Bioorthogonal prodrugs, compositions, and uses thereof

Abstract

The present application relates to a kind of biological ortho prodrug, composition and its application, specifically disclose a kind of drug prodrug with substituted acrylamide or substituted vinyl sulfonamide structure, is obtained by the reaction of covalent warhead comprising active drug and N-alkyl hydroxylamine.The drug prodrug can be released by Retro-Cope elimination reaction and Cope elimination reaction with tension alkyne, and the release rate can reach more than 90%, to realize on-demand activation active warhead, effectively solve the off-target problem of covalent inhibitor.In addition, by introducing the drug or fluorophore containing hydroxyl / amine group in the propargyl position of tension alkyne, the intermediate produced after Cope elimination reaction of tension alkyne can further undergo elimination reaction, release the drug or fluorophore containing hydroxyl / amine group, realize combined therapy or real-time monitoring of drug release.The above-mentioned drug prodrug and the composition formed by the combination of tension alkyne have good application prospect in the preparation of drug delivery system or active drug.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a biological orthogonal prodrug, a composition, and its application. Background Technology

[0002] Covalent inhibitors, as an important drug design strategy, have received widespread attention in the field of drug development. They achieve sustained and potent regulation of disease-related target protein function through a "two-step action mode"—first, binding specifically to the target protein via non-covalent interactions, and then forming an irreversible covalent bond between the warhead group in the molecule and specific amino acid residues (such as cysteine ​​and lysine) at the target's active site. This mechanism endows covalent inhibitors with significant technical advantages over traditional non-covalent inhibitors, including higher affinity and inhibitory efficacy, longer target occupancy time, stronger anti-target mutation ability (helping to overcome drug resistance), and the potential to target traditionally "undruggable" targets, providing new therapeutic hope for various refractory diseases such as cancer.

[0003] However, the clinical application of covalent inhibitors, especially their expansion into broader therapeutic areas, is severely constrained by a key bottleneck: off-target risk and the resulting potential toxicity. This risk stems primarily from two aspects: First, off-target: A large number of structurally similar non-target proteins exist in the physiological environment, and their active sites may contain homologous nucleophilic amino acids, leading to unexpected covalent binding of the covalent inhibitor warhead to these proteins, resulting in unpredictable toxic side effects. Second, on-target (off-tissue): Covalent inhibitors typically lack tissue selectivity. While effectively inhibiting the function of target proteins in diseased tissues (such as tumors), they may also irreversibly inhibit the same target proteins in normal tissues, leading to severe tissue damage and other adverse reactions. Existing technologies mainly improve selectivity for target proteins by optimizing the molecular structure of the non-covalent binding portion or by modulating the chemical reactivity of the warhead group to enhance its specificity in reacting with target amino acids. However, these strategies primarily focus on improving binding specificity at the molecular level and cannot fundamentally solve the problem of tissue selectivity at the in vivo distribution level, i.e., they cannot ensure that the active form of the drug is released and exerts its effect only at the lesion site.

[0004] To address the challenge of tissue selectivity, prodrug strategies are considered a highly promising solution. This strategy aims to design precursor molecules that are inactive or poorly active in normal tissues, allowing them to be activated only at the lesion site by specific stimuli, releasing the original drug with covalent inhibitory activity. However, successfully applying prodrug strategies to highly reactive covalent inhibitors faces significant challenges, the core difficulty being how to achieve effective and controllable "masking" and "activation" of the active warhead. Current research explores utilizing endogenous stimuli from the tumor microenvironment (such as highly expressed hydrogen peroxide (H2O2)) to activate prodrugs. For example, some techniques use H2O2 to oxidize selenide ether groups, thereby initiating an elimination reaction to generate an acrylamide warhead with covalent inhibitory activity. However, this type of prodrug strategy triggered by endogenous stimuli has inherent limitations: (1) The triggering signal is heterogeneous and uncontrollable: The level of endogenous stimuli (such as H2O2) varies significantly among individuals and changes dynamically with the stage of disease development, resulting in unstable and heterogeneous activation efficiency of prodrugs in patients or lesions, affecting the reliability and reproducibility of the therapeutic effect; (2) Insufficient tissue selectivity: The abundance difference of certain endogenous stimuli (such as specific pH, enzymes or reactive oxygen species) between diseased tissues and normal tissues is limited, or normal tissues may also have similar microenvironment characteristics under certain physiological and stress states, resulting in the prodrug being mistakenly activated at non-target sites, causing off-target toxicity, and failing to completely solve the core problem of tissue selectivity; (3) Limited universality of activation mechanism: It relies on the chemical reaction of specific endogenous substances (such as H2O2) to generate active warheads. Its chemical design is often deeply coupled with specific warhead types and release mechanisms, making it difficult to promote its application to a wide range of covalent inhibitors with diverse structures and mechanisms of action, resulting in poor technical universality.

[0005] Therefore, there is an urgent need to develop a novel prodrug platform technology that is universal, controllable, and highly tissue-selective. This technology should effectively "shut down" the reactivity of covalent inhibitor warheads in the circulatory system, significantly reducing their non-specific binding to free nucleophilic groups in plasma and normal tissues. Simultaneously, it should utilize an exogenous, precisely regulated activation mechanism to efficiently and selectively restore the activity of covalent inhibitors at specific times and locations (such as diseased tissues). This would allow for the maximization of the potent therapeutic advantages of covalent inhibitors while minimizing off-target toxicity, significantly improving the safety window for clinical application. Summary of the Invention

[0006] Existing covalent inhibitor technologies face safety and selectivity bottlenecks, and current prodrug strategies are limited by inherent defects in their application to endogenous stimuli, failing to achieve precise, controllable, and broad-spectrum drug release. To address these issues, this invention provides a bioorthogonal prodrug, a composition, and its application. By directly modifying the active proton site of the covalent inhibitor, it reduces the non-specific binding of free thiol / nucleophilic groups in plasma. Furthermore, by targeted delivery, it reduces the toxicity issues caused by the inability to accurately distinguish between lesion and normal tissues, thereby achieving efficient and controllable release of covalent inhibitors and other drugs.

[0007] Specifically, the following technical solutions are provided: The first aspect of this invention provides a pharmaceutical prodrug comprising a substituted acrylamide or a substituted vinyl sulfonamide structure, the structure of which is as follows: , Where Y is or ; R1 is the ligand portion of the drug, which is a covalent inhibitor or molecular glue with a substituted acrylamide or substituted vinyl sulfonamide as the warhead. R2 is selected from H, F, CN, C 1-6 Alkyl, F-substituted C 1-6 One of the alkyl groups; R3 is selected from H and C. 1-6 Alkyl, amino-substituted C 1-6 One of the alkyl groups; R4 is selected from C 1-6 Alkyl and aryl substituted C 1-6 One of the alkyl groups; the aryl group includes, but is not limited to, phenyl and trifluoromethyl-substituted phenyl groups; R5 and R6 are H and C respectively. 1-6 The alkyl group or the N group in the adjacent substituent R1 may cyclize to form a substituted or unsubstituted piperazine ring; the substituents of the substituted piperazine ring include, but are not limited to, C. 1-6 alkyl.

[0008] Furthermore, the prodrug is one of the compounds shown in the following structures: , , , , , .

[0009] A second aspect of this invention provides a method for preparing the pharmaceutical prodrug comprising a substituted acrylamide or a substituted vinyl sulfonamide structure as described in the first aspect, wherein the compound represented by formula (I) is reacted with an N-alkyl hydroxylamine represented by formula (II) in the presence of an organic amine and a solvent to obtain the pharmaceutical prodrug; the structures of formulas (I) and (II) are shown below: , Where Y is or ; R1 is the ligand portion of the drug, which is a covalent inhibitor or molecular glue with a substituted acrylamide or substituted vinyl sulfonamide as the warhead. R2 is selected from H, F, CN, C 1-6 Alkyl, F-substituted C 1-6 One of the alkyl groups; R3 is selected from H and C. 1-6 Alkyl, amino-substituted C 1-6 One of the alkyl groups; R4 is selected from C 1-6 Alkyl and aryl substituted C 1-6 One of the alkyl groups; R5 and R6 are H and C respectively. 1-6 The alkyl group or the N group in the adjacent substituent R1 can cyclize to form a substituted or unsubstituted piperazine ring.

[0010] Furthermore, the organic amine includes, but is not limited to, triethylamine.

[0011] Furthermore, the solvent includes, but is not limited to, dichloromethane.

[0012] A third aspect of the present invention provides a biological orthogonal prodrug composition comprising the drug prodrug described in the first aspect and an angiolyne derivative; wherein the angiolyne derivative is an eight-membered cycloalkyne derivative.

[0013] Furthermore, the strainyne derivative is selected from one of the following structures: , .

[0014] The fourth aspect of the present invention provides the use of the prodrug comprising substituted acrylamide or substituted vinyl sulfonamide as described in the first aspect or the bioorthogonal prodrug composition as described in the third aspect in the preparation of a drug delivery system or an active drug.

[0015] The fifth aspect of the present invention provides a prodrug having a tensiyne structure, the structure of which is as follows: , Where X is -O-, -OC(O)NR'-, or -OC(O)O-; R8 is a monovalent group formed by removing one hydrogen atom from an active drug containing hydroxyl, amino, or alkylamino groups. When R8 is a monovalent group formed by removing one hydrogen atom from the hydroxyl group of an active drug containing a hydroxyl group, X is -O- or OC(O)O-. When R8 is a monovalent group formed by removing one hydrogen atom from an amino or alkylamino active drug, X is -OC(O)NR'-. R' is H or C 1-6 alkyl; The alkyl group in the above alkylamino group is C. 1-6 alkyl.

[0016] Furthermore, the prodrug is one of the following structures: .

[0017] A sixth aspect of the present invention provides a bio-orthogonal prodrug composition comprising the prodrug described in the fifth aspect and a hydroxylamine derivative; the structure of the hydroxylamine derivative is shown below: , Where Y is or ; R1 is selected from H, substituted or unsubstituted aryl group, substituted or unsubstituted C group. 1-6 One of the alkyl groups; R2 is selected from H, F, CN, C 1-6 Alkyl, F-substituted C 1-6 One of the alkyl groups; R3 is selected from H and C. 1-6 Alkyl, amino-substituted C 1-6 One of the alkyl groups; R4 is selected from C 1-6 Alkyl and aryl substituted C 1-6 One of the alkyl groups; R5 and R6 are selected from H and C, respectively. 1-6 One of the alkyl groups.

[0018] The seventh aspect of the present invention provides the use of a prodrug having a strained alkyne structure as described in the fifth aspect or a bioorthogonal prodrug composition as described in the sixth aspect in the preparation of a drug delivery system or an active drug.

[0019] In this invention, the mechanism by which a drug prodrug having a substituted acrylamide or substituted vinyl sulfonamide structure and a drug prodrug having a tolyne structure triggers the release of the drug is as follows:

[0020] When a prodrug with a substituted acrylamide or substituted vinylsulfonamide structure comes into contact with an anthryne derivative (a prodrug with an anthryne structure), a click-like retro-Cope elimination reaction occurs, generating a nitride intermediate. This intermediate then undergoes a Cope elimination reaction to generate a covalent inhibitor / molecular glue containing a substituted acrylamide or vinylsulfonamide active tip. When a drug containing a hydroxyl or amino group is introduced at the propargyl position of the anthryne derivative, the intermediate after the Cope elimination reaction can further undergo a 1,4 elimination reaction, activating the corresponding drug and thus achieving combination therapy.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. This invention prepares a covalent inhibitor prodrug with a substituted acrylamide or substituted vinyl sulfonamide structure by modifying the active warhead. The covalent inhibitor prodrug has tonicyne response specificity, which can realize the efficient and controllable release of the covalent inhibitor at a specific time and at a specific site, effectively solving the tissue toxicity problem and off-target toxicity problem caused by the active warhead of the covalent inhibitor.

[0022] 2. The bioorthogonal retro-cope elimination and cope elimination strategy provided by this invention, which involves a drug prodrug with a substituted acrylamide or substituted vinylsulfonamide structure in tandem with angiolyne derivatives, can be successfully applied to EGFR inhibitors, BTK inhibitors, KRAS inhibitors, and BRD4 molecular glue degrading agents. Moreover, the covalent inhibitor release exceeds 90%, and it has the potential to become a universal covalent inhibitor prodrug strategy.

[0023] 3. The tonolyne derivative of the present invention used to trigger the above-mentioned covalent inhibitor prodrug can be linked to a secondary functional molecule at the propyne position. For example, linking a fluorophore can monitor drug release in real time, and linking a drug can achieve dual-drug combination therapy. When the tonolyne derivative is linked to a drug with a hydroxyl or amino group at the propyne position, it can also be used as a drug prodrug in drug delivery systems or in the preparation of active drugs. Attached Figure Description

[0024] Figure 1 The reaction mechanism diagram of the tensionyne SA-1f / SA-1g / SA-1i / SA-1j and hydroxylamine derivative 1d; Figure 2 The diagram shows the cytotoxicity of cell-selective activation covalent inhibitor prodrugs based on a pre-targeting triggering strategy. A) is a schematic diagram of the mechanism of action, and B) is a diagram of the cell activity of A549 cells (top) / HCT116 cells (bottom) after different treatments. Figure 3The graph shows the degradation effect of BRD4 protein based on click release. A) is a protein band graph, and B) is a protein content graph after different treatments. Detailed Implementation

[0025] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0026] 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 invention pertains. The terminology used herein in the specification of this invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The terms “comprising” or “including” used in this invention may also be replaced with the closed form “is” or “consisting of”.

[0027] The raw materials used in the following examples are all commercially available, and the specific preparation operations and testing methods involved are all conventional methods in the field.

[0028] Example 1: This example relates to the preparation of molecular gel prodrug 6c, as detailed below:

[0029] 7 (1 mmol) was dissolved in dichloromethane (5 mL), and N-methylhydroxylamine hydrochloride (1.2 mmol) and triethylamine (3 mmol) were added to the solution. The reaction mixture was stirred overnight at room temperature and the reaction was monitored by TLC (DCM: MeOH = 10: 1). After the reaction was complete, the reaction solution was washed twice with saturated brine, the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and the crude product was purified by silica gel column chromatography using dichloromethane: methanol = 20: 1 as the eluent to give the target compound 6c in 40% yield. 1 H NMR (400 MHz, Methanol-d4) δ 7.47 – 7.40 (m, 4H), 4.70 (t, J= 6.9 Hz, 1H), 3.94 – 3.87 (m, 1H), 3.84 – 3.77 (m, 2H), 3.69 – 3.61 (m, 3H), 3.47 (t, J = 5.1 Hz, 2H), 3.36 – 3.32 (m, 4H), 3.00 (t, J = 7.0 Hz, 2H), 2.70 (s, 3H), 2.63 (s, 3H), 2.45 (s, 3H), 1.70 (s, 3H).

[0030] Example 2: This example relates to the preparation of EGFR inhibitor prodrugs 6-9a, as detailed below:

[0031] Referring to the reaction route in this embodiment, A (1.0 mmol, 1.0 equivalent) was dissolved in DCM (5 mL), and B (1.2 mmol, 1.2 equivalent) and TEA (3 mmol, 3 equivalent) were added with stirring. The reaction was stirred at room temperature, and after the reaction was confirmed to be complete by TLC, the mixture was washed three times with saturated brine, the organic phase was collected, concentrated under reduced pressure, and the crude product was purified by column chromatography using (dichloromethane:methanol = 40:1~10:1) as the eluent to obtain the target product C, namely compounds 6a, 7a, 8a, and 9a.

[0032] Compound 6a: Yield 70%, 1 H NMR (300 MHz, Methanol-d4) δ 9.20 (s, 1H), 8.47(s, 1H), 8.28 – 8.06 (m, 2H), 7.41 (d, J = 7.9 Hz, 1H), 7.30 – 7.10 (m, 3H), 6.92 (s, 1H), 3.91 (s, 3H), 3.87 (s, 3H), 3.09 (t, J = 6.0 Hz, 2H), 2.96 (t,J = 6.3 Hz, 2H), 2.80 – 2.60 (m, 8H), 2.54 (t, J = 6.0 Hz, 2H), 2.34 (s, 6H).

[0033] Compound 7a: Yield 50%, 1 H NMR (500 MHz, Chloroform-d) δ 8.55 – 8.47 (m,2H), 8.18 – 8.12 (m, 1H), 8.00 (s, 1H), 7.54 – 7.48 (m, 1H), 7.41 (d, J = 4.8Hz, 1H), 7.35 – 7.17 (m, 8H), 6.44 (s, 1H), 3.89 (s, 3H), 3.74 (s, 3H), 3.58(t, J = 6.5 Hz, 2H), 3.12 (t, J = 6.4 Hz, 2H), 3.00 (s, 3H), 2.84 – 2.77 (m,4H), 2.70 – 2.59 (m, 4H), 2.36 (s, 6H), 1.93 – 1.84 (m, 2H).

[0034] Compound 8a: Yield 30%, 1 H NMR (500 MHz, Chloroform-d) δ 8.55 – 8.47 (m,2H), 8.18 – 8.12 (m, 1H), 8.00 (s, 1H), 7.59 – 7.53 (m, 2H), 7.53 – 7.48 (m,1H), 7.41 (d, J = 4.8 Hz, 1H), 7.35 – 7.28 (m, 1H), 7.22 (s, 2H), 7.16 – 7.12(m, 2H), 6.44 (s, 1H), 3.89 (s, 3H), 3.74 (s, 3H), 3.58 (t, J = 6.5 Hz, 2H),3.12 (t, J = 6.4 Hz, 2H), 3.00 (s, 3H), 2.84 – 2.77 (m, 4H), 2.70 – 2.59 (m, 4H), 2.36 (s, 6H), 1.93 – 1.84 (m, 2H).

[0035] Compound 9a: Yield 45%, 1 H NMR (500 MHz, Chloroform-d) δ 8.55 – 8.47 (m,2H), 8.18 – 8.12 (m, 1H), 8.00 (s, 1H), 7.51 – 7.50 (m, 1H), 7.41 (d, J = 4.8Hz, 1H), 7.35 – 7.17 (m, 13H), 6.44 (s, 1H), 3.92 – 3.89 (m, 1H), 3.89 (s,3H), 3.74 (s, 3H), 3.58 (t, J = 6.5 Hz, 2H), 3.12 (t, J = 6.4 Hz, 2H), 3.00(s, 3H), 2.87 (t, J = 8.3 Hz, 2H), 2.80 (t, J = 6.5 Hz, 2H), 2.62 (t, J = 6.4Hz, 2H), 2.36 (s, 6H), 2.12 – 2.03 (m, 2H).

[0036] Example 3: This example involves the preparation of EGFR inhibitor prodrugs 6-9b, as detailed below:

[0037] Referring to the reaction route in this embodiment, A (1.0 mmol, 1.0 equivalent) was dissolved in DCM (5 mL), and B (1.2 mmol, 1.2 equivalent) and TEA (3 mmol, 3 equivalent) were added with stirring. The reaction was stirred at room temperature, and after the reaction was confirmed to be complete by TLC, the mixture was washed three times with saturated brine, the organic phase was collected, concentrated under reduced pressure, and the crude product was purified by column chromatography using (dichloromethane:methanol = 40:1~10:1) as the eluent to obtain the target product C, namely compounds 6b, 7b, 8b, and 9b.

[0038] Compound 6b: Yield 70%, 1 H NMR (300 MHz, Chloroform-d) δ 10.51 (s, 1H), 8.81 (s, 1H), 8.48 (s, 1H), 8.44 (s, 1H), 7.53 (d, J = 6.6 Hz, 1H), 6.98 (t,J = 8.8 Hz, 1H), 6.87 (s, 1H), 4.17 – 3.93 (m, 2H), 3.84 – 3.65 (m, 4H), 2.93– 2.81 (m, 2H), 2.73 (s, 3H), 2.66 – 2.23 (m, 8H), 2.15 – 1.95 (m, 2H).

[0039] Compound 7b: Yield 45%, 1 H NMR (500 MHz, Chloroform-d) δ 9.00 (s, 1H),8.64 (s, 1H), 8.31 (s, 1H), 7.91 – 7.87 (m, 1H), 7.51 – 7.45 (m, 1H), 7.37(s, 1H), 7.30 – 7.17 (m, 5H), 7.14 – 7.10 (m, 1H), 4.09 (t, J = 6.5 Hz, 2H), 3.74 – 3.68 (m, 4H), 3.12 (t, J = 6.4 Hz, 2H), 2.81 (t, J = 6.9 Hz, 2H), 2.75(t, J = 6.5 Hz, 2H), 2.70 – 2.58 (m, 4H), 2.58 – 2.52 (m, 4H), 1.93 – 1.84 (m, 4H).

[0040] Compound 8b: Yield 60%, 1H NMR (500 MHz, Chloroform-d) δ 9.00 (s, 1H), 8.64 (s, 1H), 8.31 (s, 1H), 7.91 – 7.87 (m, 1H), 7.59 – 7.53 (m, 2H), 7.51 –7.45 (m, 1H), 7.37 (s, 1H), 7.16 – 7.14 (m, 2H), 7.14 – 7.08 (m, 1H), 4.09(t, J = 6.5 Hz, 2H), 3.74 – 3.68 (m, 4H), 3.12 (t, J = 6.4 Hz, 2H), 2.84 –2.72 (m, 4H), 2.70 – 2.58 (m, 4H), 2.58 – 2.52 (m, 4H), 1.93 – 1.84 (m, 4H).

[0041] Compound 9b: Yield 50%, 1 H NMR (500 MHz, Chloroform-d) δ 9.00 (s, 1H),8.64 (s, 1H), 8.31 (s, 1H), 7.91 – 7.87 (m, 1H), 7.50 – 7.45 (m, 1H), 7.37(s, 1H), 7.29 – 7.25 (m, 8H), 7.24 – 7.19 (m, 2H), 7.16 – 7.08 (m, 1H), 4.09(t, J = 6.5 Hz, 2H), 3.93 – 3.87 (m, 1H), 3.74 – 3.68 (m, 4H), 3.12 (t, J =6.4 Hz, 2H), 2.87 (t, J = 8.3 Hz, 2H), 2.78 – 2.72 (m, 2H), 2.61 (t, J = 6.4Hz, 2H), 2.58 – 2.52 (m, 4H), 2.12 – 2.03 (m, 2H), 1.93 – 1.84 (m, 2H).

[0042] Example 4: This example relates to the preparation of BRD4 degrading agent prodrugs 7-9c, as detailed below:

[0043] Referring to the reaction route in this embodiment, A (1.0 mmol, 1.0 equivalent) was dissolved in DCM (5 mL), and B (1.2 mmol, 1.2 equivalent) and TEA (3 mmol, 3 equivalent) were added with stirring. The reaction was stirred at room temperature, and after the reaction was confirmed to be complete by TLC, the mixture was washed three times with saturated brine, the organic phase was collected, concentrated under reduced pressure, and the crude product was purified by column chromatography using (dichloromethane:methanol = 40:1~10:1) as the eluent to obtain the target product C, namely compounds 7c, 8c, and 9c.

[0044] Compound 7c: Yield 40%, 1 H NMR (500 MHz, Chloroform-d) δ 7.69 – 7.60 (m,2H), 7.52 – 7.46 (m, 2H), 7.30 – 7.17 (m, 5H), 4.95 (t, J = 8.7 Hz, 1H), 3.70– 3.62 (m, 4H), 3.50 (t, J = 8.9 Hz, 2H), 3.41 – 3.35 (m, 4H), 3.21 – 3.09(m, 3H), 2.93 – 2.84 (m, 1H), 2.79 (t, J = 6.8 Hz, 2H), 2.67 (t, J = 1.0 Hz,2H), 2.55 (s, 3H), 2.33 (s, 3H), 2.28 (s, 3H), 1.93 – 1.84 (m, 2H).

[0045] Compound 8c: Yield 55%, 1 H NMR (500 MHz, Chloroform-d) δ 7.64 – 7.60 (m,2H), 7.59 – 7.53 (m, 2H), 7.52 – 7.46 (m, 2H), 7.18 – 7.12 (m, 2H), 4.95 (t,J = 8.7 Hz, 1H), 3.70 – 3.62 (m, 4H), 3.50 (t, J = 8.9 Hz, 2H), 3.41 – 3.35(m, 4H), 3.21 – 3.09 (m, 3H), 2.92 – 2.84 (m, 1H), 2.79 (t, J = 6.8 Hz, 2H),2.67 (t, J = 0.9 Hz, 2H), 2.55 (s, 3H), 2.33 (s, 3H), 2.28 (s, 3H), 1.93 –1.84 (m, 2H).

[0046] Compound 9c: Yield 45%, 1 H NMR (500 MHz, Chloroform-d) δ 7.66 – 7.60 (m,2H), 7.52 – 7.47 (m, 2H), 7.31 – 7.23 (m, 8H), 7.23 – 7.17 (m, 2H), 4.95 (t,J = 8.7 Hz, 1H), 3.93 – 3.87 (m, 1H), 3.70 – 3.62 (m, 4H), 3.53 – 3.46 (m,2H), 3.41 – 3.35 (m, 4H), 3.23 – 3.09 (m, 3H), 2.92 – 2.84 (m, 1H), 2.80 (t,J = 8.3 Hz, 2H), 2.55 (s, 3H), 2.33 (s, 3H), 2.28 (s, 3H), 2.13 – 2.04 (m, 2H).

[0047] Example 5: This example involves the preparation of KRAS inhibitor prodrugs 6-9 days in advance, as detailed below:

[0048] Referring to the reaction route in this embodiment, A (1.0 mmol, 1.0 equivalent) was dissolved in DCM (5 mL), and B (1.2 mmol, 1.2 equivalent) and TEA (3 mmol, 3 equivalent) were added with stirring. The reaction was stirred at room temperature. After the reaction was complete as detected by TLC, the mixture was washed three times with saturated brine, the organic phase was collected, concentrated under reduced pressure, and the crude product was purified by column chromatography using (dichloromethane:methanol = 40:1~10:1) as the eluent to obtain the target product C, namely compounds 6d, 7d, 8d, and 9d.

[0049] Compound 6d: Yield 50%, 1H NMR (400 MHz, Methanol-d4) δ 8.39 (d, J = 5.0Hz, 1H), 8.28 – 8.19 (m, 1H), 7.27 – 7.18 (m, 2H), 6.68 – 6.56 (m, 2H), 5.08– 4.95 (m, 1H), 4.60 – 4.32 (m, 2H), 4.18 – 3.90 (m, 1H), 3.88 – 3.58 (m,2H), 3.28 – 3.22 (m, 1H), 3.01 – 2.90 (m, 2H), 2.89 – 2.78 (m, 2H), 2.77 –2.68 (m, 1H), 2.65 (d, J = 5.9 Hz, 3H), 2.01 (s, 3H), 1.52 (dd, J =27.2 Hz, 3H), 1.20 (d, J = 6.8 Hz, 3H), 1.03 (d, J = 1.1 Hz, 3H).

[0050] Compound 7d: Yield 70%, 1 H NMR (500 MHz, Chloroform-d) δ 8.65 (d, J = 12.1Hz, 1H), 8.24 (d, J = 5.3 Hz, 1H), 7.32 – 7.14 (m, 8H), 6.78 (dd, J = 10.0Hz, 1H), 4.23 – 4.13 (m, 1H), 3.96 – 3.82 (m, 3H), 3.81 – 3.73 (m, 1H), 3.55 – 3.49 (m, 1H), 3.40 – 3.32 (m, 1H), 3.18 – 3.07 (m, 3H), 2.81 (t, J = 6.9Hz, 2H), 2.67 (t, J = 1.0 Hz, 2H), 2.56 (t, J = 7.2 Hz, 2H), 2.42 (s, 3H), 1.93 – 1.84 (m, 2H), 1.36 – 1.33 (m, 6H), 1.33 (s, 3H).

[0051] Compound 8d: Yield 60%, 1H NMR (500 MHz, Chloroform-d) δ 8.65 (d, J = 12.1Hz, 1H), 8.24 (d, J = 5.3 Hz, 1H), 7.59 – 7.53 (m, 2H), 7.32 – 7.24 (m, 1H),7.19 – 7.12 (m, 4H), 6.78 (dd, J =10.0 Hz, 1H), 4.23 – 4.13 (m, 1H), 3.96 –3.82 (m, 3H), 3.81 – 3.73 (m, 1H), 3.55 – 3.49 (m, 1H), 3.40 – 3.32 (m, 1H),3.18 – 3.07 (m, 3H), 2.81 (t, J = 6.9 Hz, 2H), 2.67 (t, J = 0.9 Hz, 2H), 2.56(t, J = 7.2 Hz, 2H), 2.42 (d, J = 0.7 Hz, 3H), 1.93 – 1.84 (m, 2H), 1.36 –1.33 (m, 6H), 1.33 (s, 3H).

[0052] Compound 9d: Yield 50%, 1 H NMR (500 MHz, Chloroform-d) δ 8.65 (d, J = 12.1Hz, 1H), 8.24 (d, J = 5.3 Hz, 1H), 7.32 – 7.09 (m, 13H), 6.78 (dd, J = 10.0Hz, 1H), 4.23 – 4.13 (m, 1H), 3.96 – 3.82 (m, 4H), 3.81 – 3.73 (m, 1H), 3.55 – 3.49 (m, 1H), 3.40 – 3.32 (m, 1H), 3.12 (t, J = 7.1 Hz, 3H), 2.87 (t, J =8.3 Hz, 2H), 2.56 (t, J = 7.3 Hz, 2H), 2.42 (d, J = 0.7 Hz, 3H), 2.12 – 2.03(m, 2H), 1.36 – 1.33 (m, 6H), 1.33 (s, 3H).

[0053] Example 6: This example relates to the preparation of EGFR inhibitor prodrugs 6-9e, as detailed below:

[0054] Referring to the reaction route in this embodiment, A (1.0 mmol, 1.0 equivalent) was dissolved in DCM (5 mL), and B (1.2 mmol, 1.2 equivalent) and TEA (3 mmol, 3 equivalent) were added with stirring. The reaction was stirred at room temperature. After the reaction was complete as detected by TLC, the mixture was washed three times with saturated brine, the organic phase was collected, concentrated under reduced pressure, and the crude product was purified by column chromatography using (dichloromethane:methanol = 40:1~10:1) as the eluent to obtain the target product C, namely compounds 6e, 7e, 8e, and 9e.

[0055] Compound 6e: Yield 55%, 1 H NMR (300 MHz, Methanol- d 4) δ 8.65 (s, 1H), 8.35 (s, 1H), 7.90 (dd, J = 6.7, 2.6 Hz, 1H), 7.62 – 7.54 (m, 1H), 7.14 (t, J =2.1 Hz, 1H), 7.03 (s, 1H), 3.96 (s, 3H), 3.72 – 3.62 (m, 1H), 3.30 – 3.13 (m,6H), 3.12 – 3.04 (m, 1H), 2.73 (s, 3H), 2.63 – 2.51 (m, 1H), 1.97 – 1.74 (m, 4H), 1.71 – 1.58 (m, 2H).

[0056] Compound 7e: Yield 60%, 1 H NMR (500 MHz, Chloroform- d ) δ 9.02 (s, 1H),8.64 (s, 1H), 7.91 – 7.87 (m, 1H), 7.51 – 7.45 (m, 1H), 7.30 – 7.17 (m, 6H),7.16 – 7.08 (m, 1H), 3.99 (s, 3H), 3.24 – 3.15 (m, 1H), 2.89 – 2.78 (m, 2H), 2.78 – 2.46 (m, 10H), 1.93 – 1.79 (m, 2H), 1.63 – 1.52 (m, 4H), 1.50 – 1.41(m, 2H).

[0057] Compound 8e: Yield 55%, 1H NMR (500 MHz, Chloroform- d ) δ 9.02 (s, 1H),8.64 (s, 1H), 7.91 – 7.87 (m, 1H), 7.59 – 7.53 (m, 2H), 7.51 – 7.45 (m, 1H),7.29 (s, 1H), 7.18 – 7.13 (m, 2H), 7.13 – 7.08 (m, 1H), 3.99 (s, 3H), 3.24 –3.15 (m, 1H), 2.89 – 2.78 (m, 2H), 2.78 – 2.66 (m, 4H), 2.66 – 2.56 (m, 4H),2.56 – 2.52 (m, 1H), 2.52 – 2.46 (m, 1H), 1.93 – 1.79 (m, 2H), 1.61 – 1.52 (m, 4H), 1.50 – 1.41 (m, 2H).

[0058] Compound 9e: Yield 50%, 1 H NMR (500 MHz, Chloroform- d ) δ 9.02 (s, 1H),8.64 (s, 1H), 7.91 – 7.87 (m, 1H), 7.51 – 7.45 (m, 1H), 7.29 – 7.26 (m, 9H),7.24 – 7.19 (m, 2H), 7.16 – 7.08 (m, 1H), 3.99 (s, 3H), 3.94 – 3.87 (m, 1H), 3.26 – 3.17 (m, 1H), 2.92 – 2.83 (m, 2H), 2.80 – 2.70 (m, 2H), 2.66 – 2.52(m, 5H), 2.52 – 2.46 (m, 1H), 2.25 – 2.10 (m, 2H), 1.61 – 1.52 (m, 4H), 1.50 – 1.41 (m, 2H).

[0059] Example 7: This example relates to the preparation of BTK inhibitor prodrugs 6-9f, as detailed below:

[0060] Referring to the reaction route in this embodiment, A (1.0 mmol, 1.0 equivalent) was dissolved in DCM (5 mL), and B (1.2 mmol, 1.2 equivalent) and TEA (3 mmol, 3 equivalent) were added with stirring. The reaction was stirred at room temperature. After the reaction was complete as detected by TLC, the mixture was washed three times with saturated brine, the organic phase was collected, concentrated under reduced pressure, and the crude product was purified by column chromatography using (dichloromethane:methanol = 40:1~10:1) as the eluent to obtain the target product C, namely compounds 6f, 7f, 8f, and 9f.

[0061] Compound 6f: Yield 60%, 1 H NMR (400 MHz, Methanol- d 4) δ 8.18 (d, J = 19.6Hz, 1H), 7.65 – 7.56 (m, 2H), 7.39 – 7.29 (m, 2H), 7.17 – 7.09 (m, 1H), 7.09– 6.99 (m, 4H), 4.78 – 4.65 (m, 1H), 4.61 – 4.27 (m, 1H), 4.20 – 3.85 (m,1H), 3.70 – 3.52 (m, 1H), 3.26 – 3.13 (m, 1H), 3.02 – 2.66 (m, 4H), 2.59 (d, J = 4.5 Hz, 3H), 2.51 – 2.29 (m, 1H), 2.20 – 2.08 (m, 1H), 2.03 – 1.85 (m,1H), 1.75 – 1.51 (m, 1H).

[0062] Compound 7f: Yield 70%, 1 H NMR (500 MHz, Chloroform- d) δ 8.34 (s, 1H),7.81 – 7.75 (m, 2H), 7.39 – 7.31 (m, 2H), 7.30 – 7.18 (m, 5H), 7.14 – 6.98(m, 5H), 4.72 – 4.65 (m, 1H), 4.07 – 2.67 (t, J = 1.0 Hz, 2H), 2.55 (t, J = 7.2 Hz, 2H), 2.23 – 2.13 (m, 1H), 2.09 – 1.84 (m, 5H).

[0063] Compound 8f: Yield 65%, 1 H NMR (500 MHz, Chloroform- d ) δ 8.34 (s, 1H),7.81 – 7.75 (m, 2H), 7.59 – 7.53 (m, 2H), 7.39 – 7.31 (m, 2H), 7.16 – 7.13(m, 2H), 7.13 – 7.08 (m, 1H), 7.08 – 7.04 (m, 2H), 7.04 – 6.98 (m, 2H), 4.72 – 4.65 (m, 1H), 4.07 – 4.00 (m, 1H), 3.86 – 3.80 (m, 1H), 3.58 – 3.50 (m,1H), 3.37 – 3.30 (m, 1H), 3.14 (t, J = 7.2 Hz, 2H), 2.81 (t, J = 6.9 Hz, 2H), 2.67 (t, J = 0.9 Hz, 2H), 2.55 (t, J = 7.2 Hz, 2H), 2.23 – 2.13 (m, 1H), 2.09– 2.00 (m, 1H), 2.00 – 1.96 (m, 1H), 1.96 – 1.84 (m, 3H).

[0064] Compound 9f: Yield 60%, 1H NMR (500 MHz, Chloroform-d) δ 8.34 (s, 1H),7.81 – 7.75 (m, 2H), 7.39 – 7.31 (m, 2H), 7.31 – 7.23 (m, 8H), 7.23 – 7.17(m, 2H), 7.15 – 7.10 (m, 1H), 7.10 – 7.06 (m, 2H), 7.04 – 6.98 (m, 2H), 4.72 – 4.65 (m, 1H), 4.07 – 4.00 (m, 1H), 3.93 – 3.87 (m, 1H), 3.86 – 3.80 (m,1H), 3.58 – 3.50 (m, 1H), 3.37 – 3.30 (m, 1H), 3.12 (t, J = 7.3 Hz, 2H), 2.87 (t, J = 8.3 Hz, 2H), 2.55 (t, J = 7.2 Hz, 2H), 2.23 – 2.13 (m, 1H), 2.12 –1.86 (m, 5H).

[0065] Example 8: This example involves the preparation of 6-9g of BTK inhibitor prodrug, as detailed below:

[0066] Referring to the reaction route in this embodiment, A (1.0 mmol, 1.0 equivalent) was dissolved in DCM (5 mL), and B (1.2 mmol, 1.2 equivalent) and TEA (3 mmol, 3 equivalent) were added with stirring. The reaction was stirred at room temperature. After the reaction was complete as detected by TLC, the mixture was washed three times with saturated brine, the organic phase was collected, concentrated under reduced pressure, and the crude product was purified by column chromatography using (dichloromethane:methanol = 40:1~10:1) as the eluent to obtain the target product C, namely compounds 6 g, 7 g, 8 g, and 9 g.

[0067] Compound 6g: Yield 55%, 1 H NMR (300 MHz, Chloroform- d) δ 8.32 (s, 1H),7.76 – 7.61 (m, 2H), 7.58 – 7.42 (m, 2H), 7.42 – 7.28 (m, 1H), 7.28 – 7.07(m, 4H), 5.24 – 4.99 (m, 1H), 4.19 – 3.88 (m, 2H), 3.59 – 3.29 (m, 3H), 3.27– 3.08 (m, 2H), 3.07 – 2.87 (m, 1H), 2.76 (s, 3H), 2.45 – 2.19 (m, 2H), 2.14– 1.78 (m, 2H).

[0068] Compound 7g: yield 60%, 1 H NMR (500 MHz, Chloroform- d ) δ 8.34 (s, 1H),7.81 – 7.75 (m, 2H), 7.39 – 7.31 (m, 2H), 7.30 – 7.17 (m, 5H), 7.15 – 7.10(m, 1H), 7.10 – 7.04 (m, 2H), 7.04 – 6.98 (m, 2H), 4.65 – 4.59 (m, 1H), 3.59 – 3.52 (m, 1H), 3.46 – 3.36 (m, 3H), 3.35 – 3.21 (m, 2H), 3.21 – 3.14 (m, 2H), 2.79 (t, J = 6.8 Hz, 2H), 2.70 – 2.63 (m, 2H), 2.19 – 2.09 (m, 1H), 2.04 – 1.81 (m, 5H).

[0069] Compound 8g: Yield 70%, 1 H NMR (500 MHz, Chloroform- d) δ 8.34 (s, 1H),7.81 – 7.75 (m, 2H), 7.59 – 7.53 (m, 2H), 7.39 – 7.31 (m, 2H), 7.18 – 7.04(m, 5H), 7.04 – 6.98 (m, 2H), 4.65 – 4.59 (m, 1H), 3.59 – 3.52 (m, 1H), 3.46 – 3.36 (m, 3H), 3.35 – 3.21 (m, 2H), 3.21 – 3.14 (m, 2H), 2.79 (t, J = 6.8Hz, 2H), 2.67 (t, J = 0.9 Hz, 2H), 2.19 – 2.09 (m, 1H), 2.04 – 1.81 (m, 5H).

[0070] Compound 9g: Yield 65% ，1 H NMR (500 MHz, Chloroform-d) δ 8.34 (s, 1H), 7.81– 7.75 (m, 2H), 7.39 – 7.32 (m, 2H), 7.32 – 7.24 (m, 8H), 7.24 – 7.17 (m,2H), 7.15 – 7.10 (m, 1H), 7.07 (d, J = 8.7 Hz, 2H), 7.04 – 6.98 (m, 2H), 4.65– 4.59 (m, 1H), 3.93 – 3.87 (m, 1H), 3.59 – 3.52 (m, 1H), 3.46 – 3.36 (m,3H), 3.35 – 3.16 (m, 4H), 2.80 (t, J = 8.3 Hz, 2H), 2.19 – 2.10 (m, 1H), 2.10 – 2.04 (m, 2H), 2.04 – 1.90 (m, 2H), 1.90 – 1.81 (m, 1H).

[0071] Example 9: This example involves the preparation of a BTK inhibitor prodrug 6-9 hours prior, as detailed below:

[0072] Referring to the reaction route in this embodiment, A (1.0 mmol, 1.0 equivalent) was dissolved in DCM (5 mL), and B (1.2 mmol, 1.2 equivalent) and TEA (3 mmol, 3 equivalent) were added with stirring. The reaction was stirred at room temperature. After the reaction was complete as detected by TLC, the mixture was washed three times with saturated brine, the organic phase was collected, concentrated under reduced pressure, and the crude product was purified by column chromatography using (dichloromethane:methanol = 40:1~10:1) as the eluent to obtain the target product C, namely compounds 6h, 7h, 8h, and 9h.

[0073] Compound 6h: Yield 45%, 1 H NMR (300 MHz, Methanol- d 4 ) δ 8.17 (s, 1H), 7.30 (t, J = 7.8 Hz, 2H), 7.10 – 6.92 (m, 3H), 5.07 (s, 2H), 4.63 – 4.56 (m, 1H), 3.87 (dd, J = 11.8, 4.4 Hz, 1H), 3.73 (dd, J = 12.2 Hz, 1H), 3.38 – 3.18 (m,7H), 3.01 – 2.84 (m, 3H), 2.58 (s, 3H).

[0074] Compound 7h: Yield 70%, 1 H NMR (500 MHz, Chloroform- d ) δ 8.36 (s, 1H),7.32 – 7.17 (m, 7H), 7.04 – 6.94 (m, 3H), 5.00 (s, 2H), 4.63 – 4.56 (m, 1H),3.59 – 3.52 (m, 1H), 3.46 – 3.39 (m, 2H), 3.35 – 3.28 (m, 1H), 3.28 – 3.14 (m, 4H), 2.79 (t, J = 6.8 Hz, 2H), 2.67 (t, J = 1.0 Hz, 2H), 2.19 – 2.09 (m,1H), 1.98 – 1.84 (m, 4H), 1.84 – 1.73 (m, 1H).

[0075] Compound 8h: Yield 80%, 1H NMR (500 MHz, Chloroform- d ) δ 8.36 (s, 1H),7.59 – 7.53 (m, 2H), 7.32 – 7.25 (m, 2H), 7.18 – 7.12 (m, 2H), 7.04 – 6.93(m, 3H), 5.00 (s, 2H), 4.63 – 4.56 (m, 1H), 3.59 – 3.52 (m, 1H), 3.46 – 3.39 (m, 2H), 3.35 – 3.28 (m, 1H), 3.28 – 3.14 (m, 4H), 2.79 (t, J = 6.8 Hz, 2H), 2.70 – 2.63 (m, 2H), 2.19 – 2.09 (m, 1H), 1.98 – 1.84 (m, 4H), 1.84 – 1.73 (m, 1H).

[0076] Compound 9h: Yield 65%, 1 H NMR (500 MHz, Chloroform- d ) δ 8.36 (s, 1H),7.32 – 7.24 (m, 10H), 7.24 – 7.18 (m, 2H), 7.04 – 6.97 (m, 2H), 6.97 – 6.93(m, 1H), 5.00 (s, 2H), 4.63 – 4.56 (m, 1H), 3.93 – 3.87 (m, 1H), 3.59 – 3.52(m, 1H), 3.43 (t, J = 0.7 Hz, 2H), 3.35 – 3.26 (m, 1H), 3.26 – 3.16 (m, 4H), 2.80 (t, J = 8.3 Hz, 2H), 2.19 – 2.10 (m, 1H), 2.10 – 2.04 (m, 2H), 1.98 –1.84 (m, 2H), 1.84 – 1.73 (m, 1H).

[0077] Example 10: This example relates to the preparation of EGFR inhibitor prodrug 6i, as detailed below:

[0078] Referring to the reaction route in this embodiment, A (1.0 mmol, 1.0 equivalent) was dissolved in DCM (5 mL), and B (1.2 mmol, 1.2 equivalent) and TEA (3 mmol, 3 equivalent) were added with stirring. The reaction was stirred at room temperature, and after the reaction was confirmed to be complete by TLC, the mixture was washed three times with saturated brine, the organic phase was collected, concentrated under reduced pressure, and the crude product was purified by column chromatography using (dichloromethane:methanol = 40:1~10:1) as the eluent to obtain the target product C, i.e., compound 6i.

[0079] Compound 6i: Yield 55%, 1 H NMR (500 MHz, Chloroform-d) δ 9.87 (s, 1H), 8.11 (d, J = 1.8 Hz, 1H), 7.74 – 7.70 (m, 2H), 7.67 (s, 1H), 7.61 (d, J = 1.6Hz, 1H), 7.42 – 7.36 (m, 1H), 7.29 – 7.23 (m, 1H), 4.97 – 4.90 (m, 1H), 3.86 – 3.79 (m, 1H), 3.61 – 3.54 (m, 1H), 3.37 – 3.25 (m, 2H), 3.13 – 3.06 (m,4H), 2.64 (s, 6H), 2.61 – 2.52 (m, 4H), 2.40 (d, J = 0.7 Hz, 3H), 2.06 – 1.95 (m, 1H), 1.81 – 1.73 (m, 1H), 1.73 – 1.61 (m, 3H), 1.61 – 1.49 (m, 1H).

[0080] Example 11: This example relates to the preparation of the angiolyne derivative SA-1b, as detailed below:

[0081] Referring to the reaction route in this embodiment, A (1.0 mmol, 1.0 equivalent) was dissolved in THF (2 ml). B (1.3 mmol, 1.3 equivalent) and PPh3 (1.3 mmol, 1.3 equivalent) were added with stirring. Nitrogen was used to purge the mixture, and a diluted solution of DEAD (1.3 mmol, 1.3 equivalent) was injected into an ice bath. After the addition was complete, the mixture was allowed to react at room temperature. After TLC detection to confirm complete reaction, the reaction mixture was diluted with ethyl acetate (10 mL) and washed twice with saturated brine. The organic phase was dried, filtered, and concentrated. The crude product was purified by column chromatography using petroleum ether:ethyl acetate = 5:1 as the eluent to obtain the target product C, compound SA-1b.

[0082] Compound SA-1b: Yield 75%. 1 H NMR (300 MHz, Chloroform- d ) δ 7.62 (d, J =9.5 Hz, 1H), 7.36 (d, J = 8.4 Hz, 1H), 6.93 – 6.76 (m, 2H), 6.24 (d, J = 9.5Hz, 1H), 4.82 (t, J = 5.8 Hz, 1H), 2.34 – 2.14 (m, 4H), 1.97 – 1.83 (m, 3H), 1.77 – 1.58 (m, 3H).

[0083] Example 12: This example relates to the preparation of the strainyne derivative SA-1c, as detailed below:

[0084] Referring to the reaction route in this embodiment, B (1.5 mmol, 1.5 equivalents) and TEA (3 mmol, 3.0 equivalents) were dissolved in anhydrous THF (5 mL), and N2 was used for displacement. The solution was cooled to 0°C, and A (1 mmol, 1.0 equivalents) was added. The mixture was heated to room temperature and the reaction was continued with stirring. After the reaction was confirmed to be complete by TLC, the reaction mixture was concentrated, diluted with ethyl acetate (10 mL), and washed twice with saturated brine. The organic phase was dried, filtered, and concentrated. The crude product was purified by column chromatography using (dichloromethane:methanol = 10:1) as the eluent to obtain the target product C.

[0085] C (1.0 mmol, 1.0 equivalent) was dissolved in DCM (2 mL), and an equal volume of TFA (2 mL) was added dropwise. The reaction mixture was stirred at room temperature. After the reaction was complete as detected by TLC, the reaction mixture was concentrated to obtain D, which was used directly without further purification. A solution of D (1.0 mmol, 1.0 equivalent) and TEA (3.0 mmol, 1.2 equivalent) in DMF (3 mL) was cooled to 0°C, and then a diluted solution of E (1.2 mmol, 1.2 equivalent) was added dropwise. After the addition was complete, the reaction mixture was stirred at room temperature. After the reaction was complete as detected by TLC, the reaction mixture was diluted with ethyl acetate (10 mL) and washed twice with saturated brine. The organic phase was dried, filtered, and concentrated. The crude product was purified by column chromatography using (dichloromethane:methanol = 30:1) as the eluent to obtain the target product F, namely compound SA-1c.

[0086] Compound SA-1c: Yield 50%. 1 H NMR (300 MHz, Chloroform-d) δ 6.81 (s, 1H), 6.57 (s, 1H), 5.68 (s, 1H), 5.49 (s, 1H), 5.29 (t, J = 6.0 Hz, 1H), 4.55 –4.44 (m, 1H), 4.36 – 4.25 (m, 1H), 3.69 – 3.59 (m, 8H), 3.58 – 3.49 (m, 4H), 3.47 – 3.39 (m, 2H), 3.39 – 3.29 (m, 2H), 3.18 – 3.09 (m, 1H), 2.94 – 2.84(m, 1H), 2.79 – 2.69 (m, 1H), 2.34 – 2.28 (m, 1H), 2.27 – 2.16 (m, 4H), 2.15 – 2.09 (m, 1H), 2.03 – 1.95 (m, 1H), 1.94 – 1.84 (m, 2H), 1.80 – 1.59 (m,7H), 1.49 – 1.38 (m,2H).

[0087] Example 13: This example relates to the preparation of the strainyne derivative SA-1d, as detailed below:

[0088] Referring to the reaction route in this embodiment, sodium hydride (1.2 mmol, 1.2 equivalents) was dissolved in toluene, and A (1.0 mmol, 1.0 equivalents) was added under ice bath conditions. The reaction was carried out at room temperature for 10 minutes, and then B (1.2 mmol, 1.2 equivalents) was added. The reaction was continued with stirring, and after the reaction was confirmed to be complete by TLC, the mixture was concentrated under reduced pressure. The crude product was purified by column chromatography using petroleum ether: ethyl acetate = 5:1 as the eluent to obtain the target product C, namely compound SA-1d.

[0089] Compound SA-1d: Yield 55%. 1 H NMR (300 MHz, Chloroform-d) δ 7.27 (d, J =7.8 Hz, 2H), 7.11 (d, J = 7.2 Hz, 2H), 6.55 (s, 1H), 5.40 (s, 1H), 2.31 (s,3H), 2.28 – 2.18 (m, 2H), 2.14 – 2.05 (m, 1H), 2.01 – 1.89 (m, 2H), 1.84 –1.76 (m, 1H), 1.71 – 1.53 (m, 4H).

[0090] Example 14: This example relates to the preparation of the angiolyne derivative SA-1e, as detailed below:

[0091] Referring to the reaction route in this embodiment, A (1.2 mmol, 1.2 equivalents) and TBAI (0.25 mmol, 0.25 equivalents) were dissolved in toluene (5 ml). While stirring, 50% NaOH solution was added in portions to the reaction mixture. After the addition was complete, B (2.2 mmol, 2.2 equivalents) was added. The reaction was stirred at room temperature. After the reaction was complete as detected by TLC, the pH of the reaction mixture was adjusted to neutral with dilute hydrochloric acid. The reaction solution was diluted with ethyl acetate (10 mL), and the organic phase was washed with saturated brine. After drying, the organic phase was filtered and concentrated. The crude product was purified by column chromatography using (dichloromethane:methanol = 20:1) as the eluent to obtain the target product C, i.e., compound SA-1e.

[0092] Compound SA-1e: Yield 55%. 1 H NMR (300 MHz, Chloroform- d ) δ 4.39 (s, 1H), 4.08 (d, J = 1.8 Hz, 1H), 3.94 (d, J= 1.7 Hz, 1H), 2.28 – 2.10 (m, 3H), 2.09 – 2.00 (m, 1H), 1.98 – 1.74 (m, 4H), 1.71 – 1.60 (m, 2H), 1.47 (s, 9H).

[0093] Example 15: This example relates to the preparation of the strainyne derivative SA-1f, as detailed below:

[0094] Referring to the reaction route in this embodiment, A (1.0 mmol, 1.0 equivalent) and TEA (2.0 mmol, 2.0 equivalent) were dissolved in THF (3 mL), purged with nitrogen, and the reaction mixture was cooled to -10°C. A diluted solution of B was injected into the reaction flask, and the reaction was allowed to proceed for 10 minutes. The mixture was then allowed to react at room temperature. After TLC detection of complete reaction, the reaction mixture was diluted with ethyl acetate (10 mL) and washed twice with saturated brine. The organic phase was dried, filtered, and concentrated. The resulting crude product was purified by column chromatography using petroleum ether:ethyl acetate = 5:1 as the eluent to obtain the target product C.

[0095] C (1.0 mmol, 1.0 equivalent) was dissolved in methanol, and D (1.2 mmol, 1.2 equivalent) and DIPEA (3.0 mmol, 3.0 equivalent) were added with stirring. The reaction was allowed to proceed at room temperature. After the reaction was confirmed to be complete by TLC, the solvent was removed by concentration under reduced pressure. The product was dissolved in ethyl acetate (10 mL) and washed twice with saturated brine. The organic phase was dried, filtered, and concentrated. The crude product was purified by column chromatography using dichloromethane:methanol = 10:1 as the eluent to obtain the target product E, i.e., compound SA-1f.

[0096] Compound SA-1f: Yield 45%. 1 H NMR (400 MHz, DMSO) δ 14.02 (s, 1H), 13.26 (s, 1H), 7.89 (d, J = 4.7 Hz, 2H), 7.66 – 7.61 (m, 1H), 6.82 (d, J = 7.9 Hz,1H), 5.46 (s, 1H), 5.19 (s, 1H), 5.10 – 5.01 (m, 1H), 4.95 – 4.89 (m, 1H),4.85 (t, J = 6.0 Hz, 1H), 4.72 – 4.65 (m, 1H), 4.56 (d, J= 6.0 Hz, 2H), 4.17– 4.09 (m, 1H), 3.97 (s, 3H), 3.70 – 3.57 (m, 1H), 3.43 – 3.38 (m, 1H), 3.04– 2.88 (m, 2H), 2.23 – 1.94 (m, 6H), 1.87 – 1.73 (m, 4H), 1.57 – 1.40 (m,4H), 1.10 (d, J = 6.4 Hz, 3H).

[0097] Example 16: This example relates to the preparation of the tonicyne derivative SA-1g, as detailed below:

[0098] Referring to the reaction route in this embodiment, A (1.0 mmol, 1.0 equivalent) was dissolved in THF (4 ml). B (1.0 mmol, 1.0 equivalent) and PPh3 (1.1 mmol, 1.1 equivalent) were added with stirring. Nitrogen was used to purge the mixture, and a diluted solution of DEAD (1.1 mmol, 1.1 equivalent) was injected into an ice bath. After the addition was complete, the mixture was allowed to react at room temperature. After TLC detection to confirm complete reaction, the reaction mixture was diluted with ethyl acetate (10 mL) and washed twice with saturated brine. The organic phase was dried, filtered, and concentrated. The crude product was purified by column chromatography using petroleum ether:ethyl acetate = 10:1 as the eluent to obtain the target product C, i.e., compound SA-1g.

[0099] Compound SA-1g: Yield 20%. 1 H NMR (500 MHz, Chloroform-d) δ 7.13 – 7.08(m, 1H), 7.02 (d, J = 1.9 Hz, 1H), 6.88 (d, J = 8.4 Hz, 1H), 6.79 – 6.75 (m,3H), 6.75 – 6.69 (m, 1H), 4.90 – 4.83 (m, 1H), 3.89 – 3.82 (m, 12H), 2.36 –2.24 (m, 2H), 2.24 – 2.14 (m, 1H), 1.99 – 1.89 (m, 1H), 1.79 – 1.68 (m, 1H),1.65 – 1.48 (m, 5H).

[0100] Example 17: This example relates to the preparation of the tonicyne derivative SA-1h, as detailed below:

[0101] Referring to the reaction route in this embodiment, A (1.0 mmol, 1.0 equivalent) and DMAP (1.0 mmol, 1.0 equivalent) were dissolved in dry toluene (4 ml), triphosgene was added, and the reaction was stirred at room temperature. The reaction was monitored by TLC until completion. The reaction solution was filtered and the filter cake was collected to obtain crude product B. Crude product B was dissolved in dichloromethane (4 ml), and C (1.0 mmol, 1.0 equivalent) and triethylamine (1.0 mmol, 1.0 equivalent) were added under stirring. The reaction was stirred at room temperature. After the reaction was completed, the pH was adjusted to neutral with dilute hydrochloric acid, and the product was extracted with dichloromethane and water. The organic phase was dried, filtered, and concentrated. The crude product was purified by column chromatography using petroleum ether: ethyl acetate = 5:1 as the eluent to obtain the target product D, namely compound SA-1h.

[0102] Compound SA-1h: Yield 25%. 1 H NMR (500 MHz, Chloroform- d ) δ 7.69 (d, J =2.2 Hz, 1H), 7.63 (d, J = 8.2 Hz, 1H), 7.48 – 7.43 (m, 1H), 6.15 (s, 1H), 5.47 – 5.40 (m, 1H), 2.44 (s, 3H), 2.36 – 2.24 (m, 2H), 2.12 – 2.01 (m, 1H), 1.87 – 1.76 (m, 1H), 1.76 – 1.64 (m, 1H), 1.64 – 1.47 (m, 5H).

[0103] Example 18: This example relates to the preparation of the tonicyne derivative SA-1i, as detailed below:

[0104] Referring to the reaction route in this embodiment, A (1.0 mmol, 1.0 equivalent) was dissolved in THF (4 ml). B (1.0 mmol, 1.0 equivalent) and PPh3 (1.1 mmol, 1.1 equivalent) were added with stirring. Nitrogen was used for purging, and a diluted solution of DIAD (1.1 mmol, 1.1 equivalent) was injected into an ice bath. After the addition was complete, the mixture was allowed to react at room temperature. After TLC detection of complete reaction, the reaction mixture was diluted with ethyl acetate (10 mL) and washed twice with saturated brine. The organic phase was dried, filtered, and concentrated. The crude product was purified by column chromatography using petroleum ether:ethyl acetate = 20:1 as the eluent to obtain the target product C, compound SA-1i.

[0105] Compound SA-1i: yield 45%. 1 H NMR (500 MHz, Chloroform- d ) δ 8.24 – 8.20(m, 1H), 7.90 (d, J = 8.4 Hz, 1H), 7.29 (s, 1H), 7.18 (dd, J = 8.6, 2.4 Hz, 1H), 6.91 (t, J = 2.4 Hz, 1H), 5.45 – 5.35 (m, 2H), 5.01 (d, J = 1.0 Hz, 2H),4.82 – 4.75 (m, 1H), 4.64 (s, 1H), 2.36 – 2.24 (m, 2H), 2.21 – 2.10 (m, 1H),2.03 – 1.85 (m, 2H), 1.79 – 1.67 (m, 2H), 1.65 – 1.56 (m, 2H), 1.56 – 1.48(m, 3H), 0.90 (t, J = 8.0 Hz, 3H).

[0106] Example 19: This example relates to the preparation of the strainyne derivative SA-1j, as detailed below:

[0107] Referring to the reaction route in this embodiment, A (1.0 mmol, 1.0 equivalent) and TEA (2.0 mmol, 2.0 equivalent) were dissolved in THF (3 mL), purged with nitrogen, and the reaction mixture was cooled to -10°C. A diluted solution of B was injected into the reaction flask, and the reaction was allowed to proceed for 10 minutes. The mixture was then allowed to react at room temperature. After TLC detection of complete reaction, the reaction mixture was diluted with ethyl acetate (10 mL) and washed twice with saturated brine. The organic phase was dried, filtered, and concentrated. The resulting crude product was purified by column chromatography using petroleum ether:ethyl acetate = 5:1 as the eluent to obtain the target product C.

[0108] C (1.0 mmol, 1.0 equivalent) was dissolved in methanol, and D (1.2 mmol, 1.2 equivalent) and DIPEA (3.0 mmol, 3.0 equivalent) were added with stirring. The reaction was allowed to proceed at room temperature. After the reaction was confirmed to be complete by TLC, the solvent was removed by concentration under reduced pressure. The product was dissolved in ethyl acetate (10 mL) and washed twice with saturated brine. The organic phase was dried, filtered, and concentrated. The crude product was purified by column chromatography using dichloromethane:methanol = 20:1 as the eluent to obtain the target product E, i.e., compound SA-1j.

[0109] Compound SA-1j: Yield 30%. 1 H NMR (500 MHz, Chloroform- d ) δ 7.38 – 7.27(m, 6H), 6.90 (d, J = 8.8 Hz, 1H), 5.39 – 5.31 (m, 1H), 4.91 – 4.84 (m, 1H), 4.46 – 4.37 (m, 2H), 4.19 – 4.14 (m, 1H), 4.14 – 4.09 (m, 1H), 4.07 – 3.93(m, 2H), 3.83 – 3.75 (m, 1H), 3.73 – 3.64 (m, 1H), 3.62 – 3.55 (m, 1H), 3.50– 3.42 (m, 1H), 3.34 (d, J = 1.5 Hz, 3H), 3.32 (d, J = 1.5 Hz, 3H), 2.89 (d, J = 1.5 Hz, 3H), 2.88 (d, J= 1.5 Hz, 3H), 2.75 – 2.61 (m, 2H), 2.57 – 2.49(m, 1H), 2.36 – 2.24 (m, 2H), 2.24 – 2.12 (m, 1H), 2.12 – 1.98 (m, 2H), 1.97– 1.64 (m, 7H), 1.64 – 1.53 (m, 5H), 1.53 – 1.42 (m, 2H), 1.28 (dd, J = 6.2,1.5 Hz, 3H), 1.25 – 1.18 (m, 1H), 1.15 (dd, J = 7.0, 1.5 Hz, 3H), 0.97 – 0.79(m, 18H).

[0110] Example 20: This example relates to the preparation of hydroxylamine derivatives 1a and 1d, as detailed below:

[0111] Referring to the reaction route in this embodiment, A (1.0 mmol, 1.0 equivalent) was dissolved in DCM (5 ml), and B (1.2 mmol, 1.2 equivalent) and TEA (3 mmol, 3 equivalent) were added with stirring. After the addition was complete, the mixture was allowed to react at room temperature. After the reaction was confirmed to be complete by TLC, the reaction mixture was extracted with dichloromethane and saturated brine. The organic phase was dried, filtered, and concentrated. The crude product was purified by column chromatography using petroleum ether:ethyl acetate = 10:1 as the eluent to obtain the target product C, namely compounds 1a, 1d, and 1h.

[0112] Compound 1a: Yield 70%. 1 H NMR (300 MHz, Chloroform- d ) δ 8.89 (s, 1H),7.57 – 7.36 (m, 2H), 7.21 – 7.15 (m, 2H), 7.07 – 6.97 (m, 1H), 2.74 –2.65 (m,2H), 2.69 (s, 3H), 2.64 –2.50 (m, 2H). Compound 1d: Yield 70%. 1 H NMR (300 MHz, Chloroform- d) δ 7.48 – 7.28 (m, 3H), 7.20 – 7.06 (m, 2H), 3.17 (s, 3H), 2.86 – 2.63 (m, 2H), 2.36 (s, 3H), 2.32 – 2.20 (m, 2H). Compound 1h: yield 70%. 1 H NMR (300 MHz, Chloroform-d) δ 7.43 – 7.16 (m,5H), 3.45 – 3.31 (m, 2H), 3.13 – 2.99 (m, 2H), 2.65 (s, 3H).

[0113] Test Example 1: The release rate of the prodrugs prepared in Examples 1-10 above was tested, and the test method is as follows: The kinetics of tolenyne and hydroxylamine derivatives were determined using a pseudo-first-order kinetic method: both were dissolved in 20% acetonitrile / phosphate buffer (pH 7.4) with the tolenyne concentration at least 10 times that of the hydroxylamine derivative. After incubation at 37 °C, samples were taken periodically for HPLC analysis, and the peak area of ​​the hydroxylamine derivative was monitored. The pseudo-first-order kinetic constant (k1) was obtained by fitting the peak area against time. The experiment was repeated with different ratios of the two to obtain four sets of k1. The slope of the obtained straight line was plotted against the tolenyne concentration, and it was the second-order kinetic constant (k2) of the reverse Cope elimination reaction.

[0114] Prodrug release was determined using high-performance liquid chromatography (HPLC): a solution of tonicine and the prodrug in 20% acetonitrile / phosphate buffer (pH 7.4) was incubated at 37 °C. Samples of the mixture were taken at periodic intervals for HPLC analysis. The released parent drug was quantified according to a calibration curve.

[0115] (1) Using tension acetylene BCN ( ) and hydroxylamine derivative 1a ( ), 1d ( ), 1h Taking an example, the reaction kinetics between tonicylene and hydroxylamine derivatives and the release rate of covalent compounds were studied. The results are shown in Table 1 below: Table 1

[0116] (2) Taking hydroxylamine derivative 1d as an example, the reaction kinetics with tonichematides of different structures and the release rate of covalent compounds were studied. The results are shown in Table 2 below: Table 2

[0117] As shown in Tables 1 and 2, by modifying the structure of the tonicine, for example, by using tonicine SA-1b, SA-1d, and SA-1h compared to BCN, the present invention not only effectively improves the reaction kinetics between hydroxylamine derivatives and tonicine, but also significantly increases the drug release rate.

[0118] (3) Taking styryne SA-1b as an example, the reaction kinetics with different hydroxylamine derivatives and the release rate of covalent compounds were studied. The results are shown in Tables 3-7 below: Table 3

[0119] Table 4

[0120] Table 5

[0121] Table 6

[0122] Table 7

[0123] As shown in Table 3-7, different hydroxylamine derivatives and tonicine SA-1b not only have high reaction kinetics, but also the drug release rate after the reaction can reach more than 90%, which is far better than the release rate (~60%) of endogenously triggered covalent inhibitor prodrugs reported in the prior art.

[0124] (4) The reaction kinetics (1,4-elimination reaction kinetics) and drug release rate of different prodrugs with strained alkyne structures with hydroxylamine derivatives were studied 1 day later. The reaction mechanism diagram is shown below. Figure 1 As shown in Table 8 below: Table 8

[0125] As shown in Table 8, different drug prodrugs with anisodylene structures can release drugs by reacting with hydroxylamine derivatives, and the release rate can reach more than 90%.

[0126] Test Example 2: Using known active pharmaceutical ingredients as references, the cytotoxicity of some prodrugs prepared in Examples 1-9 and the compositions of each prodrug with SA-1b were studied, as follows: Cytotoxicity assay: Cells were cultured for 24 h in medium containing 10% fetal bovine serum and 1% penicillin-streptomycin. Cells were then seeded into 96-well plates at a density of 5000 cells / well and cultured for another 24 h. The old medium was discarded, and medium containing the reference sample and prodrug or SA-1b and prodrug was added to each well. The plates were then incubated at 37 ℃ in a 5% CO2 incubator (6–9a, b: 24 h; 6–9c, h; 6i: 72 h). The old medium was discarded, and 100 μL of fresh medium containing 10% CCK-8 was added to each well. The plates were incubated for 2 h, and the absorbance (OD value) at 450 nm was measured using a microplate reader to calculate cell viability. The results are shown in Table 9 below. Table 9 ,

[0127] As shown in Table 9, the cytotoxicity of the prodrug prepared in this invention is significantly lower than that of the active drug. However, by introducing the trigger SA-1b and treating the cells together with the prodrug, the cytotoxicity is significantly improved, and the effect is comparable to that of direct treatment with the active drug.

[0128] Test Example 3: Taking prodrug compound 6a and tonoethynyl SA-1c as examples, the feasibility of a pre-targeted triggering strategy for selective cell activation was studied, as detailed below: Cytotoxicity assay of the pre-targeting trigger: A549 or HCT116 cells were seeded at a density of 5000 cells per well in 96-well plates and incubated at 37 °C in a 5% CO2 incubator for 24 hours. Subsequently, cells were treated with 50 μM SA-1c for 1 hour; one group of cells was pretreated with 50 μM biotin for 1 hour, followed by 50 μM SA-1c for 1 hour. The original culture medium was discarded and replaced with fresh culture medium containing graded concentrations of compound 6a, and incubation continued for 24 hours. After washing the cells twice with PBS, 10% CCK-8 solution was added, and the cells were incubated at 37 °C for 1 hour. Finally, the absorbance (OD value) of each well was measured at 450 nm using a microplate reader to calculate cell viability.

[0129] Test results are as follows Figure 2 As shown, in biotin receptor-positive A549 cells, the combined use of SA-1c and 6a significantly inhibited cell proliferation, with an IC50 value of [missing information]. 50The concentration was approximately 1 μM, and the inhibitory activity was comparable to that of the positive control drug Om. However, in biotin-receptor-negative HCT116 cells, the observed antiproliferative effect was significantly weakened, with cell viability still exceeding 80% at the maximum administered concentration of 5 μM. Furthermore, in biotin-receptor-positive A549 cells, pretreatment with biotin (50 μM) to saturate the receptor also significantly reduced the antiproliferative effect of the combined use of SA-1c and 6a, with cell viability still exceeding 75% at a administered concentration of 5 μM. In conclusion, these results confirm the feasibility of using a pre-targeting strategy to achieve cell-selective activation of covalent inhibitors.

[0130] As shown in Table 9, the cytotoxicity of the prodrug prepared in this invention is significantly lower than that of the active drug. However, by introducing the trigger SA-1b and treating the cells together with the prodrug, the cytotoxicity is significantly improved, and the effect is comparable to that of direct treatment with the active drug.

[0131] Test Example 4: Taking prodrug compound 6c and tonichexene SA-1b as examples, the degradation effect of this bioorthogonal prodrug composition on proteins was studied, as follows: Protein extraction: MDA-MB-231 cells were seeded in 10 cm cell culture dishes. After complete confluence, the cells were seeded at an appropriate density into 6-well plates. When the cell confluence reached 80%, the original culture medium was discarded, and drug-containing culture medium (containing 1% DMSO) was slowly added along the well wall, and incubation continued for 24 hours. Subsequently, cells were collected by cell scraping and transferred to centrifuge tubes, and centrifuged at 1000 rpm for 5 minutes. The supernatant was discarded, and the cells were washed twice with 1 mL PBS. 100 μL of high-efficiency lysis buffer (containing 1% protease inhibitor) was added to the cell pellet, and the cells were incubated on ice for 30 minutes, with intermittent pipetting to promote lysis. The lysis buffer was centrifuged at 12,000 rpm for 15 minutes at 4 °C, and the supernatant was collected. The protein concentration was determined using the BCA method and normalized to 1 μg / μL. Finally, loading buffer was added, and the cells were heated at 95 °C for 10 minutes to denature the protein.

[0132] Protein separation and detection: Proteins were separated using SDS-PAGE and then transferred to PVDF membranes. The membranes were placed in blocking buffer containing 5% skim milk and blocked at room temperature for 1 hour. Primary antibodies (anti-BRD4, 1:200; anti-β-actin, 1:1000) were then added, and the membranes were incubated at room temperature for 4 hours. The membranes were washed three times with TBST for 10 minutes each time; then HRP-labeled secondary antibody (1:2000) was added, and the membranes were incubated at room temperature for 1 hour. After repeated washing, the membranes were developed using ECL reagent to observe the protein bands.

[0133] Test results are as follows Figure 3As shown, compound 7 effectively induced the degradation of BRD4, while the prodrug 6c, lacking the vinylsulfonamide moiety, failed to degrade BRD4 under the same conditions. This result indicates that the introduction of the N-methylhydroxylamine can effectively eliminate its ability to recruit DCAF16. Notably, the combination of alkyne SA-1b and compound 6c effectively degraded BRD4, while alkyne SA-1b alone did not affect BRD4 levels. In conclusion, these results strongly demonstrate that the tandem bioorthogonal reaction of reverse Cope and Cope elimination can be used to activate vinylsulfonamide warheads in cells.

[0134] The above-described embodiments are merely preferred embodiments provided to fully illustrate the present invention, and the scope of protection of the present invention is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are all within the scope of protection of the present invention. The scope of protection of the present invention is defined by the claims.

Claims

1. A pharmaceutical prodrug having a substituted acrylamide or substituted vinylsulfonamide structure, characterized in that, The structure of the prodrug is as follows: ， Where Y is or ; R1 is the ligand portion of the drug, which is a covalent inhibitor or molecular glue with a substituted acrylamide or substituted vinyl sulfonamide as the warhead. R2 is selected from H, F, CN, C 1-6 Alkyl, F-substituted C 1-6 One of the alkyl groups; R3 is selected from H and C. 1-6 Alkyl, amino-substituted C 1-6 One of the alkyl groups; R4 is selected from C 1-6 Alkyl and aryl substituted C 1-6 One of the alkyl groups; R5 and R6 are H and C respectively. 1-6 The alkyl group or the N group in the adjacent substituent R1 can cyclize to form a substituted or unsubstituted piperazine ring.

2. The prodrug according to claim 1, characterized in that, The prodrug is one of the compounds shown in the following structures: ， ， ， ， ， 。 3. A method for preparing a prodrug as described in claim 1 or 2, characterized in that, The compound represented by formula (I) is reacted with the N-alkylhydroxylamine represented by formula (II) in the presence of an organic amine and a solvent to obtain the prodrug; the structures of formulas (I) and (II) are shown below: ； Where Y is or ; R1 is the ligand portion of the drug, which is a covalent inhibitor or molecular glue with a substituted acrylamide or substituted vinyl sulfonamide as the warhead. R2 is selected from H, F, CN, C 1-6 Alkyl, F-substituted C 1-6 One of the alkyl groups; R3 is selected from H and C. 1-6 Alkyl, amino-substituted C 1-6 One of the alkyl groups; R4 is selected from C 1-6 Alkyl and aryl substituted C 1-6 One of the alkyl groups; R5 and R6 are H and C respectively. 1-6 The alkyl group or the N group in the adjacent substituent R1 can cyclize to form a substituted or unsubstituted piperazine ring.

4. A biological orthogonal prodrug composition, characterized in that, Includes the drug prodrug as described in claim 1 or 2 and the tonichexyne derivative, wherein the tonichexyne derivative is an eight-membered cyclohexyne derivative.

5. The bioorthogonal prodrug composition according to claim 1, characterized in that, The tensiylene derivative is selected from one of the following structures: ， 。 6. The use of a prodrug having substituted acrylamide or substituted vinylsulfonamide as described in claim 1 or 2, or a bioorthogonal prodrug composition as described in claim 4 or 5, in the preparation of a drug delivery system or an active pharmaceutical ingredient.

7. A prodrug having a tensiylene structure, characterized in that, The structure of the prodrug is as follows: ， Where X is -O-, -OC(O)NR'-, or -OC(O)O-; R8 is a monovalent group formed by removing one hydrogen atom from an active drug containing hydroxyl, amino, or alkylamino groups. When R8 is a monovalent group formed by removing one hydrogen atom from the hydroxyl group of an active drug containing a hydroxyl group, X is -O- or OC(O)O-. When R8 is a monovalent group formed by removing one hydrogen atom from an amino or alkylamino active drug, X is -OC(O)NR'-. R' is H or C 1-6 alkyl.

8. The prodrug having a tensiyne structure according to claim 7, characterized in that, The prodrug has one of the following structures: 。 9. A biological orthogonal prodrug composition, characterized in that, Includes the prodrug as described in claim 7 or 8 and the hydroxylamine derivative; the structure of the hydroxylamine derivative is shown below: ， Where Y is or ; R1 is selected from H, substituted or unsubstituted aryl group, substituted or unsubstituted C group. 1-6 One of the alkyl groups; R2 is selected from H, F, CN, C 1-6 Alkyl, F-substituted C 1-6 One of the alkyl groups; R3 is selected from H and C. 1-6 Alkyl, amino-substituted C 1-6 One of the alkyl groups; R4 is selected from C 1-6 Alkyl and aryl substituted C 1-6 One of the alkyl groups; R5 and R6 are selected from H and C, respectively. 1-6 One of the alkyl groups.

10. The use of a prodrug having a strained alkyne structure as described in claim 6 or 7, or a bioorthogonal prodrug composition as described in claim 8 or 9, in the preparation of a drug delivery system or an active pharmaceutical ingredient.

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