Enzyme-activated probe precursor for tumor diagnosis and treatment, preparation method and application thereof

Abstract

The present application relates to the field of biological medicine, in particular to an enzyme-activated probe precursor for tumor diagnosis and treatment, a preparation method and application thereof, the enzyme-activated probe precursor has the chemical structure shown in the following formula I: The present application synthesizes a fluorescent molecular precursor which can be used for tumor diagnosis and treatment, the fluorescent molecular precursor itself has weak fluorescence and low cytotoxicity, can be specifically activated by CPG2 at the tumor site, releases active fluorescent molecules, the fluorescent molecules produced after activation have fluorescence enhancement effect and also have anti-tumor effect, and are expected to display tumors and anti-tumor active molecule concentration in real time through fluorescence imaging technology. The present application can combine tumor targeting technology to direct enzyme delivery to the lesion area, so that the precursor is specifically activated only in the tumor, and real-time fluorescence imaging and targeted treatment of the tumor can be achieved simultaneously.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to an enzyme-activated probe precursor for tumor diagnosis and treatment, its preparation method and application; the core of this invention is to develop a novel fluorescent molecular precursor, which can be activated and enhance the fluorescence signal under the action of a specific biological enzyme, and is mainly used for tumor fluorescence imaging and anti-tumor therapy at the cellular and in vivo levels. Background Technology

[0002] Malignant tumors are a major threat to human health, and treatment effectiveness highly depends on early diagnosis and the use of anti-tumor drugs. Traditional chemotherapy is limited by its systemic toxicity, restricting the safe dosage that can be administered, and real-time monitoring of drug concentrations within tumors is impossible. Therapeutic molecules, such as fluorescent molecules with anti-tumor activity, can display tumor boundaries and real-time drug concentrations through fluorescence imaging. By preparing anti-tumor fluorescent molecules as precursors with low fluorescence and low cytotoxicity, and introducing enzyme activation, the precursors can be activated by specific enzymes to generate highly fluorescent and significantly cytotoxic active anti-tumor fluorescent molecules. Through tumor-targeting technology, the enzymes can be delivered to the tumor site, where the precursors are specifically activated, enabling real-time fluorescence imaging and treatment of tumors.

[0003] The reliance on endogenous enzymes to activate precursors still hinders clinical translation due to their inherent off-target risks. Activating precursors with exogenous enzymes combined with exogenous enzyme-targeted tumor technology represents a cutting-edge approach. By introducing an exogenous enzyme as an activation trigger, the precursor cannot be activated in normal tissues due to the lack of this enzyme, thus fundamentally avoiding the non-specific activation of precursors caused by co-expression of endogenous enzymes in non-tumor sites. For example, exogenous enzyme activation of fluorescent precursor molecules can maximally suppress background fluorescence, significantly improving the specificity and accuracy of imaging. Furthermore, exogenous enzyme activation of precursor drugs can reduce the cytotoxic side effects caused by the activation of precursor drugs in normal tissues.

[0004] Exogenous enzyme-activated prodrug therapies, such as antibody-directed enzyme prodrug therapy (ADEPT), have entered the clinical research stage, with carboxypeptidase G2 (CPG2) activating prodrugs being extensively studied. However, the activation and distribution of traditional antitumor drugs used in ADEPT are difficult to track in real time.

[0005] Therefore, it is necessary to improve existing technologies to provide more reliable solutions. Summary of the Invention

[0006] The technical problem this invention aims to solve is to address the shortcomings of the prior art by providing an enzyme-activated probe precursor for tumor diagnosis and treatment, its preparation method, and its application. This invention prepares a fluorescent molecule with anti-tumor activity, capable of depicting tumor boundaries and reflecting local drug concentration in real time through fluorescence signals. After being modified into a precursor, it initially exhibits low fluorescence and low toxicity. By introducing a CPG2 activation strategy, this precursor can be converted into an active molecule with high fluorescence intensity and significant cytotoxicity under CPG2 catalysis. This precursor, combined with CPG2, enables cellular-level tumor imaging and anti-tumor therapy. This invention can be combined with tumor-targeting technologies, such as the antibody-enzyme tumor-targeting technology in ADEPT, to achieve CPG2-catalyzed activation of the probe precursor at tumor tissue, potentially enabling specific tumor fluorescence imaging and concentration-visualized tumor drug therapy.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides an enzyme-activated probe precursor for tumor diagnosis and treatment, having a chemical structure as shown in Formula I: .

[0008] Preferably, the enzyme-activated probe precursor can undergo enzymatic hydrolysis to remove the glutamate protecting group and spontaneously remove carbon dioxide under the action of carboxypeptidase G2, thereby converting into the active molecule: cresol purple; the reaction process is as follows: .

[0009] A second aspect of the present invention provides a method for preparing the enzyme-activated probe precursor as described above, comprising the following steps: S1. Protect the amino group on cresol purple or the salt of cresol purple with a tert-butylglutarcarbamoyl group to obtain compound II; The chemical structure of compound II is shown in Formula II below: ; S2. The tert-butyl protecting group on glutamic acid in compound II is removed by using trifluoroacetic acid and water as solvents to obtain the enzyme-activated probe precursor.

[0010] The synthetic route for compound I is shown below: .

[0011] Preferably, step S1 specifically involves reacting cresol purple or a salt of cresol purple with L-glutamic acid di-tert-butyl hydrochloride, N,N′-carbonyldiimidazole, and N-ethyldiisopropylamine in an organic solvent to prepare compound II; Specifically, the salt of cresol purple is cresol purple acetate.

[0012] Preferably, step S1 further includes a step of purifying the reaction product: the reaction product is extracted with ethyl acetate and then purified by silica gel column chromatography to finally obtain compound II.

[0013] Preferably, step S1 specifically includes: S1-1. First, dissolve cresol purple or a salt of cresol purple in N,N-dimethylformamide to prepare a cresol purple solution. S1-2. Add L-glutamic acid di-tert-butyl hydrochloride, N,N′-carbonyldiimidazole, and N-ethyldiisopropylamine to the cresol purple solution and heat to react. S1-3. After the reaction is complete, the product is poured into deionized water and extracted with ethyl acetate. The organic phases are combined and concentrated under vacuum. The concentrated product is purified by silica gel column chromatography, concentrated and dried to obtain compound II. In the silica gel column chromatography purification, a mixture of petroleum ether and ethyl acetate was used as the eluent.

[0014] Preferably, step S1 specifically includes: S1-1. Dissolve 1-4 mmol of cresol purple acetate in 5-20 mL of N,N-dimethylformamide to prepare a cresol purple solution. S1-2, Add 1.2-4.8 mmol of L-glutamic acid di-tert-butyl hydrochloride, 1.2-4.8 mmol of N,N′-carbonyldiimidazole, and 2-8 mmol of N-ethyldiisopropylamine to the cresol purple solution, and heat at 40-80℃ for 8-32 h. S1-3. After the reaction is complete, the product is poured into 100-400 mL of deionized water and extracted with ethyl acetate. The extraction is repeated 1-4 times, each time using 100-400 mL of ethyl acetate. The organic phases are combined and concentrated under vacuum. The concentrated product is purified by silica gel column chromatography. The purification process uses a mixture of petroleum ether and ethyl acetate in a volume ratio of (0.5-2):(0.5-2) as the eluent. The eluted product is concentrated and dried to obtain compound II.

[0015] Preferably, step S2 specifically includes: Compound II was dissolved in a mixed solvent of trifluoroacetic acid and deionized water, stirred at room temperature, and then deionized water was added to stop the reaction. The reaction product was separated and purified to obtain compound I.

[0016] Preferably, step S2 specifically includes: 0.12-0.48 mmol of compound II was dissolved in a mixed solvent of 1.2-4.8 mL of trifluoroacetic acid and deionized water in a volume ratio of (2-8):1. The mixture was stirred at room temperature for 2 h, and then 10.8-43.2 mL of deionized water was added to stop the reaction. The reaction product was separated and purified by high performance liquid chromatography to obtain compound I.

[0017] A third aspect of the present invention provides the application of the enzyme-activated probe precursor as described above or the enzyme-activated probe precursor prepared by the method described above in tumor imaging and anti-tumor therapy, as well as its application in the preparation of tumor imaging agents and anti-tumor drugs.

[0018] The beneficial effects of this invention are: This invention synthesizes a fluorescent molecular precursor that can be used for tumor diagnosis and treatment. This fluorescent molecular precursor itself has weak fluorescence and low cytotoxicity. It can be specifically activated at the tumor site by CPG2 to release active fluorescent molecules. The fluorescent molecules generated after activation have both fluorescence enhancement and anti-tumor effects. It is expected to be able to display the tumor and the concentration of anti-tumor active molecules in real time through fluorescence imaging technology.

[0019] This invention can combine tumor-targeting technology (such as antibody-enzyme tumor-targeting technology in ADEPT) to deliver enzymes to the lesion area, so that the precursor is specifically activated only within the tumor, thereby achieving real-time fluorescence imaging and targeted therapy of the tumor simultaneously. Attached Figure Description

[0020] Figure 1 The image shows the 1H NMR (600 MHz, CDCl3) spectrum of compound II prepared in Example 1. Figure 2 The image shows the 1H NMR (151 MHz, CDCl3) spectrum of compound II prepared in Example 1. Figure 3 The HRMS spectrum of compound II prepared in Example 1; Figure 4 The image shows the 1H NMR (600 MHz, DMSO-d6) spectrum of compound I prepared in Example 1. Figure 5 The image shows the 13C NMR (151 MHz, DMSO-d6) spectrum of compound I prepared in Example 1. Figure 6 The HRMS spectrum of compound I prepared in Example 1; Figure 7 The reaction characterization results are for compound I prepared in Example 1, which is activated by CPG2 catalysis. Figure 8The images show cell imaging results of compound I prepared in Example 1 at different concentrations of CPG2. Figure 9 The inhibitory effects of different concentrations of cresol purple and compound I prepared in Example 1 on the growth of HeLa cells; Figure 10 The inhibitory effects of different concentrations of cresol purple and compound I prepared in Example 1 on the growth of mouse 4T1 cells; Figure 11 The inhibitory effect of compound I prepared in Example 1 combined with different concentrations of CPG2 on the growth of mouse 4T1 cells is shown. Detailed Implementation

[0021] The present invention will be further described in detail below with reference to embodiments, so that those skilled in the art can implement it based on the description.

[0022] It should be understood that terms such as “having,” “comprising,” and “including” as used herein do not exclude the presence or addition of one or more other elements or combinations thereof.

[0023] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available. For examples where specific conditions are not specified, conventional conditions or conditions recommended by the manufacturer are followed. For reagents or instruments whose manufacturers are not specified, they are all commercially available products.

[0024] Example 1 An enzyme-activated probe precursor for tumor diagnosis and treatment has the chemical structure shown in Formula I: .

[0025] Its preparation method includes the following steps: S1, Synthetic compound II: The chemical structure of compound II is shown in Formula II below: ; The synthesis method is as follows: S1-1. First, 2 mmol of cresol purple acetate (642.66 mg) was dissolved in 10 mL of N,N-dimethylformamide to prepare a cresol purple solution. S1-2, Add L-glutamic acid di-tert-butyl hydrochloride (709.92 mg, 2.4 mmol), N,N′-carbonyldiimidazole (389.16 mg, 2.4 mmol), and N-ethyldiisopropylamine (DIPEA, 696.8 μL, 4 mmol) to the cresol purple solution, and heat and stir at 60°C for 16 h; S1-3. After the reaction was completed, the product was poured into 200 mL of deionized water and extracted with ethyl acetate twice, each time using 200 mL of ethyl acetate. The organic phases were combined and concentrated under vacuum. The product was purified by silica gel column chromatography (eluting with a mixture of petroleum ether and ethyl acetate in a volume ratio of 1:1) and then concentrated and dried to give compound II (184 mg, 0.34 mmol), with a yield of 16.8%. The product was a purple oily liquid.

[0026] The spectral data for compound II are as follows: 1 H NMR (600 MHz, CDCl3) δ 8.53 (d, J = 7.8 Hz, 1H), 8.37 (d, J = 7.8 Hz, 1H), 7.60 (dt, J = 33.6, 7.1 Hz, 2H), 7.33 (d, J = 8.5 Hz, 1H), 6.73 (s, 1H), 6.42 (d, J = 6.5 Hz, 1H), 6.33 (s, 1H), 5.98 (d, J = 7.5 Hz, 1H), 4.57 (td, J =8.2, 5.0 Hz, 1H), 4.37 (s, 1H), 2.42 (m, 2H), 2.26 (s, 1H), 2.04 (s, 1H), 1.53 (s, 9H), 1.46 (s, 9H).

[0027] 13 C NMR (151 MHz, CDCl3) δ 172.4, 171.6, 150.4, 146.2, 131.7, 130.8,130.8, 130.3, 126.5, 125.3, 124.1, 112.4, 100.7, 100.0, 82.4, 80.9, 77.2, 60.5, 53.6, 31.9, 28.3, 28.2, 28.1, 14.3.

[0028] S2, Synthetic compound I: 0.24 mmol of compound II (131.2 mg) was dissolved in a mixed solvent of 2.4 mL trifluoroacetic acid and deionized water (v:v = 4:1), and stirred at room temperature for 2 h. Then, 21.6 mL of deionized water was added to stop the reaction. The reaction product was separated and purified by high performance liquid chromatography to obtain compound I (46.8 mg, 0.108 mmol), with a yield of 44.9%. The product was a purple solid, which is the fluorescent molecular precursor of cresol purple.

[0029] The spectral data of compound I are as follows: 1 H NMR (600 MHz, DMSO- d 6) δ 8.51 (d, J = 8.0 Hz, 1H), 8.33 (d, J = 7.9 Hz, 1H), 7.86 (d, J = 7.5 Hz, 1H), 7.75 (t, J = 7.6 Hz, 1H), 7.69 (t, J = 7.6 Hz, 1H), 7.44 (d, J = 8.6 Hz, 1H), 6.63 – 6.56 (m, 2H), 6.46 (s, 1H), 6.43 (d, J = 2.3 Hz,1H), 4.20 (s, 1H), 2.41 – 2.34 (m, 2H), 2.03 (qd, J = 8.0, 7.6, 3.8 Hz, 1H),1.84 (dtd, J = 14.2, 8.8, 6.1 Hz, 1H).

[0030] 13 C NMR (151 MHz, DMSO- d 6) δ 173.9, 173.8, 164.7, 157.3, 148.0, 146.1,139.5, 131.3, 131.1, 130.7, 130.6, 129.9, 124.6, 124.4, 123.4, 112.0, 99.5,97.9, 30.4, 26.0.

[0031] Performance testing 1. Characterization of the reaction of compound I activated by CPG2 catalysis: For the activation of compound I by CPG2 catalysis, the general reaction conditions are as follows: 1.6-200 μM (10 μM unless otherwise specified) of compound I, a buffer solution (PBS, pH 7.3) containing 10 mM phosphate, 50 μM zinc chloride, and a certain concentration (1 U / mL unless otherwise specified) of CPG2 are added to a 2 mL reaction system. The reaction is carried out at 37 degrees Celsius for 3 h, and the fluorescence spectrum under 590 nm excitation light is detected by a fluorescence spectrometer.

[0032] Experimental results are as follows Figure 7 As shown, after adding CPG2, the fluorescence value of the reaction system at 1 U / mL CPG2 was measured as a function of time. Figure 7 a) The results showed that the fluorescence of the solution increased with time; different concentrations of CPG2 (0-0.64 U / mL) were added and reacted at 37℃ for 3 h, and the fluorescence spectra of the solution were measured under excitation at 590 nm wavelength. Figure 7 b) To confirm the specificity of CPG2 in catalyzing the reaction of compound I, several selected substances were added to 2 mL of phosphate buffer (pH 7.3): 1. Blank control, no substance added; 2. 1 mM Mg 2+ 3.1 mM Ca 2+ 4.1 mM Zn 2+ 5. 1 mM DTT; 6. 1 mM cysteine; 7. 1 mM GSH; 8. 1 mM Vitamin C; 9. 1 mM H2O2; 10. 0.4 U / mL CPG2 plus 50 μM zinc chloride) were reacted at 37℃ for 3 h, and the fluorescence spectrum of the solution was measured under excitation at 590 nm wavelength. Figure 7 c, n = 3), the fluorescence value of the group with added CPG2 was significantly different from that of other groups; the kinetic parameters of the activation of compound I by CPG2 were measured, and the results were obtained. k cat and K M The values ​​were 3.18 ± 0.21 min. −1 With 53.7 ± 5.7 μM ( Figure 7 d, n = 3).

[0033] 2. Cell fluorescence imaging effect experiment of CPG2-compound I: Add 2×10 to a 35 mm confocal dish 5Mouse 4T1 cells were cultured overnight in 1 mL of medium to allow cell adhesion. Then, compound I at a final concentration of 10 μM and different concentrations of CPG2 (0–1.6 U / mL) were added. The cells were incubated at 37°C for 3 h in a 5% CO2 incubator before being imaged using a confocal microscope. Results are as follows: Figure 8 As shown, the fluorescence value of cells increased after the addition of CPG2. The fluorescence became stronger after the addition of 0.4 U / mL CPG2, and the fluorescence value did not increase significantly after further increasing the amount of enzyme added.

[0034] 3. Cellular level anti-tumor experiments: Add 5,000 cells / well and 100 μL of culture medium to 96-well plates and incubate overnight to allow cell adhesion. Then add the specified compound or CPG2 simultaneously. Continue incubation in an incubator for 48 h, discard the culture medium, add fresh culture medium containing 10% WST-1, and incubate at 37°C for 1 h. Then measure the absorbance at 450 nm using a microplate reader to analyze cell viability.

[0035] Compared to the addition of cresol purple, compound I alone showed minimal cytotoxicity against HeLa and mouse 4T1 cells at the maximum tested concentration (16 μM), while cresol purple exhibited cytotoxic effects on the tested tumor cells. Figure 9 , Figure 10 Subsequently, different concentrations of CPG2 were added to a 4T1 cell culture system containing 10 μM compound I. With increasing CPG2 concentration, the survival rate of 4T1 cells significantly decreased, indicating that compound I, after being activated by CPG2, has the ability to inhibit cancer cell growth. Figure 11 ).

[0036] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details.

Claims

1. An enzyme-activated probe precursor for tumor diagnosis and treatment, characterized in that, It has the chemical structure shown in Formula I: 。 2. The enzyme-activated probe precursor according to claim 1, characterized in that, The enzyme-activated probe precursor can be hydrolyzed by carboxypeptidase G2 to remove the glutamate protecting group and spontaneously remove carbon dioxide, thereby converting into cresol purple.

3. A method for preparing the enzyme-activated probe precursor as described in claim 1 or 2, characterized in that, Includes the following steps: S1. Protect the amino group on cresol purple or the salt of cresol purple with a tert-butylglutarcarbamoyl group to obtain compound II; S2. The tert-butyl protecting group on glutamic acid in compound II is removed by using trifluoroacetic acid and water as solvents to obtain the enzyme-activated probe precursor. The chemical structure of compound II is shown in Formula II below: 。 4. The method for preparing the enzyme-activated probe precursor according to claim 3, characterized in that, Step S1 specifically involves reacting cresol purple or a salt of cresol purple with L-glutamic acid di-tert-butyl hydrochloride, N,N′-carbonyldiimidazole, and N-ethyldiisopropylamine in an organic solvent to prepare compound II. Specifically, the salt of cresol purple is cresol purple acetate.

5. The method for preparing the enzyme-activated probe precursor according to claim 4, characterized in that, Step S1 also includes a step of purifying the reaction product: the reaction product is extracted with ethyl acetate and then purified by silica gel column chromatography to finally obtain compound II.

6. The method for preparing the enzyme-activated probe precursor according to claim 5, characterized in that, Step S1 is as follows: S1-1. First, dissolve cresol purple or a salt of cresol purple in N,N-dimethylformamide to prepare a cresol purple solution. S1-2. Add L-glutamic acid di-tert-butyl hydrochloride, N,N′-carbonyldiimidazole, and N-ethyldiisopropylamine to the cresol purple solution and heat to react. S1-3. After the reaction is complete, the product is poured into deionized water and extracted with ethyl acetate. The organic phases are combined and concentrated under vacuum. The concentrated product is purified by silica gel column chromatography, concentrated and dried to obtain compound II. In the silica gel column chromatography purification, a mixture of petroleum ether and ethyl acetate was used as the eluent.

7. The method for preparing the enzyme-activated probe precursor according to claim 6, characterized in that, Step S1 is as follows: S1-1. Dissolve 1-4 mmol of cresol purple acetate in 5-20 mL of N,N-dimethylformamide to prepare a cresol purple solution. S1-2, Add 1.2-4.8 mmol of L-glutamic acid di-tert-butyl hydrochloride, 1.2-4.8 mmol of N,N′-carbonyldiimidazole, and 2-8 mmol of N-ethyldiisopropylamine to the cresol purple solution, and heat at 40-80℃ for 8-32 h. S1-3. After the reaction is complete, the product is poured into 100-400 mL of deionized water and extracted with ethyl acetate. The extraction is repeated 1-4 times, each time using 100-400 mL of ethyl acetate. The organic phases are combined and concentrated under vacuum. The concentrated product is purified by silica gel column chromatography. The purification process uses a mixture of petroleum ether and ethyl acetate in a volume ratio of (0.5-2):(0.5-2) as the eluent. The eluted product is concentrated and dried to obtain compound II.

8. The method for preparing the enzyme-activated probe precursor according to claim 3, characterized in that, Step S2 is as follows: Compound II was dissolved in a mixed solvent of trifluoroacetic acid and deionized water, stirred at room temperature, and then deionized water was added to stop the reaction. The reaction product was separated and purified to obtain compound I.

9. The method for preparing the enzyme-activated probe precursor according to claim 8, characterized in that, Step S2 is as follows: 0.12-0.48 mmol of compound II was dissolved in a mixed solvent of 1.2-4.8 mL of trifluoroacetic acid and deionized water in a volume ratio of (2-8):

1. The mixture was stirred at room temperature for 2 h, and then 10.8-43.2 mL of deionized water was added to stop the reaction. The reaction product was separated and purified by high performance liquid chromatography to obtain compound I.

10. The application of the enzyme-activated probe precursor as described in claims 1-2 or the enzyme-activated probe precursor prepared by the method described in any one of claims 3-9 in tumor imaging and anti-tumor therapy, and in the preparation of tumor imaging agents and anti-tumor drugs.

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