CN117304176A - Enzyme-activated prodrug compound and preparation method and application thereof - Google Patents

Enzyme-activated prodrug compound and preparation method and application thereof Download PDF

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CN117304176A
CN117304176A CN202311262863.1A CN202311262863A CN117304176A CN 117304176 A CN117304176 A CN 117304176A CN 202311262863 A CN202311262863 A CN 202311262863A CN 117304176 A CN117304176 A CN 117304176A
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compound
reaction
general formula
enzyme
prodrug
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彭孝军
张寒
韩富平
樊江莉
杜健军
孙文
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Dalian University of Technology
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Abstract

The invention disclosesAn enzyme-activated prodrug compound has the following structural general formula I:the invention discloses a class of enzyme-sensitive chlorambucil analog prodrugs. The prodrug has high selectivity to hypoxic tumor and can be effectively activated in hypoxic tumor cells. When activated specifically, the prodrug releases the free chemotherapeutic drug chlorambucil and releases the photosensitizer cyanine dye to realize the conversion of the photosensitive property of OFF-ON and generate a large amount of active oxygen species to realize the unification and the synergy of photodynamic therapy and activatable chemotherapy.

Description

Enzyme-activated prodrug compound and preparation method and application thereof
Technical Field
The invention relates to release of targeted antitumor drugs, in particular to an enzyme-activated prodrug compound, a preparation method and application thereof.
Background
For a long time, the conventional chemotherapy drugs have poor prognosis effect on cancer patients due to the limitation of factors such as poor enrichment power in tumors, serious toxic and side effects, frequent drug resistance and the like. In contrast, selective activation of prodrugs changes the state of the art of traditional chemotherapy. Prodrugs are nontoxic in normal cells, but can be specifically activated as highly toxic factors in tumor cells. This not only improves the selectivity of the tumor, reduces cell killing to non-target spots, but also minimizes the risk of drug resistance.
Existing prodrugs can be activated by a variety of triggers in the tumor microenvironment, such as specific pH values, reactive oxygen species, reactive thiols, and the like. Among them, prodrugs activated by enzymes endogenous to cells are most attractive because of their advantages of high affinity, high specificity, and rapid responsiveness. The enzyme activated prodrug not only greatly improves the targeting force on tumors, but also overcomes the complexity of manually introducing exogenous animals. However, the range of applications of existing enzyme-activated prodrugs is generally limited by tumor heterogeneity. Furthermore, most enzyme-activated prodrugs involve only a single chemotherapy.
Therefore, research and development of enzyme-activated prodrugs that can accurately identify tumors and can be combined with other therapeutic modalities is of great significance.
Chlorambucil (chloramucil) is used clinically in a variety of malignancies including chronic lymphocytic leukemia, ovarian cancer, non-hodgkin's lymphoma and the like. It induces apoptosis by causing inter-strand cross-linking in DNA. However, the drawbacks associated with chemotherapy have limited their widespread use, including relatively poor selectivity for tumor cells, irreversible damage to normal cells and tissues, and the like. Therefore, the development of a chlorambucil-based multimode enzyme-activated prodrug is of great significance. Cyanine dye-based photosensitizers have a number of special physicochemical properties, such as: the fluorescent dye has the advantages of larger molar extinction coefficient and fluorescence quantum yield, good thermal stability and photo stability, better phototoxicity, capability of controlling the photosensitive property of OFF-ON and the like through chemical modification, and capability of efficiently performing photodynamic therapy (PDT) and realizing tumor inhibition.
Disclosure of Invention
Based on the current situation of lack of enzyme-activated prodrugs capable of realizing accurate chemotherapy and considering excellent tumor selectivity and inhibition effect of photodynamic therapy, the invention constructs a class of enzyme-sensitive chlorambucil analogue prodrugs. The prodrug has high selectivity to hypoxic tumor and can be effectively activated in hypoxic tumor cells. When activated specifically, the prodrug releases the free chemotherapeutic drug chlorambucil and releases the photosensitizer cyanine dye to realize the conversion of the photosensitive property of OFF-ON and generate a large amount of active oxygen species to realize the unification and the synergy of photodynamic therapy and activatable chemotherapy.
In order to achieve the above object, the technical scheme of the present invention is as follows: an enzyme-activated prodrug compound has the following structural general formula I:
in the general formula I, the components are shown in the specification,
R 1 is-N [ (CH) 2 CH 2 ) m X] 2 Wherein X is selected from halogen, hydroxy, mercapto or nitro; m is an integer of 1 to 4;
R 2 selected from nitro or any of the groups of formulae i to iii;
R 3 selected from-NH or O;
R 4 selected from O or S;
R 5 selected from hydrogen, alkyl having 1-6 carbons, carboxyalkyl having 1-6 carbons, hydroxyalkyl having 1-6 carbons, or alkylsulfonate having 1-6 carbons;
R 6 is optionally substituted on the 6-membered ring by hydrogen, halogen, hydroxy, mercapto, cyano, nitro, alkyl having 1 to 6 carbons, carboxyalkyl having 1 to 6 carbons, hydroxyalkyl having 1 to 6 carbons or alkylsulfonate having 1 to 6 carbons.
Further, the stimulus release factor of the compound is the highly expressed nitroreductase in the anaerobic tumor cells, and the compound simultaneously releases free chlorambucil and activated cyanine dye under the hydrolysis of nitroreductase in the anaerobic tumor.
Further, the compounds release chlorambucil and activated cyanine dye in a 1:1 relationship.
A method for preparing an enzyme-activated prodrug compound comprising the steps of:
(1) 2-hydroxy-5-methyl m-xylylene glycol reacts with a compound with a general formula S-1 according to a molar ratio of 1:1-3 to prepare a compound with a general formula S-2;
the reaction time is 4-12 h, and the reaction solvent is at least one of acetone, N-dimethylformamide, dichloromethane, chloroform and ethyl acetate;
the reaction temperature is the boiling point of the corresponding reaction solvent; the catalyst is at least one of potassium carbonate, cesium carbonate, sodium carbonate, triethylamine, 4-dimethylaminopyridine, N' -diisopropylethylamine and pyridine;
(2) Reacting the compound prepared in the step (1) with a compound with a general formula S-3 according to a molar ratio of 1:1.2-2 to prepare a compound with a general formula S-4;
the reaction time is 12-36 h, the reaction solvent is N, N-dimethylformamide, acetonitrile, methylene dichloride, ethanol, ethyl acetate or a mixture thereof, the reaction temperature is 0-40 ℃, and the catalyst is at least one of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, 2- (7-azabenzotriazol) -N, N, N ', N ' -tetramethylurea hexafluorophosphate, O-benzotriazol-tetramethylurea hexafluorophosphate, 4-dimethylaminopyridine, N, N ' -diisopropylethylamine, triethylamine and pyridine;
(3) Reacting the compound prepared in the step (2) with benzyl p-nitrochloroformate in a molar ratio of 1:1.5-3 in a nitrogen atmosphere to prepare a compound with a 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 ℃, and the catalyst is at least one of pyridine, piperidine, triethylamine, 4-dimethylaminopyridine, N' -diisopropylethylamine, potassium carbonate and cesium carbonate;
(4) Reacting the compound prepared in the step (3) with a compound of the general formula Y-4 according to a molar ratio of 1:1.1-1.5 to prepare an enzyme activated prodrug of the general formula I;
the reaction time is 12-48 h, the reaction solvent is at least one of dichloromethane, chloroform, N-dimethylformamide and acetonitrile, the reaction temperature is 0-40 ℃, and the catalyst is at least one of triethylamine, N' -diisopropylethylamine, 4-dimethylaminopyridine, pyridine, potassium carbonate and aniline;
further, the compound of the formula Y-4 is prepared by the following method:
s1: the compound of the general formula Y-1 and the compound containing R 5 Reacting the substituted haloalkane compound according to the mol ratio of 1:2-10 to prepare a compound with the general formula of 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 ℃;
s2: reacting the compound prepared in the step S1 with 2-chloro-3- (hydroxy methylene) -1-cyclohexene-1-formaldehyde in a molar ratio of 1:0.5-0.75 in a nitrogen atmosphere to prepare a compound with a general formula of 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 ℃, and the catalyst is at least one of sodium acetate, potassium acetate or potassium carbonate;
s3: reacting the compound prepared in the step S2 with a phenol compound containing a substituent group according to the molar ratio of 1:3-4 to prepare a compound with a general formula of 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 ℃, and the catalyst is one or a mixture of potassium carbonate, sodium carbonate, triethylamine, DIPEA, DMAP and pyridine;
further, the phenol compound containing the substituent is one of resorcinol, m-aminophenol, m-nitrophenol, m-hydroxysulfur phenol, m-nitrophenol or m-aminophenol.
The application of a compound shown in a formula I in preparing a tumor diagnosis and treatment reagent.
Further, the nitroreductase is a specific reductase highly expressed in hypoxic tumor cells.
Further, fluorescence excitation and emission wavelengths of the nitroreductase activated prodrug are both greater than 660nm.
In summary, the invention has the following beneficial effects:
the invention skillfully fuses tumor fluorescent diagnosis, photodynamic therapy and targeted chemotherapy. In the invention, the diagnosis and treatment prodrug based on cyanine dye and chlorambucil shows high sensitivity and selectivity to the enzyme highly expressed in the anoxic microenvironment of the tumor. Wherein Nitroreductase (NTR) is a specific reductase highly expressed in hypoxic tumor cells, and the activation and release of the prodrug is achieved by an enzymatic reaction caused by the NTR. When the prodrug is not activated, fluorescence and ROS production capacity of the cyanine dye are quenched due to the inhibition of Intramolecular Charge Transfer (ICT) effect, and the alkalinity of nitrogen atoms of chlorambucil is effectively reduced due to covalent closure of the carboxyl terminal (COOH-) of chlorambucil, so that non-target toxicity of the chlorambucil is reduced; the prodrug molecule simultaneously releases free chlorambucil and activated cyanine dye under the NTR hydrolysis in hypoxic tumor, at this time, chlorambucil chemotherapy activity is activated, ICT effect of the cyanine dye is recovered, and fluorescence and ROS production capability are recovered, so that chemotherapy, PDT (photodynamic therapy) and fluorescence imaging multimode tumor diagnosis and treatment are realized.
The invention provides application of the cyanine dye-based enzyme-activated diagnosis and treatment prodrug in hypoxia tumor multi-mode diagnosis and treatment. According to the proposed mechanism, in normoxic cells, the release of the prodrug from the free chlorambucil is retarded and the toxic side effects on normal cells are reduced; in hypoxic tumor cells, uptake of the pro-drugs by the cells is significantly enhanced and can be effectively activated, thus exhibiting higher targeted toxicity. Therefore, the prodrug is hopeful to be used for accurate diagnosis and multi-mode chemotherapy of tumors, and is hopeful to improve the prognosis effect of tumor patients.
Based on this, the invention further provides the use of said enzyme-activated prodrug compounds for the preparation of a diagnostic and therapeutic agent for tumors. Specifically, the tumor is NTR high expression tumor. More specifically, the tumor diagnosis and treatment reagent is a biological sample identification marking preparation or a tumor diagnosis and treatment drug.
In conclusion, the fluorescence excitation and emission wavelengths of the NTR activated prodrug compound are both larger than 660nm, and the NTR activated prodrug compound has excellent near infrared fluorescent dye characteristics, and is beneficial to imaging and phototherapy with high signal-to-noise ratio in living bodies; the introduction of specific targeting groups in the compound ensures that the compound has sensitive and selective response to NTR enzyme, and can identify and screen tumor cells; the prodrug molecule is highly selective and has low toxic side effects on hypoxic cells relative to free chlorambucil. In view of this, the enzyme-activated prodrugs based on cyanine dyes according to the invention can be used for the identification and treatment of tumor cells. Moreover, the compound has the advantages of easily available raw materials, simple preparation and industrialization prospect.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions of the prior art, the drawings that are needed in the embodiments or the description of the prior art will be briefly described below, it will be obvious that the drawings in the following description are some embodiments of the present invention, and that other drawings can be obtained according to these drawings without inventive effort to a person skilled in the art.
FIG. 1 is a nuclear magnetic resonance hydrogen spectrum of a prodrug molecule Cy-NTR-CB disclosed in example 1 of the present invention;
FIG. 2 is a nuclear magnetic resonance spectrum of a prodrug molecule Cy-NTR-CB disclosed in example 1 of the present invention;
FIG. 3 is an ultraviolet-visible absorption spectrum of the prodrug molecule Cy-NTR-CB and intermediate 6 disclosed in example 1 of the present invention;
FIG. 4 is a graph showing fluorescence emission spectra of the prodrug molecule Cy-NTR-CB and intermediate 6 disclosed in example 1 of the present invention;
FIG. 5 is a graph of the UV-visible absorption spectrum of the synthesized prodrug molecules of examples 2-4 of the present invention;
FIG. 6 is a fluorescence emission spectrum of the synthesized prodrug molecules of examples 2-4 of the present invention;
FIG. 7 is a graph showing the fluorescence response of the prodrug molecules Cy-NTR-CB and NTR disclosed in example 1 of the present invention;
FIG. 8 is a graph showing the selective response of the prodrug molecules Cy-NTR-CB and NTR disclosed in example 1 of the present invention;
FIG. 9 is a graph showing the change in absorption spectrum of a mixed solution of the compound Cy-NTR-CB and 1, 3-diphenylisobenzofuran under light;
FIG. 10 is a graph showing the change in absorbance spectra of a mixed solution of 1,3 diphenyl isobenzofuran after reaction with NTR and NADH, for the compound Cy-NTR-CB;
FIG. 11 is a graph showing the fluorescence response of the compound Cy-NTR-CB to NTR in cells under different incubation conditions, wherein: a and c are confocal imaging patterns of intermediate 6 and compound Cy-NTR-CB in normoxic cells, respectively, and b and d are confocal imaging patterns of compound Cy-NTR-CB in hypoxic cells and in dicoumarol-inhibited NTR cells, respectively;
FIG. 12 is the results of toxicity experiments of the compounds Cy-NTR-CB and chlorambucil on different cells.
Detailed Description
The present invention will be described in further detail below.
Unless otherwise indicated, the terms used herein have the following meanings.
The term "halogen" as used herein includes fluorine, chlorine, bromine and iodine.
The term "alkyl" as used herein includes both straight chain alkyl and branched alkyl groups.
In the preparation method, the solvent is preferably a water-removing solvent.
The purification method in the above-mentioned preparation method of the present invention is not particularly limited by conventional methods, and is preferably a column separation using methylene chloride-methanol as an eluent, recrystallization or a combination of both. And the resulting intermediates and final products can be recovered by isolation and purification techniques well known in the art to the desired purity.
The raw materials used in the preparation method of the invention can be obtained by the methods which are commercially available or are well known in the art.
The compounds synthesized in the preparation method of the invention all adopt mass spectrum, nuclear magnetic resonance hydrogen spectrum and nuclear magnetic resonance carbon spectrum to confirm the structure.
The DMF of the 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-azabenzotriazol) -N, N, N ', N ' -tetramethylurea hexafluorophosphate, and HBTU is O-benzotriazol-tetramethylurea hexafluorophosphate.
Instruments and devices employed in the examples:
dye absorption and emission spectra were measured using a Cary 60 UV visible spectrophotometer and a Cary Eclipse fluorescence spectrophotometer from Agilent corporation. The absolute fluorescence quantum yield of the dye was measured using a C11347 absolute fluorescence quantum yield meter from the company of the trade, photonics, china.
Examples
Example 1
(1) Synthesis of intermediate 3
1.1 Synthesis of intermediate 1
2-hydroxy-5-methyl-m-xylylene glycol (1.68 g,10mmol,1.0 eq), p-nitrobenzyl bromide (4.28 g,20mmol,2.0 eq) and potassium carbonate (6.91 g,50mmol,5.0 eq) were dissolved in acetone (50 mL) and the reaction temperature was 57℃and stirred at reflux for 4h. 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%).
1.2 Synthesis of intermediate 2
Intermediate 1 (303 mg,1mmol,1.0 eq), chlorambucil (264 mg,1.2mmol,1.2 eq), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDCl, 230mg,1.2mmol,1.2 eq), p-dimethylaminopyridine (DMAP, 25mg,0.2mmol,0.2 eq) were dissolved in dry DMF and reacted at room temperature under nitrogen with stirring for 12h. After the reaction, the reaction solution is 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%).
1.3 Synthesis of intermediate 3
Intermediate 2 (120 mg,0.2mmol,1.0 eq) and pyridine (100 μl) were dissolved in dichloromethane (20 mL) and a dichloromethane solution containing paranitrobenzoyl chloride (70 mg,0.35mmol,1.75 eq) was added dropwise to the reaction solution under stirring at 0 ℃ under nitrogen atmosphere. The reaction was stirred at 0 ℃ for 2h and then warmed to room temperature and stirred overnight. After the completion of the reaction, the solvent was removed under reduced pressure to give a yellow viscous liquid as a crude product (about 200 mg) of intermediate 3, which was immediately taken into the next reaction without purification.
(2) Synthesis of intermediate 6
2.1 Synthesis of intermediate 4
2, 3-trimethyl-5-bromo-3H-indole (1 g,4.2mmol,1.0 eq) was dissolved in toluene (20 mL) and heated to reflux, and 2g (12.8 mmol,3.0 eq) of ethyl iodide was added in portions with stirring, followed by stirring under reflux until a large amount of precipitate had precipitated. After the reaction solution was cooled to room temperature, the precipitate was obtained by filtration under reduced pressure. The precipitate was washed 3 times with ethyl acetate and dried in vacuo to give intermediate 4 as a pink solid (1.32 g, 80.4% yield).
2.2 Synthesis of intermediate 5
Intermediate 4 (500 mg,1.27mmol,1.0 eq), 2-chloro-3- (hydroxymethyl) cyclohexyl-1-enal (110 mg,0.64mmol,0.5 eq) and anhydrous sodium acetate (130 mg,1.59mmol,1.25 eq) were dissolved in anhydrous ethanol (20 mL). The reaction was carried out overnight at 80℃under a nitrogen atmosphere. After the reaction was completed, the reaction solution was cooled to room temperature, and the solvent was removed under reduced pressure. The residue was dissolved in methylene chloride, washed with saturated brine, and the organic phase was dried over anhydrous sodium sulfate. The residue was chromatographed on a column of silica gel (DCM/meoh=100/1-20/1) to give green solid as intermediate 5 (345 mg, 66.5% yield).
2.3 Synthesis of intermediate 6
3-nitrophenol (123 mg,0.88mmol,3.5 eq) and potassium carbonate (120 mg,0.88mmol,3.5 eq) were dissolved in anhydrous acetonitrile (20 mL) and stirred at room temperature for 30min. Intermediate 5 (200 mg,0.25mmol,1.0 eq) was dissolved in acetonitrile (5 mL) and added dropwise to the reaction solution above and the reaction was stirred at room temperature for 4h. After the solvent was removed under reduced pressure, the residue was dissolved in methylene chloride and washed with water to remove potassium carbonate. After the solvent was removed under reduced pressure, the residue was redissolved in anhydrous methanol and the next reaction was carried out. A methanol solution (5 mL) in which stannous chloride dihydrate (3995 mg,1.75mmol,7.0 eq) and hydrochloric acid (3 mL) were dissolved was added dropwise to the reaction solution, and the mixture was stirred overnight at 70 ℃. After the reaction solution was cooled to room temperature, the reaction solution was neutralized with a 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 chromatographed on a column of silica gel (DCM/meoh=50/1-10/1) to give compound as intermediate 6 as a blue solid (85 mg, yield 56.8%).
(3) Synthesis of prodrug molecule Cy-NTR-CB
Intermediate 3 (60 mg,0.08mmol,1.0 eq) and N, N-diisopropylethylamine (DIPEA, 50. Mu.L) were dissolved in a mixed solution of dichloromethane and DMF (volume ratio 1:1) and stirred for 30min at 0deg.C under nitrogen atmosphere. Subsequently, a dichloromethane solution in which intermediate 6 (40 mg,0.08mmol,1.0 eq) was dissolved was added dropwise to the reaction solution, and the temperature was raised to 40 ℃ and the reaction was carried out overnight. The reaction solution was treated with water and extracted with dichloromethane. The organic phase was washed 3 times with water, the solvent was removed under reduced pressure, and the residue was chromatographed on a silica gel column (DCM/meoh=15/1-10/1) to give the compound Cy-NTR-CB as a blue solid (24 mg, yield 29.7%).
1 H NMR(400MHz,CD 2 Cl 2 ) δ8.56 (d, j=12.6 hz, 1H), δ8.26 (d, j=8.7 hz, 2H), δ 07.69 (d, j=8.4 hz, 2H), δ17.60 (s, 1H), δ27.55 (d, j=12.1 hz, 2H), δ37.38 (d, j=8.7 hz, 1H), δ 47.31 (s, 1H), δ 57.22 (s, 1H), δ 67.11 (s, 1H), δ 77.07 (d, j=7.3 hz, 2H), δ 87.03 (d, j=13.5 hz, 2H), δ96.70 (d, j=8.2 hz, 2H), δ5.97 (d, j=11.8 hz, 1H), δ 05.17 (s, 2H), δ 34.07 (d, j=6.5 hz), δ52 (s, 2H), δ37.7.7 hz, 2H), δ37.5 hz (d, 5 hz), δ52 (d, j=6.7.7 hz, 2H), δ37.5 hz (d, 3hz, 2H), δ96.7.7 hz, 2H), δ5.97 (d, j=7.7 hz, 2H), δ5.7 hz, 2H), δ5.97 (d, j=7.7 hz, 2H), δ5.7.7, 1H;
13 C NMR(125MHz,CD 2 Cl 2 ) Delta 173.05,171.20,164.30,156.69,155.72,152.92,147.54,144.86,144.47,141.33,141.14,140.48,134.67,134.43,131.47,130.62,130.45,129.74,129.51,129.14,127.80,125.95,123.63,122.79,117.55,116.48,115.63,114.45,112.13,111.27,98.34,97.96,75.34,61.37,60.17,49.23,40.75,33.84,33.56,28.52,28.26,26.82,24.28,20.70,20.57, see fig. 2.
Example 2
The difference from example 1 is only that intermediate 3 is different, and this example uses intermediate 10 instead of intermediate 3.
(1) Synthesis of intermediate 10
1.1 Synthesis of intermediate 7
Para-aminobenzyl bromide (1.85 g,10mmol,1.0 eq), glycine (1.78 g,20mmol,2.0 eq), HATU (4.56 g,12mmol,1.2 eq) and DIPEA (3.87 g,30mmol,3.0 eq) were dissolved in dichloromethane and reacted at 25 ℃ for 12h. 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%).
1.2 Synthesis of intermediate 8
2-hydroxy-5-methyl-m-xylylene glycol (1.68 g,10mmol,1.0 eq), intermediate 7 (5.13 g,20mmol,2.0 eq) and potassium carbonate (6.91 g,50mmol,5.0 eq) were dissolved in acetone (50 mL) and stirred at reflux for 4h. 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, 82.3% yield).
1.3 Synthesis of intermediate 9
Intermediate 8 (345 mg,1mmol,1.0 eq), chlorambucil (264 mg,1.2mmol,1.2 eq), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDCl, 230mg,1.2mmol,1.2 eq), p-dimethylaminopyridine (DMAP, 25mg,0.2mmol,0.2 eq) were dissolved in dry DMF and the reaction stirred at room temperature under nitrogen for 36h. After the reaction, the reaction solution is 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%).
1.4 Synthesis of intermediate 10
Intermediate 9 (123 mg,0.2mmol,1.0 eq) and pyridine (100 μl) were dissolved in dichloromethane (20 mL) and a dichloromethane solution containing paranitrobenzoyl chloride (70 mg,0.35mmol,1.75 eq) was added dropwise to the reaction solution under stirring at 0 ℃ under nitrogen atmosphere. The reaction was stirred at 0deg.C for an additional 4h, then warmed to room temperature and stirred overnight. After the completion of the reaction, the solvent was removed under reduced pressure to give a yellow viscous liquid as a crude product (about 150 mg) of intermediate 10, which was immediately taken into the next reaction without purification.
(2) Synthesis of intermediate 6
Same as in example 1
(3) Synthesis of prodrug molecule Cy-APN-CB
Intermediate 10 (64 mg,0.08mmol,1.0 eq) and N, N-diisopropylethylamine (DIPEA, 50. Mu.L) were dissolved in a mixed solution of dichloromethane and DMF and stirred under nitrogen at 0deg.C for 30min. Subsequently, a dichloromethane solution in which intermediate 6 (40 mg,0.08mmol,1.0 eq) was dissolved was added dropwise to the reaction solution, and the temperature was raised to 35 ℃ and the reaction was carried out overnight. The reaction solution was treated with water and extracted with dichloromethane. The organic phase was washed 3 times with water, the solvent was removed under reduced pressure, and the residue was chromatographed on a silica gel column (DCM/meoh=15/1-10/1) to give the blue solid compound Cy-APN-CB (19 mg, yield 21.5%).
1 H NMR(500MHz,CD 2 Cl 2 )δ9.45(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),7.62-7.53(m,4H),7.51-7.43(m,2H),7.34(dt,J＝7.8,1.0Hz,2H),7.14(d,J＝1.7Hz,1H),6.95-6.87(m,3H),6.70-6.64(m,2H),6.44(d,J＝15.0Hz,1H),5.16-5.05(m,6H),4.61(qd,J＝7.0,1.8Hz,2H),4.14(h,J＝5.4Hz,1H),3.69(t,J＝3.3Hz,4H),3.61-3.51(m,5H),3.32(dd,J＝7.8,5.6Hz,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＝8.5,5.5Hz,2H),1.71-1.61(m,3H),1.64-1.58(m,1H),1.55(t,J＝7.0Hz,3H),1.35(d,J＝5.2Hz,3H)；
13 C NMR(125MHz,CD 2 Cl 2 )δ172.85,170.91,167.30,154.85,154.61,153.62,145.77,145.54,144.55,143.44,140.77,139.47,138.90,137.27,135.37,132.02,131.40,131.19,130.45,129.64,129.62,129.52,129.50,128.21,128.01,126.95,126.93,126.83,120.92,120.74,117.52,116.71,116.37,116.03,113.13,103.64,74.08,63.12,62.99,52.60,50.04,44.10,40.71,39.91,34.20,33.88,30.57,28.49,27.52,25.83,24.18,20.37,18.61,13.19.。
Example 3
The difference from example 1 is only that intermediate 3 is different, and this example uses intermediate 14 instead of intermediate 3.
(1) Synthesis of intermediate 14
1.1 Synthesis of intermediate 11
P-aminobenzyl bromide (1.85 g,10mmol,1.0 eq), t-butyl L-glutamate (4.06 g,20mmol,2.0 eq), HATU (4.56 g,12mmol,1.2 eq) and DIPEA (3.87 g,30mmol,3.0 eq) were dissolved in dichloromethane and reacted at 25℃for 12h. 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 and reacted at 25℃for 1h. 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 column chromatography on silica gel (DCM/meoh=25/1) to give intermediate 11 as a yellow solid (1.64 g, 52.3% yield).
1.2 Synthesis of intermediate 12
2-hydroxy-5-methyl-m-xylylene glycol (1.68 g,10mmol,1.0 eq), intermediate 11 (4.71 g,15mmol,1.5 eq) and potassium carbonate (6.91 g,50mmol,5.0 eq) were dissolved in acetone (50 mL) and stirred at reflux for 4h. 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).
1.3 Synthesis of intermediate 13
Intermediate 12 (402 mg,1mmol,1.0 eq), chlorambucil (264 mg,1.2mmol,1.2 eq), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDCl, 230mg,1.2mmol,1.2 eq), p-dimethylaminopyridine (DMAP, 25mg,0.2mmol,0.2 eq) were dissolved in dry DMF and reacted at room temperature under nitrogen with stirring for 24h. After the reaction, the reaction solution is 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%).
1.4 Synthesis of intermediate 14
Intermediate 13 (138 mg,0.2mmol,1.0 eq) and pyridine (100. Mu.L) were dissolved in dichloromethane (20 mL) and a dichloromethane solution containing paranitrobenzoyl chloride (70 mg,0.35mmol,1.75 eq) was added dropwise to the reaction solution under stirring at 0℃under nitrogen. The reaction was stirred at 0deg.C for a further 12h, then warmed to room temperature and stirred overnight. After the completion of the reaction, the solvent was removed under reduced pressure to give a yellow viscous liquid as a crude product (about 120 mg) of intermediate 14, which was immediately taken into the next reaction without purification.
(2) Synthesis of intermediate 6
As in example 1;
(3) Synthesis of prodrug molecule Cy-GGT-CB
Intermediate 14 (70 mg,0.08mmol,1.0 eq) and N, N-diisopropylethylamine (DIPEA, 50. Mu.L) were dissolved in a mixed solution of dichloromethane and DMF and stirred under nitrogen at 0deg.C for 30min. Subsequently, a dichloromethane solution in which intermediate 6 (40 mg,0.08mmol,1.0 eq) was dissolved was added dropwise to the reaction solution, and the temperature was raised to 35 ℃ and the reaction was carried out overnight. The reaction solution was treated with water and extracted with dichloromethane. The organic phase was washed 3 times with water, the solvent was removed under reduced pressure, and the residue was chromatographed on a silica gel column (DCM/meoh=15/1-10/1) to give the blue solid compound Cy-GGT-CB (17 mg, yield 17.9%).
1 H NMR(500MHz,CD 2 Cl 2 )δ9.17(s,1H),9.11(s,1H),7.80(dd,J＝8.8,2.2Hz,1H),7.67(d,J＝17.3Hz,1H),7.66(s,1H),7.62-7.43(m,6H),7.34(dt,J＝7.9,1.1Hz,2H),7.14(d,J＝1.6Hz,1H),6.96-6.87(m,3H),6.70-6.64(m,2H),6.44(d,J＝15.0Hz,1H),5.16-5.05(m,5H),4.61(qd,J＝7.1,1.8Hz,2H),3.82(p,J＝6.4Hz,1H),3.69(t,J＝3.3Hz,4H),3.58(s,1H),3.59-3.51(m,4H),3.41(dd,J＝7.2,6.5Hz,1H),2.74-2.63(m,3H),2.63-2.55(m,3H),2.50-2.36(m,4H),2.30(s,2H),2.13-1.96(m,2H),1.88(pd,J＝8.5,5.5Hz,2H),1.69(s,2H),1.68-1.58(m,2H),1.55(t,J＝7.0Hz,3H).
13 C NMR(125MHz,CD 2 Cl 2 )δ175.86,173.46,172.85,167.30,154.85,154.61,153.62,145.77,145.54,144.55,143.44,140.77,139.33,138.90,137.27,135.37,132.02,131.38,131.19,130.45,129.64,129.62,129.52,129.50,128.21,128.01,126.95,126.93,126.83,120.74,120.65,117.52,116.71,116.37,116.03,113.13,103.64,74.08,63.12,62.99,56.95,52.60,44.10,40.71,39.91,34.20,33.88,32.88,30.57,28.49,27.52,27.10,25.83,24.18,20.37,13.19.。
Example 4
The difference from example 1 is only that intermediate 3 is different, and this example uses intermediate 18 instead of intermediate 3.
(1) Synthesis of intermediate 18
1.1 Synthesis of intermediate 15
P-aminobenzyl bromide (1.85 g,10mmol,1.0 eq), L-leucine (2.62 g,20mmol,2.0 eq), HATU (4.56 g,12mmol,1.2 eq) and DIPEA (3.87 g,30mmol,3.0 eq) were dissolved in dichloromethane and reacted at 25℃for 12h. 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 column chromatography on silica gel (DCM/meoh=15/1) to give intermediate 15 as a yellow solid (2.67 g, 52.3% yield).
1.2 Synthesis of intermediate 16
2-hydroxy-5-methyl-m-xylylene glycol (1.68 g,10mmol,1.0 eq), intermediate 15 (4.48 g,15mmol,1.5 eq) and potassium carbonate (6.91 g,50mmol,5.0 eq) were dissolved in acetone (50 mL) and stirred at reflux for 4h. 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, yield 85.2%).
1.3 Synthesis of intermediate 17
Intermediate 16 (383 mg,1mmol,1.0 eq), chlorambucil (284 mg,1.2mmol,1.2 eq), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDCl, 230mg,1.2mmol,1.2 eq), p-dimethylaminopyridine (DMAP, 25mg,0.2mmol,0.2 eq) were dissolved in dry DMF and reacted at room temperature under nitrogen with stirring for 24h. After the reaction, the reaction solution is 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%).
1.4 Synthesis of intermediate 18
Intermediate 17 (135 mg,0.2mmol,1.0 eq) and pyridine (100 μl) were dissolved in dichloromethane (20 mL) and a dichloromethane solution containing paranitrobenzoyl chloride (70 mg,0.35mmol,1.75 eq) was added dropwise to the reaction solution under stirring at 0 ℃ under nitrogen atmosphere. The reaction was stirred at 0deg.C for a further 12h, then warmed to room temperature and stirred overnight. After the completion of the reaction, the solvent was removed under reduced pressure to give a yellow viscous liquid as a crude product (about 200 mg) of intermediate 18, which was immediately taken into the next reaction without purification.
(2) Synthesis of intermediate 6
As in example 1;
(3) Synthesis of prodrug molecule Cy-LAP-CB
Intermediate 18 (67 mg,0.08mmol,1.0 eq) and N, N-diisopropylethylamine (DIPEA, 50. Mu.L) were dissolved in a mixed solution of dichloromethane and DMF and stirred under nitrogen at 0℃for 30min. Subsequently, a dichloromethane solution in which intermediate 6 (40 mg,0.08mmol,1.0 eq) was dissolved was added dropwise to the reaction solution, and the temperature was raised to 35 ℃ and the reaction was carried out overnight. The reaction solution was treated with water and extracted with dichloromethane. The organic phase was washed 3 times with water, the solvent was removed under reduced pressure, and the residue was chromatographed on a silica gel column (DCM/meoh=15/1-10/1) to give the compound Cy-LAP-CB as a blue solid (12 mg, yield 12.9%).
1 H NMR(500MHz,CD 2 Cl 2 )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).
13 C NMR(125MHz,CD 2 Cl 2 )δ173.17,172.85,167.30,154.85,154.61,153.62,145.77,145.54,144.55,143.44,140.77,139.74,138.90,137.27,135.37,132.02,131.40,131.19,130.45,129.64,129.62,129.52,129.50,128.21,128.01,126.95,126.93,126.83,120.97,120.74,117.52,116.71,116.37,116.03,113.13,103.64,74.08,63.12,62.99,52.64,52.60,44.10,42.31,40.71,39.91,34.20,33.88,30.57,28.49,27.52,25.83,25.03,24.18,22.39,20.37,13.19.
Test case
The prodrug molecules provided in examples 1-4 above were tested as follows.
Test example 1 photophysical property test
Accurately weighing the prodrug molecules or intermediates 6 after vacuum drying by a ten-thousandth balance, preparing mother solution of 2mmol/L by DMSO, placing the mother solution into a brown sample bottle, and storing the brown sample bottle in a refrigerator at the temperature of 4 ℃ for later use.
When the ultraviolet-visible absorption spectrum and the fluorescence spectrum are tested, 45 mu L of mother solution is sucked by a micropipette, and is dissolved in a quartz cuvette containing 3mL of methanol, and the mixture is uniformly mixed to obtain the molecule concentration of 30 mu mol/L for testing the absorption spectrum and the fluorescence emission spectrum. All tests were completed at 25 ℃. The instruments used for the test were an AgIIlent 8453 ultraviolet spectrophotometer and a AgIIlent Cary EclIIpse fluorescence spectrophotometer, respectively.
FIG. 3 is an ultraviolet-visible absorption spectrum of the prodrug molecule Cy-NTR-CB and intermediate 6 prepared in example 1, and FIG. 4 is a fluorescence emission spectrum of the prodrug molecule Cy-NTR-CB and intermediate 6 prepared in example 1;
FIG. 5 is a graph of the UV-visible absorption spectra of the synthesized prodrug molecules of examples 2-4; FIG. 6 is a fluorescence emission spectrum of the prodrug molecules synthesized in examples 2-4;
as can be seen from fig. 3 and fig. 4, the maximum absorption and emission wavelengths of the prodrug molecule Cy-NTR-CB before and after activation (intermediate 6) are 675nm and 720nm, respectively, which are both in the near infrared region, have good photophysical properties, and are suitable for biological imaging applications.
As can be seen in conjunction with fig. 3-6, each prodrug molecule has a similar absorption spectrum, and both the maximum absorption and emission wavelengths lie in the near infrared region, indicating good photophysical properties.
Test example 2 test of the responsiveness of prodrug molecules to NTR in vitro
The test was performed taking the prodrug molecule Cy-NTR-CB as an example: 15. Mu.L of mother liquor is sucked by a micropipette and dissolved in a quartz cuvette containing 3mL of an equal ratio of DMSO and phosphoric acid buffer solution, the mixture is uniformly mixed to obtain a molecular concentration of 10. Mu. Mol/L, NTR and 50, 100, 150, 200, 250 and 300. Mu.M of reduced Nicotinamide Adenine Dinucleotide (NADH) with concentrations of 1,2,3,4,5 and 6. Mu.g/mL are respectively added into the system, and the mixture is placed in a constant temperature shaker at 37 ℃ for reaction for 90min, and a fluorescence spectrum is tested by taking 675nm as an excitation wavelength. The results are shown in FIG. 7.
As can be seen from fig. 7, the fluorescence intensity of the molecules gradually increased with increasing concentration of NTR, indicating that the reference compound has good responsiveness to NTR.
Test example 3 Selective testing of prodrug molecules for NTR in vitro
The test was performed taking the prodrug molecule Cy-NTR-CB as an example: the mother solution of 15. Mu.L was aspirated by a micropipette and dissolved in a quartz cuvette containing 3mL of an equal ratio of DMSO and phosphate buffer, mixed well to give a molecular concentration of 10. Mu. Mol/L, then different substrate interferons (sodium chloride, calcium chloride, hydrogen peroxide, aminopeptidase N, transglutaminase, glutathione, asparagine, tryptophan, proline, glycine, valine, leucine, tyrosine, glutamic acid, histidine, asparagine, cysteine, methionine, arginine, NTR and NADH) were added to the system to be tested, and reacted in a thermostatical shaker at 37℃for 90min, and fluorescence spectra were measured at an excitation wavelength of 675 nm. The results are shown in FIG. 8.
As can be seen from fig. 8, the prodrug molecule Cy-NTR-CB has good selectivity for NTR, but no response to other substrates.
Test example 4 in vitro activation of prodrug molecules by NTR to generate reactive oxygen species
The test was performed taking the prodrug molecule Cy-NTR-CB as an example:
45. Mu.L of mother liquor was sucked up by a micropipette and dissolved in a quartz cuvette containing 3mL of methanol, and mixed uniformly to give a molecular concentration of 30. Mu. Mol/L, 1, 3-diphenylisobenzofuran (DPBF, 10. Mu. Mol/L) was added thereto as a capturing agent for active oxygen, and then the mixed solution was exposed to light of 660nm (5 mW/cm 2), and the decrease in absorption intensity at 415nm was monitored at various times to evaluate the active oxygen generating ability of the prodrug molecule Cy-NTR-CB before being activated. As a result, as shown in FIG. 9, the prodrug molecule Cy-NTR-CB was not activated, and thus the ability to generate active oxygen was greatly inhibited, and the degradation of DPBF was not evident.
15. Mu.L of mother liquor was aspirated by a micropipette and dissolved in a quartz cuvette containing 3mL of an equal ratio of DMSO and phosphate buffer solution, mixed well to give a molecular concentration of 10. Mu. Mol/L, then NTR at a concentration of 6. Mu.g/mL and 300. Mu.M of reduced Nicotinamide Adenine Dinucleotide (NADH) were added to the system, respectively, and reacted in a thermostatic shaker at 37℃for 90min. After completion of the reaction, DPBF (10. Mu. Mol/L) was added thereto, and the mixed solution was then exposed to 660nm (5 mW/cm 2 ) The decrease in absorbance at 415nm at various times was monitored to evaluate the active oxygen generating capacity of the prodrug molecule Cy-NTR-CB after activation. As shown in fig. 10, the prodrug molecule Cy-NTR-CB is activated by NTR, releasing an activated photosensitizer with photodynamic therapy effect, capable of producing a large amount of active oxygen under illumination conditions, resulting in rapid degradation of DPBF.
As can be seen from fig. 9 and 10, when the prodrug Cy-NTR-CB is not contacted with NTR, the ability of generating active oxygen is weak due to masking of the end capping group, which indicates that the killing ability and toxic and side effects of the prodrug Cy-NTR-CB in an unactivated state are low; and after the active oxygen reacts with NTR and is activated, the capability of generating active oxygen is greatly improved, and the active oxygen shows a strong target killing effect. Therefore, cy-NTR-CB can be expected to realize the accurate targeted activation and cancer cell killing of tumor focus.
Test example 5 Selective imaging test of prodrug molecules on normoxic and hypoxic cells
Confocal laser imaging was performed on tumor cells incubated under different oxygen atmospheres using the prodrug compound Cy-NTR-CB prepared in example 1.
2 mu L of mother solution is sucked by a micropipette and added respectivelyInto an atmosphere of normal oxygen (20% O) 2 ) Or anoxic atmosphere (2%O) 2 ) In HepG-2 cell culture dishes (cell density 10) 5 Cells/mL, 70-80% coverage of the bottom of the dish), staining was incubated at 37 ℃ for 60min, then medium was discarded, and Cells were rinsed 3 times with phosphate buffer, and fresh medium was added. For negative control, the specific inhibitor biscoumarin of NTR was added to cells incubated in an anaerobic atmosphere, and the same culture and treatment were performed. Representative regions were selected for imaging using an Olympus FV1000-IX81 confocal laser microscope with an excitation wavelength of 635nm and an acceptance band of 700-750nm, and the results are shown in fig. 11.
From fig. 11 (a) and 11 (c), it can be seen that fluorescence of the prodrug molecule Cy-NTR-CB is strongly inhibited compared to intermediate 6 in normoxic cells due to barely expressed NTR, and from fig. 11 (b) and 11 (d), cy-NTR-CB is significantly activated and fluorescence is significantly restored in hypoxic cells, whereas fluorescence is quenched after addition of the inhibitor biscoumarin of NTR, which demonstrates that activation of Cy-NTR-CB is mediated by hypoxia-induced NTR.
Test example 6 Selective cytotoxicity assay of prodrug molecules on normoxic and hypoxic cells
The prodrug compound Cy-NTR-CB prepared in example 1 was tested for selective cytotoxicity on tumor cells incubated under different oxygen atmospheres using the MTT method.
To an atmosphere of normal oxygen (20% O) 2 ) Or anoxic atmosphere (2%O) 2 ) Adding a culture medium containing a prodrug compound Cy-NTR-CB at a certain concentration into HepG-2 cells which are well incubated, and simultaneously adding chlorambucil which is a free chemotherapeutic agent and contains the same concentration into cells of a control group. After further incubation for 2h, the cells of the illumination group were subjected to illumination with near infrared light (wavelength 660nm, optical density 20mW cm) -2 Illumination time 10 min). Then, MTT was added after further culturing for 24 hours, after the blue-violet precipitate was generated, the precipitate was dissolved with DMSO, absorbance values at 570nm and 630nm were measured, cell viability was calculated, and the size of cytotoxicity of each treatment was characterized by cell viability. As shown in FIG. 12, in normoxic cells, cy-NTR-CB was toxic to cells due to the extremely low NTR contentThe sex is very low. In hypoxic cells, cy-NTR-CB is significantly activated to exhibit an obvious killing effect on cells due to rapid increase of NTR expression, whereas Cy-NTR-CB exhibits excellent killing effect on hypoxic tumor cells by virtue of combination of active chemotherapy and PDT after illumination. Furthermore, cy-NTR-CB has lower toxic side effects compared to free chlorambucil. Experimental results show that Cy-NTR-CB can selectively kill cells with high NTR expression.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.

Claims (9)

1. An enzyme-activated prodrug compound characterized by having the following structural formula i:
in the general formula I, the components are shown in the specification,
R 1 is-N [ (CH) 2 CH 2 ) m X] 2 Wherein X is selected from halogen, hydroxy, mercapto or nitro; m is an integer of 1 to 4;
R 2 selected from nitro or any of the groups of formulae i to iii;
R 3 selected from-NH or O;
R 4 selected from O or S;
R 5 selected from hydrogen, having 1-6 carbonsAlkyl, carboxyalkyl having 1 to 6 carbons, hydroxyalkyl having 1 to 6 carbons or alkylsulfonate having 1 to 6 carbons;
R 6 is optionally substituted on the 6-membered ring by hydrogen, halogen, hydroxy, mercapto, cyano, nitro, alkyl having 1 to 6 carbons, carboxyalkyl having 1 to 6 carbons, hydroxyalkyl having 1 to 6 carbons or alkylsulfonate having 1 to 6 carbons.
2. The enzyme activated prodrug compound of claim 1 wherein the compound is stimulated release of highly expressed nitroreductase enzyme in anaerobic tumor cells, which compound simultaneously releases free chlorambucil and activated cyanine dye upon nitroreductase hydrolysis in hypoxic tumors.
3. The enzyme activated prodrug compound of claim 2 wherein the compound releases chlorambucil and activated cyanine dye in a 1:1 relationship.
4. A process for the preparation of a class of enzyme-activated prodrug compounds according to any of claims 1 to 3, comprising the steps of:
(1) 2-hydroxy-5-methyl m-xylylene glycol reacts with a compound with a general formula S-1 according to a molar ratio of 1:1-3 to prepare a compound with a general formula S-2;
the reaction time is 4-12 h, and the reaction solvent is at least one of acetone, N-dimethylformamide, dichloromethane, chloroform and ethyl acetate;
the reaction temperature is the boiling point of the reaction solvent; the catalyst is at least one of potassium carbonate, cesium carbonate, sodium carbonate, triethylamine, 4-dimethylaminopyridine, N' -diisopropylethylamine and pyridine;
(2) Reacting the compound prepared in the step (1) with a compound with a general formula S-3 according to a molar ratio of 1:1.2-2 to prepare a compound with a general formula S-4;
the reaction time is 12-36 h, the reaction solvent is N, N-dimethylformamide, acetonitrile, methylene dichloride, ethanol, ethyl acetate or a mixture thereof, the reaction temperature is 0-40 ℃, and the catalyst is at least one of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, 2- (7-azabenzotriazol) -N, N, N ', N ' -tetramethylurea hexafluorophosphate, O-benzotriazol-tetramethylurea hexafluorophosphate, 4-dimethylaminopyridine, N, N ' -diisopropylethylamine, triethylamine and pyridine;
(3) Reacting the compound prepared in the step (2) with benzyl p-nitrochloroformate in a molar ratio of 1:1.5-3 in a nitrogen atmosphere to prepare a compound with a 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 ℃, and the catalyst is at least one of pyridine, piperidine, triethylamine, 4-dimethylaminopyridine, N' -diisopropylethylamine, potassium carbonate and cesium carbonate;
(4) Reacting the compound prepared in the step (3) with a compound of the general formula Y-4 according to a molar ratio of 1:1.1-1.5 to prepare an enzyme activated prodrug of the general formula I;
the reaction time is 12-48 h, the reaction solvent is at least one of dichloromethane, chloroform, N-dimethylformamide and acetonitrile, the reaction temperature is 0-40 ℃, and the catalyst is at least one of triethylamine, N' -diisopropylethylamine, 4-dimethylaminopyridine, pyridine, potassium carbonate and aniline;
5. the process of claim 4, wherein the compound of formula Y-4 is prepared by:
s1: the compound of the general formula Y-1 and the compound containing R 5 Reacting the substituted haloalkane compound according to the mol ratio of 1:2-10 to prepare a compound with the general formula of 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 ℃;
s2: reacting the compound prepared in the step S1 with 2-chloro-3- (hydroxy methylene) -1-cyclohexene-1-formaldehyde in a molar ratio of 1:0.5-0.75 in a nitrogen atmosphere to prepare a compound with a general formula of 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 ℃, and the catalyst is at least one of sodium acetate, potassium acetate or potassium carbonate;
s3: reacting the compound prepared in the step S2 with a phenol compound containing a substituent group according to the molar ratio of 1:3-4 to prepare a compound with a general formula of 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 ℃, and the catalyst is one or a mixture of potassium carbonate, sodium carbonate, triethylamine, DIPEA, DMAP and pyridine;
6. the method according to claim 5, wherein the phenol compound having a substituent is one of resorcinol, m-aminophenol, m-nitrophenol, m-hydroxysulfur phenol, m-nitrophenol or m-aminophenylsulfol.
7. Use of a compound according to any one of claims 1-3 for the preparation of a diagnostic tumor therapeutic agent that is a biological sample identification marker formulation or a drug responsive to nitroreductase to kill a diagnostic tumor therapeutic.
8. The use according to claim 7, wherein the nitroreductase is a specific reductase highly expressed in hypoxic tumor cells.
9. The use according to claim 7, wherein the nitroreductase-activated prodrug has fluorescence excitation and emission wavelengths of greater than 660nm.
CN202311262863.1A 2023-09-27 2023-09-27 Enzyme-activated prodrug compound and preparation method and application thereof Pending CN117304176A (en)

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