Novel click release system based on monoalkyl hydroxylamine and cyclooctyne to realize time-space controllable delivery of drugs

Abstract

The invention discloses a space-time controllable activatable click release system, which comprises the following components: (a) a trigger component, the structure of which is monoalkyl hydroxylamine, a nitrogen atom is connected with a responsive bond breaking group through a carbamate bond, and click activity is shielded through an electron withdrawing group connected with the nitrogen atom; (b) a cargo carrier component comprising a cyclooctyne structural unit, wherein cyclooctyne is covalently linked to a load molecule through a cleavable linking bond; under the action of external or endogenous stimulation, the responsive bond breaking group is specifically broken, the shielding effect of a carbamic acid bond with an electron withdrawing effect is relieved, and a click reaction between monoalkyl hydroxylamine and cyclooctyne is triggered; the cleavable connecting bond is cleaved through a 1, 4-elimination mechanism in the click reaction, so that space-time selective release of the loaded molecules is realized. The invention discloses a novel'click release 'strategy based on monoalkyl hydroxylamine and cyclooctyne, which can realize quick and almost complete payload release.

Description

Technical Field

[0001] This invention belongs to the field of bioorthogonal chemistry and drug delivery technology, and relates to a click release system based on the reaction of monoalkyl hydroxylamine with cyclooctyne (COT) to achieve spatiotemporally controlled release of drug molecules. Background Technology

[0002] Bioorthogonal reactions enable specific chemical reactions to occur in complex biological matrices without interfering with natural biological processes within the organism. Click chemistry, due to its high efficiency and selectivity, has long been a focus of bioorthogonal chemistry. The 2022 Nobel Prize in Chemistry was awarded to three scientists (Carolyn R. Bertozzi, Morten Meldal, and K. Barry Sharpless) for their pioneering contributions to click chemistry and bioorthogonal chemistry. Initially, click chemistry was widely used to label biomolecules in vivo, enabling their visualization, isolation, or manipulation, due to its rapid and complete bonding properties under physiological conditions. In recent years, click-release strategies have developed into a powerful new approach, initiating the breaking of chemical bonds immediately after the initial click reaction, thereby releasing signaling molecules. Tetraazine and trans-ene have stood out due to their rapid reaction kinetics and high release efficiency, and have been used in many fields such as tumor imaging, enzyme activity control, and drug delivery. Especially in prodrug activation, it is considered one of the most effective strategies for achieving the activation of specific drugs in cells and in vivo. Encouragingly, one of these bioorthogonal prodrugs has successfully entered phase II clinical trials.

[0003] In vivo applications of this click-release strategy typically require precise release of signaling molecules at specific locations or times. However, traditional biological orthogonal reaction handles and their counterparts often lack spatial and temporal controllability, making it impossible to effectively manipulate the location and timing of the reaction. Wang proposed a concentrated click-release strategy to enrich and activate reactants at specific locations, but this often faces problems such as low reactivity or off-target effects. Recently, there have been studies on protecting the tetrazine precursor dihydrotetrazine, followed by removal of the protecting group using a specific enzyme, ultimately oxidizing it to tetrazine. 1 However, this process requires lengthy oxidation steps or additional oxidation conditions, limiting its in vivo application. Furthermore, the emergence of photocage dihydrotetrazine and bilocked dihydrotetrazine allows for direct tetrazine formation after deprotection without intermediates, but this depends on specific tetrazine structures or reactive groups, thus lacking universality. 2-3 Therefore, the development of new, readily available, and universally applicable bioorthogonal elements with spatiotemporally controllable activation functions has become an urgent need. Summary of the Invention

[0004] The inventors extended the ring-strain-promoted hydrogenamination reaction of hydroxylamines with alkynes, inventing a novel click chemistry—the intermolecular Cope-type hydrogenamination reaction of monosubstituted hydroxylamines with cyclooctyne to generate nitrones—and applied it to click-release strategies. This strategy exhibits rapid reaction kinetics (k2 values ​​up to 40 M). -1 S -1 It features high area selectivity, allowing for rapid and near-complete release upon click, and is suitable for breaking different bonds. Figure 1 (A). In further applications, the inventors modified the monosubstituted hydroxylamine to lock its clickability (OFF), and activated its clickability (ON) by high concentrations of glutathione (GSH) or overexpressed nitroreductase at hypoxic tumor sites, or by light irradiation at specific times, thereby achieving control over the location and timing of the reaction. Figure 1 (B).

[0005] To demonstrate the effectiveness of this invention strategy in vivo, the inventors first developed a bio-orthogonal anticancer prodrug that responds to hypoxic tumor sites. This prodrug is specifically triggered only in the presence of high concentrations of GSH at the tumor site, releasing the active drug upon click. A significant tumor-suppressing effect was observed in a 4T1 mouse mammary tumor model. Subsequently, a photoresponsive bio-orthogonal local anesthetic prodrug was developed. By irradiating the rat paw with 405nm LED light, the amount of active drug released upon click can be controlled as needed by adjusting the light intensity and duration, thereby modulating the anesthetic effect and enabling multiple anesthesias. Figure 1 (C).

[0006] By attaching an electron-withdrawing group to the nitrogen atom of a monoalkyl hydroxylamine, rendering it non-clicking, the inventors utilized this property to link various responsive bond-breaking groups to the hydroxylamine via urethane bonds, thereby achieving shielding of click activity (click ability OFF). Through specific stimulation, its click activity can be reactivated (click ability ON), allowing for effective control over the location and timing of the click release reaction. Figure 2 The inventors have developed a novel controlled-activation click release strategy. They designed and synthesized three compounds with different activation mechanisms: thiol-responsive compound 6a, light-activated compound 6b, and nitroreductase (NTR)-specific compound 6c. The click activity of all these compounds was effectively masked (click ability OFF).

[0007] The inventors have developed a novel activatable click-release system based on monoalkyl hydroxylamine and cyclooctyne chemistry, which can be applied to achieve spatiotemporally controlled drug release.

[0008] The objective of this invention is achieved through the following technical solution:

[0009] A spatiotemporally controllable, click-to-release system comprising the following components:

[0010] (a) Trigger component: Its structure is monoalkyl hydroxylamine, in which the nitrogen atom is connected to a responsive bond-breaking group through a urethane bond, and the click activity is shielded by the electron-withdrawing group (i.e., urethane bond) connected to the nitrogen atom;

[0011] (b) Cargo carrier component: contains cyclooctyne structural units, which are covalently linked to the load molecules via cleavable linkages;

[0012] Under external or endogenous stimulation, the responsive bond-breaking group specifically breaks, releasing the shielding effect of the carbamate bond with electron-withdrawing effect, triggering a click reaction between monoalkyl hydroxylamine and cyclooctyne; the click reaction cleaves the linker bond through a 1,4-elimination mechanism, achieving spatiotemporal selective release of the loaded molecule.

[0013] The responsive bond-breaking group can be one of the following: a photo-cleaving group, a thiol-sensitive disulfide bond, or a nitro reductase (NTR) responsive group.

[0014] The photolytic group is o-nitrobenzyl. Or its derivatives, with a response wavelength of 300-405nm, wherein irradiation with 405nm blue light for 60 minutes can achieve a release efficiency of over 90%.

[0015] The o-nitrobenzyl derivative is an o-nitrobenzyl group in which the hydrogen in -CH2- is replaced by a C1 to C3 alkyl group.

[0016] Specifically, the photolytic group is (2-nitrophenyl)ethyl.

[0017] The thiol-sensitive disulfide bonds break at high intracellular glutathione (GSH) concentrations. In normal cells, the GSH concentration is <10 μM, so the activation rate is negligible. However, in the tumor microenvironment, the activation efficiency is more than 100 times higher than that in normal tissues.

[0018] The nitro reductase response group is catalytically reduced to an amino derivative under hypoxic conditions, triggering a self-elimination reaction with a release efficiency of 98% within 50 minutes.

[0019] The nitro reductase responsive group can be a nitroaromatic group, specifically a p-nitrobenzyl group.

[0020] The electron-withdrawing groups in the trigger component are urethane bonds.

[0021] The trigger component and the cargo carrier component do not react with each other until the responsive cleavage group is specifically broken under external or endogenous stimulation, which activates the click activity of the trigger component and causes the cargo carrier component to release the loaded molecules. The release rate of the loaded molecules is as high as 99%.

[0022] The loaded molecules are selected from chemotherapy drugs, fluorescent probes, or local anesthetics.

[0023] The supported molecule contains groups such as -NH2, -NH-, and -OH that can form cleavable linkages with cyclooctylene. R2 can be selected from butyl.

[0024] The cleavable linkages are urethane bonds and ether bonds (-O-).

[0025] Preferably, the cleavable linker is a carbamate bond formed by the hydroxyl group (-OH) at the 3-position of cyclooctyne and -NH2 or -NHR2 contained in the supporting molecule, or an ether bond (-O-) formed by the -OH contained in the supporting molecule.

[0026] Specifically, the cargo carrier component is a compound with a carbamate bond formed by the hydroxyl group (-OH) at the 3-position of cyclooctyne and a loading molecule containing -NH2 (primary amine) or -NH- (secondary amine). Compounds with ether bonds formed by the hydroxyl group (-OH) at the 3-position of cyclooctyne and the supported molecule containing the -OH group. Where R represents the group remaining after the supported molecule loses hydrogen from -NH2 or -NHR2, or the group remaining after the supported molecule loses hydrogen from -OH.

[0027] Specifically, a spatiotemporally controllable, activatable click-to-release system comprises the following components:

[0028] (a) The trigger component with the structure shown below:

[0029]

[0030] (b) Cargo carrier components with the structure shown below:

[0031]

[0032] Or (a) the trigger component with the structure shown below:

[0033]

[0034] (b) Cargo carrier components with the structure shown below:

[0035]

[0036] Or (a) the trigger component with the structure shown below:

[0037]

[0038] (b) Cargo carrier components with the structure shown below:

[0039]

[0040] When the responsive bond-breaking group is a thiol-sensitive disulfide bond and the loaded molecule is a chemotherapeutic drug, the spatiotemporally controllable activatable click-release system is used in the preparation of drugs for treating diseases related to abnormal glutathione levels.

[0041] Specifically, the cargo carrier component is a conjugate formed by the covalent linkage of doxorubicin (DOX) with cyclooctyne via carbamate bonds. The triggering component is a thiol-responsive monoalkyl hydroxylamine derivative, which triggers click activity in the tumor microenvironment via glutathione (GSH≥1mM). The cargo carrier component and the triggering component are administered together via intravenous injection. In a 4T1 tumor-bearing mouse model, the tumor inhibition effect after 12 days of treatment was significantly higher than that of the free DOX control, and no toxic damage was observed in the histopathological analysis of the heart, liver, spleen, lung, and kidney tissues.

[0042] When the responsive bond-breaking group is a photolytic group, a thiol-sensitive disulfide bond, or a nitro reductase responsive group, and the loaded molecule is a fluorescent probe, the spatiotemporally controllable activatable click-release system is used in the preparation of diagnostic reagents for diseases related to abnormal glutathione levels.

[0043] The use of the spatiotemporally controllable, activatable click-release system in the preparation of imaging agents for diseases associated with abnormal glutathione levels, when the responsive bond-breaking group is a photolytic cleavage group, a thiol-sensitive disulfide bond, or a nitro reductase responsive group, and the loaded molecule is a fluorescent probe. The disease associated with abnormal glutathione levels is cancer. The cancer is specifically breast cancer.

[0044] When the responsive bond-breaking group is a photolytic group and the loaded molecule is a local anesthetic, the spatiotemporally controllable activatable click-release system is used in the preparation of anesthetic reagents.

[0045] The anesthetic reagent is in the form of a thermosensitive hydrogel.

[0046] Specifically, the cargo carrier component is an inactive prodrug formed by covalently linking tetracaine to cyclooctyne via a carbamate bond. The triggering agent component is a photoactivated monoalkyl hydroxylamine derivative, which is activated by 405nm blue light (200-300mW / cm²).2 Irradiation for 5 minutes removes the click activity shield; the prodrug and trigger components are encapsulated in a thermosensitive hydrogel and injected into the target site. After light-triggered activation, the duration of nerve block lasts for (48.8±7.5) minutes, and at least 4 effective activations can be achieved through repeated light irradiation.

[0047] When the responsive bond-breaking group is a nitroreductase (NTR) responsive group and the loaded molecule is a fluorescent probe, the spatiotemporally controllable activatable click-release system can be used in the preparation of diagnostic reagents for diseases related to abnormal nitroreductase levels, or in reagents for detecting nitroreductase levels.

[0048] The diseases associated with abnormal nitroreductase levels are cancers caused by nitroreductase overexpression.

[0049] The beneficial effects of this invention are:

[0050] This invention discloses a novel "click-release" strategy based on monoalkyl hydroxylamine and cyclooctyne, enabling rapid and near-complete payload release. The inventors demonstrated that the reaction of monoalkyl hydroxylamine with cyclooctyne to generate nitrone facilitates a multifunctional and efficient release mechanism. By conjugating the monoalkyl hydroxylamine with various responsive cleavage groups, the inventors transformed its inherent sustained reactivity into an on-demand activation system. Compared to parent anticancer drugs, this strategy exhibits significantly enhanced tumor inhibition in vivo. Furthermore, in local anesthesia, the anesthetic effect can be modulated by light exposure and repeated activation is possible. This click-release system combines rapid kinetics, high release efficiency, and precise spatiotemporal control, showing broad application prospects in the fields of chemical biology and drug delivery. Attached Figure Description

[0051] Figure 1 This is bioorthogonal click chemistry; where A is monoalkyl hydroxylamine / cyclooctyne bond-breaking chemistry, B is spatiotemporally controlled click release chemistry, and C is bioorthogonal prodrug design.

[0052] Figure 2 To achieve a spatiotemporally controllable "click-release" chemical reaction through specific stimulation.

[0053] Figure 3 In response to specific experimental results; where a represents the release of compound 4d in a mixture of compounds 4b and 6a with or without GSH; b represents the cell imaging results of compound 6a + compound 4c in 4T1 cells; c represents the design of the bioorthogonal anticancer prodrug 5c; and d represents the activity of compound 5c in combination with compound 6b in inhibiting 4T1 cell proliferation.

[0054] Figure 4This experiment investigated the click activity release of compound 4d by sodium sulfite activating compound 6c; where A is the fluorescence spectrum of the Na2S2O4 + compound 6c + compound 4b system over time; B is a comparison of fluorescence intensity between the experimental group (containing Na2S2O4) and the control group (without Na2S2O4).

[0055] Figure 5 Experiments were conducted to investigate the click-activity release of compounds 4d or 4c from compound 6b by external light activation; where 'a' represents the effect of tracking the presence or absence of a 405nm blue LED lamp (100mW / cm²) using high-performance liquid chromatography. -2 (b) shows the release of compound 4d; (b) shows the cell imaging results of compound 6b + compound 4c in 4T1 cells.

[0056] Figure 6 The graph shows the effect of GSH-triggered orthogonal prodrugs on targeted cancer therapy; where a represents the experimental protocol for in vivo antitumor therapy; b represents the growth curves of subcutaneous 4T1 tumors in mice under different treatments; c represents the weight change of mice during treatment; and d represents a photograph of tumor samples obtained 12 days after treatment. Data are presented as mean ± standard deviation (sd) (n = 5), *p < 0.05, ****p < 0.0001.

[0057] Figure 7 The results of the investigation of phototriggered bioorthogonal prodrugs for local anesthesia are shown; where a represents the release of tetracaine tracked by high performance liquid chromatography (HPLC); b represents the presence or absence of a 300 mW / cm² release rate after injection of tetracaine, compound 7a, or p407-(6b+7a). -2 The time course of nerve block after irradiation; c represents the time of nerve block under different light intensities and exposure times (1. Green: 200mW / cm). -2 1. Irradiate with light for 2 minutes; 2. Blue: 300mW / cm -2 2 min light exposure; 3. Red: 300 mW / cm -2 The nerve block induced after 5 minutes of light irradiation; d represents the time sequence of the nerve block, including multiple light-triggered events (blue arrows indicate the start of light irradiation at 300 mW / cm). -2 (Irradiation for 3 minutes); e is the effective duration of nerve block after each light irradiation in d. Detailed Implementation

[0058] The structures of some of the known compounds used in this invention are as follows:

[0059] Compound 4b:

[0060] Compound 4d:

[0061] Example 1

[0062]

[0063] Preparation of compound 2c-2: Compound 2c-1 (3-phenylpropyl bromide, 1.0 g, 5.02 mmol) was dissolved in DMF (10 mL), and K2CO3 (1.7 g, 12.04 mmol) and N,O-di-tert-butoxycarbonylhydroxylamine (BocO-NHBoc, 1.4 g, 6.03 mmol) were added. The mixture was heated to 50 °C and stirred for 14 hours. After the reaction was completed, the reaction solution was diluted with ethyl acetate (120 mL) and washed with saturated brine (10 mL × 5). The organic layer was dried over anhydrous Na2SO4 and concentrated under reduced pressure. The crude product was purified by normal-phase silica gel column chromatography (eluent: PE:EA = 10:1 V / V) to give compound 2c-2 (white solid, yield: 92%).

[0064] 1 H NMR (300MHz, CDCl3) δ7.42-7.21(m,5H),3.71(t,J=6.9Hz,2H),2.78(t,J=7.8Hz,2H),2.01(p,J=7.0Hz,2H),1.60(s,9H)),1.54(s,9H).

[0065] 13 C NMR (75MHz, CDCl3) δ154.63,152.01,141.16,128.14,128.04,125.57,84.43,81.94,49.30,32.45,28.44,27.80,27.30.

[0066] HRMS(ESI): calculated for C 19 H 29 NO5[M+Na] + :m / z 374.1944; found:374.19534.

[0067] Preparation of compound 2c: Compound 2c-2 (300 mg, 0.85 mmol) was dissolved in DCM (2 mL), and then TFA (1 mL) was added dropwise. The mixture was stirred at room temperature for 3 hours. After the reaction was completed, the reaction solution was diluted with toluene (5 mL) and concentrated under vacuum. Saturated NaHCO3 solution (20 mL) was added, and the mixture was extracted with EA (50 mL × 3). The organic layer was washed with saturated brine, dried with anhydrous Na2SO4, and concentrated under reduced pressure. Compound 2c (colorless transparent liquid, yield: 68%) was purified by normal phase silica gel column chromatography (eluent: PE:EA = 3:1 V / V). Compound 2c turned into a white solid after being left at room temperature for one day.

[0068] 1 H NMR (300MHz, MeOD) δ7.45-6.95(m,5H),2.86(t,J=7.3Hz,2H),2.65(t,J=7.8Hz,2H),1.84(p,J=7.5Hz,2H).

[0069] 13 C NMR (75MHz, MeOD) δ143.31,129.39,129.34,126.81,54.22,34.35,29.77.

[0070] HRMS(ESI): calculated for C9H 13 NO[M+H] + :m / z 152.1075; found:152.10775.

[0071]

[0072] Preparation of compound 6a-2: According to literature [ 4 Preparation of compound 6a-2.

[0073] Preparation of compound 6a: Under N2 protection, pyridine (68 mg, 0.86 mmol) and compound 2c (128 mg, 0.84 mmol) were added sequentially to anhydrous DCM (20 mL) solution of compound 6a-2 (200 mg, 0.41 mmol). The mixture was heated to room temperature and stirred for 2 hours. After the reaction was complete, the reaction solution was concentrated under reduced pressure and purified by normal-phase silica gel column chromatography (eluting agent: PE:EA 5:1 V / V) to give compound 6a (a clear oil, yield: 42%).

[0074] 1H NMR (300MHz, CDCl3) δ7.44-7.15(m,10H),6.95(t,J=6.0Hz,2H),4.50(t,J=6.5Hz,4H),3 .17(q,J=6.6Hz,4H),3.03(t,J=6.5Hz,4H),2.77(t,J=7.7Hz,4H),1.98(q,J=7.4Hz,4H).

[0075] 13 C NMR (75MHz, CDCl3) δ151.08,136.28,128.95,122.95,68.41,53.45,37.00,31.25,28.99.

[0076] HRMS(ESI): calculated for C 24 H 32 N₂O₆S₂[M+Na] + :m / z 531.16; found:531.15960.

[0077] Example 2

[0078]

[0079] Preparation of compound 6b-2: According to literature [ 5 Preparation of compound 6b-2.

[0080] Preparation of compound 6b: Under N2 protection, pyridine (213 mg, 2.69 mmol) and compound 2c (297 mg, 1.97 mmol) were sequentially added to an anhydrous DCM (40 mL) solution of compound 6b-2 (595 mg, 1.79 mmol) at 0 °C. The mixture was heated to room temperature and stirred for 2 hours. After the reaction was complete, the reaction solution was concentrated under reduced pressure and purified by normal-phase silica gel column chromatography (eluent: PE:EA = 5:1 V / V) to give compound 6b (white oil, yield: 46%).

[0081] 1 H NMR (300MHz, CDCl3) δ7.99(d,J=8.1Hz,1H),7.76-7.58(m,2H),7.52-7.40(m,1H),7.38-7.08(m,5H),6.92(t,J=6.0Hz ,1H),6.31(q,J=6.4Hz,1H),3.07(q,J=6.7Hz,2H),2.69(t,J=7.7Hz,2H),1.86(p,J=7.3Hz,2H),1.73(d,J=6.4Hz,3H).

[0082] 13 C NMR (75MHz, CDCl3) δ157.09,146.73,141.39,137.20,133.99,130.02,128.50,127.10,126.07,124.63,72.81,51.96,37.18,28.48,24.68.

[0083] HRMS(ESI): calculated for C 18 H 20 N₂O₅[M+Na] + :m / z 376.1270; found:367.12809.

[0084] Example 3

[0085]

[0086] Preparation of compound 4e: According to literature [ 6 Compound 4e was prepared.

[0087] Preparation of compound 4c: Under N2 protection, compound 4e (80 mg, 0.30 mmol), triphenylphosphine (PPh3, 94 mg, 0.36 mmol), and diethyl azodicarbonate (DEAD, 57 μL) were sequentially added to an anhydrous THF (3 mL) solution of cyclooctane-2-yn-1-ol (37 mg, 0.30 mmol). The mixture was kept at 0 °C for 30 min, then heated to room temperature and stirred for 2 h. After the reaction was complete, the reaction solution was concentrated under reduced pressure and purified by normal-phase silica gel column chromatography (eluent: pure DCM) to give compound 4c (yellow solid, yield: 77%).

[0088] 1 H NMR (600MHz, CDCl3) δ8.56 (d, J=7.3Hz, 1H), 8.52 (dd, J=8.5, 3.0Hz, 2H), 7.66 (t, J= 7.8Hz,1H),7.10(d,J=8.2Hz,1H),5.06(t,J=5.8Hz,1H),4.15(t,J=7.6Hz,2H),2.5 2-2.35(m,2H),2.31-2.17(m,2H),2.07-1.89(m,3H),1.87-1.79(m,1H),1.76(dd,J =15.9,8.3Hz,1H),1.74-1.66(m,3H),1.44(h,J=7.4Hz,2H),0.97(t,J=7.4Hz,3H).

[0089] 13C NMR (151MHz, CDCl3) δ168.24,163.57,158.91,133.31,131.52,129.53,128.76,125.93,123.87,122.60 ,115.44,107.56,104.00,93.47,67.81,42.36,40.20,34.23,30.41,29.79,26.26,20.82,20.54,13.98.

[0090] HRMS(ESI): calculated for C 24 H 25 NO3[M+H] + :m / z 376.1912; found:376.19137.

[0091] Example 4

[0092]

[0093] Preparation of compound 5c-1: According to literature [ 7 Preparation of compound 5c-1.

[0094] Preparation of compound 5c: Compound 5c-1 (24 mg, 0.08 mmol) was completely dissolved in DMF (1 mL), and then doxorubicin hydrochloride (DOX-HCl, 58 mg, 0.10 mmol) was added. DIPEA (44 μL, 0.26 mmol) was slowly added dropwise at 0 °C. The mixture was stirred at room temperature for 3 hours in the dark, concentrated under reduced pressure, and purified by normal-phase silica gel column chromatography (eluent: DCM:MeOH = 20:1 V / V) to give compound 5c (red solid, yield: 95%).

[0095] 11H NMR (300 MHz, CDCl3) δ 13.98 (s, 1H), 13.25 (s, 1H), 8.04 (d, J = 7.6 Hz, 1H), 7.79 (t, J = 8.1 Hz, 1H), 7.39 (d, J = 8.5 Hz, 1H), 5.50 (s, 1H), 5.29 (s, 1H), 5.20 (s, 1H), 5.07 (d, J = 8.5 Hz, 1H), 4.76 (d, J = 4.9 Hz, 2H), 4.55 (s, 1H), 4.20 - 4.08 (m, 4H), 3.85 (s, 1H), 3.67 (s, 1H), 3.28 (d, J = 18.9 Hz, 1H), 3.12 - 2.89 (m, 2H), 2.33 (d, J = 14.8 Hz, 1H), 2.16 (d, J = 14.4 Hz, 4H), 1.95 (m, 7H), 1.29 (d, J = 6.8 Hz, 5H), 0.87 (d, J = 8.4 Hz, 1H).

[0096] 13 13C NMR (101 MHz, CDCl3) δ 212.85, 187.92, 186.49, 161.04, 156.22, 155.58, 154.95, 137.21, 134.82, 134.39, 121.32, 119.86, 118.07, 111.51, 110.15, 101.73, 100.75, 91.71, 69.58, 68.67, 67.27, 65.64, 56.71, 45.72, 41.02, 35.68, 34.75, 33.97, 30.74, 28.84, 25.71, 20.73, 17.70.

[0097] HRMS (ESI): calculated for C 36 H 39 NO 13 [M + Na] + : m / z 716.2319; found: 716.23113.

[0098] Example 5

[0099]

[0100] Preparation of compound 7a: Triphosgene (BTC, 47 mg, 0.16 mmol) was added to an anhydrous DCM solution of tetracaine (120 mg, 0.45 mmol) in 5 mL under N2 protection at 0 °C; subsequently, an anhydrous DCM solution of triethanolamine (76 μL, 0.54 mmol) in 2 mL was added dropwise; after the addition was complete, the mixture was heated to room temperature and stirred for 30 minutes; after the reaction was completed, the reaction solution was concentrated under reduced pressure to obtain the crude product of compound 7a-1; the crude product of compound 7a-1 was dissolved in anhydrous DCM (5 mL), and under N2 protection, the mixture was stirred according to the following... Cyclooctyl-2-yn-1-ol (61 mg, 0.49 mmol), DMAP (11 mg, 0.09 mmol), and DBU (82 mg, 0.54 mmol) were added one after another, and the mixture was stirred at room temperature for 4 hours. After the reaction was completed, DCM (30 mL) was added to the reaction solution for dilution, and the mixture was washed with saturated saline (10 mL × 3). The organic layer was dried over Na2SO4, concentrated under reduced pressure, and the crude mixture was purified by normal phase silica gel column chromatography (eluent: PE:EA = 1:1 V / V) to give compound 7a (colorless oil, yield: 46%).

[0101] 1 H NMR (300MHz, CDCl3) δ8.14-7.95(m,2H),7.37-7.24(m,2H),5.33(tt,J=5.0,2.3Hz,1H ),4.45(t,J=5.8Hz,2H),3.72(td,J=7.1,2.2Hz,2H),2.74(t,J=5.8Hz,2H),2.37(s,1H ),2.32-2.17(m,2H),2.13(td,J=8.2,7.6,3.6Hz,1H),2.05-1.82(m,3H),1.66(td,J= 10.3,8.4,4.7Hz,3H),1.59-1.45(m,3H),1.30(t,J=7.5Hz,2H),0.90(t,J=7.3Hz,3H).

[0102] 13 C NMR (75MHz, CDCl3) δ166.07,154.24,146.20,130.35,127.55,126.27,101.61,91.03,68. 02,62.99,57.85,49.79,45.85,41.65,34.25,30.46,29.55,26.19,20.75,19.86,13.75.

[0103] HRMS(ESI): calculated for C 24 H 34 N₂O₄[M+H]+ :m / z 415.2597; found:415.26114.

[0104] Example 6

[0105] (I) Response Specificity Experiment

[0106] 1. Compounds 6a and 4b were prepared separately in dimethyl sulfoxide (DMSO) to form 50 mM stock solutions of compound 6a and 4b, respectively. A 50 mM glutathione (GSH) stock solution was prepared using phosphate-buffered saline (PBS). The experimental group (4b+6a (With GSH)) consisted of: compound 6a stock solution, appropriate amounts of DMSO and PBS, compound 4b stock solution, and a quantitative amount of GSH stock solution, resulting in a final system containing 5 mM GSH, 500 μM compound 6a, 50 μM compound 4b, and 20% DMSO / PBS (v / v). A control group (4b+6a (With GSH)) containing the same concentration of compounds 6a and 4b but without GSH was also prepared. Both systems were incubated at 37℃ using GSH. The release of compound 4d was quantitatively detected by high performance liquid chromatography (HPLC). In the experimental group, the release of compound 4d exceeded 98% within 2 hours, which was significantly higher than that of the control group (release rate <5% within 2 hours). This demonstrates that GSH triggers the click activity of compound 6a, causing it to react with compound 4b, resulting in the effective release of compound 4d. Figure 3 (a).

[0107] 2. Based on the biological characteristic that the concentration of glutathione (GSH) in the cytoplasm of cancer cells (2-10 mM) is significantly higher than that in the normal extracellular environment (<10 μM), compounds 6a and 4c were dissolved in dimethyl sulfoxide (DMSO) to prepare 50 mM stock solutions of compound 6a and 4c, respectively. The compound 6a and 4c stock solutions were then mixed and diluted in DMEM medium to a final concentration of 100 μM for compound 6a and 100 μM for compound 4c. A concentration of 20 μM was added to a confocal culture dish containing 4T1 cells and incubated at 37°C for 2 hours. Significant green fluorescence was observed using a confocal microscope. A control group (NEM + 2 h) was also set up. This control group was pre-treated with DMEM medium containing 500 μM of the strong thiol reagent N-ethylmaleimide (NEM) for 1 hour to deplete endogenous GSH. The medium was then replaced with DMEM medium containing the same concentrations of 6a and compound 4c, and incubated at 37°C for 2 hours. Only a weak fluorescence signal was detected. Figure 3 (b). This confirms that the click-release activity of compound 6a can be specifically activated by high concentrations of GSH within tumor cells.

[0108] 3. 4T1 cells in logarithmic growth phase were injected with 5 × 10⁻⁶ cells. 3 Cells were seeded at a density of 96-well plates and cultured at 37°C and 5% CO2 for 24 hours. Compounds 5c and 6a were weighed and prepared into 50 mM solutions of compound 5c and 6a respectively using DMSO. These solutions were then added to DMEM medium to obtain solutions of the corresponding concentrations of 5c and 6a. The experimental groups were treated with gradient concentrations of the test compound 5c and synergistically with a final concentration of 200 μM of compound 6a. The control group was treated with either compound 6a alone, a masked prodrug 5c with masked anticancer activity, or free DOX. Cells were incubated for 24 hours. After discarding the drugs, cells were washed twice with PBS, and 10% CCK-8 solution was added. The reaction was carried out at 37°C for 1 hour. The optical density at 450 nm was measured using a microplate reader to assess cell viability and calculate the IC50. 50 This indicates that neither prodrug 5c nor compound 6a alone has an anticancer effect. When compound 6a is used in combination with prodrug 5c, the click-release activity of compound 6a can be specifically activated by high concentrations of GSH within tumor cells, causing it to react with prodrug 5c and effectively release DOX. Figure 3 c), the inhibitory effect on the 4T1 breast cancer cell line is equivalent to that of free DOX ( Figure 3 d).

[0109] Cell viability = (Experimental group optical density - background) / (Control group optical density - background)

[0110] 4. To verify whether compound 6c could be successfully activated for its click activity, the inventors used an aqueous solution of sodium sulfite (Na2S2O4) to perform a mild reduction on compound 6c. Compounds 6c and 4b were dissolved in DMSO to prepare 50 mM stock solutions of compound 6c and compound 4b, respectively. These solutions were then diluted with PBS to obtain target concentrations of compound 6c and compound 4b solutions. A 5 mM sodium sulfite aqueous solution was prepared. The experimental group (With Na2S2O4): the compound 6c and compound 4b solutions were mixed, and Na2S2O4 aqueous solution was added to make the final system contain 5 mM Na2S2O4, 500 μM compound 6c, 50 μM compound 4b, and 20% DMSO / PBS (v / v). A control group (No Na3S2O4): a mixed solution of 6c and compound 4b with the same concentration but without Na2S2O4 was prepared, similar to the experimental group. Both systems were incubated at 37°C. Fluorescence spectral changes were monitored in real time at an excitation wavelength of 397 nm. Figure 4 In the experimental group, the fluorescence intensity increased with increasing incubation time, reaching a peak at 50 minutes. At this time point, the spectra of the experimental group and the control group were compared. Figure 4The fluorescence intensity of the experimental group was significantly higher than that of the control group (*P<0.01), which proved that the reduction of sodium sulfite successfully activated the clicking activity of compound 6c, causing the cyclooctyne group to decage and release compound 4d.

[0111] 5. Compound 6b and compound 4b were dissolved separately in dimethyl sulfoxide (DMSO) to prepare 50 mM stock solutions of compound 6b and 4b, respectively. These solutions were then diluted to the target concentrations with PBS and mixed to form a final system containing 500 μM compound 6b, 50 μM compound 4b, and 20% DMSO / PBS (v / v). Illumination group (4b+6b (With Light): A 405 nm blue LED lamp (100 mW / cm²) was used at 37 °C. -2 The reaction system was irradiated for 100 minutes. The light-protected group (4b + 6b (No Light)) was incubated under the same light-protected conditions. HPLC quantitative detection tracked the release of compound 4d, showing that the release rate of compound 4d in the light-protected group was >98%, significantly higher than the release rate of compound 4d in the light-protected group (<2%). Figure 5 (a).

[0112] Compounds 6b and 4c were dissolved in DMSO to prepare stock solutions of compound 6b and compound 4c, each with a concentration of 50 mM. Compounds 6b and 4c were diluted to working concentrations (200 μM for compound 6b and 20 μM for compound 4c) in DMEM medium and added to a 4T1 cell culture system. The light-illuminated group was exposed to 405 nm light for 5 minutes, while the dark-protected group was kept in the dark. Confocal microscopy showed that only the light-illuminated group exhibited significant green fluorescence in the cytoplasm. Figure 5 (b). This confirms that external light can precisely control the timing of the click response.

[0113] (II) In vivo anti-tumor effects

[0114] To verify the effectiveness of this strategy in vivo, the inventors tested the tumor-suppressive effect of the combination of compound 6a and compound 5c in a 4T1 mouse subcutaneous tumor model.

[0115] All compounds were dissolved in physiological saline containing 5% Tween 80 and 5% DMSO before administration, with each administration volume being 100 μL.

[0116] Experimental protocol as follows Figure 6 As shown in a, firstly, 4T1 cells were subcutaneously implanted into mice. After 7 days, when the tumor volume reached 60-100 mm, 3Mice were randomly divided into five groups (n=5 / group): a combination therapy (6a+5c) group, a compound 6a group, a prodrug 5c group, and a positive control (DOX) group. The mice received the first dose, followed by doses every other day for a total of four doses, all administered via tail vein injection. In the combination therapy group, compound 5c (2.4 mg / kg, equivalent to DOX) was mixed with compound 6a (17 mg / kg) solution before administration and injected into mice via tail vein. In the positive control group, DOX (2 mg / kg) was injected via tail vein. In the compound 6a group, compound 6a (17 mg / kg) was injected via tail vein. In the prodrug 5c group, prodrug 5c (2.4 mg / kg) was injected via tail vein. In the negative control (PBS) group, PBS (100 μL) was injected via tail vein. Throughout the experiment, tumor volume growth in mice was observed daily. Figure 6 b) and weight change Figure 6 c), until day 12. When the tumor volume in the PBS group mice reached approximately 1500 mm². 3 At that time, the mice were euthanized and the tumors were collected. Figure 6 The results showed that tumors in the 6a group and the PBS group grew rapidly, while the prodrug 5c group showed a slight tumor-inhibiting effect. Encouragingly, the combined use of compound 5c and compound 6a significantly inhibited tumor growth, with a significantly better effect than the negative control group (p<0.0001), and showed a stronger tumor-inhibiting effect than the DOX group (p<0.05). This indicates that preparing DOX as a prodrug 5c can effectively mask its toxicity and reduce its toxicity to normal cells. Only when it reaches the tumor site will the high concentration of GSH specifically activate the responsiveness of compound 6a, thereby activating the toxicity of 5c and killing cancerous tissue. Compared with direct intravenous injection of free DOX, this method is safer and more effective.

[0117] (III) Tetracaine was linked to cyclooctyne alcohol via a carbamate bond to construct a phototriggered bioorthogonal prodrug 7a to mask its local anesthetic activity; Compound 7a and Compound 6b were dissolved separately in DMSO to prepare a 50 mM stock solution of Compound 7a and a 50 mM stock solution of Compound 6b. The stock solutions of Compound 7a and Compound 6b were mixed in 50% DMSO / PBS (v / v), with the concentration of Compound 7a being 10 mM and the concentration of Compound 6b being 30 mM; Illumination (7a+6b illumination) group: at room temperature, exposed to a 405 nm LED light source (100 mW / cm²). -2 Irradiation for 30 minutes; control group (7a+6b, no light exposure): treated in the same way while protected from light; release of tetracaine was tracked by high performance liquid chromatography (HPLC), results are shown in [Figure number missing]. Figure 7The results showed that the release rate of tetracaine in the light-exposed group was 89.4 ± 2.4%, significantly higher than that in the control group (release rate < 5%). This confirms that exogenous light irradiation can precisely activate the prodrug release.

[0118] (IV) Methods for evaluating the efficacy of light-controlled anesthetics in vivo

[0119] At 0°C, 4 g of poloxamer 407 (P407) was dissolved in 16 mL of phosphate buffer (PBS) to prepare a 20% P407 solution. Compound 7a was dissolved in ethanol, and an equimolar amount of 2 M hydrochloric acid was added. The solvent was removed by vacuum distillation to obtain compound 7a hydrochloride. Compound 7a hydrochloride was dissolved in the above P407 solution. It was then mixed with a dimethyl sulfoxide (DMSO) solution of compound 6b to make the final system contain 50 mM compound 7a, 60 mM compound 6b and 10% DMSO (v / v) to prepare a p407-(6b+7a) thermosensitive hydrogel formulation. This formulation is in liquid state when stored at 0-4°C and can reversibly transform into a gel state at body temperature (37°C) and room temperature (25°C) to prolong the drug retention time.

[0120] Based on the tissue penetration characteristics of 405nm light, the anesthetic effect was evaluated by injection into the paw pads of rats. The rats were divided into four groups: tetracaine group, compound 7a group, p407-(6b+7a) light exposure group, and p407-(6b+7a) light-protected group, with four rats in each group (n=4). In the p407-(6b+7a) light exposure group and the p407-(6b+7a) light-protected group, each rat was injected with 100 μL of the compound into the paw pad of its hind limb under isoflurane-oxygen mixed anesthesia. The p407-(6b+7a) thermosensitive hydrogel formulation was used in two ways. In the tetracaine group, each rat was anesthetized with isoflurane-oxygen mixture and injected with 100 μL of tetracaine solution (tetracaine dissolved in PBS to prepare a 60 mM tetracaine solution). In the compound 7a group, each rat was anesthetized with isoflurane-oxygen mixture and injected with 100 μL of compound 7a solution (compound 7a dissolved in PBS to prepare a 60 mM compound 7a solution). At set time points, the paw pads were stimulated using a tactile sensory evaluation device (target force value 180g), and the withdrawal response was observed. No withdrawal response after six stimulations was defined as complete nerve block (100% MPE), and the duration of nerve block was defined as the period when MPE > 50%. In the phototriggered experiment, after injection, the rats were irradiated with a 2.5 cm diameter LED light source (completely covering the paw pads) at a set intensity for 2-5 minutes under anesthesia. The efficacy of photocontrolled drug release was verified by quantifying the nerve block intensity and duration.

[0121] No local anesthesia was induced after injection of the pre-drug 7a, indicating that its anesthetic effect was effectively inhibited. No local anesthesia was induced after injection of the p407-(6b+7a) thermosensitive hydrogel preparation. Ten minutes after injection, the anesthesia was observed under a 405nm LED lamp (300mW / cm²). -2 Irradiating the drug delivery site for 5 minutes can effectively activate its anesthetic effect, and the effective anesthetic time is comparable to that of tetracaine. Figure 7 (b).

[0122] Subsequently, the inventors adjusted the anesthetic effect by regulating the light intensity and irradiation time. Rats were divided into three groups of four (n=4) each. Each rat was anesthetized with isoflurane-oxygen mixture and injected with 100 μL of p407-(6b+7a) thermosensitive hydrogel into the paw pads of its hind limbs. Ten minutes after injection, the anesthetic effect was adjusted by regulating the light intensity and irradiation time. 200 mW / cm² -2 Irradiation of the drug delivery site for 2 minutes yields a sustained local anesthetic effect, lasting approximately 26.3 ± 7.5 minutes. When the light intensity is increased to 300 mW / cm², a more sustained local anesthetic effect is achieved. -2 At that time, irradiating the drug delivery site for 2 minutes extended the local anesthesia time to 33.8 ± 7.5 minutes. If 300 mW / cm -2 The irradiation time was extended to 5 minutes, and the local anesthesia time was further extended to 48.8 ± 7.5 minutes. Figure 7 (c).

[0123] To meet practical needs and treat pain for extended periods, the ability of p407-(6b+7a) to provide repetitive, on-demand local anesthesia was evaluated in a group of four rats. Under isoflurane-oxygen mixed anesthesia, 150 μL of the p407-(6b+7a) thermosensitive hydrogel formulation was injected into the hind limb paw pads of the rats. Ten minutes after injection, the rats were exposed to light at an intensity of 300 mW / cm². -2 Irradiate for 3 minutes. Once the nerves are blocked by the first irradiation, perform the next irradiation after the anesthetic effect of the previous irradiation wears off. Repeat this process five times (300mW / cm² each time). -2 Irradiation for 3 minutes, each irradiation successfully induced effective nerve block. Figure 7 (d), and the duration of each block is stable ( Figure 7 e) confirms that the system has the capability for on-demand cyclical anesthesia.

[0124] References:

[0125] 1.Zheng,Y.et al.Enrichment-triggered prodrug activation demonstratedthrough mitochondria-targeted delivery of doxorubicin and carbon monoxide.NatChem10,787-794(2018).

[0126] 2.Liu,L.,Zhang,D.,Johnson,M.&Devaraj,N.K.Light-activated tetrazinesenable precision live-cell bioorthogonal chemistry.Nat Chem14,1078-1085(2022).

[0127] 3.Wang,X.et al.Dual-Locked Enzyme-ActivatableBioorthogonalFluorescence Turn-On Imaging of Senescent Cancer Cells.Journal of theAmerican Chemical Society146,22689-22698(2024).

[0128] 4.Amano,Y.et al.Activation of lysine-specific demethylase 1 inhibitorpeptide by redox-controlled cleavage of a tracelesslinker.Bioorg.Med.Chem.25,1227-1234(2017).

[0129] 5.Li,P.,Song,Y.&Dong,C.-M.Hyperbranched polypeptides synthesized fromphototriggered ROP of a photocaged Nε-[1-(2-nitrophenyl)ethoxycarbonyl]-l-lysine-N-carboxyanhydride:microstructures and effects of irradiationintensity and nitrogen flow rate.Polymer Chemistry 9,3974-3986(2018).

[0130] 6.Shu,W.et al.Highly Selective Fluorescent Probe for the SensitiveDetection of Inorganic and Organic Mercury Species Assisted byH2O2.Industrial&Engineering Chemistry Research 54,8056-8062(2015).

[0131] 7.Kang,D.&Kim,J.Bioorthogonal Retro-Cope Elimination Reaction of N,N-Dialkylhydroxylamines and Strained Alkynes.Journal of the American ChemicalSociety 143,5616-5621(2021).

Claims

1. A spatiotemporally controllable, click-to-release system, characterized in that: It contains the following components: (a) Trigger component: Its structure is a monoalkyl hydroxylamine, wherein the nitrogen atom is connected to a responsive bond-breaking group through a urethane bond, and the click activity is shielded by the electron-withdrawing group connected to the nitrogen atom; (b) Cargo carrier component: contains cyclooctyne structural units, which are covalently linked to the load molecules via cleavable linkages; Under external or endogenous stimulation, the responsive bond-breaking group specifically breaks, releasing the shielding effect of the carbamate bond with electron-withdrawing effect, triggering a click reaction between monoalkyl hydroxylamine and cyclooctyne; the click reaction cleaves the linker bond through a 1,4-elimination mechanism, achieving spatiotemporal selective release of the loaded molecule.

2. The spatiotemporally controllable, activatable click-to-release system according to claim 1, characterized in that: The responsive bond-breaking group is one of the following: a photo-cleaving group, a thiol-sensitive disulfide bond, or a nitro reductase responsive group.

3. The spatiotemporally controllable, activatable click-to-release system according to claim 2, characterized in that: The photolytic group is o-nitrobenzyl or its derivative, with a response wavelength of 300-405 nm; The thiol-sensitive disulfide bond can be cleaved by high concentrations of glutathione; The nitro reductase response group is catalytically reduced to an amino derivative under anaerobic conditions, triggering a self-elimination reaction.

4. The spatiotemporally controllable, activatable click-to-release system according to claim 3, characterized in that: The o-nitrobenzyl derivative is an o-nitrobenzyl group in which the hydrogen in -CH2- is replaced by a C1-C3 alkyl group; the nitro reductase responsive group is a nitroaromatic group.

5. The spatiotemporally controllable, activatable click-to-release system according to claim 1, characterized in that: The trigger component and the cargo carrier component do not react with each other until, under external or endogenous stimulation, the responsive bond-breaking group specifically breaks, activating the click activity of the trigger component and causing the cargo carrier component to release the loaded molecules.

6. The spatiotemporally controllable, activatable click-to-release system according to claim 1, characterized in that: The loaded molecule is selected from chemotherapy drugs, fluorescent probes, or local anesthetics; the loaded molecule contains a group that can form a cleavable linker bond with cyclooctyne; the cleavable linker bond is a carbamate bond or an ether bond.

7. The spatiotemporally controllable, activatable click-to-release system according to claim 1 or 6, characterized in that: The cleavable linker is a carbamate bond formed by the hydroxyl group at the 3-position of cyclooctyne and -NH2 or -NH- contained in the supporting molecule, or an ether bond formed by the -OH contained in the supporting molecule.

8. A spatiotemporally controllable, click-to-release system, characterized in that: It contains the following components: (a) The trigger component with the structure shown below: (b) Cargo carrier components with the structure shown below: Or (a) the trigger component with the structure shown below: (b) Cargo carrier components with the structure shown below: Or (a) the trigger component with the structure shown below: (b) Cargo carrier components with the structure shown below:

9. The use of the spatiotemporally controllable, activatable click-to-release system as described in claim 1, characterized in that: The use of the spatiotemporally controllable activated click-release system in the preparation of diagnostic reagents for diseases related to abnormal glutathione levels when the responsive bond-breaking group is a photolytic group, a thiol-sensitive disulfide bond, or a nitro reductase responsive group, and the loaded molecule is a fluorescent probe. When the responsive bond-breaking group is a photolytic group, a thiol-sensitive disulfide bond, or a nitro reductase responsive group, and the loaded molecule is a fluorescent probe, the spatiotemporally controllable activatable click-release system is used in the preparation of imaging agents for diseases related to abnormal glutathione levels. When the responsive bond-breaking group is a photolytic group and the loaded molecule is a local anesthetic, the use of the spatiotemporally controllable activatable click-release system in the preparation of anesthetic reagents; When the responsive bond-breaking group is a nitroreductase responsive group and the loaded molecule is a fluorescent probe, the spatiotemporally controllable activatable click-release system can be used in the preparation of diagnostic reagents for diseases related to abnormal nitroreductase levels, or in reagents for detecting nitroreductase levels.

10. The use according to claim 9, characterized in that: The disease associated with abnormal glutathione levels is cancer; the anesthetic reagent is in the form of a thermosensitive hydrogel; and the disease associated with abnormal nitroreductase levels is cancer with nitroreductase overexpression.

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