).

[0009] Furthermore, the present invention inserts a self-eliminating linker between the above N-oxide prodrug and the parent drug, which can further expand the tolerance of the functional group for releasing the parent drug. The N-oxide prodrug has the following chemical structural formula:

[0010] After the N-oxide prodrug is reduced by a silicon boron reagent to a tertiary amine and cyclized, a self-eliminating linker (L part) is released, and the self-eliminating linker undergoes a self-eliminating reaction under physiological conditions to release the parent drug, and the parent drug is represented as Payload-YH.

[0011] The present invention discloses a method for bioorthogonal bond cleavage reaction, reacting the above N-oxide or N-oxide prodrug with an organosilicon boronic acid derivative to complete the bioorthogonal bond cleavage reaction; the present invention discloses a bioorthogonal bond cleavage reaction prodrug system, including the above N-oxide prodrug and an organosilicon boronic acid derivative. Preferably, the reaction is carried out under quasi-physiological conditions or physiological conditions.

[0012] In the present invention, the connection between the original drug (parent drug) and the N-oxide or L self-eliminating linker is a conventional method, and an N-oxide prodrug can be formed by conventional chemical bond binding. The parent drug itself is an existing product. The creativity of the present invention lies in disclosing a bioorthogonal bond cleavage reaction, in which the N-oxide rapidly reacts with an organosilicon boronic acid derivative under quasi-physiological conditions, solving two key problems of low reaction kinetics and poor stability of prodrugs and activators in the prior art. Preferably, in the formula, R1-R7 are independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted aryl; R8-R 10 are independently selected from substituted or unsubstituted alkyl, substituted or unsubstituted aryl; R 11 is selected from H, alkyl or substituted cycloalkyl; X includes O, S, -SS-, etc.; X` includes O, S, -OOC-, -O-CO-O-, -O-CO-NR-, -N + (R)2-, -O-CO-S-, etc.; Y includes O, -NR 12 -, N(R)2, S, -COO-, etc.

[0013] The present invention discloses the application of the above bioorthogonal bond cleavage reaction system, N-oxide prodrug or bioorthogonal bond cleavage reaction prodrug system in the preparation of bioorthogonal prodrugs, or in the preparation of drug delivery systems, or in the preparation of active drugs, and the active drugs include anti-tumor drugs and cell imaging drugs.

[0014] In the existing types of bioorthogonal reactions, the kinetic constant of the bioorthogonal reaction between tetrazine and trans-ene is in the range of 10 2 ~10 3 M -1 s -1, is the best system currently applied to drug delivery, but it has defects. The present invention discloses a new class of bioorthogonal bond-breaking reactions. N-oxides can rapidly react with organosilicon boronic acid derivatives under physiological conditions to obtain corresponding tertiary amines and silanols. This reaction features fast reaction kinetics and simple availability of reaction components. Its second-order reaction kinetic constant k2 can reach 10 3 M -1 s -1 . By using the bioorthogonal bond-breaking reaction of the present invention, corresponding intelligent fluorescent probes or prodrugs containing tertiary amine functional groups are designed, which are very stable under physiological conditions and generate corresponding tertiary amines after click reaction with organosilicon boron reagents, thereby turning on fluorescence or producing corresponding pharmacological activities. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 are the structures of some existing bioorthogonal prodrugs and their second-order reaction kinetic constants.

[0016] Figure 2 are the chemical structural formulas of N-oxides and organosilicon boronic acids and the schematic diagram of the bioorthogonal bond-breaking reaction.

[0017] Figure 3 are the schematic diagrams of N-oxide prodrugs and the bioorthogonal bond-breaking reaction.

[0018] Figure 4 are the schematic diagrams of the specific chemical structural formulas of N-oxides and organosilicon boronic acids.

[0019] Figure 5 are the schematic diagrams of the specific chemical structural formulas of N-oxide prodrugs.

[0020] Figure 6 is the schematic diagram of the chemical structural formula of the self-eliminating linker L.

[0021] In the formula, R1-R7 are independently selected from H, substituted or unsubstituted alkyl groups, substituted or unsubstituted aryl groups; R8-R 10 are independently selected from substituted or unsubstituted alkyl groups, substituted or unsubstituted aryl groups; R 11 is selected from H, alkyl groups, substituted cycloalkyl groups; X includes O, S, -SS-, etc.; X` includes O, S, -OOC-, -O-CO-O-, -O-CO-NR-, -N + (R)2-, -O-CO-S-, etc.; Y includes O, N(R)2, -NR 12 -, S, -COO-, etc.; R 12 -R 15 are independently selected from H, substituted or unsubstituted alkanes, -NO2, -CF3, etc., and Z is selected from O, S.

[0022] Figure 7Schematic diagram for the general synthesis of organosilicon boron derivatives and product data.

[0023] Figure 8 Product data and structure diagram of organosilicon boron derivatives.

[0024] Figure 9 Schematic diagram for the general synthesis of tertiary amine prodrugs, product data and structure diagram.

[0025] Figure 10 Fluorescence intensity change curve of the reaction of 8-Si and 9-Si with NO-1 and its second-order reaction kinetic constant.

[0026] Figure 11 Kinetic constant of the reaction of 1-Si with NO-1 in a simulated tumor environment.

[0027] Figure 12 1-Si cytotoxicity and NO-1 cell imaging at 24 hours.

[0028] Figure 13 Synthesis schematic diagram of prodrug 1a, reaction process of prodrug 1a with 1-Si, and change diagrams of intermediate and drug release peak areas.

[0029] Figure 14 Cytotoxicity diagram of prodrug 1a.

[0030] Figure 15 Synthesis schematic diagram of prodrug 1b.

[0031] Figure 16 Particle size distribution diagram of prodrug 1b nanoparticles.

[0032] Figure 17 Electron microscopy image of prodrug 1b nanoparticles.

[0033] Figure 18 Electron microscopy image of the reaction solution of prodrug 1b nanoparticles with boron reagent.

[0034] Figure 19 Cytotoxicity of prodrug 1b.

[0035] Figure 20 Reaction schematic diagram of structures 1c - 1l, product data and structure diagrams.

[0036] Figure 21 Synthesis schematic diagrams of compounds 1h, 1i, 1m, 2a, 2b.

[0037] Figure 22 Synthesis schematic diagrams of compounds 2c, 2d, 2e, 2f.

[0038] Figure 23 Reaction phenomena of compound 2a with compound 1-Si and ultraviolet absorption changes of the reaction solution at different times.

[0039] Figure 24 It is the reaction phenomenon of prodrug 2b and compound 1-Si and the fluorescence intensity change curve of the reaction solution from 0 min to 60 min.

[0040] Figure 25 It is the cell imaging diagram of compound 2b.

[0041] Figure 26 It is the cytotoxicity diagram of prodrugs 2c, 2d, and 2e. Specific implementation mode

[0042] The raw materials involved in the present invention are all existing products, and the specific preparation operations and performance tests are conventional technologies; for example, activating magnesium chips with acid. Specifically, dissolve 1 g of magnesium chips in 10 mL of water, dropwise add 4 mL of hydrochloric acid under ice bath, stir for 10 min, filter, and dry to obtain the activated magnesium chips.

[0043] Among the existing bioorthogonal reaction types, the kinetic constant of the bioorthogonal reaction between tetrazine and trans-ene is in the range of 10 2 ~10 3 M -1 s -1 , which is currently the best system applied to drug delivery. See Figure 1 ; The present invention discloses a new type of bioorthogonal bond-breaking reaction. N-oxide can rapidly react with organosilicon boronic acid derivatives under physiological-like conditions to obtain the corresponding tertiary amine and silanol. This reaction has the characteristics of fast reaction kinetics and simple and easily available reaction components. Its second-order reaction kinetic constant k2 can reach 10 3 M -1 s -1 , see Figure 2 The N-oxide is R1R2R3N + O - , and the organosilicon boronic acid derivative is R8R9R 10 SiB(OR 11 ). The substituents involved are conventional substituents. The present invention utilizes the nitrogen oxide and silicon boron click reaction, which has the characteristics of fast reaction kinetics and simple and easily available reaction components. Its second-order reaction kinetic constant k2 can reach 10 3 M -1 s -1 . Using this bioorthogonal bond-breaking reaction, corresponding intelligent fluorescent probes or prodrugs containing tertiary amine functional groups can be designed. These probes and prodrugs are very stable under physiological conditions and generate the corresponding tertiary amine after click reaction with organosilicon boron reagents, thereby turning on fluorescence or generating the corresponding pharmacological activity. See Figure 3 , and the ethylenediamine skeleton is a self-eliminating linker, and L is another self-eliminating linker.

[0044] As an example, the chemical structural formulas of N-oxides and organosilicon borates are shown in Figure 4 ; the chemical structural formula of the N-oxide prodrug is shown in Figure 5 ; the self-eliminating linker L can adopt a conventional chemical structure in the art. Preferably, the chemical structural formula of the self-eliminating linker L is shown in Figure 6 . The present invention designs a series of self-eliminating linking structures (N-oxides based on the ethylenediamine backbone) based on the reaction of nitroxy-silicon borate and uses them in the design of prodrugs of parent drugs containing multiple functional groups. The nitroxy functional group can selectively respond to the silicon boron reagent. After the prodrug reacts with the silicon boron reagent, the N-oxide is reduced to a tertiary amine intermediate. Utilizing the nucleophilicity of the tertiary amine nitrogen atom, this intermediate undergoes intramolecular cyclization under physiological conditions to release the parent drug. In addition, by further inserting another self-eliminating linker between the above self-eliminating linking structure and the parent drug, the tolerance of the functional group for releasing the parent drug can be further expanded. After the N-oxide is reduced to a tertiary amine and cyclized by the silicon boron reagent, the self-eliminating linker (part L) is released, and this self-eliminating linker undergoes a self-eliminating reaction under physiological conditions to release the parent drug.

[0045] Based on the click reaction of N-oxides and organosilicon boron reagents, the present invention designs several types of prodrug delivery systems, synthesizes a series of derivatives, verifies the release of the parent drug, and tests the in vitro biological activity of the prodrugs in vitro. Moreover, the molecule connected with biotin-PEG-N3 in Structure 1 can self-assemble into nanoparticles, which can better target tumor tissues.

[0046] (1) General preparation method of nanoparticles:

[0047] Prepare nanoparticles by the ultrasonic emulsification method. Weigh 0.5 mg of the compound and dissolve it in dichloromethane, then drop it into 5 mL of deionized water and perform ultrasonic emulsification for 30 min. At this time, the solution is a milky turbid solution. Concentrate it under vacuum at 40 °C until the solution is clear and transparent to obtain an aqueous solution containing 0.5 mg / mL of the nanoparticle drug.

[0048] (2) General method for measuring the drug release half-life

[0049] The prodrug releases the drug through two processes: First, the prodrug reacts with the boron reagent to form an intermediate; Second, the intermediate releases the drug through processes such as cyclization and elimination. Fix the prodrug concentration at 100 μM (20% CH3CN / PBS) and the boron reagent at 500 μM (20% CH3CN / PBS). Take the reaction solutions at different time points, freeze the reaction solutions at -80 °C, and use HPLC to analyze the changes in the peak areas of the drug and the intermediate. The eluent is acetonitrile and water containing 0.1% phosphoric acid. After quickly thawing the reaction solutions at different time points at 40 °C, inject the samples for analysis in sequence to determine the peak areas of each compound. Set the compound peak area as the y-axis and time as the x-axis, use prism to fit to obtain the first-order reaction kinetic constant, and use the first-order reaction formula to calculate the drug release half-life.

[0050] (3)General method for evaluating the anti-tumor activity of prodrugs

[0051] Set up three groups of experiments using a 96-well plate: the original drug control group, the prodrug group, and the prodrug group + 100 μM boron reagent. The number of cells in each well is 5000, each group of experiments contains 3 replicates, the maximum dosing concentration is 10 μM, and it is diluted 3 times in sequence, with a total of 8 concentrations. First, culture the cells in a cell incubator for 24 h and then administer the drug. After administration, continue to incubate for 48 h, aspirate the drug solution, wash the well plate 2 times with PBS, and then use the CCK-8 method to measure the absorbance at 460 nm to determine the cell viability and thus reflect the anti-tumor activity of the compound.

[0052] Example 1 Synthesis of organosilicon boric acid derivatives

[0053] Under nitrogen protection, the activated magnesium chips (304 mg, 12.5 mmol, 5 equiv) were added to anhydrous tetrahydrofuran (15 mL), and then elemental iodine (0.25 mmol, 0.1 equiv) was added. Then, dimethylchlorosilane (710 mg, 7.5 mmol, 3 equiv) was added dropwise to the above reaction solution under stirring at 40 °C. Then, the halogenated hydrocarbon (2.5 mmol, 1 equiv) was added, and the reaction was refluxed for 2 h. After the reaction was completed, it was filtered through diatomaceous earth, and the filtrate was separated by silica gel column chromatography (PE) to obtain the corresponding substituted dimethylsilane. Substituted dimethylsilane (0.5 mmol, 1 equiv), B2pin2 (bis(pinacolato)diboron) (317 mg, 1.25 mmol, 2.5 equiv) were added to a sealed tube, then Pt / C (5%) (2 mg, 0.01 mmol, 0.02 equiv) was added, and then 0.5 mL of cyclohexane solution was added. The reaction was carried out at 80 °C, and the reaction progress was monitored by TLC plate. After the reaction was completed, it was cooled to room temperature, filtered through diatomaceous earth, and the filtrate was separated and purified by silica gel column chromatography (PE:EA = 20:1) to obtain the corresponding organosilicon boron derivative. The general synthetic schematic diagram and product data of the organosilicon boron derivative are shown in Figure 7 and Figure 8 .

[0054] Example 2 Synthesis of NO-1

[0055]

[0056] 4-Methyl-7-aminocoumarin (175 mg, 0.998 mmol, 1 equiv) was added to 5 mL of DMF solution, and then K2CO3 (553 mg, 3.99 mmol, 4 equiv) was added. Stirring was carried out at room temperature, and methyl iodide (1.135 g, 7.99 mmol, 8 equiv) was added dropwise to the above reaction solution. The reaction was carried out at 60 °C for 6 h. After the reaction was completed, the reaction was cooled to room temperature, extracted with H2O / EA (v / v, 1 / 2), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, and separated and purified by silica gel column chromatography (PE:EA = 5:1) to obtain a pale yellow powder with a yield of 51%. 1 H NMR (300 MHz, Chloroform- d ) δ 7.50 – 7.32 (m, 1H),6.69 – 6.56 (m, 1H), 6.51 (q, J = 3.9, 3.3 Hz, 1H), 5.97 (s, 1H), 3.06 (d, J= 4.4 Hz, 6H), 2.45 – 2.28 (m, 3H).

[0057] Dissolve the obtained 7-(dimethylamino)-4-methylcoumarin (40 mg, 0.197 mmol, 1 equiv) in 5 mL of DCM, add mCPBA (meta-chloroperoxybenzoic acid) (59 mg, 0.295 mmol, 1.5 equiv) at 0 °C, stir at room temperature for 2 h. After the reaction is completed, concentrate the reaction solution, and separate and purify it by silica gel column chromatography (DCM:MeOH = 10:1) to obtain an off-white solid (NO-1) with a yield of 86%. 1 H NMR (300 MHz, DMSO- d 6 ) δ 8.05 (d, J = 8.7 Hz, 1H), 7.88 (s,1H), 7.73 (d, J = 8.9 Hz, 1H), 6.37 (s, 1H), 3.64 (d, J = 2.6 Hz, 6H), 2.47 (s,3H).

[0058] Example 3 General synthetic method of tertiary amine prodrugs

[0059] Dissolve the tertiary amine drug (0.51 mmol, 1 equiv) in 5 mL of acetic acid, dropwise add 30% aqueous hydrogen peroxide solution (2.5 mmol, 5 equiv) to the mixture, stir the reaction solution at 80 °C for 3 h. After the reaction is complete, cool to room temperature, add ice water to the crude product, filter, and dry the filter cake to obtain each tertiary amine prodrug. See the general synthetic schematic diagram and product data of the tertiary amine prodrugs Figure 9 .

[0060] Example 4 Reaction kinetics study of organosilicon boron derivatives and NO-1

[0061] 7-(Dimethylamino)-4-coumarin itself has fluorescence properties. Under the irradiation of light at 397 nm, it exhibits blue fluorescence (λ em = 450 nm). NO-1 obtained by introducing a nitrogen-oxygen bond at the tertiary amine position can effectively mask its fluorescence properties. In this invention, fluorescence spectrophotometry is used to monitor the change in the fluorescence intensity of 7-(dimethylamino)-4-coumarin, thereby reflecting the second-order reaction rate of the reaction between organosilicon boron derivatives and NO-1 compounds.

[0062] First, the second-order reaction rates of 1-Si, 8-Si, and 9-Si with NO-1 in acetonitrile were tested. The final concentration of NO-1 was fixed at 2 μM, and then the mother liquors of five different concentrations of organosilicon boron derivatives were prepared. The two were mixed in a fluorescence cuvette, and the fluorescence intensity changes at an excitation wavelength of 397 nm and an emission wavelength of 450 nm were set. The obtained data were fitted using prism to obtain the reaction kinetic constants at each concentration. With the concentration as the x-axis and the reaction kinetic constants at each concentration as the y-axis, the slope of the obtained straight line is the second-order reaction kinetic constant of the organosilicon boron compound with NO-1.

[0063] As Figure 10 shown, the second-order reaction kinetic constants of 8-Si and 9-Si with NO-1 can both exceed 10 3 M -1 s -1 . Similarly, setting the final concentration of 1-Si (10 μM, 20 μM, 30 μM, 40 μM, 50 μM) and the final concentration of NO-1 at 2 μM, 1-Si was tested in the same way. Since the reaction rate between the two is too fast and the fluorescence intensity immediately reaches the peak after mixing, the second-order reaction kinetic constant cannot be accurately obtained. It is faster than others, and it can be determined that K2 > 10 3 M -1 s -1 .

[0064] Using the same method, the second-order reaction kinetic constants of other organosilicon boron compounds with NO-1 in acetonitrile were tested. Table 1 shows that the second-order reaction kinetic constants of each silicon boron derivative with NO-1 are all above 10 3 M -1 s -1 above.

[0065]

[0066] 10 vol% acetonitrile was added to PBS (pH 5), and the reaction kinetics of 1-Si with NO-1 in this system were tested. The results are shown in Figure 11 , indicating that the orthogonal bond-breaking reaction system of the present invention has excellent reaction performance in simulating the tumor environment.

[0067] Example 5 Determination of the drug release efficiency of 1-Si activating N-oxide prodrugs

[0068] The peak area changes of the drugs in the reaction of each N-oxide prodrug with 1-Si were monitored by HPLC, and the standard curve between the drug concentration and the peak area was drawn at the same time. The release efficiency of each N-oxide prodrug was obtained by calculation, and the data were summarized into Table 2. Table 2 shows that the release efficiency of each N-oxide under the trigger of 1-Si is above 90%.

[0069]

[0070] Example VI Cell Imaging Experiment of Compound NO-1

[0071] Verify whether the click reaction between compound NO-1 and 1-Si can occur in living cells through a cell imaging experiment. Determine the cytotoxicity of compound 1-Si against the A549 lung cancer cell line within 24 h. Set the dosing concentrations of compound 1-Si as 150 μM, 200 μM, 250 μM, 300 μM, and 350 μM. Measure the absorbance at 450 nm using the CCK-8 method to indirectly reflect the cell viability. Figure 12 The results show that the cell viability is above 80% after treating the cells with 200 μM of the drug for 24 h. Based on this, fix the final concentration of NO-1 at 10 μM and the final concentration of 1-Si at 80 μM. Co-incubate the two with the cells for 3 h, wash the well plate 3 times with PBS, add 1 mL of culture medium to the well plate, and then take corresponding pictures with a fluorescence confocal microscope. Figure 12 Analysis shows that within 3 h, compound NO-1 does not show obvious fluorescence in the cells, while after adding 1-Si, obvious blue fluorescence can be observed, proving that the template compound NO-1 can respond to the trigger of 1-Si in living cells and release 7-(dimethylamino)-4-coumarin.

[0072] Example VII Synthesis of Prodrug 1a

[0073] Synthesis of compound 1. Dissolve N1,N2,N3-trimethylethylenediamine (1 equiv) in tetrahydrofuran, add a solution of Boc2O (1.2 equiv) dropwise at 0 °C, react under this condition for 2 h, monitor the reaction by TLC. After the raw materials are completely reacted, spin-dry the reaction solution and separate it by silica gel column chromatography. The eluent is DCM:MeOH = 18:1, and a colorless oily compound is obtained with a yield of 98%. 1 H NMR (600MHz, Chloroform- d ) δ 3.28 (d, J = 22.2 Hz, 2H), 2.84 (s, 3H), 2.41 – 2.33 (m,2H), 2.23 (s, 6H), 1.43 (s, 9H).

[0074] Synthesis of compound 2. Dissolve compound 1 (1 equiv) in dichloromethane solution, add m-chloroperoxybenzoic acid (1.5 equiv) at 0 °C, stir under this condition for 30 min, monitor the reaction by TCL. After the raw materials are completely reacted, spin-dry the reaction solution and separate it by silica gel column chromatography. The eluent is DCM:MeOH = 18:1, and a colorless oily liquid is obtained with a yield of 85%.1 H NMR (600 MHz, Chloroform- d ) δ 3.72 – 3.67 (m, 2H), 3.34 (d, J = 4.2 Hz, 2H), 3.16 – 3.12 (m, 6H), 2.86 (d, J = 2.8 Hz, 3H), 1.41 – 1.34 (m, 9H).

[0075] Synthesis of Compound 3. At 0 °C, a hydrochloric acid solution of ethyl acetate (20 equiv) was added to the reaction flask containing 2. Monitored by TLC, the solution was white and turbid at this time. After filtration, a white solid was obtained with a yield of 99%. 1 H NMR (400 MHz, D2O) δ 4.13 – 4.01 (m, 2H), 3.71 (d, J = 6.7 Hz, 2H), 3.60 (d, J = 7.7 Hz, 6H), 2.79 (q, J = 7.1, 6.4 Hz, 3H).

[0076] Synthesis of Combretastatin active ester 4. Under nitrogen protection, CA-4 (Combretastatin, 1 equiv) and 4-nitrophenyl chloroformate (1.5 equiv) were dissolved in anhydrous tetrahydrofuran. At 0 °C, DIPEA (diisopropylethylamine) (2 equiv) was added dropwise to the above reaction solution. After the addition was completed, the reaction was transferred to room temperature for 2.5 h. Monitored by TLC, after the Combretastatin reaction was complete, the reaction solution was washed with saturated ammonium chloride, extracted 3 times with dichloromethane, the organic phases were combined, dried over anhydrous sodium sulfate, filtered and concentrated by rotary evaporation, and separated by silica gel column chromatography. The eluent was PE:EA = 5:1, and a white solid was obtained with a yield of 95%. 1 H NMR (400 MHz, Chloroform- d ) δ 8.31 – 8.28 (m, 2H), 7.47 – 7.43 (m, 2H), 7.18 (s, 2H), 6.92 (d, J = 8.3 Hz, 1H), 6.49 (s, 2H), 3.88 (s, 3H), 3.84 (d, J = 0.9 Hz, 3H), 3.70 (d, J = 0.9 Hz, 6H).

[0077] Synthesis of Prodrug 1a: Dissolve compound 3 (1 equiv) in DMF, add DIPEA (3 equiv) dropwise at room temperature. After stirring for 10 min, dissolve the active ester 4 (1.2 equiv) in DMF and add it dropwise to the above reaction solution. The solution changes from colorless and clear to yellow and clear. Monitor the reaction by TLC. After the reaction is completed, rotary evaporate the DMF and perform column chromatography on aluminum oxide. The eluent is DCM:MeOH = 15:1 to obtain a white solid (which is prone to moisture absorption at room temperature). 1 H NMR (600 MHz, Chloroform- d ) δ 7.10 (dd, J = 8.4, 2.3 Hz, 1H), 6.94 (s, 1H), 6.83 (d, J = 8.4 Hz, 1H), 6.49 (s, 2H), 6.44(s, 2H), 3.88 – 3.75 (m, 10H), 3.71 (s, 6H), 3.45 (d, J = 7.5 Hz, 6H), 3.10 (d, J =65.5 Hz, 3H).

[0078] Drug release half-life of prodrug 1a. Prodrug 1a reacts with compound 1-Si to form the drug CA-4 and by-product 18, Figure 13 indicating that at 0 min, prodrug 1a reacts rapidly with an excess of compound 1-Si to form intermediate A and by-product 18. Intermediate A releases the drug molecule CA-4 through an intramolecular cyclization reaction. The peak area of intermediate A gradually decreases within 100 min, and the peak area of CA-4 gradually increases until intermediate A is completely converted to CA-4, proving that prodrug 1a can release the drug according to the above mechanism. Fit the curves of the peak area changes of intermediate A and CA-4, and obtain the first-order reaction kinetic constant k as 7.33×10 -4 s -1 by monitoring the peak area change of CA-4. Using the half-life formula 0.693 / k, the calculated half-life of drug release is 15.8 min.

[0079] Using the A549 cell line as a model, the A549 cell line is a human non-small cell lung cancer cell line, and the drug combretastatin (CA-4) is an antitumor drug that exerts its antitumor effect by inhibiting tubulin in tumor cells. Analysis Figure 14 shows that compared with CA-4, the IC 50 of the prodrug 1a group is 0.3746 μM, compared with CA-4 (IC 50was increased by about 20 times (to 0.01738 μM). In the experimental group, the addition of compound 1-Si could make the prodrug 1a produce cytotoxicity equivalent to that of the drug CA-4 (IC 50 was 0.01921 μM), proving that the drug CA-4 was released after the reaction of compound 1-Si with the prodrug 1a.

[0080] Example VIII Synthesis of nanoparticle monomer prodrug 1b

[0081] Synthesis of compound 5. Under nitrogen protection, 3-butyn-1-ol (1 equiv) was dissolved in anhydrous dichloromethane. p-Toluenesulfonyl chloride (1.2 equiv) was slowly added at 0 °C, and then TEA (triethylamine) (1.5 equiv) was added dropwise. The reaction was then transferred to room temperature for 3 h. Monitored by TLC. After the alcohol reaction was completed, it was washed with saturated ammonium chloride, and the aqueous phase was extracted 3 times with dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate, filtered and concentrated by evaporation. Silica gel column chromatography was used with an eluent polarity of PE:EA = 20:1 to obtain a colorless oily compound with a yield of 93%. 1 H NMR (400 MHz, Chloroform- d ) δ 7.76 – 7.69 (m, 2H),7.51 – 7.45(m, 2H), 4.51 – 4.45 (m, 2H), 2.74 (td, J J = 6.2, 2.6 Hz, 2H), 2.41 (d, J J = 0.8Hz, 3H), 2.31 (t, J J = 2.5 Hz, 1H).

[0082] Synthesis of compound 6. Compound 5 (1.2 equiv) and N1,N2-ethylenediamine (1 equiv) were added to anhydrous potassium carbonate (2 equiv). Monitored by TLC. After the amine reaction was complete, the reaction solution was cooled to room temperature, filtered, and the filtrate was concentrated by evaporation and directly used in the next step. The crude product was dissolved in tetrahydrofuran, and a Boc2O solution was added dropwise at room temperature. The reaction was carried out for 2 h. Monitored by TLC. Then the reaction solution was concentrated. Silica gel column chromatography was used with an eluent polarity of DCM:MeOH = 40:1 to obtain a colorless oily compound with a yield of 43%. 1 H NMR (400MHz, Chloroform- d ) δ 3.42 (td, J J = 5.7, 1.2 Hz, 2H), 2.88 (t, J J = 5.8 Hz, 1H),2.60 (td, J= 5.5, 2.5 Hz, 1H), 2.49 (s, 2H), 2.11 (t, J = 2.6 Hz, 0H), 1.46 (s, 3H).

[0083] Synthesis of Compound 7. Similar to Compound 2, a colorless oily liquid was obtained with a yield of 87%. 1 H NMR (400 MHz, Chloroform- d ) δ 3.51 (t, J = 5.4 Hz, 1H), 3.38 (td, J = 5.6, 1.0 Hz, 2H), 2.98 (s, 2H), 2.61 (d, J = 2.5 Hz, 0H), 2.16 (s, 0H), 1.46 (s, 3H).

[0084] Synthesis of Compound 8. Similar to Compound 3, a white solid was obtained with a yield of 96%. 1 H NMR (400 MHz, Chloroform- d ) δ 4.14 (t, J = 5.9 Hz, 2H), 3.81 (t, J = 5.9 Hz, 2H), 3.65 (s, 6H), 3.37 (t, J = 6.6 Hz, 2H), 2.74 (td, J = 6.7, 2.7 Hz, 2H), 2.55 (t, J = 2.7 Hz, 1H).

[0085] Synthesis of Compound 9. Similar to Prodrug 1a, a pale yellow oily compound was obtained with a yield of 76%. 1 H NMR (400 MHz, Chloroform- d ) δ 7.11 (dd, J = 8.4, 2.1 Hz, 1H), 7.00 (t, J = 5.2 Hz, 1H), 6.82 (d, J = 8.5 Hz, 1H), 6.50 (s, 2H), 6.44 (s, 2H), 3.90 (d, J = 7.1 Hz, 2H), 3.84 (d, J = 3.0 Hz, 1H), 3.80 (d, J= 3.5 Hz, 5H), 3.71 (s, 6H), 3.62 (t, J = 6.9 Hz,2H), 3.56 (dd, J = 9.4, 5.2 Hz, 2H), 3.45 (s, 3H), 3.27 (d, J = 6.7 Hz, 6H).

[0086] Synthesis of prodrug 1b. See Figure 15 , under nitrogen protection, bio-PEG-N3 (1 equiv), active ester 4 (1.1 equiv), pentamethyldiethylenetriamine (PMDETA) (1.2 equiv), and copper(I) iodide (0.32 equiv) were mixed and dissolved in ultrasonically degassed DMF, and the reaction was carried out at room temperature for 24 h. Monitored by TLC. After the raw materials were reacted completely, concentrated, and purified by neutral alumina column chromatography with eluent DCM:MeOH = 9:1 to obtain an off-white solid with a yield of 56%. 1 1H NMR (400 MHz, Chloroform- d ) δ7.71 (s, 1H), 7.29 (s, 1H), 7.11 (dd, J = 8.5, 2.1 Hz, 1H), 6.96 (s, 1H), 6.82(d, J = 8.5 Hz, 1H), 6.52 (d, J = 4.0 Hz, 3H), 6.44 (s, 2H), 6.11 (s, 1H), 4.52(t, J = 5.2 Hz, 2H), 4.45 (dd, J = 7.8, 4.9 Hz, 1H), 4.27 (t, J = 6.3 Hz, 1H), 3.86(q, J = 7.5, 6.3 Hz, 4H), 3.80 (d, J = 1.8 Hz, 6H), 3.71 (s, 7H), 3.66 – 3.63 (m,1H), 3.60 (s, 3H), 3.56 (d, J = 9.3 Hz, 6H), 3.53 (d, J = 4.9 Hz, 2H), 3.40 (d, J = 5.8 Hz, 2H), 3.26 (d, J = 4.3 Hz, 6H), 3.12 (d,J = 6.9 Hz, 1H), 2.86 (dd, J =12.8, 4.9 Hz, 1H), 2.62 (s, 2H), 2.24 – 2.17 (m, 2H), 1.66 (t, J = 21.9, 14.7,6.8 Hz, 4H), 1.42 (q, J = 7.7 Hz, 2H).

[0087] Preparation and characterization of nanoparticles. The preparation method adopts the above general method. The aqueous solution of nanoparticles is colorless and transparent. The particle size of the nanoparticles is 87nm and the PDI is 0.3. Figure 16 . Figure 17 It can be observed that the nanoparticles have obvious internal hollow distribution. Figure 18 In the experiment, when the nanoparticles reacted with 1-Si, the target drug was released. The electron microscope image showed that the morphology of the nanoparticles changed significantly, proving that the prodrug structure of the present invention can self-assemble into nanoparticles in water and can also effectively respond to the triggering of boron reagents. Figure 19 As shown, compared with the drug CA-4, the cell (A549) toxicity of the prodrug 1b group was significantly reduced, while in the experimental group, the addition of compound 1-Si caused the prodrug 1b to produce cytotoxicity comparable to that of the drug CA-4, proving that compound 1-Si reacted with the prodrug 1b to successfully release the drug CA-4.

[0088] Example 9 General Synthesis of Structures 1c to 11

[0089] Under nitrogen protection, thiol / phenol (1 equiv) was dissolved in 5 mL of anhydrous dichloromethane, and a dichloromethane solution of triphosgene (0.35 equiv) was added dropwise. The mixture was kept at 0°C, and triethylamine (1.5 equiv) was added dropwise. The reaction was monitored by TLC. After the thiol / phenol reaction was complete, the reaction solution was reserved for use. Under nitrogen protection, compound 3 / 8 (1 equiv) was dissolved in 3 mL of anhydrous dichloromethane, and the prepared acyl chloride was added dropwise to compound 3 / 8 using a double-ended needle. The reaction was carried out at 0°C for 30 min and then at room temperature for 8 h. The reaction was monitored by TLC. After the reaction was completed, the crude product was concentrated and separated and purified by silica gel column chromatography with DCM:MeOH=18:1 to obtain the corresponding derivative. For the reaction schematic diagram and product NMR data and structure, see Figure 20 .

[0090] Synthesis of compounds 1h, 1i, and 1m.

[0091] Compound 15 was synthesized in the same manner as compound 6 to obtain a colorless oily compound with a yield of 86%. 11H NMR (600 MHz, Chloroform- d ) δ 3.67 – 3.58 (m, 4H), 3.57 – 3.51 (m, 6H), 3.42 (t, J J = 5.8 Hz, 2H), 3.40 (s, 3H), 2.95 (s, 3H), 2.68 (t, J J = 6.4 Hz, 2H), 2.60 (t, J J = 5.8 Hz, 2H), 2.31 (s, 3H), 1.45 (s, 9H).

[0092] Synthesis of Compound 16. Similar to Compound 7, a pale yellow oily compound was obtained. Yield: 78%. 1 1H NMR (600 MHz, Chloroform- d ) δ 3.75 (t, J J = 6.1 Hz, 2H), 3.66 (td, J J = 6.1, 0.9 Hz, 2H), 3.58(td, J J = 6.1, 0.9 Hz, 2H), 3.55 (d, J J = 1.1 Hz, 4H), 3.52 (t, J J = 5.4 Hz, 2H), 3.40(s, 3H), 3.35 (t, J J = 5.3 Hz, 2H), 3.26 (t, J J = 6.1 Hz, 2H), 3.03 (s, 3H), 2.92(s, 3H), 1.45 (s, 9H).

[0093] Synthesis of Compound 17. Similar to Compound 8, a white solid was obtained. Yield: 95%. 1 1H NMR (600 MHz, Chloroform- d ) δ 3.71 (t, J J = 6.2 Hz, 2H), 3.68 – 3.64 (m, 4H), 3.60 – 3.53 (m, 6H), 3.45 (q, J J = 5.1, 4.0 Hz, 2H), 3.39 (s, 3H), 3.29 (t, J J = 6.1 Hz, 2H), 2.94(s, 3H), 2.54 (t, J J = 4.9 Hz, 3H).

[0094] Compound 1a was synthesized from 1h / 1i.

[0095] 1h: A pale yellow oily compound. 1 H NMR (600 MHz, Chloroform- d ) δ 6.95 – 6.92 (m, 2H), 6.87 – 6.84 (m, 2H), 3.80 (s, 3H), 3.73 (t, J J = 6.1 Hz, 2H), 3.68 – 3.64 (m, 2H), 3.58 – 3.52 (m, 8H), 3.40 (s, 3H), 3.35 (t, J J = 5.4 Hz, 2H), 3.26 (t, J J = 6.1 Hz, 2H), 3.05 (s, 3H), 2.93 (s, 3H).

[0096] 1i: A white solid. 1 H NMR (600 MHz, Chloroform- d ) δ 7.10 (dd, J J = 8.5, 2.0 Hz, 1H), 7.01 (d, J J = 8.4 Hz, 1H), 6.90 (d, J J = 2.0 Hz, 1H), 6.80 (s, 2H), 6.71 (d, J J = 16.3 Hz, 1H), 6.62 (d, J J = 16.3 Hz, 1H), 3.86 (d, J J = 15.4 Hz, 7H), 3.79 (s, 2H), 3.73 – 3.65 (m, 4H), 3.62 – 3.49 (m, 8H), 3.41 – 3.32 (m, 4H), 3.26 (t, J J = 6.1 Hz, 2H), 3.07 (s, 2H), 2.95 (s, 2H).

[0097] Synthesis of Compound 1m. Under nitrogen protection, chlorocarbonylsulfenyl chloride (1.1 equiv) was dissolved in anhydrous solvent, and a dichloromethane solution of benzyl mercaptan (1 equiv) was added dropwise at 0 °C. The reaction was carried out at 0 °C for 1.5 h, and the reaction was monitored by TLC. After the benzyl mercaptan reacted completely, the reaction solution was concentrated by rotary evaporation. Then 3 mL of anhydrous dichloromethane was added, and the mixture was dropped into a dichloromethane solution of Compound 3 (1 equiv). Diisopropylethylamine (3 equiv) was added dropwise at room temperature, and the reaction was carried out for 12 h. The reaction was monitored by TLC. Then the crude product was concentrated by rotary evaporation and purified by silica gel column chromatography (DCM:MeOH = 20:1) to obtain Compound 1m with a yield of 23%. 1 H NMR (600MHz, Chloroform- d ) δ 7.40 (q, J = 7.8, 1.1 Hz, 2H), 7.35 – 7.31 (m, 2H), 7.25(dd, J = 8.2, 6.6, 1.5 Hz, 1H), 3.97 (t, J = 1.0 Hz, 2H), 3.53 (t, J = 5.3 Hz, 2H),3.38 (t, J = 5.4 Hz, 2H), 3.00 (s, 3H), 2.98 (s, 6H).

[0098] Half-life of drug release of 1c - 1m. After the reaction of each prodrug with 1-Si, the changes in the peak areas of the tertiary amine intermediate and the leaving group were monitored by HPLC. The peak areas were plotted against time, and the first-order reaction kinetic constants were obtained by Prism fitting. The release half-lives of each prodrug were calculated according to the formula. The data were summarized in Table 3.

[0099]

[0100] Example X Synthesis of Derivatives of Structure 2

[0101] Synthesis of Compound 10. p-Hydroxybenzyl alcohol (1 equiv) and imidazole (1.2 equiv) were mixed in DMF and stirred at room temperature for 10 min. Then tert-butyldimethylchlorosilane (1.2 equiv) was added slowly, and the reaction was carried out for 4 h. The reaction was monitored by TLC. After p-hydroxybenzyl alcohol reacted completely, an equal volume of saturated ammonium chloride solution was added to the reaction solution, and the mixture was stirred for 10 min. It was extracted with ethyl acetate three times, and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and separated by silica gel column chromatography. The eluent was PE:EA = 8:1 to obtain a colorless oily compound with a yield of 85%. 1 H NMR (400 MHz, Chloroform-d ) δ 7.65 (s, 1H), 7.18 (s, 2H), 6.80 (d, J = 8.4 Hz, 2H), 4.91 (t, J = 1.0 Hz, 2H), 0.89 (s, 9H), 0.08 (s, 6H).

[0102] Synthesis of Compound 11. The same as Compound 4, with PE:EA = 15:1 to obtain a white solid with a yield of 78%. 1 1H NMR (400 MHz, Chloroform- d ) δ 8.32 (d, J = 8.8 Hz, 2H), 7.49 (d, J = 8.9 Hz, 2H), 7.39 (d, J = 8.7 Hz, 2H), 7.24 (d, J = 9.4 Hz, 2H), 4.76 (s, 2H), 0.95 (s, 9H), 0.11 (s, 6H).

[0103] Synthesis of Compound 12. Dissolve Compound 11 (1.1 equiv) in anhydrous toluene. Add DMAP (0.1 equiv) and diisopropylethylenediamine (1.5 equiv) to the reaction solution in sequence, stir at room temperature for 10 min, then slowly dropwise add N1,N2,N3-trimethylethylenediamine (1 equiv), stir at room temperature for 2.5 h, monitor the reaction by TLC, and perform post-treatment by extraction with water and ethyl acetate. Combine the organic phases, dry with anhydrous sodium sulfate, filter, and perform silica gel column chromatography with an eluent of EA:MeOH = 40:1 to obtain a yellow oily compound with a yield of 79%. 1 1H NMR (300 MHz, Chloroform- d ) δ 7.27 (d, J = 8.3 Hz, 2H), 7.04 (d, J = 8.2 Hz, 2H), 4.69 (s, 2H), 3.52 – 3.37 (m, 2H), 3.03 (d, J = 26.3 Hz, 3H), 2.50 (d, J = 6.8 Hz, 2H), 2.26 (d, J = 1.8 Hz, 6H), 0.91 (d, J = 1.9 Hz, 9H), 0.06 (d, J= 1.8 Hz, 6H).

[0104] Synthesis of Compound 13. Similar to Compound 2, a pale yellow oily liquid was obtained with a yield of 81%. 1 H NMR (400 MHz, Chloroform- d ) δ 7.31 (d, J = 8.1 Hz, 2H), 7.05 (d, J = 8.1 Hz, 2H), 4.72 (s, 2H), 3.94 (t, J = 7.4 Hz, 2H), 3.55 (t, J = 7.3 Hz, 2H), 3.27 (d, J = 8.8 Hz, 6H), 3.19 (s, 3H), 0.93 (s, 9H), 0.09 (s, 6H).

[0105] Synthesis of Compound 14. Compound 13 (1 equiv) was dissolved in tetrahydrofuran, and tetrabutylammonium fluoride (1.5 equiv) was added dropwise at 0 °C. The reaction was carried out for 1 h under this condition, monitored by TLC. After the raw materials reacted completely, the solvent was evaporated, and silica gel column chromatography was performed. The polarity of the eluent was DCM:MeOH = 9:1, and a white solid was obtained with a yield of 65%. 1 H NMR (400 MHz, Chloroform- d ) δ 7.37 (s, 2H), 7.08 (s, 2H), 4.68 (s, 2H), 3.98 (d, J = 48.3 Hz, 2H), 3.52 (s, 2H), 3.23 (d, J = 15.2 Hz, 6H), 3.08 (s, 3H).

[0106] Synthesis of Compound 2a. Under a nitrogen atmosphere, Compound 14 (1 equiv) and 4-nitrophenyl isocyanate (1.5 equiv) were dissolved in anhydrous dichloromethane, and (diisopropylethylamine) DIPEA (1.5 equiv) was added dropwise at room temperature. The reaction was carried out for 2.5 h under this condition, monitored by TLC. After Compound 14 reacted completely, the reaction solution was concentrated, and silica gel column chromatography was performed. The polarity of the eluent was DCM:MeOH = 15:1, and a white solid was obtained with a yield of 52%. 1 H NMR (400 MHz, Chloroform- d ) δ 10.28 (s, 1H), 8.22 – 8.11 (m, 2H), 7.70 (d,J = 11.3 Hz, 2H), 7.44 (d, J = 8.6 Hz, 2H), 7.11(d, J = 8.9 Hz, 2H), 5.21 (d, J = 11.0 Hz, 2H), 3.81 (d, J = 40.8 Hz, 2H), 3.32 (s,2H), 3.15 (s, 3H), 3.12 – 3.04 (m, 6H).

[0107] Synthesis of Compound 2b. Under a nitrogen atmosphere, Compound 19 (1 equiv) and triphosgene (0.35 equiv) were dissolved in anhydrous dichloromethane, and triethylamine (2 equiv) was added dropwise at 0 °C. The mixture was stirred for 2 h, and the reaction was monitored by TLC. After Compound 19 reacted completely, the solvent was removed under nitrogen protection. The prepared crude acyl chloride product was dissolved in dichloromethane and slowly dropped into a reaction flask containing Compound 14 (0.9 equiv). Triethylamine (2 equiv) was added dropwise at room temperature, and the reaction was carried out for 24 h. The reaction was monitored by TLC. After the reaction was complete, the solvent was removed, and the product was separated by silica gel column chromatography. The polarity of the eluent was DCM:MeOH = 12:1, and a yellow solid was obtained with a yield of 35%. 1 HNMR (600 MHz, Chloroform- d ) δ 8.79 (dd, J = 8.4, 1.4 Hz, 1H), 7.68 (t, J = 8.5,7.1, 1.5 Hz, 1H), 7.53 (s, 1H), 7.51 – 7.48 (m, 2H), 7.45 (q, J = 8.7 Hz, 4H),7.38 (d, J = 1.3 Hz, 1H), 7.35 (dd, J = 8.9, 2.3 Hz, 2H), 7.04 (t, J = 8.1 Hz, 2H),6.68 (s, 1H), 6.65 (s, 1H), 5.11 (s, 2H), 3.90 (t, J = 7.3 Hz, 1H), 3.77 (t, J =7.1 Hz, 2H), 3.53 (t, J = 7.2 Hz, 2H), 3.21 (d, J= 10.0 Hz, 6H), 3.05 (d, J = 77.1Hz, 3H).

[0108] Synthesis of prodrug 2c. Under nitrogen protection, compound 14 (1 equiv) and CPT active ester (1.5 equiv) were dissolved in anhydrous dichloromethane. Triethylamine (TEA) (1.5 equiv) was added dropwise at room temperature, and 4-dimethylaminopyridine (DMAP) (0.1 equiv) dissolved in dichloromethane was slowly added dropwise to the above solution. The reaction was carried out for 2.5 h under this condition and monitored by TLC. After the reaction of compound 14 was completed, the reaction solution was concentrated and purified by silica gel column chromatography (DCM:MeOH = 15:1) to obtain a white solid with a yield of 40%. 1 H NMR (400MHz, Chloroform-d) δ 8.41 (s, 1H), 8.24 (d, J = 8.6 Hz, 1H), 7.96 (d, J = 8.2Hz, 1H), 7.85 (t, J = 7.7 Hz, 1H), 7.69 (t, J = 7.5 Hz, 1H), 7.36 (d, J = 8.2 Hz,2H), 7.31 (s, 1H), 7.05 (d, J = 8.2 Hz, 2H), 5.69 (d, J = 17.1 Hz, 1H), 5.38 (d, J = 17.3 Hz, 1H), 5.30 (s, 2H), 5.16 (d, J = 12.3 Hz, 1H), 5.07 (d, J = 12.3 Hz,1H), 3.99 – 3.85 (m, 2H), 3.69 – 3.58 (m, 2H), 3.32 (s, 6H), 3.08 (d, J = 23.3Hz, 3H), 2.28 (dd, J = 14.1, 7.4 Hz, 2H), 0.99 (t, J = 7.5 Hz, 3H).

[0109] Synthesis of prodrug 2d. Under nitrogen protection, compound 14 (1 equiv) and combretastatin active ester (1.5 equiv) were dissolved in anhydrous dichloromethane. TEA (1.5 equiv) was added dropwise at room temperature, and a solution of DMAP (0.1 equiv) in dichloromethane was slowly added dropwise to the above solution. The reaction was carried out under these conditions for 2.5 h. Monitored by TLC, after the reaction of compound 14 was completed, the reaction solution was concentrated, and purified by silica gel column chromatography (DCM:MeOH = 15:1) to obtain a white solid with a yield of 40%. 1 H NMR (600 MHz, Chloroform- d ) δ 7.10 (dd, J = 8.4, 2.3 Hz, 1H), 6.94 (s, 1H), 6.83 (d, J = 8.4 Hz, 1H), 6.49(s, 2H), 6.44 (s, 2H), 3.88 – 3.75 (m, 10H), 3.71 (s, 6H), 3.45 (d, J = 7.5 Hz,6H), 3.10 (d, J = 65.5 Hz, 3H).

[0110] Synthesis of prodrugs 2e and 2f. Under nitrogen protection, aniline compound (1 equiv) was dissolved in 5 mL of anhydrous dichloromethane, and a dichloromethane solution of triphosgene (0.35 equiv) was added dropwise. The mixture was placed at 0 °C, and triethylamine (1.5 equiv) was added dropwise. Monitored by TLC, after the reaction of aniline was completed, the reaction solution was reserved for later use; under nitrogen protection, compound 14 (1 equiv) was dissolved in 3 mL of anhydrous dichloromethane, and the prepared acyl chloride was slowly added dropwise to compound 14 using a double-headed needle. After reacting at 0 °C for 30 min, the reaction was transferred to room temperature and continued for 8 h. Monitored by TLC, after the reaction was completed, the crude product was rotary evaporated to dryness, and purified by silica gel column chromatography (DCM:MeOH = 18:1) to obtain the corresponding derivatives.

[0111] Schematic diagrams for the synthesis of compounds 1h, 1i, 1m, 2a, and 2b are shown in Figure 21 ; schematic diagrams for the synthesis of compounds 2c, 2d, 2e, and 2f are shown in Figure 22 .

[0112] Example XI

[0113] Compound 2a reacts with compound 1-Si and releases p-nitroaniline. Compound 2a uses p-nitroaniline as a template drug and can react with compound 1-Si to generate p-nitroaniline. p-Nitroaniline is yellow in solution, and its maximum absorption wavelength is 400 nm. Set the final concentration of compound 2a to 50 μM (10% CH3CN / PBS), and the final concentration of the boron reagent to 250 μΜ. Figure 23 In A, prodrug 2a is colorless and transparent in 10% CH3CN / PBS. When compound 1-Si is added, the reaction occurs in the solution at 37 °C for 75 min, and the solution changes from colorless to yellow, proving the formation of p-nitroaniline. Figure 23 In B, the maximum absorption wavelength of the control group of compound 2a is 320 nm, and its absorbance decreases continuously with time, while the increase in absorbance at 400 nm shows a time-dependent trend. From the experimental phenomena and the ultraviolet absorption changes of the reaction solution, it can be proved that compound 2a reacts with compound 1-Si to slowly release p-nitroaniline.

[0114] Compound 2b reacts with compound 1-Si to release a red fluorescent probe. Compound 2b uses a fluorescent molecule as a template drug, with an excitation wavelength of 450 nm and an emission wavelength of 650 nm. Prepare a mother liquor of prodrug 2b with a final concentration of 10 μM (10% CH3CN / PBS), and at the same time prepare 100 (10% CH3CN / PBS) of compound 1-Si. Mix the two in equal volumes and incubate at 37 °C for 60 min. Set the excitation wavelength of the fluorescence spectrophotometer to 460 nm and the emission wavelength to 650 nm, and take fluorescence spectra at each time point and collect spectral data. Figure 24 It shows that prodrug 2b is yellow in the mixed solution. When compound 1-Si is added, the solution color changes from yellow to red after 60 min. At the same time, the fluorescence spectrum shows that the fluorescence intensity at 650 nm increases in a time-dependent manner, proving that prodrug 2b slowly releases a red fluorescent molecule under the trigger of compound 1-Si, resulting in a change in the solution color.

[0115] Cell imaging experiment of compound 2b. Use the A549 lung cancer cell line to verify whether compound 2b can be triggered by compound 1-Si in living cells and release a fluorescent group. Fix the concentration of prodrug 2b at 10 μM (0.5% DMSO / 1640 medium), and the concentration of compound 1-Si at 80 μM (0.5% DMSO / 1640 medium). Set two wells in a 6-well plate, with 5000 cells in each well. After culturing for 24 h, aspirate the medium, and add two groups of medicated solutions, 2b and 2b + 1-Si, respectively. Place the well plate in a cell culture incubator and incubate for 3 h. Then aspirate the medicated solution, wash the wells 3 times with PBS, and add 1 mL of medium, and take pictures under a fluorescence microscope. As Figure 25As shown, the 2b compound group shows almost no red fluorescence under the red light channel, while obvious red fluorescence can be observed in the 2b + 1-Si group. This is because after the compound 2b is triggered by 1-Si, the fluorescent group is released, so that red fluorescence can be shown under the fluorescence microscope, indicating that the compound 2b can be activated by the compound 1-Si in living cells and release the loaded fluorescent group.

[0116] Drug release half-life of prodrugs 2c - 2f. The peak area changes of the intermediates and drugs generated by the reaction of the prodrugs with 1-Si were monitored by HPLC. The first-order reaction kinetic constant of the intermediate degradation was obtained by fitting with Prism, and the drug release half-life t of each prodrug was obtained by substituting it into the first-order reaction half-life calculation formula 0.693 / k. 1 / 2 , and they were summarized into Table 4.

[0117]

[0118] Antitumor activity test of prodrugs 2c - 2e. The antitumor activities of three prodrugs 2c, 2d, and 2e within 48 h were evaluated using the A549 lung cancer cell line. Figure 26 Analysis shows that the cytotoxicity of the prodrug group has decreased significantly compared with that of the original drug group. In addition, the cytotoxicity shown after adding 100 μΜ of 1-Si to the prodrug is comparable to that of the original drug group, indicating that the click reaction occurs between the prodrug and 1-Si in the cell to successfully release the drug.

[0119] The present invention has developed a brand-new bioorthogonal bond-breaking reaction, which has the advantages of fast reaction kinetics, easy availability of raw materials, and good stability of raw materials. The prodrugs designed based on this reaction can release drugs quickly, specifically, and with high spatiotemporal resolution, and can be used for the specific activation and release of cytotoxic drugs and analgesic drugs at the target site, effectively improving the drug efficacy and reducing the toxic and side effects.

Claims

1. An N-oxide prodrug having one of the following chemical structural formulas: ； 。 2. A bioorthogonal bond-cleavage reaction prodrug system comprising the N-oxide prodrug according to claim 1 and an organosilicon boronic acid derivative; the organosilicon boronic acid derivative having one of the following chemical structural formulas: 。 3. Use of the N-oxide prodrug according to claim 1 in the preparation of a bioorthogonal prodrug, a drug delivery system or an active drug.

4. Use of the bioorthogonal bond-cleavage reaction prodrug system according to claim 2 in the preparation of a bioorthogonal prodrug, a drug delivery system or an active drug.

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