Fluorescent probe for specifically detecting fatty acid amide hydrolase, and preparation method and application thereof

Abstract

This invention discloses a fluorescent probe for the specific detection of fatty acid amide hydrolases, its preparation method, and its applications, relating to the field of biomedical detection technology. The probe, named TMBID, has the structure shown in formula (I): (I). The structure includes a FAAH-specific recognition unit and a large Stokes shift red fluorophore. This probe has been demonstrated for the first time to selectively and sensitively visualize FAAH activity in renal cell carcinoma cells (ACHN, A498) and a renal ischemia-reperfusion injury (RIRI) model. In vitro experiments showed that TMBID's response to FAAH is not interfered with by other hydrolases (such as FAP-α, aminopeptidase N, etc.), and the URB597 inhibitor significantly suppressed the fluorescence signal by 67%. In cell experiments, TMBID could distinguish renal cell carcinoma cells (high fluorescence intensity) from normal kidney / hepatocytes (fluorescence intensity as low as 20%). Animal models confirmed its specific enrichment in renal cell carcinoma and RIRI tissues. This probe provides a revolutionary tool for early diagnosis of renal cell carcinoma, dynamic monitoring of renal injury, and FAAH-targeted therapy.

Description

Technical Field

[0001] This invention relates to the field of biomedical detection technology, specifically to a fluorescent probe for the specific detection of fatty acid amide hydrolase, its preparation method, and its application. Background Technology

[0002] Early, non-invasive diagnostic tools are lacking for renal cell carcinoma (such as renal cell carcinoma) and renal ischemia-reperfusion injury (RIRI), with current technologies relying on invasive biopsies or non-specific imaging examinations. FAAH enzymes play a crucial role in renal cell carcinoma progression and oxidative stress in RIRI, but there are currently no FAAH-targeting probes for kidney diseases. Furthermore, existing FAAH probes are only used in neurological research and fail in the complex renal environment due to poor selectivity and significant background interference. Therefore, no probe currently can correlate FAAH detection with the diagnosis of kidney diseases (especially renal cell carcinoma and RIRI). Summary of the Invention

[0003] The present invention provides a fluorescent probe for the specific detection of fatty acid amide hydrolase, its preparation method and application, aiming to solve the problems existing in the above-mentioned background art.

[0004] To achieve the above-mentioned technical objectives, the present invention mainly adopts the following technical solutions:

[0005] In a first aspect, the present invention discloses a fluorescent probe for the specific detection of fatty acid amide hydrolase (FAAH), the probe being named TMBID and having the structure shown in formula (I):

[0006] (I).

[0007] In a second aspect, the present invention discloses a method for preparing a fluorescent probe as described in the first aspect, comprising the following steps:

[0008] Step 1: Compound 1, N,N-dimethylisopropylamine and decanoyl chloride are reacted in dichloromethane to obtain the crude product;

[0009] Step 2: After separation and purification, the crude product is used to obtain compound 2, which is the fluorescent probe.

[0010] In a preferred embodiment of the present invention, the molar ratio of compound 1 to decanoyl chloride is 1:1-3.

[0011] In a preferred embodiment of the present invention, the mass-volume ratio of compound 1 to N,N-dimethylisopropylamine and dichloromethane is 1:2:5.

[0012] In a preferred embodiment of the present invention, in step 2, the method for separating and purifying the crude product is as follows: after removing the solvent by vacuum distillation, the product is separated and purified by silica gel column chromatography.

[0013] Thirdly, the present invention discloses the use of a fluorescent probe as described in the first aspect in the preparation of reagents or kits for diagnosing kidney diseases.

[0014] In a preferred embodiment of the present invention, the kidney disease includes renal cell carcinoma or renal ischemia-reperfusion injury.

[0015] Fourthly, the present invention discloses a reagent or kit for diagnosing kidney diseases, comprising a fluorescent probe as described in the first aspect.

[0016] Fifthly, the present invention discloses a method for detecting FAAH activity in kidney samples, comprising the following steps:

[0017] (a) Contacting a kidney tissue sample with the fluorescent probe as described in the first aspect;

[0018] (b) Detect the changes in fluorescence signal generated by the reaction of the probe with FAAH;

[0019] The samples were derived from patients with renal cell carcinoma or renal ischemia-reperfusion injury models.

[0020] In a preferred embodiment of the present invention, the sample is an ex vivo kidney tissue slice or a living kidney tissue.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] The TMBID fluorescent probe structure prepared by this invention includes a FAAH specific recognition unit and a large Stokes shift red fluorophore. On the one hand, the large Stokes shift reduces fluorescence background interference during excitation and emission due to the greater distance. On the other hand, the TMBID fluorescent probe prepared by this invention is an ionic salt with good water solubility and good biocompatibility.

[0023] The TMBID fluorescent probe prepared in this invention has been demonstrated for the first time to selectively and sensitively visualize FAAH activity in renal cell carcinomas (ACHN, A498) and renal ischemia-reperfusion injury (RIRI) models. In vitro experiments showed that the TMBID response to FAAH was not interfered with by other hydrolases (such as FAP-α, aminopeptidase N, etc.), and the URB597 inhibitor significantly suppressed the fluorescence signal by 67%. In cell experiments, TMBID could distinguish renal cell carcinomas (high fluorescence intensity) from normal kidney / hepatocytes (fluorescence intensity as low as 20%). Animal models confirmed its specific enrichment in renal cell carcinoma and RIRI tissues. This probe provides a revolutionary tool for early diagnosis of renal cell carcinoma, dynamic monitoring of renal injury, and targeted therapy for FAAH. Attached Figure Description

[0024] Figure 1 The chemical structural formula of the TMBID fluorescent probe provided by this invention;

[0025] Figure 2 The proton spectrum of the TMBID fluorescent probe provided by this invention;

[0026] Figure 3 The carbon spectrum of the TMBID fluorescent probe provided by this invention;

[0027] Figure 4 Here is a high-resolution mass spectrum of the TMBID fluorescent probe provided by this invention;

[0028] Figure 5 The diagram shows the ability of the TMBID fluorescent probe provided by this invention to detect FAAH activity in live cells; wherein, (a) the time-dependent fluorescence response of TMBID in ACHN cells; and (b) the inhibitory effect of the inhibitor URB597 on the fluorescence signal.

[0029] Figure 6 The TMBID fluorescent probe provided by this invention is shown in the fluorescence imaging comparison of different cell lines (A498, HK-2, LO2); wherein, (a) fluorescence imaging image; (b) quantitative comparison of fluorescence intensity;

[0030] Figure 7 Vivo imaging of the TMBID fluorescent probe provided by this invention in mouse xenografts of renal cell carcinoma; wherein, (a) fluorescence image of a live nude mouse injected with TMBID (50 μM); (b) fluorescence images of dissected organs after incubation with TMBID (50 μM) for 40 minutes (tumor tissue; heart; intestine; liver; spleen; lung; kidney); (c) quantitative analysis of fluorescence intensity of each organ in (b);

[0031] Figure 8The images show the kidney-specific fluorescence signal of the TMBID fluorescent probe provided by this invention in a model of renal ischemia-reperfusion injury (RIRI); (a) fluorescence images of anatomical organs after incubation with TMBID (50 μM) for 60 min; (b) quantitative bar chart of fluorescence intensity of multiple organs; (c) time series of in vivo fluorescence imaging of the kidney; (d) curve of kidney fluorescence intensity changing over time; and (e) histopathological section of kidney tissue (HE staining, 100X). Detailed Implementation

[0032] The present invention will be further illustrated below through embodiments, the purpose of which is to better understand the content of the present invention, but the embodiments given do not limit the scope of protection of the present invention.

[0033] The TMBID fluorescent probe prepared in this invention exhibits far higher-than-expected specificity for FAAH, with no interference from other hydrolases, and can be used for specific detection of FAAH. The high-contrast imaging of this probe in renal cancer cells, renal cancer tissues, and renal ischemia-reperfusion injury models provides a research direction for the diagnosis of kidney diseases (especially renal cancer and RIRI).

[0034] The following is a description through specific embodiments.

[0035] Example 1: Synthesis of probe TMBID

[0036]

[0037] (E)-2-(4-aminostyryl)-1,1,3-trimethyl-1H-benzo[e]indole-3-onium (hereinafter referred to as compound 1, 327 mg, 1 mmol), N,N-diisopropylethylamine (259 mg, 2 mmol), and decanoyl chloride (381 mg, 2 mmol) were reacted in dichloromethane at room temperature for 30-60 min to obtain a crude product. The crude product was then purified by silica gel column chromatography after removing the solvent under reduced pressure. Compound 2, i.e., the fluorescent probe TMBID, was obtained with a yield of 59%. The eluents in the column chromatography purification were dichloromethane and methanol, with a volume ratio of dichloromethane to methanol of 20:1.

[0038] TMBID: 1H NMR (400 MHz, DMSO-d6) δ 9.99 (s, 1H), 8.45 (dd, J = 22.3,12.4 Hz, 2H), 8.26 (dt, J = 16.8, 8.7 Hz, 4H), 8.12 (d, J = 9.0 Hz, 1H), 7.93(d, J = 8.3 Hz, 2H), 7.82 (d, J = 8.0 Hz, 1H), 7.74 – 7.59 (m, 2H), 4.29 (s,3H), 3.40 (s, 2H), 3.11 (s, 2H), 2.51 (s, 2H), 2.02 (s, 4H), 1.60 (s, 2H),1.31 – 1.26 (m, 10H), 0.86 (s, 3H).

[0039] 13 C NMR (101 MHz, DMSO-d6) δ 182.72, 172.76, 152.33, 144.77, 139.94,138.20, 133.54, 132.32, 131.25, 130.49, 129.51, 128.82, 127.45, 127.11,123.56, 119.33, 113.74, 110.99, 53.99, 53.60, 41.89, 36.97, 35.30, 31.74,29.29, 29.14, 25.73, 25.43, 22.56, 18.37, 17.14, 14.43, 12.52. Calcd. [M] + :481.3213, find 481.3209.

[0040] Example 2

[0041] To verify the application performance of the designed fluorescent probe and its response behavior in living cells, this invention selected two renal cancer cell lines, human renal cell adenocarcinoma (ACHN) and human renal cancer cell (A498), one normal human renal tubular epithelial cell line (HK-2), and one normal human hepatocyte (LO2), to conduct live-cell fluorescence imaging experiments.

[0042] Cell culture conditions were as follows: All cells were cultured in a cell culture incubator at 37°C and 5% CO2 saturated humidity using RPMI-1640 medium (suitable for ACHN, A498, and HK-2 cells) containing 10% fetal bovine serum (FBS) or DMEM medium (suitable for LO2 cells). The cells were seeded in 15 mm glass-bottomed culture dishes (NEST). After cell adhesion and reaching approximately 70%-80% confluence, the medium was discarded, and the cells were washed three times with PBS (pH 7.4). Then, medium containing fluorescent probes was added for incubation. The final concentration of the fluorescent probes was 5 μM, and incubation was performed for different time periods or for 90 minutes. Subsequently, the cells were washed three more times with PBS to remove unbound probes, and fluorescence microscopy was performed directly on live cells.

[0043] To further investigate the response mechanism of the fluorescent probe, cells were pre-incubated with different metabolic enzyme inhibitors as control experiments. Cells were incubated with specific concentrations of inhibitors (Bestatin, 50 μM; BNPP, 500 μM; Biochanin A, 100 μM; URB597, 20 μM) at 37°C for 1 hour, followed by incubation in serum-free medium containing the fluorescent probe (final concentration 5 μM) for an additional 90 minutes. Subsequently, the cells were washed three times with PBS, and changes in fluorescence signal were observed and recorded.

[0044] The fluorescence imaging instrument used was a Leica DM 4 B (Germany), with excitation and emission wavelengths of 490 nm and 550 nm, respectively. Image acquisition and analysis were performed using ImageJ software to analyze the average IOD and quantify the intensity normalization. All parameters were kept consistent throughout the experiment to ensure the comparability of the results.

[0045] To further verify the ability of the designed fluorescent probe to detect FAAH activity in live cells, this invention used ACHN renal cancer cells with high FAAH expression as experimental cells. After the addition of the fluorescent probe, a stable fluorescence signal gradually appeared in the cells, and the fluorescence intensity increased over time, reaching a plateau phase after approximately 90 minutes of incubation. Figure 5 a). The results showed that the probe could enter living cells and respond to FAAH activity in real time, further confirming its ability to detect FAAH activity levels. To verify the probe's specificity for FAAH, an enzyme inhibitor intervention experiment was designed. Cells were co-incubated with the FAAH-specific inhibitor URB597 (20 μM) and other metabolic enzyme inhibitors (Bestatin, 50 μM; BNPP, 10 μM; Biochanin A, 25 μM), and then the fluorescent probe was added for further incubation.

[0046] The results showed that after treatment with URB597, the intracellular fluorescence signal was significantly reduced, with a decrease of approximately 67%. Figure 5 (b) Other inhibitors had no significant effect on the fluorescence signal. This indicates that the probe can specifically respond to FAAH activity and is not affected by other metabolic enzymes.

[0047] In summary, the designed fluorescent probe can specifically detect FAAH activity in living cells and exhibits a sensitive fluorescent response to cells with high FAAH expression. This characteristic provides a solid foundation for the application of the probe in the study of FAAH-related biological processes and the detection of potential disease biomarkers.

[0048] Example 3

[0049] After verifying that the probe could specifically detect FAAH activity, this invention further applied the probe to study the FAAH activity levels in different renal cancer cell lines and normal cell lines. Renal cancer cell line A498, normal human renal tubular epithelial cells HK-2, and normal human hepatocytes LO2 were selected as models for the experiment. The probe was added to the above cells respectively, and after incubation for 90 minutes, the differences in probe signals were analyzed by fluorescence microscopy and fluorescence intensity quantification. Figure 6 .a).

[0050] The results showed that the probe produced a significant fluorescent signal in the renal cell carcinoma cell line (A498), indicating high intracellular FAAH activity; while in normal cell lines (HK-2 and LO2), the fluorescent signal was significantly weaker, only about 20% of the fluorescence intensity of the renal cell carcinoma cells. Figure 6 (b) further validated the selectivity and sensitivity of the probe. In particular, the fluorescence signal of the probe was extremely weak in normal renal tubular epithelial cells (HK-2), indicating that the probe can accurately reflect the actual activity level of FAAH in different cell types without producing non-specific responses due to interference from other background enzymes.

[0051] In summary, the probe can be used to distinguish the difference in FAAH activity between renal cell carcinoma cells and normal cells. Its significant fluorescence enhancement effect provides an effective tool for the study of FAAH-related tumor markers, and also offers potential application value for the early diagnosis and targeted therapy of renal cell carcinoma.

[0052] Example 4

[0053] To assess the tissue distribution of TMBID in animals and the signal differences in tumor / kidney tissues, in vitro fluorescence imaging of major organs was performed after in vivo imaging. The specific steps were as follows: Mice were anesthetized at predetermined imaging time points and in vivo fluorescence imaging was completed; subsequently, the mice were euthanized and rapidly dissected, and tumor tissue and major organs (heart, liver, spleen, lung, kidney, etc.) were collected. The tissues were gently rinsed with pre-cooled PBS to remove surface blood and residual body fluids, and after abscission, placed on a black background for in vitro fluorescence imaging. Excitation / emission filters, exposure times, and other parameters were maintained consistent with those used in in vivo imaging to ensure comparability of results between different samples.

[0054] Quantitative analysis of ex vivo fluorescence images involved delineating regions of interest (ROIs) in the software to obtain average fluorescence intensity (average radiant efficiency or average gray value, as output by the instrument software) and performing normalized comparisons between different tissues. To assess tumor targeting enrichment capacity, the tumor-to-kidney fluorescence intensity ratio (T / K) was calculated and used for subsequent statistical analysis.

[0055] To further validate the in vivo imaging capability of TMBID in a renal cell carcinoma model, a subcutaneous xenograft model of ACHN cells was established and fluorescence in vivo imaging was performed. ACHN cells were cultured in RPMI-1640 medium containing 10% FBS at 37°C and 5% CO2. The cultured ACHN cells were uniformly dispersed in the medium and placed in 75 cm² culture flasks. The medium was changed after 24 h. When the cells adhered to the culture flask and covered approximately 80% of the surface area, they were passaged. After 24 h of passage, the cells were digested with trypsin and centrifuged. The supernatant was discarded, and the cells were resuspended in 1 mL of 0.01 mol / L PBS to obtain a cell suspension for later use. Cell counting showed that the total number of cells in the culture flask was approximately 1 × 10⁻⁶. 7 indivual.

[0056] BALB / c-nu nude mice aged 4–6 weeks were selected, and the right hind limb region was shaved before tumor implantation. The cell suspension was gently shaken to mix, and 100 μL of the cell suspension was drawn up using an insulin syringe and subcutaneously injected into the subcutaneous cavity above the muscle of the mouse's right hind limb. The injection was slow and the needle was gently withdrawn to avoid extravasation of the cell suspension. After injection, the mice were numbered and housed separately, and tumor growth was monitored regularly. Approximately 2 weeks later, when the tumor volume reached about 80 mm³, the mice were anesthetized and in vivo fluorescence imaging was performed. Immediately after imaging, the tumor was dissected and compared with tissues such as the kidney for ex vivo imaging, and the fluorescence intensity was quantitatively compared.

[0057] In the ACHN subcutaneous xenograft model, in vivo fluorescence imaging was performed on mice after administration of TMBID. The imaging results are as follows: Figure 7The results showed that the fluorescence signal was mainly concentrated in the subcutaneous tumor area, with the tumor site exhibiting significant brightness and high contrast with the surrounding normal tissue, suggesting that TMBID can effectively visualize the tumor area at the in vivo level. This result is consistent with the phenomenon observed in the aforementioned cell experiments where "ACHN cells showed high FAAH activity and significantly enhanced TMBID signal," indicating that TMBID can not only be used to detect endogenous FAAH activity at the cellular level but also has the potential to reflect tumor-related FAAH activity at the whole animal level.

[0058] To further validate the in vivo signal source and assess tissue distribution differences, mice were dissected after in vivo imaging, and major organs and tumor tissues were collected for ex vivo fluorescence imaging and quantitative analysis. The results showed that the fluorescence signal in tumor tissue was significantly stronger than that in kidney tissue, with a fluorescence intensity difference of approximately 2.4 times (T / K≈2.4) between the tumor and kidney. This result suggests that TMBID has a higher signal output in tumor tissue and can be used to differentiate tumor from kidney background signals to some extent. Considering that the kidney, as a metabolic and excretory organ, may have some physiological fluorescence accumulation, a tumor / kidney signal ratio of 2.4 still indicates that the probe has a good imaging window in the tumor region.

[0059] Based on combined results from cell imaging, inhibitor validation, and in vivo / ex vivo imaging of xenograft tumors, TMBID exhibited a consistent targeting response trend in renal cell carcinoma-related models: producing stronger fluorescence signals in tumor cells and tissues with high FAAH activity. This characteristic provides experimental evidence for the subsequent application of TMBID in renal cell carcinoma-related biological research (such as tumor heterogeneity assessment and monitoring of FAAH activity changes after drug intervention). Further research could involve increasing the time-signal curve, expanding the sample size, and combining FAAH immunohistochemistry / Western blot techniques to more systematically validate the in vivo targeting mechanism and imaging threshold of TMBID.

[0060] Example 5

[0061] To evaluate the tissue distribution of TMBID in vivo and the differences in fluorescence signals among different tissues, mice were sacrificed and rapidly dissected after in vivo treatment at predetermined time points. Tissues from both kidneys, heart, liver, spleen, lungs, and intestines were harvested. Each tissue was gently rinsed with pre-cooled PBS to remove surface blood, blotted dry, and placed on a black background for in vitro fluorescence imaging. To ensure comparability of results between different tissues, excitation / emission filters and exposure times were kept consistent throughout the imaging process.

[0062] The average fluorescence intensity of the ex vivo images was obtained by delineating the ROI using software, and the ratio of fluorescence intensity of the kidney to other organs (Kidney-to-Organ Ratio) or normalized fluorescence intensity was used for comparison and statistical analysis.

[0063] To verify the kidney-specific fluorescence signal of TMBID in a renal ischemia-reperfusion injury model, this invention established a mouse RIRI model and performed in vitro fluorescence imaging evaluation. Mice were anesthetized and placed in a supine position. Routine disinfection and draping were performed, and the renal pedicle was exposed by an incision in the abdomen / lumbar region. The renal artery was clamped with a non-invasive vascular clamp for 45 minutes to establish an ischemia model. The clamp was then released to restore blood flow (reperfusion), and the incision was sutured layer by layer. The mice were then fed for one week post-surgery.

[0064] One week later, the mice were euthanized and dissected, and tissues including the kidneys, heart, liver, spleen, lungs, and intestines were collected for in vitro fluorescence imaging. To assess signal changes over time, in vitro fluorescence images were acquired at 60 min and 90 min after the addition of TMBID, and the ROI fluorescence intensity of the kidneys and other organs was quantitatively analyzed.

[0065] Pathological verification: Kidney tissue was fixed, embedded in paraffin, and sectioned. H&E staining was performed to assess histological damage caused by ischemia-reperfusion, and the results were used for correlation analysis with changes in fluorescence signals.

[0066] To evaluate the organ distribution characteristics and renal signal dominance of TMBID under renal ischemia-reperfusion injury, this invention performed in vitro fluorescence imaging comparisons of major organs one week after establishing an RIRI model and feeding the patient post-surgery. The results are as follows: Figure 8 As shown in a and b, compared to tissues such as the heart, liver, spleen, lungs, and intestines, the fluorescence signal in both kidneys is the most significant, with obvious tissue contrast, suggesting that TMBID exhibits prominent kidney-biased fluorescence output in the RIRI model.

[0067] Further quantitative analysis of time points, such as Figure 8 c and d showed that TMBID increased the renal fluorescence signal intensity relative to control tissue by approximately 2.6 times at 60 min, and this difference further increased to approximately 3.0 times at 90 min, indicating that TMBID can produce a high-contrast fluorescence signal that increases over time in damaged kidneys. This phenomenon suggests that TMBID can be used for the visual monitoring of RIRI-related renal pathological conditions, such as... Figure 8 This provides imaging evidence for subsequent studies on damage severity grading and drug intervention efficacy evaluation.

[0068] To verify the authenticity of the model construction and tissue damage, the kidney tissue was subjected to H&E pathological staining. The results showed that the kidney tissue had obvious ischemia-reperfusion-related injury manifestations (such as renal tubular epithelial damage, luminal dilation / brush border changes, and interstitial changes, etc., which were determined by observation of sections). This was corroborated by the significant increase in kidney signal in the imaging, further supporting the correlation between TMBID fluorescence signal and RIRI pathological state.

[0069] It should be understood that the appended claims summarize the scope of the invention, and those skilled in the art, guided by the inventive concept, should realize that any changes made to the various embodiments of the invention will be covered by the spirit and scope of the claims.

Claims

1. A fluorescent probe for the specific detection of fatty acid amide hydrolase (FAAH), characterized in that, The probe is named TMBID and has the structure shown in equation (I): （I）。 2. The method for preparing the fluorescent probe as described in claim 1, characterized in that, Includes the following steps: Step 1: Compound 1, N,N-dimethylisopropylamine and decanoyl chloride are reacted in dichloromethane to obtain the crude product; Step 2: After separation and purification, the crude product is used to obtain compound 2, which is the fluorescent probe.

3. The preparation method according to claim 2, characterized in that, The molar ratio of compound 1 to decanoyl chloride is 1:1-3.

4. The preparation method according to claim 2, characterized in that, The mass-to-volume ratio of compound 1 to N,N-dimethylisopropylamine and dichloromethane is 1:2:

5.

5. The preparation method according to claim 2, characterized in that, In step 2, the method for separating and purifying the crude product is as follows: after removing the solvent by vacuum distillation, the product is separated and purified by silica gel column chromatography.

6. Use of the fluorescent probe as described in claim 1 in the preparation of reagents or kits for diagnosing kidney diseases.

7. The use according to claim 6, characterized in that, The kidney diseases mentioned include renal cell carcinoma or renal ischemia-reperfusion injury.

8. A reagent or kit for diagnosing kidney diseases, characterized in that, Includes the fluorescent probe as described in claim 1.

9. A method for detecting FAAH activity in kidney samples, characterized in that, Includes the following steps: (a) Contacting the fluorescent probe as described in claim 1 with a kidney tissue sample; (b) Detect the changes in fluorescence signal generated by the reaction of the probe with FAAH; The samples were derived from patients with renal cell carcinoma or renal ischemia-reperfusion injury models.

10. The method according to claim 9, characterized in that, The sample is an ex vivo kidney tissue slice or a living kidney tissue.

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