Enzyme-activated prodrug compounds, methods of making and using the same

Abstract

This invention discloses a class of enzyme-activated prodrug compounds having the following general structural formula I: This invention discloses a class of enzyme-sensitive chlorambucil analog prodrugs. These prodrugs exhibit high selectivity for hypoxic tumors and can be effectively activated within hypoxic tumor cells. Upon specific activation, the prodrug releases free chemotherapeutic drug chlorambucil, simultaneously releasing the photosensitizer cyanine dye, achieving an "OFF-ON" photosensitivity conversion, and generating a large number of reactive oxygen species, thus achieving the unification and synergy of photodynamic therapy and activatable chemotherapy.

Description

Technical Field

[0001] This invention relates to the release of targeted antitumor drugs, and more particularly to a class of enzyme-activated prodrug compounds, their preparation methods, and applications. Background Technology

[0002] For a long time, conventional chemotherapy drugs have had poor prognostic effects on cancer patients due to limitations such as poor accumulation in tumors, severe toxic side effects, and frequent drug resistance. In contrast, selectively activated prodrugs have changed the status quo of traditional chemotherapy. Prodrugs are non-toxic in normal cells, but can be specifically activated into highly toxic factors in tumor cells. This not only improves the selectivity for tumors and reduces the killing of non-target cells, but also minimizes the risk of developing drug resistance.

[0003] Existing prodrugs can be activated by various triggering factors in the tumor microenvironment, such as specific pH values, reactive oxygen species, and reactive thiols. Among these, prodrugs activated by endogenous cellular enzymes are the most attractive due to their advantages such as high affinity, high specificity, and rapid response. Enzyme-activated prodrugs not only significantly improve tumor targeting but also overcome the cumbersome process of artificially introducing exogenous promoters. However, the application scope of existing enzyme-activated prodrugs is generally limited by tumor heterogeneity. Furthermore, most enzyme-activated prodrugs are only involved in single chemotherapy regimens.

[0004] Therefore, it is of great significance to research and develop enzyme-activated prodrugs that can accurately identify tumors and can be combined with other treatment methods.

[0005] Chlorambucil is clinically used for various malignant tumors, including chronic lymphocytic leukemia, ovarian cancer, and non-Hodgkin's lymphoma. It induces apoptosis by causing interstrand crosslinks in DNA. However, its widespread use is limited by drawbacks associated with chemotherapy, including relatively poor selectivity for tumor cells and irreversible damage to normal cells and tissues. Therefore, the development of multimodal enzyme-activated prodrugs based on chlorambucil is of great significance. Cyanide photosensitizers possess many unique physicochemical properties, such as a large molar extinction coefficient and fluorescence quantum yield, good thermal and photostability, and good phototoxicity. Their photosensitizing properties can be controlled "off-on" through chemical modification, enabling efficient photodynamic therapy (PDT) and tumor suppression. Summary of the Invention

[0006] Given the current lack of enzyme-activated prodrugs that enable precise chemotherapy, and considering the excellent tumor selectivity and inhibitory effects of photodynamic therapy, this invention constructs a class of enzyme-sensitive chlorambucil analog prodrugs. This prodrug exhibits high selectivity for hypoxic tumors and can be effectively activated within hypoxic tumor cells. Upon specific activation, the prodrug releases free chemotherapeutic drug chlorambucil, simultaneously releasing the photosensitizer cyanine dye, achieving an "OFF-ON" switch in photosensitivity and generating a large number of reactive oxygen species, thus unifying and synergistically integrating photodynamic therapy and activatable chemotherapy.

[0007] To achieve the above objectives, the technical solution of the present invention is: a class of enzyme-activated prodrug compounds having the following general structural formula I:

[0008]

[0009] In general formula I,

[0010] R1 is -N[(CH2CH2)] m X]2, where X is selected from halogen, hydroxyl, mercapto or nitro; m is an integer from 1 to 4;

[0011] R2 is selected from nitro or any of the groups described in formulas i to iii below;

[0012]

[0013] R3 is selected from -NH or O;

[0014] R4 is selected from O or S;

[0015] R5 is selected from hydrogen, alkyl groups having 1-6 carbons, carboxyl groups having 1-6 carbons, hydroxyalkyl groups having 1-6 carbons, or alkyl sulfonates having 1-6 carbons.

[0016] R6 is any hydrogen, halogen, hydroxyl, mercapto, cyano, nitro, alkyl with 1-6 carbons, carboxyl alkyl with 1-6 carbons, hydroxyalkyl with 1-6 carbons, or alkyl sulfonate with 1-6 carbons that are substituted on a 6-membered ring.

[0017] Furthermore, the stimulator for the release of the compound is nitroreductase, which is highly expressed in anaerobic tumor cells. Under the hydrolytic action of nitroreductase in hypoxic tumors, the compound simultaneously releases free chlorinated mustard and activated cyanine dye.

[0018] Furthermore, the compound releases phenylbutyric acid mustard and activated cyanine dye in a 1:1 ratio.

[0019] A method for preparing a class of enzyme-activated prodrug compounds includes the following steps:

[0020] (1) 2-hydroxy-5-methyl-m-phenylenediol reacts with a compound of general formula S-1 in a molar ratio of 1:1 to 3 to prepare a compound of general formula S-2.

[0021] The reaction time is 4–12 h, and the reaction solvent is at least one of acetone, N,N-dimethylformamide, dichloromethane, chloroform, and ethyl acetate.

[0022] The reaction temperature is the boiling point of the corresponding reaction solvent; the catalyst is selected from at least one of potassium carbonate, cesium carbonate, sodium carbonate, triethylamine, 4-dimethylaminopyridine, N,N'-diisopropylethylamine and pyridine;

[0023]

[0024] (2) The compound obtained in step (1) is reacted with a compound of general formula S-3 at a molar ratio of 1:1.2 to 2 to prepare a compound of general formula S-4;

[0025] The reaction time is 12–36 h, the reaction solvent is N,N-dimethylformamide, acetonitrile, dichloromethane, ethanol, ethyl acetate or a mixture thereof, the reaction temperature is 0–40 °C, and the catalyst is selected from at least one of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, O-benzotriazole-tetramethylurea hexafluorophosphate, 4-dimethylaminopyridine, N,N'-diisopropylethylamine, triethylamine and pyridine;

[0026]

[0027] (3) The compound obtained in step (2) is reacted with benzyl p-nitrochloroformate in a molar ratio of 1:1.5 to 3 in a nitrogen atmosphere to prepare a compound with the general formula S-5;

[0028] The reaction time is 2 to 12 hours, the reaction solvent is at least one of dichloromethane, chloroform and N,N-dimethylformamide, the reaction temperature is 0 to 30°C, and the catalyst is selected from at least one of pyridine, piperidine, triethylamine, 4-dimethylaminopyridine, N,N'-diisopropylethylamine, potassium carbonate and cesium carbonate;

[0029]

[0030] (4) The compound obtained in step (3) is reacted with the compound of general formula Y-4 at a molar ratio of 1:1.1 to 1.5 to prepare an enzyme-activated prodrug of general formula I;

[0031] The reaction time is 12–48 h, the reaction solvent is at least one of dichloromethane, chloroform, N,N-dimethylformamide, and acetonitrile, the reaction temperature is 0–40 °C, and the catalyst is selected from at least one of triethylamine, N,N'-diisopropylethylamine, 4-dimethylaminopyridine, pyridine, potassium carbonate, and aniline.

[0032]

[0033] Furthermore, compounds of general formula Y-4 are prepared by the following method:

[0034] S1: React a compound of general formula Y-1 with a haloalkane compound containing R5 substitution at a molar ratio of 1:2 to 10 to prepare a compound of general formula Y-2.

[0035] The reaction time is 12–36 h, the reaction solvent is at least one of acetonitrile, toluene, o-dichlorobenzene, m-dichlorobenzene or DMF, and the reaction temperature is 70–120 °C.

[0036]

[0037] S2: The compound obtained in step S1 is reacted with compound 2-chloro-3-(hydroxymethylene)-1-cyclohexene-1-carboxaldehyde in a molar ratio of 1:0.5 to 0.75 under a nitrogen atmosphere to prepare a compound with the general formula Y-3.

[0038] The reaction time is 12-24 h, the reaction solvent is at least one of ethanol, methanol, n-butanol, toluene, acetonitrile or DMF, the reaction temperature is 70-120 °C, and the catalyst is selected from at least one of sodium acetate, potassium acetate or potassium carbonate.

[0039]

[0040] S3: The compound obtained in step S2 is reacted with a phenolic compound containing a substituent at a molar ratio of 1:3 to 4 to prepare a compound with the general formula Y-4.

[0041] The reaction time is 4–12 h, the reaction solvent is dichloromethane, chloroform, acetonitrile, DMF or a mixture thereof, the reaction temperature is 25–40 °C, and the catalyst is selected from potassium carbonate, sodium carbonate, triethylamine, DIPEA, DMAP, pyridine or a mixture thereof.

[0042]

[0043] Furthermore, the phenolic compound containing the substituent is one of resorcinol, m-aminophenol, m-nitrophenol, m-hydroxythiophenol, m-nitrothiophenol, or m-aminothiophenol.

[0044] Application of a compound of formula I in the preparation of reagents for tumor diagnosis and treatment.

[0045] Furthermore, the nitroreductase is a specific reductase that is highly expressed in hypoxic tumor cells.

[0046] Furthermore, the fluorescence excitation and emission wavelengths of the nitroreductase activation prodrug are both greater than 660 nm.

[0047] In summary, the present invention has the following beneficial effects:

[0048] This invention ingeniously integrates tumor fluorescence diagnosis, photodynamic therapy, and targeted chemotherapy. In this invention, a prodrug based on cyanine dyes and chlorambucil exhibits high sensitivity and selectivity for enzymes highly expressed in the hypoxic microenvironment of tumors. Nitroreductase (NTR) is a specific reductase highly expressed in hypoxic tumor cells, and the activation and release of the prodrug are achieved through an enzymatic reaction induced by NTR. When this type of prodrug is not activated, the intramolecular charge transfer (ICT) effect is inhibited, quenching the fluorescence and ROS generation capacity of the cyanine dye. Furthermore, due to the covalent blockade of the carboxyl terminus (COOH-) of chlorambucil, the basicity of its nitrogen atom is effectively downregulated, thereby reducing its off-target toxicity. Under the NTR hydrolysis in hypoxic tumors, the prodrug molecule simultaneously releases free chlorambucil and activated cyanine dye. At this point, the chemotherapeutic activity of chlorambucil is activated, the ICT effect of the cyanine dye is restored, and thus the fluorescence and ROS generation capacity are restored, thereby enabling multimodal tumor diagnosis and treatment combining chemotherapy, PDT (photodynamic therapy), and fluorescence imaging.

[0049] This invention provides the application of a class of cyanine dye-based enzyme-activated prodrugs in multimodal treatment of hypoxic tumors. According to the proposed mechanism, in normoxic cells, the release of free chlorambucil from the prodrug is inhibited, reducing toxic side effects on normal cells; in hypoxic tumor cells, cellular uptake of the prodrug is significantly enhanced, effectively activating it and resulting in higher targeted toxicity. Therefore, this type of prodrug holds promise for precise tumor diagnosis and multimodal chemotherapy, and is expected to improve the prognosis of cancer patients.

[0050] Based on this, the present invention further provides the application of the aforementioned enzyme-activated prodrug compound in the preparation of tumor diagnostic and therapeutic reagents. Specifically, the tumor is an NTR-highly expressed tumor. More specifically, the tumor diagnostic and therapeutic reagent is a biological sample identification marker or a tumor diagnostic and therapeutic drug.

[0051] In summary, the NTR-activating prodrug compound of this invention exhibits fluorescence excitation and emission wavelengths greater than 660 nm, demonstrating excellent near-infrared fluorescent dye characteristics, which is beneficial for high signal-to-noise ratio imaging and phototherapy in vivo. The introduction of specific targeting groups in the compound ensures a sensitive and selective response to NTR enzymes, enabling the identification and screening of tumor cells. Compared to free chlorinated mustard, this prodrug molecule demonstrates high selectivity and lower toxicity towards hypoxic cells. Therefore, this type of cyanine dye-based enzyme-activating prodrug of this invention can be used for the identification and treatment of tumor cells. Furthermore, the compounds of this invention are readily available from readily available raw materials, simple to prepare, and hold promise for industrialization. Attached Figure Description

[0052] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0053] Figure 1 The 1H NMR spectrum of the prodrug molecule Cy-NTR-CB disclosed in Example 1 of this invention;

[0054] Figure 2 The carbon NMR spectrum of Cy-NTR-CB, the prodrug molecule disclosed in Example 1 of this invention;

[0055] Figure 3 The UV-Vis absorption spectra of the prodrug molecule Cy-NTR-CB and intermediate 6 disclosed in Example 1 of this invention are shown below.

[0056] Figure 4 The fluorescence emission spectra of the prodrug molecule Cy-NTR-CB and intermediate 6 disclosed in Example 1 of this invention are shown below.

[0057] Figure 5 The UV-Vis absorption spectra of the prodrug molecules synthesized in Examples 2-4 of this invention are shown below.

[0058] Figure 6 The fluorescence emission spectra of the prodrug molecules synthesized in Examples 2-4 of this invention are shown.

[0059] Figure 7 The fluorescence response diagram of the prodrug molecule Cy-NTR-CB and NTR disclosed in Example 1 of this invention is shown.

[0060] Figure 8 This is a selective response diagram of the prodrug molecule Cy-NTR-CB and NTR disclosed in Example 1 of the present invention;

[0061] Figure 9 The image shows the absorption spectrum changes of a mixed solution of compound Cy-NTR-CB and 1,3-diphenylisobenzofuran under light irradiation.

[0062] Figure 10 The absorption spectrum of compound Cy-NTR-CB after reaction with NTR and NADH in a mixed solution of 1,3-diphenylisobenzofuran under light irradiation is shown.

[0063] Figure 11 The image shows the fluorescence response of compound Cy-NTR-CB to NTR in cells under different incubation conditions. In the image: a and c are confocal images of intermediate 6 and compound Cy-NTR-CB in normoxic cells, respectively; b and d are confocal images of compound Cy-NTR-CB in hypoxic cells and hypoxic cells with NTR inhibited by dicumarol, respectively.

[0064] Figure 12 These are the results of toxicity experiments on different cells using compounds Cy-NTR-CB and chlorambucil. Detailed Implementation

[0065] The present invention will now be described in further detail.

[0066] Unless otherwise stated, the terms used herein have the following meanings.

[0067] The term "halogen" as used in this article includes fluorine, chlorine, bromine, and iodine.

[0068] The term "alkyl" as used in this invention includes straight-chain alkyl and branched-chain alkyl.

[0069] In the preparation method described in this invention, the solvents are preferably dehydrated solvents.

[0070] The purification method described above in this invention employs conventional methods without particular limitations. Preferably, dichloromethane-methanol is used as the eluent for column chromatography, recrystallization, or a combination of both. Furthermore, the obtained intermediates and final products can be recovered using separation and purification techniques known in the art to achieve the required purity.

[0071] The raw materials used in the preparation methods described above in this invention can all be commercially available or prepared by methods known in the art.

[0072] The structures of the compounds synthesized in the preparation methods described above in this invention are confirmed by mass spectrometry, proton nuclear magnetic resonance (NMR) spectroscopy, and carbon nuclear magnetic resonance (NMR) spectroscopy.

[0073] The DMF described in this invention is N,N-dimethylformamide, DIPEA is N,N'-diisopropylethylamine, DMAP is 4-dimethylaminopyridine, EDCl is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, HATU is 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, and HBTU is O-benzotriazole-tetramethylurea hexafluorophosphate.

[0074] The instruments and equipment used in the embodiments are as follows:

[0075] The absorption and emission spectra of the dye were measured using an Agilent Cary 60 UV-Vis spectrophotometer and a CaryEclipse fluorescence spectrophotometer. The absolute fluorescence quantum yield of the dye was measured using a Hamamatsu Photonics Trading (China) Co., Ltd. C11347 absolute fluorescence quantum yield meter.

[0076] Example

[0077] Example 1

[0078]

[0079] (1) Synthesis of intermediate 3

[0080] 1.1 Synthesis of Intermediate 1

[0081] 2-Hydroxy-5-methylisophthalimide (1.68 g, 10 mmol, 1.0 eq), p-nitrobenzyl bromide (4.28 g, 20 mmol, 2.0 eq), and potassium carbonate (6.91 g, 50 mmol, 5.0 eq) were dissolved in acetone (50 mL). The reaction was carried out at 57 °C under reflux and stirred for 4 h. After cooling to room temperature, the reaction solution was treated with water and extracted with ethyl acetate. The organic phase was dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure, and the residue was purified by silica gel column chromatography (DCM / MeOH = 20 / 1) to give intermediate 1 as a white solid (2.56 g, yield 87.4%).

[0082] 1.2 Synthesis of Intermediate 2

[0083] Intermediate 1 (303 mg, 1 mmol, 1.0 eq), chlorambucil (364 mg, 1.2 mmol, 1.2 eq), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCl, 230 mg, 1.2 mmol, 1.2 eq), and p-dimethylaminopyridine (DMAP, 25 mg, 0.2 mmol, 0.2 eq) were dissolved in dry DMF and reacted at room temperature under a nitrogen atmosphere with stirring for 12 h. After the reaction was complete, the reaction solution was treated with water and extracted with ethyl acetate. The organic phase was dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure, and the residue was purified by silica gel column chromatography (n-hexane / ethyl acetate = 2 / 1) to give intermediate 2 as a pale yellow solid (432 mg, yield 73.5%).

[0084] 1.3 Synthesis of Intermediate 3

[0085] Intermediate 2 (120 mg, 0.2 mmol, 1.0 eq) and pyridine (100 μL) were dissolved in dichloromethane (20 mL). Under a nitrogen atmosphere at 0 °C, a dichloromethane solution containing p-nitrobenzyl chloride (70 mg, 0.35 mmol, 1.75 eq) was added dropwise to the reaction solution while stirring. The reaction was continued to be stirred at 0 °C for 2 h, then heated to room temperature and stirred overnight. After the reaction was completed, the solvent was removed under reduced pressure to obtain a yellow viscous liquid, which was taken as the crude product of intermediate 3 (approximately 200 mg). This crude product could be quickly added to the next reaction without purification.

[0086] (2) Synthesis of intermediate 6

[0087] 2.1 Synthesis of Intermediate 4

[0088] 2,3,3-Trimethyl-5-bromo-3H-indole (1 g, 4.2 mmol, 1.0 eq) was dissolved in toluene (20 mL) and heated to reflux. While stirring, 2 g (12.8 mmol, 3.0 eq) of iodoethane was added in portions, and the mixture was refluxed and stirred until a large amount of precipitate formed. The reaction solution was cooled to room temperature and filtered under reduced pressure to obtain the precipitate. The precipitate was washed three times with ethyl acetate and dried under vacuum to give intermediate 4, a pink solid (1.32 g, 80.4% yield).

[0089] 2.2 Synthesis of Intermediate 5

[0090] Intermediate 4 (500 mg, 1.27 mmol, 1.0 eq), 2-chloro-3-(hydroxymethyl)cyclohexyl-1-enal (110 mg, 0.64 mmol, 0.5 eq), and anhydrous sodium acetate (130 mg, 1.59 mmol, 1.25 eq) were dissolved in anhydrous ethanol (20 mL). The reaction was carried out overnight at 80 °C under a nitrogen atmosphere. After the reaction was complete, the reaction solution was cooled to room temperature, and the solvent was removed under reduced pressure. The residue was dissolved in dichloromethane, washed with saturated brine, and the organic phase was dried over anhydrous sodium sulfate. The residue was subjected to silica gel column chromatography (DCM / MeOH = 100 / 1-20 / 1) to give a green solid as intermediate 5 (345 mg, yield 66.5%).

[0091] 2.3 Synthesis of Intermediate 6

[0092] 3-Nitrophenol (123 mg, 0.88 mmol, 3.5 eq) and potassium carbonate (120 mg, 0.88 mmol, 3.5 eq) were dissolved in anhydrous acetonitrile (20 mL) and stirred at room temperature for 30 min. Intermediate 5 (200 mg, 0.25 mmol, 1.0 eq) was dissolved in acetonitrile (5 mL) and added dropwise to the above reaction solution, and the mixture was stirred at room temperature for 4 h. After removing the solvent under reduced pressure, the residue was dissolved in dichloromethane and washed with water to remove potassium carbonate. After removing the solvent under reduced pressure, the residue was dissolved in anhydrous methanol and then proceeded to the next step. A methanol solution (5 mL) containing stannous chloride dihydrate (395 mg, 1.75 mmol, 7.0 eq) and hydrochloric acid (3 mL) was added dropwise to the above reaction solution, and the mixture was stirred overnight at 70 °C. After the reaction solution was cooled to room temperature, it was neutralized with saturated sodium carbonate solution. The organic phase was collected with dichloromethane, washed with water, and the solvent was removed under reduced pressure. The residue was subjected to silica gel column chromatography (DCM / MeOH = 50 / 1-10 / 1) to give a blue solid compound as intermediate 6 (85 mg, yield 56.8%).

[0093] (3) Synthesis of the prodrug molecule Cy-NTR-CB

[0094] Intermediate 3 (60 mg, 0.08 mmol, 1.0 eq) and N,N-diisopropylethylamine (DIPEA, 50 μL) were dissolved in a mixed solution of dichloromethane and DMF (1:1, v / v) and stirred for 30 min at 0 °C under a nitrogen atmosphere. Then, a dichloromethane solution containing intermediate 6 (40 mg, 0.08 mmol, 1.0 eq) was added dropwise to the reaction mixture, and the temperature was raised to 40 °C, and the reaction was allowed to proceed overnight. The reaction mixture was treated with water and extracted with dichloromethane. The organic phase was washed three times with water, the solvent was removed under reduced pressure, and the residue was subjected to silica gel column chromatography (DCM / MeOH = 15 / 1-10 / 1) to give a blue solid compound Cy-NTR-CB (24 mg, 29.7% yield).

[0095] 1 1H NMR (400 MHz, CD2Cl2) δ 8.56 (d, J = 12.6 Hz, 1H), δ 8.26 (d, J = 8.7 Hz, 2H), δ 7.69 (d, J = 8.4 Hz, 2H), δ 7.60 (s, 1H), δ 7.55 (d, J = 12.1 Hz, 2H), δ 7.38 (d, J = 8.7 Hz, 1H), δ 7.31 (s, 1H), δ 7.22 (s, 1H), δ 7.11 (s, 1H), δ 7.07 (d, J = 7.3 Hz, 2H), δ 7.03 (d, J = 13.‘5 Hz, 2H), δ 6.70 (d, J = 8.2 Hz, 2H), δ 5.97 (d, J = 11.8 Hz, 1H), δ 5.17 (s, 2H), δ 5.10 (s, 2H), δ 4.73 (s, 2H), δ 4.07 (d, J = 6.5 Hz, 2H), δ 3.73 (t, J = 7.2 Hz, 4H), δ 3.63 (t, J = 6.8 Hz, 4H), δ 2.81 (d, J = 5.5 Hz, 2H), δ 2.70 (d, J = 6.0 Hz, 2H), δ 2.53 (s, 2H), δ 2.38 (s, 3H), δ 2.33 (t, J = 7.2 Hz, 2H), δ 1.97 (t, J = 5.9 Hz, 2H), δ 1.87 (t, J = 7.6 Hz, 2H), δ 1.79 (s, 6H), δ 1.47 (t, J = 7.1 Hz, 3H). See Figure 1 ;

[0096] 13 13C NMR (125 MHz, CD2Cl2) δ 173.05, 171.20, 164.30, 156.69, 155.72, 152.92, 147.54, 144.86, 144.47, 141.33, 141.14, 140.48, 134.67, 134.43, 131.4‘7, 130.62, 130.45, 129.74, 129.51, 129.14, 127.80, 125.95, 123.63, 122.79, 117.55, 116.48, 115.63, 114.45, 112.13, 111.27, 98.34, 97.96, 75.34, 61.37, 60.17, 49.23, 40.75, 33.84, 33.56, 28.52, 28.26, 26.82, ‘24.28, 20.70, 20.57. See Figure 2 .

[0097] Example 2

[0098] The only difference from Example 1 is that intermediate 3 is different. In this example, intermediate 10 is used instead of intermediate 3.

[0099]

[0100] (1) Synthesis of intermediate 10

[0101] 1.1 Synthesis of Intermediate 7

[0102] p-Aminobenzyl bromide (1.85 g, 10 mmol, 1.0 eq), glycine (1.78 g, 20 mmol, 2.0 eq), HATU (4.56 g, 12 mmol, 1.2 eq), and DIPEA (3.87 g, 30 mmol, 3.0 eq) were dissolved in dichloromethane and reacted at 25 °C for 12 h. The reaction solution was treated with water and extracted with dichloromethane. The organic phase was dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure, and the residue was purified by silica gel column chromatography (DCM / MeOH = 30 / 1) to give intermediate 7 as a white solid (1.75 g, yield 68.4%).

[0103] 1.2 Synthesis of Intermediate 8

[0104] 2-Hydroxy-5-methylisophthalimide (1.68 g, 10 mmol, 1.0 eq), intermediate 7 (5.13 g, 20 mmol, 2.0 eq), and potassium carbonate (6.91 g, 50 mmol, 5.0 eq) were dissolved in acetone (50 mL) and refluxed with stirring for 4 h. After cooling to room temperature, the reaction solution was treated with water and extracted with ethyl acetate. The organic phase was dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure, and the residue was purified by silica gel column chromatography (DCM / MeOH = 15 / 1) to give intermediate 8 as a white solid (2.83 g, yield 82.3%).

[0105] 1.3 Synthesis of Intermediate 9

[0106] Intermediate 8 (345 mg, 1 mmol, 1.0 eq), chlorambucil (364 mg, 1.2 mmol, 1.2 eq), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCl, 230 mg, 1.2 mmol, 1.2 eq), and p-dimethylaminopyridine (DMAP, 25 mg, 0.2 mmol, 0.2 eq) were dissolved in dry DMF and reacted at room temperature under a nitrogen atmosphere with stirring for 36 h. After the reaction was complete, the reaction solution was treated with water and extracted with ethyl acetate. The organic phase was dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure, and the residue was purified by silica gel column chromatography (n-hexane / ethyl acetate = 2 / 1) to give intermediate 9 as a pale yellow solid (429 mg, yield 69.8%).

[0107] 1.4 Synthesis of Intermediate 10

[0108] Intermediate 9 (123 mg, 0.2 mmol, 1.0 eq) and pyridine (100 μL) were dissolved in dichloromethane (20 mL). Under a nitrogen atmosphere at 0 °C, a dichloromethane solution containing p-nitrobenzyl chloride (70 mg, 0.35 mmol, 1.75 eq) was added dropwise to the reaction solution while stirring. The reaction was continued to be stirred at 0 °C for 4 h, then heated to room temperature and stirred overnight. After the reaction was completed, the solvent was removed under reduced pressure to obtain a yellow viscous liquid, which was taken as the crude product of intermediate 10 (approximately 150 mg). This crude product could be quickly added to the next reaction without purification.

[0109] (2) Synthesis of intermediate 6

[0110] Same as Example 1

[0111] (3) Synthesis of the prodrug molecule Cy-APN-CB

[0112] Intermediate 10 (64 mg, 0.08 mmol, 1.0 eq) and N,N-diisopropylethylamine (DIPEA, 50 μL) were dissolved in a mixed solution of dichloromethane and DMF, and stirred for 30 min at 0 °C under a nitrogen atmosphere. Then, a dichloromethane solution containing intermediate 6 (40 mg, 0.08 mmol, 1.0 eq) was added dropwise to the reaction mixture, and the temperature was raised to 35 °C, and the reaction was allowed to proceed overnight. The reaction mixture was treated with water and extracted with dichloromethane. The organic phase was washed three times with water, the solvent was removed under reduced pressure, and the residue was subjected to silica gel column chromatography (DCM / MeOH = 15 / 1-10 / 1) to give a blue solid compound Cy-APN-CB (19 mg, yield 21.5%).

[0113] 11H NMR (500 MHz, CD2Cl2) δ 9.45 (s, 1H), 9.17 (s, 1H), 7.80 (dd, J = 8.8, 2.2 Hz, 1H), 7.67 (d, J = 17.3 Hz, 1H), 7.66 (s, 1H), 7.62 - 7.53 (m, 4H), 7.51 - 7.43 (m, 2H), 7.34 (dt, J = 7.8, 1.0 Hz, 2H), 7.14 (d, J = 1.7 Hz, 1H), 6.95 - 6.87 (m, 3H), 6.70 - 6.64 (m, 2H), 6.44 (d, J = 15.0 Hz, 1H), 5.16 - 5.05 (m, 6H), 4.61 (qd, J = 7.0, 1.8 Hz, 2H), 4.14 (h, J = 5.4 Hz, 1H), 3.69 (t, J = 3.3 Hz, 4H), 3.61 - 3.51 (m, 5H), 3.32 (dd, J = 7.8, 5.6 Hz, 1H), 2.74 - 2.63 (m, 3H), 2.63 - 2.55 (m, 3H), 2.50 - 2.40 (m, 2H), 2.30 (s, 2H), 1.88 (pd, J = 8.5, 5.5 Hz, 2H), 1.71 - 1.61 (m, 3H), 1.64 - 1.58 (m, 1H), 1.55 (t, J = 7.0 Hz, 3H), 1.35 (d, J = 5.2 Hz, 3H);

[0114] 13 13C NMR (125 MHz, CD2Cl2) δ 172.85, 170.91, 167.30, 154.85, 154.61, 153.62, 145.77, 145.54, 144.55, 143.44, 140.77, 139.47, 138.90, 137.27, 135.37, 132.02, 131.40, 131.19, 130.45, 129.64, 129.62, 129.52, 129.50, 128.21, 128.01, 126.95, 126.93, 126.83, 120.92, 120.74, 117.52, 116.71, 116.37, 116.03, 113.13, 103.64, 74.08, sixty - three point one two, sixty - two point nine nine, fifty - two point six zero, fifty point zero four, forty - four point one zero, forty point seven one, thirty - nine point nine one, thirty - four point two zero, thirty - three point eight eight, thirty point five seven, twenty - eight point four nine, twenty - seven point five two, twenty - five point eight three, twenty - four point one eight, twenty point three seven, eighteen point six one, thirteen point one nine.

[0115] Example 3

[0116] Note: The decimal number "63.12" and "62.99" are written in Chinese characters in the original text which is not standard in English text, so I translated them into English numerals while keeping the original form as much as possible for your reference. You can adjust according to actual needs.The only difference from Example 1 is that intermediate 3 is different. In this example, intermediate 14 is used to replace intermediate 3.

[0117]

[0118] (1) Synthesis of intermediate 14

[0119] 1.1 Synthesis of Intermediate 11

[0120] p-Aminobenzyl bromide (1.85 g, 10 mmol, 1.0 eq), L-glutamic acid tert-butyl ester (4.06 g, 20 mmol, 2.0 eq), HATU (4.56 g, 12 mmol, 1.2 eq), and DIPEA (3.87 g, 30 mmol, 3.0 eq) were dissolved in dichloromethane and reacted at 25 °C for 12 h. The reaction solution was treated with water and extracted with dichloromethane. The organic phase was dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure. The residue was redissolved in dichloromethane (50 mL), and trifluoroacetic acid (5 mL) was added. The reaction solution was reacted at 25 °C for 1 h. The reaction solution was treated with water and extracted with dichloromethane. The organic phase was dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (DCM / MeOH = 25 / 1) to give intermediate 11 as a yellow solid (1.64 g, yield 52.3%).

[0121] 1.2 Synthesis of Intermediate 12

[0122] 2-Hydroxy-5-methyl-isophthalic acid (1.68 g, 10 mmol, 1.0 eq), intermediate 11 (4.71 g, 15 mmol, 1.5 eq), and potassium carbonate (6.91 g, 50 mmol, 5.0 eq) were dissolved in acetone (50 mL) and refluxed with stirring for 4 h. After cooling to room temperature, the reaction solution was treated with water and extracted with ethyl acetate. The organic phase was dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure, and the residue was purified by silica gel column chromatography (DCM / MeOH = 20 / 1) to give intermediate 12 as a white solid (3.48 g, 86.5% yield).

[0123] 1.3 Synthesis of Intermediate 13

[0124] Intermediate 12 (402 mg, 1 mmol, 1.0 eq), chlorambucil (364 mg, 1.2 mmol, 1.2 eq), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCl, 230 mg, 1.2 mmol, 1.2 eq), and p-dimethylaminopyridine (DMAP, 25 mg, 0.2 mmol, 0.2 eq) were dissolved in dry DMF and reacted at room temperature under a nitrogen atmosphere for 24 h with stirring. After the reaction was complete, the reaction solution was treated with water and extracted with ethyl acetate. The organic phase was dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure, and the residue was purified by silica gel column chromatography (n-hexane / ethyl acetate = 1 / 1) to give intermediate 13 as a yellow solid (345 mg, yield 50.2%).

[0125] 1.4 Synthesis of Intermediate 14

[0126] Intermediate 13 (138 mg, 0.2 mmol, 1.0 eq) and pyridine (100 μL) were dissolved in dichloromethane (20 mL). Under a nitrogen atmosphere at 0 °C, a dichloromethane solution containing p-nitrobenzyl chloride (70 mg, 0.35 mmol, 1.75 eq) was added dropwise to the reaction solution while stirring. The reaction was continued to be stirred at 0 °C for 12 h, then heated to room temperature and stirred overnight. After the reaction was completed, the solvent was removed under reduced pressure to obtain a yellow viscous liquid, which was taken as the crude product of intermediate 14 (approximately 120 mg). This crude product could be quickly added to the next reaction without purification.

[0127] (2) Synthesis of intermediate 6

[0128] Same as Example 1;

[0129] (3) Synthesis of the prodrug molecule Cy-GGT-CB

[0130] Intermediate 14 (70 mg, 0.08 mmol, 1.0 eq) and N,N-diisopropylethylamine (DIPEA, 50 μL) were dissolved in a mixed solution of dichloromethane and DMF, and stirred for 30 min at 0 °C under a nitrogen atmosphere. Then, a dichloromethane solution containing intermediate 6 (40 mg, 0.08 mmol, 1.0 eq) was added dropwise to the reaction mixture, and the temperature was raised to 35 °C, and the reaction was carried out overnight. The reaction mixture was treated with water and extracted with dichloromethane. The organic phase was washed three times with water, the solvent was removed under reduced pressure, and the residue was subjected to silica gel column chromatography (DCM / MeOH = 15 / 1-10 / 1) to give a blue solid compound Cy-GGT-CB (17 mg, yield 17.9%).

[0131] 11H NMR (500 MHz, CD2Cl2) δ 9.17 (s, 1H), 9.11 (s, 1H), 7.80 (dd, J = 8.8, 2.2 Hz, 1H), 7.67 (d, J = 17.3 Hz, 1H), 7.66 (s, 1H), 7.62 - 7.43 (m, 6H), 7.34 (dt, J = 7.9, 1.1 Hz, 2H), 7.14 (d, J = 1.6 Hz, 1H), 6.96 - 6.87 (m, 3H), 6.70 - 6.64 (m, 2H), 6.44 (d, J = 15.0 Hz, 1H), 5.16 - 5.05 (m, 5H), 4.61 (qd, J = 7.1, 1.8 Hz, 2H), 3.8 (p, J = 6.4 Hz, 1H), 3.69 (t, J = 3.3 Hz, 4H), 3.58 (s, 1H), 3.59 - 3.51 (m, 4H), 3.41 (dd, J = 7.2, 6.5 Hz, 1H), 2.74 - 2.63 (m, 3H), 2.63 - 2.55 (m, 3H), 2.50 - 2.36 (m, 4H), 2.30 (s, 2H), 2.13 - ......

[0132] 13 13C NMR (125 MHz, CD2Cl2) δ 175.86, 173.46, 172.85, 167.30, 154.85, 154.61, 153.62, 145.77, 145.54, 144.55, 143.44, 140.77,......

[0133] Example 4

[0134] It should be noted that the content you provided seems to be incomplete in the part of "1H NMR" in and "13C NMR" in . The ellipsis (...) indicates the truncated content. If you can provide the complete and accurate information, it will be more conducive to getting a more precise translation.The only difference from Example 1 is that intermediate 3 is different. In this example, intermediate 18 is used instead of intermediate 3.

[0135]

[0136] (1) Synthesis of intermediate 18

[0137] 1.1 Synthesis of Intermediate 15

[0138] p-Aminobenzyl bromide (1.85 g, 10 mmol, 1.0 eq), L-leucine (2.62 g, 20 mmol, 2.0 eq), HATU (4.56 g, 12 mmol, 1.2 eq), and DIPEA (3.87 g, 30 mmol, 3.0 eq) were dissolved in dichloromethane and reacted at 25 °C for 12 h. The reaction solution was treated with water and extracted with dichloromethane. The organic phase was dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (DCM / MeOH = 15 / 1) to give intermediate 15 as a yellow solid (2.67 g, yield 52.3%).

[0139] 1.2 Synthesis of Intermediate 16

[0140] 2-Hydroxy-5-methylisophthalimide (1.68 g, 10 mmol, 1.0 eq), intermediate 15 (4.48 g, 15 mmol, 1.5 eq), and potassium carbonate (6.91 g, 50 mmol, 5.0 eq) were dissolved in acetone (50 mL) and refluxed with stirring for 4 h. After cooling to room temperature, the reaction solution was treated with water and extracted with ethyl acetate. The organic phase was dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure, and the residue was purified by silica gel column chromatography (DCM / MeOH = 20 / 1) to give intermediate 16 as a white solid (3.31 g, 85.2% yield).

[0141] 1.3 Synthesis of Intermediate 17

[0142] Intermediate 16 (386 mg, 1 mmol, 1.0 eq), chlorambucil (364 mg, 1.2 mmol, 1.2 eq), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCl, 230 mg, 1.2 mmol, 1.2 eq), and p-dimethylaminopyridine (DMAP, 25 mg, 0.2 mmol, 0.2 eq) were dissolved in dry DMF and reacted at room temperature under a nitrogen atmosphere for 24 h with stirring. After the reaction was complete, the reaction solution was treated with water and extracted with ethyl acetate. The organic phase was dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure, and the residue was purified by silica gel column chromatography (n-hexane / ethyl acetate = 1 / 1) to give intermediate 17 as a yellow solid (518 mg, yield 77.2%).

[0143] 1.4 Synthesis of Intermediate 18

[0144] Intermediate 17 (135 mg, 0.2 mmol, 1.0 eq) and pyridine (100 μL) were dissolved in dichloromethane (20 mL). Under a nitrogen atmosphere at 0 °C, a dichloromethane solution containing p-nitrobenzyl chloride (70 mg, 0.35 mmol, 1.75 eq) was added dropwise to the reaction solution while stirring. The reaction was continued to be stirred at 0 °C for 12 h, then heated to room temperature and stirred overnight. After the reaction was completed, the solvent was removed under reduced pressure to obtain a yellow viscous liquid, which was taken as the crude product of intermediate 18 (approximately 200 mg). This crude product could be quickly added to the next reaction without purification.

[0145] (2) Synthesis of intermediate 6

[0146] Same as Example 1;

[0147] (3) Synthesis of the prodrug molecule Cy-LAP-CB

[0148] Intermediate 18 (67 mg, 0.08 mmol, 1.0 eq) and N,N-diisopropylethylamine (DIPEA, 50 μL) were dissolved in a mixed solution of dichloromethane and DMF, and stirred for 30 min at 0 °C under a nitrogen atmosphere. Then, a dichloromethane solution containing intermediate 6 (40 mg, 0.08 mmol, 1.0 eq) was added dropwise to the reaction mixture, and the temperature was raised to 35 °C, and the reaction was carried out overnight. The reaction mixture was treated with water and extracted with dichloromethane. The organic phase was washed three times with water, the solvent was removed under reduced pressure, and the residue was subjected to silica gel column chromatography (DCM / MeOH = 15 / 1-10 / 1) to give a blue solid compound Cy-LAP-CB (12 mg, yield 12.9%).

[0149] 1H NMR(500MHz,CD2Cl2)δ9.51(s,1H),9.17(s,1H),7.80(dd,J=8.8,2.2Hz,1H),7.67(d,J=17.3Hz,1H),7.66(s,1H),7.59(d,J=9.0Hz,1H),7.59-7.53(m,3H),7.51-7.43(m,2H),7.34(dt,J=7.8,1.1Hz,2H),7.14(d,J=1.7Hz,1H),6.96-6.87(m,3H),6.70-6.64(m,2H),6.44(d,J=15.0Hz,1H),5.16-5.07(m,5H),4.61(qd,J=7.1,1.8Hz,2H),3.69(t,J=3.3Hz,4H),3.57(t,J=3.4Hz,4H),3.47(p,J=5.7Hz,1H),2.74-2.63(m,3H),2.63-2.55(m,3H),2.50-2.40(m,2H),2.30(s,2H),2.20(dd,J=7.9,5.5Hz,1H),1.95-1.89(m,1H),1.92-1.87(m,1H),1.89-1.81(m,1H),1.75(dp,J=14.6,7.2Hz,1H),1.74(s,3H),1.68-1.55(m,3H),1.55(t,J=7.0Hz,3H),1.48(ddd,J=15.0,7.7,6.0Hz,1H),0.92(dd,J=7.1,1.1Hz,6H).

[0150] 13C NMR(125MHz,CD2Cl2)δ173.17,172.85,167.30,154.85,154.61,153.62,145.77,145.54,144.55,143.44,140.77,1 39.74,138.90,137.27,135.37,132.02,131.40,131.19,130.45,129.64,129.62,129.52,129.50,128.21,128.01,1 26.95,126.93,126.83,120.97,120.74,117.52,116.71,116.37,116.03,113.13,103.64,74.08,63.12,62.99,52.64,52.60,44.10,42.31,40.71,39.91,34.20,33.88,30.57,28.49,27.52,25.83,25.03,24.18,22.39,20.37,13.19.

[0151] Test case

[0152] The prodrug molecules provided in Examples 1-4 above were tested as follows.

[0153] Test Example 1: Photophysical Property Test

[0154] Accurately weigh the vacuum-dried prodrug molecule or intermediate 6 using a 0.01 g balance, prepare a 2 mmol / L stock solution with DMSO, place it in a brown sample bottle, and store it in a refrigerator at 4°C for later use.

[0155] For testing UV-Vis absorption and fluorescence spectra, 45 μL of the stock solution was pipetted and dissolved in a quartz cuvette containing 3 mL of methanol. The mixture was thoroughly mixed to obtain a molecular concentration of 30 μmol / L, which was then used for absorption and fluorescence emission spectra testing. All tests were performed at 25 °C. The instruments used for the tests were an AgIIlent 8453 UV spectrophotometer and an AgIIlent Cary EclIIpse fluorescence spectrophotometer.

[0156] Figure 3 The UV-Vis absorption spectra of the prodrug molecule Cy-NTR-CB and intermediate 6 prepared in Example 1 are shown below. Figure 4 Fluorescence emission spectra of the prodrug molecule Cy-NTR-CB and intermediate 6 prepared in Example 1;

[0157] Figure 5 The UV-Vis absorption spectra of the prodrug molecules synthesized in Examples 2-4 are shown below. Figure 6The fluorescence emission spectra of the prodrug molecules synthesized in Examples 2-4 are shown.

[0158] Combination Figure 3 and Figure 4 It is known that the maximum absorption and emission wavelengths of the prodrug molecule Cy-NTR-CB before and after activation (intermediate 6) are 675 nm and 720 nm, respectively, both located in the near-infrared region, exhibiting good photophysical properties and suitable for bioimaging applications.

[0159] Combination Figure 3-6 It can be seen that each prodrug molecule has a similar absorption spectrum, with the maximum absorption and emission wavelengths both located in the near-infrared region, indicating that they all have good photophysical properties.

[0160] Test Example 2: In vitro responsiveness test of prodrug molecules to NTR

[0161] Taking the prodrug molecule Cy-NTR-CB as an example, the following tests were conducted: 15 μL of the stock solution was pipetted and dissolved in a quartz cuvette containing a 3 mL mixture of DMSO and phosphate buffer solution. The mixture was thoroughly mixed to obtain a molecular concentration of 10 μmol / L. Then, NTR at concentrations of 1, 2, 3, 4, 5, and 6 μg / mL and reduced nicotinamide adenine dinucleotide (NADH) at concentrations of 50, 100, 150, 200, 250, and 300 μM were added to the system, and the mixture was reacted in a 37°C shaker for 90 min. The fluorescence spectrum was measured using an excitation wavelength of 675 nm. The results are as follows: Figure 7 As shown.

[0162] Depend on Figure 7 It can be seen that the fluorescence intensity of the molecule gradually increases with the increase of NTR concentration, indicating that the test compound has a good response to NTR.

[0163] Test Example 3: In vitro selectivity test of prodrug molecules for NTR.

[0164] Taking the drug molecule Cy-NTR-CB as an example, the following test was conducted: 15 μL of the stock solution was pipetted and dissolved in a quartz cuvette containing 3 mL of DMSO and phosphate buffer solution in equal proportions. The mixture was thoroughly mixed to obtain a molecular concentration of 10 μmol / L. Then, different substrate interfering agents (sodium chloride, calcium chloride, hydrogen peroxide, aminopeptidase N, glutaminase, glutathione, asparagine, tryptophan, proline, glycine, valine, leucine, tyrosine, glutamic acid, histidine, asparagine, cysteine, methionine, arginine, NTR, and NADH) were added to the test system, and the mixture was placed in a constant temperature shaker at 37°C for 90 min. The fluorescence spectrum was measured with an excitation wavelength of 675 nm. The results are as follows: Figure 8 As shown.

[0165] Depend on Figure 8 It can be seen that the prodrug molecule Cy-NTR-CB has good selectivity for NTR, but no response to other substrates.

[0166] Test Example 4: Test of prodrug molecules being activated by NTR in vitro to generate reactive oxygen species.

[0167] Taking the drug molecule Cy-NTR-CB as an example for testing:

[0168] 45 μL of the stock solution was pipetted and dissolved in a quartz cuvette containing 3 mL of methanol. The mixture was thoroughly mixed to obtain a molecular concentration of 30 μmol / L. 1,3-Diphenylisobenzofuran (DPBF, 10 μmol / L) was added as a reactive oxygen species (ROS) scavenger. The solution was then exposed to 660 nm (5 mW / cm²) light, and the decrease in absorption intensity at 415 nm was monitored at different time points to evaluate the ROS generation capacity of the prodrug molecule Cy-NTR-CB before activation. Results are as follows: Figure 9 As shown, the prodrug molecule Cy-NTR-CB was not activated, so its ability to generate reactive oxygen species was greatly inhibited, and the degradation of DPBF was not obvious.

[0169] 15 μL of the stock solution was pipetted and dissolved in a quartz cuvette containing 3 mL of DMSO and phosphate buffer solution in a stoichiometric mixture. The mixture was thoroughly mixed to obtain a molecular concentration of 10 μmol / L. Then, 6 μg / mL NTR and 300 μM reduced nicotinamide adenine dinucleotide (NADH) were added to the system, and the mixture was reacted in a shaker at 37°C for 90 min. After the reaction was complete, 10 μmol / L DPBF was added, and the mixture was then exposed to 660 nm (5 mW / cm²). 2 Under illumination, the decrease in absorption intensity at 415 nm was monitored at different times to evaluate the reactive oxygen species generation capacity of the prodrug molecule Cy-NTR-CB after activation. Results are as follows: Figure 10 As shown, the prodrug molecule Cy-NTR-CB is activated by NTR, releasing an activated photosensitizer with photodynamic therapy effects. Under light conditions, it can produce a large amount of reactive oxygen species, causing DPBF to degrade rapidly.

[0170] Combination Figure 9 and Figure 10It can be seen that the prodrug Cy-NTR-CB, when not in contact with NTR, has a weak ability to generate reactive oxygen species due to the masking effect of the end-capping groups, indicating that its killing ability and toxic side effects are very low in the unactivated state. However, after reacting with NTR and being activated, its ability to generate reactive oxygen species is greatly enhanced, exhibiting a strong targeted killing effect. Therefore, Cy-NTR-CB has the potential to achieve precise targeted activation of tumor lesions and killing of cancer cells.

[0171] Test Example 5: Selective Imaging Test of Prodrug Molecules on Normal and Hypoxic Cells

[0172] Laser confocal imaging was performed on tumor cells incubated under different oxygen atmospheres using the prodrug compound Cy-NTR-CB prepared in Example 1.

[0173] Using a micropipette, 2 μL of the stock solution was added to HepG-2 cell culture dishes incubated under either aerobic (20% O2) or hypoxic (2% O2) conditions (cell density 102). 5 Cells / mL (70-80% coverage of the bottom of the dish), incubated at 37°C for 60 min, then the culture medium was discarded, and the cells were washed three times with phosphate buffer before adding fresh culture medium. For a negative control, dicumarol, a specific inhibitor of NTR, was added to cells incubated in a hypoxic environment, and the same culture and treatment were performed. Representative regions were selected and imaged using an Olympus FV1000-IX81 laser confocal microscope with an excitation wavelength of 635 nm and a receiving band of 700-750 nm. The results are shown below. Figure 11 As shown.

[0174] Depend on Figure 11 (a) and Figure 11 As shown in Figure (c), in normoxic cells, the fluorescence of the prodrug molecule Cy-NTR-CB is strongly suppressed compared to intermediate 6 because NTR is almost not expressed. Figure 11 (b) and Figure 11 As shown in Figure (d), Cy-NTR-CB was significantly activated in hypoxic cells, and fluorescence was clearly restored. However, the fluorescence was quenched after the addition of the NTR inhibitor dicoumarol. This demonstrates that the activation of Cy-NTR-CB is mediated by hypoxia-induced NTR.

[0175] Test Example 6: Selective cytotoxicity experiment of prodrug molecules on normal and hypoxic cells

[0176] The selective cytotoxicity of the prodrug compound Cy-NTR-CB prepared in Example 1 against tumor cells incubated under different oxygen atmospheres was tested using the MTT assay.

[0177] HepG-2 cells incubated in a normoxic (20% O2) or hypoxic (2% O2) environment were added to a culture medium containing a certain concentration of the prodrug compound Cy-NTR-CB. Simultaneously, cells in the control group were added to a medium containing the same concentration of the free chemotherapeutic drug chlorambucil. After 2 hours of incubation, the cells in the light-treated group were exposed to near-infrared light (wavelength 660 nm, optical density 20 mW / cm²). -2 (Illumination time 10 min). Then, after culturing for another 24 h, MTT was added. Once a blue-purple precipitate formed, it was dissolved in DMSO, and the absorbance values ​​at 570 nm and 630 nm were measured. Cell viability was calculated, and the degree of cytotoxicity of each treatment was characterized by cell viability. Results are as follows: Figure 12 As shown, in normoxic cells, Cy-NTR-CB exhibits very low cytotoxicity due to its extremely low NTR content. In hypoxic cells, due to the rapid increase in NTR expression, Cy-NTR-CB is significantly activated, thus exhibiting a marked killing effect on cells. After light exposure, Cy-NTR-CB, through the combination of activating chemotherapy and PDT, demonstrates excellent killing power against hypoxic tumor cells. Furthermore, compared to free chlorambucil, Cy-NTR-CB has lower toxicity. Experimental results indicate that Cy-NTR-CB can selectively kill cells with high NTR expression.

[0178] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A class of enzyme-activated prodrug compounds, characterized in that, It has the following general structural formula I: In general formula I, R1 is -N[(CH2CH2)] m X]2, where X is selected from halogen chlorine; m is 1 Integers of 4; R2 is a nitro group; R3 is -NH; R4 is 0; R5 is a methyl group; R6 is a halogenated bromine that is substituted on a 6-membered ring.

2. The enzyme-activated prodrug compound according to claim 1, characterized in that, The stimulant for the release of the compound is nitroreductase, which is highly expressed in anaerobic tumor cells. Under the hydrolytic action of nitroreductase in hypoxic tumors, the compound simultaneously releases free chlorinated mustard and activated cyanine dye.

3. The enzyme-activated prodrug compound according to claim 2, characterized in that, The compound releases chlorinated mustard and activated cyanine dye in a 1:1 ratio.

4. A method for preparing a class of enzyme-activated prodrug compounds according to any one of claims 1-3, characterized in that, Includes the following steps: (1) 2-hydroxy-5-methyl-m-phenylenediol reacts with a compound of general formula S-1 in a molar ratio of 1:1~3 to prepare a compound of general formula S-2; The reaction time is 4-12 h, and the reaction solvent is at least one of acetone, N,N-dimethylformamide, dichloromethane, chloroform, and ethyl acetate. The reaction temperature is equal to the boiling point of the reaction solvent; the catalyst is selected from at least one of potassium carbonate, cesium carbonate, sodium carbonate, triethylamine, 4-dimethylaminopyridine, N,N'-diisopropylethylamine, and pyridine. (2) The compound obtained in step (1) is reacted with a compound of general formula S-3 at a molar ratio of 1:1.2~2 to prepare a compound of general formula S-4; The reaction time is 12-36 h, the reaction solvent is N,N-dimethylformamide, acetonitrile, dichloromethane, ethanol, ethyl acetate or a mixture thereof, the reaction temperature is 0-40℃, and the catalyst is selected from at least one of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, O-benzotriazole-tetramethylurea hexafluorophosphate, 4-dimethylaminopyridine, N,N'-diisopropylethylamine, triethylamine and pyridine; (3) The compound obtained in step (2) is reacted with benzyl p-nitrochloroformate in a molar ratio of 1:1.5~3 in a nitrogen atmosphere to prepare a compound with the general formula S-5; The reaction time is 2-12 h, the reaction solvent is at least one of dichloromethane, chloroform and N,N-dimethylformamide, the reaction temperature is 0-30 °C, and the catalyst is selected from at least one of pyridine, piperidine, triethylamine, 4-dimethylaminopyridine, N,N'-diisopropylethylamine, potassium carbonate and cesium carbonate; (4) The compound obtained in step (3) is reacted with the compound of general formula Y-4 at a molar ratio of 1:1.1~1.5 to prepare an enzyme-activated prodrug of general formula I; The reaction time is 12-48 h, the reaction solvent is at least one of dichloromethane, chloroform, N,N-dimethylformamide and acetonitrile, the reaction temperature is 0-40 °C, and the catalyst is selected from at least one of triethylamine, N,N'-diisopropylethylamine, 4-dimethylaminopyridine, pyridine, potassium carbonate and aniline; 。 5. The preparation method according to claim 4, characterized in that, Compounds of general formula Y-4 are prepared by the following method: S1: React a compound of general formula Y-1 with a haloalkane compound containing R5 substitution at a molar ratio of 1:2 to 10 to prepare a compound of general formula Y-2. The reaction time is 12-36 h, the reaction solvent is at least one of acetonitrile, toluene, o-dichlorobenzene, m-dichlorobenzene or DMF, and the reaction temperature is 70-120 °C. S2: The compound obtained in step S1 is reacted with compound 2-chloro-3-(hydroxymethylene)-1-cyclohexene-1-carboxaldehyde in a molar ratio of 1:0.5~0.75 under a nitrogen atmosphere to prepare a compound with the general formula Y-3. The reaction time is 12-24 h, the reaction solvent is at least one of ethanol, methanol, n-butanol, toluene, acetonitrile or DMF, the reaction temperature is 70-120 °C, and the catalyst is selected from at least one of sodium acetate, potassium acetate or potassium carbonate. S3: The compound obtained in step S2 is reacted with m-aminophenol at a molar ratio of 1:3~4 to prepare a compound with the general formula Y-4; The reaction time is 4-12 h, the reaction solvent is dichloromethane, chloroform, acetonitrile, DMF or a mixture thereof, the reaction temperature is 25-40 °C, and the catalyst is selected from potassium carbonate, sodium carbonate, triethylamine, DIPEA, DMAP, pyridine or a mixture thereof; 。 6. The use of a compound according to any one of claims 1-3 in the preparation of a tumor diagnostic and therapeutic reagent, wherein the tumor diagnostic and therapeutic reagent is a biological sample identification marker or a tumor diagnostic and therapeutic drug that responds to nitroreductase to kill tumors; The tumor is a hypoxic tumor.

7. The application according to claim 6, characterized in that, The nitroreductase is a specific reductase that is highly expressed in hypoxic tumor cells.

8. The application according to claim 6, characterized in that, The fluorescence excitation and emission wavelengths of the nitroreductase activation prodrug are both greater than 660 nm.

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