A biotinylated gefitinib probe, a preparation method and application thereof

Abstract

The application discloses a kind of biotinylated gefitinib probe and its preparation method and application, the probe described in the application is better in stability, sensitivity and accuracy are high, can be long-term preserved, can be used in the experiment needing long time preservation and multiple analysis;It can also be used to detect the binding condition of gefitinib and EGFR in various situations, treat or diagnose cancer;Determine the EGFR mutation type in combination with gene sequencing, the feasibility and effectiveness of the treatment of gefitinib to the EGFR mutation type can also be evaluated, guide drug use.Therefore, biotinylated gefitinib probe is expected to become indispensable tool in precision medicine, so that more patients benefit from gefitinib treatment.

Description

Technical Field

[0001] This invention relates to the field of cancer diagnosis and treatment in the field of biotechnology, specifically to a biotinylated gefitinib probe, its preparation method, and its application. Background Technology

[0002] Epidermal growth factor receptor (EGFR) plays a crucial role in cell proliferation, differentiation, migration, and survival. EGFR is overexpressed or mutated on the surface of many tumor cells, leading to abnormally elevated tyrosine kinase activity. This continuously activates downstream signaling pathways, such as RAS-RAF-MEK-ERK and PI3K-AKT-mTOR, promoting tumor cell growth and survival. Gefitinib is an epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) that competitively binds to the ATP-binding site of EGFR, thereby inhibiting EGFR tyrosine kinase autophosphorylation and blocking the activation of downstream signaling pathways. This results in multiple anti-tumor effects, including inhibiting tumor cell proliferation, inducing apoptosis, inhibiting angiogenesis, and suppressing invasion and metastasis. Furthermore, gefitinib can be used in combination with other treatments, such as chemotherapy drugs, to synergistically inhibit tumor growth through different mechanisms of action; or with radiotherapy to enhance the killing effect of radiotherapy on tumor cells.

[0003] Gefitinib is primarily used to treat advanced or metastatic non-small cell lung cancer (NSCLC) with EGFR mutations. Gefitinib has shown good therapeutic efficacy in NSCLC patients with EGFR-sensitive mutations (such as exon 19 deletion mutations and exon 21 L858R point mutations). Clinically, the standard of gefitinib use is mainly based on the patient's EGFR gene testing results. However, EGFR gene testing cannot quantitatively detect the level of EGFR tyrosine kinase inhibition, thus it cannot accurately assess the effectiveness of gefitinib treatment in NSCLC patients. Designing gefitinib probes can accurately detect the binding of the probe to EGFR in tumor cells in vitro, inferring the level of EGFR tyrosine kinase inhibition, and thus predicting the feasibility and effectiveness of gefitinib treatment in NSCLC patients. Currently, patent CN104212440A discloses a type of quinazoline fluorescent probe and its preparation and application. Specifically, it involves linking a fluorescent chromophore to gefitinib to prepare a fluorescent probe. Due to the targeting effect of gefitinib, this fluorescent probe can be used to investigate the binding of the probe to EGFR, and applied to the relative quantitative detection of the cellular biomarker EGFR, providing more direct information for cancer diagnosis. Simultaneously, this probe can effectively inhibit EGFR tyrosine kinase activity, thus possessing cancer therapeutic functions. However, since the optimal pH for luminescence and storage of fluorescein is usually between 7.0 and 9.0, and tumor tissues are often acidic due to the Warburg effect (tumor cells, even under aerobic conditions, primarily rely on glycolysis for energy, producing large amounts of lactic acid, leading to a decrease in pH), this is unfavorable for the use of fluorescein-labeled probes in practical detection. Furthermore, when using excitation light to detect the probe, the fluorescent label on the probe itself can cause background interference during the detection process, leading to inaccurate results. Therefore, providing a gefitinib probe with higher stability, sensitivity, and accuracy, and suitable for detection in various situations, is of great significance. Summary of the Invention

[0004] The purpose of this invention is to overcome the above-mentioned defects and deficiencies in the prior art and to provide a biotinylated gefitinib probe.

[0005] The second objective of this invention is to provide a method for preparing the above-mentioned biotinylated gefitinib probe.

[0006] A third objective of this invention is to provide the application of the above-described probe in the preparation of products for cancer treatment or diagnosis.

[0007] A fourth objective of this invention is to provide the use of the above-mentioned probe in the preparation of products that guide the use of gefitinib in cancer patients.

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

[0009] This invention provides a biotinylated gefitinib probe, the structural formula of which is shown in formula (I).

[0010]

[0011] Gefitinib is primarily used to treat advanced or metastatic non-small cell lung cancer (NSCLC) with EGFR mutations. For NSCLC patients with EGFR-sensitive mutations (such as exon 19 deletion mutations and exon 21 L858R point mutations), gefitinib has shown good therapeutic efficacy. Biotin has a strong and specific affinity interaction with avidin. When biotinylated gefitinib probes are labeled with fluorescently labeled streptavidin, even low-level mutations that can bind to gefitinib can be amplified through the streptavidin-biotin reaction, increasing detection sensitivity. Furthermore, the biotinylated gefitinib probe itself is non-fluorescent, preventing background interference during detection and resulting in higher sensitivity and accuracy. Meanwhile, streptavidin, by binding to various markers (such as enzymes and fluorescent dyes), is applicable not only to various types of experiments (such as immunohistochemistry, Western blot, and ELISA) but also to various detection systems (such as HRP, AP, and fluorescence detection systems) to determine the target site of gefitinib and whether EGFR is co-localized, thus improving detection accuracy. Therefore, biotinylated gefitinib probes, due to their better stability, high sensitivity and accuracy, and long-term storage capability, can be used in experiments requiring long-term storage and multiple analyses; they can be used to detect the binding of gefitinib to EGFR in various situations for cancer diagnosis; combined with gene sequencing to determine EGFR mutation types, they can also evaluate the feasibility and effectiveness of gefitinib treatment for these EGFR mutation types, guiding medication use.

[0012] This invention also provides a method for preparing the above-mentioned biotinylated gefitinib probe, comprising the following steps:

[0013] S1. Gefitinib is subjected to a methoxydemethylation reaction to obtain the raw material; the raw material is...

[0014]

[0015] S2. The raw materials and biotin are subjected to an esterification reaction to obtain the biotinylated gefitinib probe shown in formula (Ⅰ).

[0016] This invention first involves a methoxy-demethylation reaction of gefitinib to convert the methoxy group (-OCH3) on its benzene ring into a hydroxyl group (-OH). Then, the reaction product is esterified with biotin to prepare a biotinylated gefitinib probe.

[0017] Furthermore, in step S1, the methoxydemethylation reaction is carried out in an organic solvent using aluminum chloride as a demethylation reagent, and the pH is adjusted to alkaline before purification.

[0018] Furthermore, the organic solvent is dichloroethane, acetonitrile, or dichloromethane, etc.

[0019] Furthermore, the reaction temperature is 58–62°C.

[0020] Furthermore, the purification is performed by column purification with dichloromethane / methanol.

[0021] Furthermore, in step S2, the mass ratio of raw material to biotin is 40-45:28-30.

[0022] Preferably, the mass ratio of raw materials to biotin in step S2 is 432:293.

[0023] Furthermore, in step S2, the esterification reaction is carried out by reacting carbodiimide with 1-hydroxybenzotriazole in an organic solvent.

[0024] Furthermore, the organic solvent is N,N-dimethylformamide (DMF).

[0025] Clinically, the standard of care for gefitinib is primarily based on the patient's EGFR gene testing results. Current EGFR gene testing focuses on known exon 19 deletion mutations and exon 21 L858R point mutations. However, the EGFR gene also harbors many other rare mutation types, and the sensitivity of these rare mutations to gefitinib cannot be effectively assessed through gene testing, thus hindering a comprehensive evaluation of gefitinib's therapeutic efficacy. In practice, patients carrying other EGFR mutations (not exon 19 deletion mutations or exon 21 L858R point mutations) can still benefit from gefitinib treatment. The effectiveness of gefitinib treatment varies among patients, fundamentally due to differences in the binding affinity of gefitinib to targeted EGFR. Therefore, the binding affinity of gefitinib to EGFR is the key to evaluating treatment efficacy. Biotinylated gefitinib probes can simultaneously determine EGFR mutation types through gene sequencing and detect EGFR activity at the protein level. This comprehensive approach allows for a more precise assessment of a patient's suitability for gefitinib treatment and prediction of treatment outcomes, leading to more accurate treatment plans. Therefore, biotinylated gefitinib probes hold promise as an indispensable tool in precision medicine, enabling more patients to benefit from gefitinib therapy.

[0026] Therefore, the present invention provides the use of the above-mentioned biotinylated gefitinib probe in the preparation of products for cancer treatment or diagnosis.

[0027] The present invention also provides the use of the above-mentioned biotinylated gefitinib probe in the preparation of products that guide the use of gefitinib in cancer patients.

[0028] Furthermore, the product is used to determine gefitinib dosage for patients by detecting the ability of the probe to target the epidermal growth factor receptor.

[0029] Furthermore, the cancer is a cancer caused by overexpression or mutation of the epidermal growth factor receptor.

[0030] Preferably, the cancer is non-small cell lung cancer.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] This invention provides a biotinylated gefitinib probe, its preparation method, and its applications. The probe described in this invention exhibits better stability, higher sensitivity and accuracy, and long-term storage capability, making it suitable for experiments requiring long-term preservation and repeated analysis. It can be used to detect the binding of gefitinib to EGFR in various situations, for cancer diagnosis, to determine EGFR mutation types in conjunction with gene sequencing, and to evaluate the feasibility and effectiveness of gefitinib treatment for specific EGFR mutation types, guiding medication use. Therefore, the biotinylated gefitinib probe holds promise as an indispensable tool in precision medicine, enabling more patients to benefit from gefitinib treatment. Attached Figure Description

[0033] Figure 1 Mass spectrometry of biotinylated gefitinib probe.

[0034] Figure 2 The H spectrum of the biotinylated gefitinib probe.

[0035] Figure 3 To detect the binding ability of biotinylated gefitinib probes to EGFR. Detailed Implementation

[0036] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0037] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0038] Example 1: Preparation of biotinylated gefitinib probe

[0039] The preparation protocol for the biotinylated gefitinib probe is as follows:

[0040] (1) At room temperature, 500 mg of gefitinib and 10 mL of dichloromethane (DCM) were added to a 100 mL single-necked flask, 1.2 g of aluminum chloride was added, and the mixture was placed at 60 °C overnight. Water was added to quench the flask, sodium carbonate was added to adjust the alkali, and the mixture was filtered. The filter cake was mixed with silica gel and purified by column chromatography. Dichloromethane / methanol was used to pass the filter cake through the column to obtain 420 mg of the raw material product.

[0041]

[0042] (2) 432 mg of raw material product, 293 mg of biotin, 228 mg of carbodiimide (EDCI, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride), and 156 mg of 1-hydroxybenzotriazole (HOBT) were added to 10 mL of formamide (DMF), reacted at room temperature overnight, and evaporated to dryness to obtain 480 mg of biotinylated gefitinib probe.

[0043]

[0044] Mass spectrometry results of biotinylated gefitinib probe are as follows: Figure 1 As shown; the H-spectrum detection results are as follows Figure 2 As shown, this indicates that the biotinylated gefitinib probe was successfully synthesized.

[0045] Example 2: Detection of the binding ability of biotinylated gefitinib probe to EGFR

[0046] I. Experimental Methods

[0047] This detection protocol uses immunofluorescence assay, seeding approximately 10 [units of something] into a laser confocal culture dish. 5 Two EGFR exon 19 deletion mutant and EGFR wild-type cells were cultured and incubated overnight with biotinylated gefitinib probe after cell adhesion. The cells were washed three times with PBS the next day, fixed with 4% paraformaldehyde for 30 minutes, washed three times with PBS for 5 minutes each time, and blocked with 5% BSA for 30 minutes. Diluted EGFR primary antibody and biotinylate primary antibody (sc-53179, Santa Cruz Biotechnology) were added sequentially, and the cells were incubated overnight at 4°C. After overnight incubation, the cells were washed three times with PBS for 5 minutes each time, and diluted FITC-conjugated secondary antibody and Texas Red fluorescent secondary antibody were added sequentially. The cells were incubated at 37°C for 1 hour, washed three times with PBS for 5 minutes each time, and then DAPI (4',6-diamidinyl-2-phenylindole, blue fluorescent dye) was added and incubated for 5 minutes. The cells were then mounted with mounting medium containing anti-fluorescence quencher, and the images were observed and acquired under a fluorescence microscope.

[0048] The results are as follows Figure 3As shown, in cells with EGFR exon 19 deletion mutations, EGFR and biotinylated gefitinib probes colocalized, suggesting that EGFR and biotinylated gefitinib probes are tightly bound together. However, no EGFR and biotinylated gefitinib probes were observed in EGFR wild-type cells, indicating that gefitinib is effective in treating EGFR exon 19 deletion mutation cells.

[0049] Example 3: Detection of the binding ability of biotinylated gefitinib probes to different EGFR mutation types

[0050] I. Experimental Methods

[0051] This detection protocol uses immunofluorescence assay, seeding 10 [units of something] into a laser confocal culture dish. 5 EGFR exon 19 deletion mutation, EGFR L858R combined with T790M mutation, and EGFR wild-type cells were collected. After cell adhesion, they were co-incubated overnight with biotinylated gefitinib probe. The next day, the cells were washed three times with PBS. They were fixed with 4% paraformaldehyde for 30 minutes, washed three times with PBS for 5 minutes each time, and blocked with 5% BSA for 30 minutes. Diluted EGFR primary antibody and biotinylated primary antibody were added sequentially, and the cells were incubated overnight at 4°C. After overnight incubation, the cells were washed three times with PBS for 5 minutes each time. Diluted FITC-conjugated secondary antibody and Texas Red fluorescent secondary antibody were added sequentially, and the cells were incubated at 37°C for 1 hour. The cells were washed three times with PBS for 5 minutes each time. DAPI (4',6-diamidinyl-2-phenylindole, blue fluorescent dye) was added and the cells were incubated for 5 minutes. The cells were mounted with mounting solution containing anti-fluorescence quencher, and the images were observed and acquired under a fluorescence microscope.

[0052] The results are as follows Figure 3 As shown, in cells with EGFR exon 19 deletion mutations, EGFR and biotinylated gefitinib probes co-localized; in T790M mutant cells with gefitinib resistance, the binding of EGFR and biotinylated gefitinib probes weakened; in EGFR wild-type cells, no binding of EGFR and biotinylated gefitinib probes was observed. This indicates that gefitinib is effective in treating EGFR exon 19 deletion mutant cells, and EGFR and biotinylated gefitinib probes bind tightly; however, the therapeutic effect of gefitinib on EGFR L858R cells with T790M mutations is significantly reduced.

[0053] This experiment verified the binding ability of the biotinylated gefitinib probe provided by this invention to EGFR. Using this probe, the targeting ability of gefitinib for different EGFR mutations can be distinguished, thereby evaluating the feasibility and efficacy of gefitinib in treating different EGFR mutations and guiding patient medication. This probe is expected to become an indispensable tool in precision medicine, enabling more patients to benefit from gefitinib treatment.

Claims

1. A biotinylated gefitinib probe, characterized in that, The structural formula is shown as formula (I), 。 2. The method of preparing the biotinylated gefitinib probe according to claim 1, wherein, The method comprises the following steps: ​ S1. The methoxy demethylation reaction of gefitinib is carried out to obtain a raw material; the raw material is ； S2. The esterification reaction of the raw material and biotin is carried out to obtain the biotinylated gefitinib probe shown as formula (I).

3. The preparation method according to claim 2, characterized in that, The methoxy demethylation reaction in the step S1 is carried out by using aluminum chloride as a demethylation reagent in an organic solvent, and the pH is adjusted to be alkaline, and purification is carried out.

4. The preparation method according to claim 2, characterized in that, The mass ratio of the raw material to biotin in the step S2 is 40-45:28-30.

5. The preparation method according to claim 2, characterized in that, The esterification reaction in the step S2 is carried out by using carbodiimide and 1-hydroxybenzotriazole in an organic solvent.

6. Use of the biotinylated gefitinib probe of claim 1 in the manufacture of a product for the diagnosis of cancer, characterized in that, The cancer is non-small cell lung cancer caused by epidermal growth factor receptor mutation.

7. Use of the biotinylated gefitinib probe of claim 1 in the manufacture of a product for guiding the administration of gefitinib to a cancer patient, characterized in that, The cancer is non-small cell lung cancer caused by epidermal growth factor receptor mutation.

8. Use according to claim 7, characterized in that, The product is used for determining the gefitinib medication of a patient by detecting the ability of the probe to target epidermal growth factor receptor.

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