A drug interference-resistant urinary protein fluorescence probe, a preparation method and application thereof

Abstract

The application discloses a drug interference-resistant urine protein fluorescent probe, which has a structure shown in formula (I). The application further discloses a preparation method of the fluorescent probe, which comprises the following steps: (1) dissolving a compound with a structure shown in formula (II) and propenal in 1, 4-dioxane, adding K2CO3 as a catalyst, heating to react, and separating to obtain a compound intermediate with a structure shown in formula (III) after the reaction is completed; (2) dissolving the compound intermediate with the structure shown in formula (III) in ethanol, adding a compound with a structure shown in formula (IV) and a catalyst KOH to react, adding dilute hydrochloric acid to adjust the pH to neutral after the reaction is completed, and separating to obtain the drug interference-resistant urine protein fluorescent probe. The fluorescent probe has good anti-interference on the drugs frequently taken by CKD patients, and the fluorescent signal has high correlation and high accuracy with the added ALB concentration.

Description

Technical Field

[0001] This invention belongs to the field of point-of-care rapid detection technology, specifically, it relates to a drug-resistant urinary protein fluorescent probe, its preparation method, and its application. Background Technology

[0002] Chronic kidney disease (CKD) is a kidney abnormality that exists in the general population worldwide for a long time. It is characterized by a decline or even loss of kidney filtration function. Long-term CKD will increase the risk of cardiovascular disease and stroke. Patients with advanced CKD will develop severe uremia and kidney failure. Therefore, early diagnosis of CKD is beneficial for patients to discover the disease in time and seek medical treatment. It can also help the elderly distinguish between normal aging and kidney function decline caused by pathological damage.

[0003] Because of the decreased renal filtration function in CKD patients, a large amount of serum albumin (ALB) in the blood will enter the urine, resulting in proteinuria. According to the standards issued by the Kidney Disease Improving Global Outcomes (KDIGO) organization, a urine ALB concentration exceeding 30 mg / L and 300 mg / L are defined as microalbuminuria (A2 level) and macroalbuminuria (A3 level), respectively. Therefore, real-time monitoring of urinary protein levels can be used for point-of-care testing (POCT) in CKD.

[0004] Fluorescence analysis can provide precise quantitative results based on a high-precision spectrometer, and can also achieve rapid qualitative home testing using visible light signals. Regarding urine protein detection, several studies in recent years have reported the use of molecular fluorescent probes to determine the ALB content in real urine. The detection mechanism utilizes ALB's ability to bind to hydrophobic small molecules, inhibiting TICT and reducing the polarity of the molecular microenvironment to induce fluorescence enhancement or color change in the probe. While this mechanism ensures extremely high detection sensitivity for environmentally sensitive fluorescent probes, ALB's ability to bind to hydrophobic small molecules also makes it highly susceptible to binding to other compounds, especially exogenous drugs taken by patients. In fact, ALB's primary function in the human body is to carry and deliver drugs; therefore, if the hydrophobic sites of ALB are occupied by medications taken daily by CKD patients, conventional fluorescent probes cannot accurately reflect the urine protein content, and may even produce false negative results.

[0005] Probes designed using existing albumin (ALB) fluorescent probe design methods typically bind to drug sites on ALB. In applications targeting urinary protein detection, the signals of these probes are highly susceptible to interference from exogenous drugs. In real-world urine sample testing scenarios for CKD patients, the use of these probes requires strict control of the patient's medication status, or ensuring that any medication taken by the patient has been completely metabolized and excreted before testing; otherwise, the accuracy of the test results will be affected. Although some ALB fluorescent probes have been reported to penetrate non-drug sites, the detection efficacy of these probe molecular structures in detecting urinary albumin against drug interference has not yet been reported, and a well-defined molecular design method for such fluorescent probes is still lacking.

[0006] This invention addresses this problem by designing and obtaining a class of ALB fluorescent probes with non-drug site binding properties for accurate and rapid point-of-care detection of urinary protein. The fluorescence response signal of this probe is unaffected by various exogenous drugs and their metabolites, and remains highly correlated with ALB levels even under such exogenous interference conditions, enabling efficient determination of ALB levels in urine samples with random drug and metabolite spikes. Summary of the Invention

[0007] To address the shortcomings of existing technologies and practical needs, this invention provides a novel nano-formulation for phototherapy, its preparation method, and its application. It develops new materials that can be used in cancer photothermal therapy, further improving treatment efficacy and providing new methods and ideas for cancer photothermal therapy.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] In a first aspect, the present invention provides a drug-resistant urinary protein fluorescent probe having the structure shown in formula (I):

[0010]

[0011] Where R1 is an aromatic ring group, selected from the following structures:

[0012] R1:

[0013]

[0014] Where R2 is a nitrogen substituent, selected from the following structures:

[0015] R2:

[0016] In one or more embodiments, the drug-resistant urinary protein fluorescent probe is selected from at least one compound having the following chemical formulas:

[0017]

[0018] Secondly, the present invention provides a method for preparing the drug-resistant urinary protein fluorescent probe, comprising the following steps:

[0019] (1) The compound having the structure shown in formula (II) and acrolein were dissolved in 1,4-dioxane, K2CO3 was added as a catalyst, and the reaction was carried out by heating. After the reaction was completed, the intermediate compound having the structure shown in formula (III) was obtained by separation:

[0020]

[0021] (2) The intermediate of the compound having the structure shown in formula (III) was dissolved in ethanol, and the compound having the structure shown in formula (IV) and the catalyst KOH were added to react. After the reaction was completed, dilute hydrochloric acid was added to adjust the pH to neutral, and the compound having the structure shown in formula (I) was separated, which is the drug-resistant urinary protein fluorescent probe:

[0022]

[0023] In one or more embodiments, the molar ratio of the compound having the structure shown in formula (II) in step (1), acrolein, and the catalyst K2CO3 is 1:1:2.

[0024] In one or more embodiments, the heating temperature in step (1) is 80°C and the heating time is 8 hours.

[0025] In one or more embodiments, the separation step in step (1) is as follows: after the reaction is completed, the reaction solution is naturally cooled to room temperature, a yellow solid is precipitated in the solution, the solid is filtered out and dried to obtain a compound intermediate having the structure shown in formula (Ⅲ).

[0026] In one or more embodiments, the molar ratio of the compound having the structure shown in formula (III), the compound having the structure shown in formula (IV), and the catalyst KOH in step (2) is 1:1.2:2.

[0027] In one or more embodiments, the reaction time described in step (2) is 8 hours.

[0028] In one or more embodiments, the separation step in step (2) is as follows: after concentrating the solution adjusted to neutral, extract it with dichloromethane to obtain an organic phase, and then separate it by silica gel column chromatography to obtain a compound with the structure shown in formula (I), which is the anti-drug interference urinary protein fluorescent probe.

[0029] Thirdly, the present invention provides the application of the drug-resistant urinary protein fluorescent probe in the preparation of a point-of-care rapid urinary protein detection kit.

[0030] Based on the drug-resistant fluorescent probe for urinary protein and its applications as described above, this invention also provides a method for rapid detection of urinary protein, comprising the following steps:

[0031] (1) Prepare a portable test strip by impregnating filter paper with any of the drug-resistant urinary protein fluorescent probes described in this invention.

[0032] (2) Take random urine from healthy individuals and divide it into n groups. Add protein stock solutions with different concentration gradients to the n groups of urine to obtain n groups of samples with different urinary protein concentrations. Add the same volume of the n groups of samples to the test strip.

[0033] (3) Calculate the color difference of each group based on the luminescent photographs of each group of samples on the test strip, establish a standard curve with the color difference of each group and the corresponding urinary protein concentration, and calculate the urinary protein content in the urine to be tested based on the standard curve.

[0034] In one or more embodiments, the step of calculating the color difference of each group based on the emission photographs of each group of samples specifically includes: determining the trichromatic values ​​and color coordinates of the emission color based on the emission photographs of the two samples in each group, and calculating the color difference of each group based on the trichromatic values ​​and color coordinates of each group of samples.

[0035] In one or more implementation schemes, the step of calculating the color difference of each group based on the luminescence photographs of each group of samples can be achieved by directly taking photos of each group of samples with a mobile phone and quickly analyzing the results such as color difference (ΔE*) and color coordinate ratio (R / B) on a mobile APP, thereby realizing semi-quantitative bedside rapid detection of urinary protein.

[0036] Compared with the prior art, the present invention has the following beneficial effects:

[0037] (1) Anti-drug interference: Through the design of the probe molecular structure, it is located at the non-drug site of albumin (ALB). Its fluorescence response has good anti-interference ability against drugs commonly taken by CKD patients. The fluorescence signal shows a high correlation and high accuracy with the spiked ALB concentration.

[0038] (2) The kit prepared with this probe can realize rapid bedside detection of urine protein qualitative and semi-quantitative. The high accuracy of the probe can avoid false negative results caused by drug interference in actual applications. Attached Figure Description

[0039] Figure 1 It is a test of the spectral response, anti-interference, and accuracy of fluorescent probe C-6 to spiked albumin in real urine samples.

[0040] Figure 2 It relates to the preparation of portable test strip devices and on-site detection of urine protein. Detailed Implementation

[0041] The renal filtration function of CKD patients is reduced compared to normal individuals, leading to a significant increase in serum albumin (ALB) levels in the urine, resulting in proteinuria. According to the standards set by the Kidney Disease Improving Global Outcomes (KDIGO), urinary ALB concentrations exceeding 30 mg / L and 300 mg / L are defined as microalbuminuria (A2 level) and macroalbuminuria (A3 level), respectively. Therefore, real-time monitoring of urinary protein levels is useful for point-of-care testing (POCT) in CKD. However, probes designed using existing albumin (ALB) fluorescent probes typically bind to drug sites on ALB, making their signals highly susceptible to interference from exogenous drugs in applications targeting urinary protein detection. In real-world urine sample testing scenarios for CKD patients, the use of these probes requires strict control of whether the patient is taking medication or whether any medication taken by the patient has been completely metabolized and excreted from the body at the time of testing; otherwise, the accuracy of the test results will be affected. To address these practical problems, the inventors of this invention have developed a drug-resistant fluorescent probe for urinary protein.

[0042] Through continuous research and exploration, the inventors of this invention have discovered a drug-resistant fluorescent probe for urinary proteins, which has the structure shown in formula (Ⅰ):

[0043]

[0044] Where R1 is an aromatic ring group, selected from the following structures:

[0045] R1:

[0046]

[0047] Where R2 is a nitrogen substituent, selected from the following structures:

[0048] R2:

[0049] Drugs typically enter conventional sites, while probes located at non-drug sites can reduce or even avoid drug interference. Through research on compounds with different structures, the inventors discovered that the non-conjugated ring structure in formula (I) allows the molecule to have a torsion-restricted linear structure. This structural change may make the molecule more compatible with the narrow space of the FA1 site, ultimately producing a probe-resistant effect against drug interference.

[0050] In one or more embodiments, the drug-resistant urinary protein fluorescent probe is selected from at least one compound having the following chemical formulas:

[0051]

[0052] This invention also includes a method for preparing the drug-resistant urinary protein fluorescent probe, comprising the following steps:

[0053] (1) The compound having the structure shown in formula (II) and acrolein were dissolved in 1,4-dioxane, K2CO3 was added as a catalyst, and the reaction was carried out by heating. After the reaction was completed, the intermediate compound having the structure shown in formula (III) was obtained by separation:

[0054]

[0055] (2) The intermediate of the compound having the structure shown in formula (III) was dissolved in ethanol, and the compound having the structure shown in formula (IV) and the catalyst KOH were added to react. After the reaction was completed, dilute hydrochloric acid was added to adjust the pH to neutral, and the compound having the structure shown in formula (I) was separated, which is the drug-resistant urinary protein fluorescent probe:

[0056]

[0057] In one or more embodiments, the molar ratio of the compound having the structure shown in formula (II) in step (1), acrolein, and the catalyst K2CO3 is 1:1:2.

[0058] In one or more embodiments, the heating temperature in step (1) is 80°C and the heating time is 8 hours.

[0059] In one or more embodiments, the separation step in step (1) is as follows: after the reaction is completed, the reaction solution is naturally cooled to room temperature, a yellow solid is precipitated in the solution, the solid is filtered out and dried to obtain a compound intermediate having the structure shown in formula (Ⅲ).

[0060] In one or more embodiments, the molar ratio of the compound having the structure shown in formula (III), the compound having the structure shown in formula (IV), and the catalyst KOH in step (2) is 1:1.2:2.

[0061] In one or more embodiments, the reaction time described in step (2) is 8 hours.

[0062] In one or more embodiments, the separation step in step (2) is as follows: after concentrating the solution adjusted to neutral, extracting it with dichloromethane to obtain an organic phase, and then separating it by silica gel column chromatography to obtain a compound with the structure shown in formula (I), which is the anti-drug interference urinary protein fluorescent probe.

[0063] The present invention also includes the application of the drug-resistant urinary protein fluorescent probe in the preparation of a point-of-care rapid urinary protein detection kit.

[0064] Based on the drug-resistant fluorescent probe for urinary protein and its applications as described above, this invention also includes a method for rapid detection of urinary protein, comprising the following steps:

[0065] (1) Prepare a portable test strip by impregnating filter paper with any of the drug-resistant urinary protein fluorescent probes described in this invention.

[0066] (2) Take random urine from healthy individuals and divide it into n groups. Add protein stock solutions with different concentration gradients to the n groups of urine to obtain n groups of samples with different urinary protein concentrations. Add the same volume of the n groups of samples to the test strip.

[0067] (3) Calculate the color difference of each group based on the luminescent photographs of each group of samples on the test strip, establish a standard curve with the color difference of each group and the corresponding urinary protein concentration, and calculate the urinary protein content in the urine to be tested based on the standard curve.

[0068] In one or more embodiments, the step of calculating the color difference of each group based on the emission photographs of each group of samples specifically includes: determining the tri-color values ​​and color coordinates of the emission color based on the emission photographs of the two samples in each group, and calculating the color difference of each group based on the tri-color values ​​and color coordinates of each group of samples.

[0069] In one or more embodiments, the step of calculating the color difference of each group based on the luminescence photographs of each group of samples can be achieved by directly taking photos of each group of samples with a mobile phone and quickly analyzing the results such as color difference (ΔE*) and color coordinate ratio (R / B) on a mobile APP, thereby realizing semi-quantitative bedside rapid detection of urinary protein.

[0070] In one or more embodiments, the drug-resistant urinary protein fluorescent probe of the present invention is dissolved in an organic solvent, which may be dimethyl sulfoxide, dichloromethane, ethanol, dimethylformamide, ethyl acetate, methanol, acetonitrile, etc.

[0071] In one or more embodiments, the concentration of the drug-resistant urinary protein fluorescent probe of the present invention dissolved in the above-mentioned solvent is 1-100 mM.

[0072] In one or more embodiments, the filter paper material used to prepare the portable test strip is quantitative filter paper, qualitative filter paper, glass fiber filter paper, or other types of filter paper material.

[0073] In one or more embodiments, in the preparation of the portable test strip, the filter paper is impregnated with any of the drug-resistant urinary protein fluorescent probes of the present invention for 5 to 30 minutes, and then air-dried or freeze-dried.

[0074] It should be noted that when using the portable test strips or other kits containing the drug-interference-resistant urine protein fluorescent probes of this invention to detect urine protein, the results may not be extremely accurate due to various limitations. However, under the testing conditions of this invention, the probe can distinguish between healthy urine (ALB < 30 mg / L), microalbuminuria (30–300 mg / L), and macroalbuminuria (> 300 mg / L), and these results already meet the requirements for rapid point-of-care urine protein detection.

[0075] Example

[0076] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0077] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention.

[0078] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0079] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0080] The examples involve the addition amount, content and concentration of various substances, and unless otherwise specified, the percentage content refers to the mass percentage content.

[0081] Examples 1-6: Preparation of fluorescent probes C-1 to C-6

[0082] Compound 1 (molar ratio 1:1) and acrolein were dissolved in 1,4-dioxane. Twice the amount of K₂CO₃ was added as a base catalyst, and the mixture was heated to 80°C for 8 hours. After cooling and filtration, a yellow intermediate 2 was obtained. Intermediate 2 (1 equiv.) was dissolved in ethanol, and compounds A-1 to A-6 (1.2 equiv.) and KOH (2 equiv.) were added. The reaction was carried out for 8 hours. The pH was adjusted to neutral with dilute hydrochloric acid. The solution was concentrated, and the organic phase was extracted with dichloromethane. Separation by silica gel column chromatography yielded red compounds C-1 to C-6.

[0083] The final products were analyzed using nuclear magnetic resonance spectroscopy. C-1: MS(ESI, m / z): 305.14; C-2: MS(ESI, m / z): 319.15; C-3: MS(ESI, m / z): 323.13; C-4: MS(ESI, m / z): 335.15; C-5: MS(ESI, m / z): 335.15; C-6: MS(ESI, m / z): 355.15.

[0084]

[0085] Examples 7-12: Preparation of fluorescent probes C-7 to C-12

[0086] Compound 3 (molar ratio 1:1) and acrolein were dissolved in 1,4-dioxane. Twice the amount of K₂CO₃ was added as a base catalyst, and the mixture was heated to 80°C for 8 hours. After cooling and filtration, a yellow intermediate 4 was obtained. 4 (1 equiv.) was dissolved in ethanol, and A-7 to A-12 (1.2 equiv.) and KOH (2 equiv.) were added. The reaction was carried out for 8 hours. The pH was adjusted to neutral with dilute hydrochloric acid. The solution was concentrated, and the organic phase was extracted with dichloromethane. Separation by silica gel column chromatography yielded red compounds C-7 to C-12.

[0087] The final products were analyzed using nuclear magnetic resonance spectroscopy, and the results are as follows: C-7: MS(ESI,m / z): 399.14; C-8: MS(ESI,m / z): 365.12; C-9: MS(ESI,m / z): 397.17; C-10: MS(ESI,m / z): 469.29; C-5: MS(ESI,m / z): 371.15; C-6: MS(ESI,m / z): 388.12.

[0088]

[0089] Example 3: Testing the fluorescence performance of the fluorescent probe C-6

[0090] When albumin was added to the urine of three healthy adult volunteers, the fluorescence intensity of C-6 showed a trend of increasing with increasing albumin concentration. Figure 1 a) and individual differences are small ( Figure 1 b), Figure 1 The detection limit calculated by fitting in c is lower than the clinically determined proteinuria threshold (30 mg / L). Figure 1 The results showed that adding commonly used medications (ferulic acid, irbesartan, valsartan, sulfamethoxazole, naproxen sodium, tolbutamide) and their metabolites (VA, SA, o-DN, 4-HT) to urine did not affect the enhanced fluorescence response of C-6 to albumin. In the commercial drug test, only naproxen sodium tablets (NST) caused a certain degree of decrease in the enhancement fold. Further randomization of urine samples (albumin + drug + generic) in healthy volunteers was conducted to simulate the urine of CKD patients at different stages and taking different medications. Figure 1 The results in sample e showed that the fluorescence intensity signal was highly correlated with albumin concentration in all four urine groups. Figure 1 The results showed that C-6 exhibited high accuracy in all four groups of randomly spiked urine samples.

[0091] Example 4: Preparation of portable test strips

[0092] Methods for preparing portable test strips, such as Figure 2 As shown in (a) and (b), the steps include:

[0093] (1) The C-6 probe prepared in Example 1 was dissolved in PBS solvent to make its concentrations of 1 μM, 5 μM, 10 μM and 20 μM;

[0094] (2) Soak the filter paper in the above solution for 5 to 30 minutes, and then let it air dry naturally.

[0095] (3) Use a glass slide or plastic plate as the base plate, lay filter paper loaded with C-6 probe on it, and cover the surface of the filter paper with a plastic film material with multiple circular holes to complete the preparation of the portable test paper; when using the test paper, simply drop the urine sample onto the circular holes.

[0096] Example 5: Urine protein detection using portable test strips

[0097] The portable test strip prepared in Example 4 was used for rapid detection of urinary protein. Figure 2The results showed that after adding urine samples from three healthy adult volunteers, spiked with different concentrations of ALB (0 μM, 0.45 μM, 2 μM, 4.5 μM, 20 μM, and 45 μM), to the test strip, the fluorescent color of the strip exhibited a visually perceptible color change (blue-pink). Further analysis of color difference (ΔE*) and color coordinate ratio (R / B) using a mobile app (such as the "Color Recognizer" app) enables rapid, semi-quantitative, point-of-care detection of urinary protein.

[0098] According to the standards issued by Kidney Disease Improving Global Outcomes (KDIGO), a urine ALB concentration of more than 30 mg / L and 300 mg / L is defined as microalbuminuria (A2 level) and macroalbuminuria (A3 level), respectively. Figure 2 The concentration units in (c) are in μM, where 0.45 μM corresponds to 30 mg / L and 4.5 μM corresponds to 300 mg / L.

[0099] according to Figure 2 Data from (d) and (e) show that the probe can distinguish between healthy urine (ALB < 30 mg / L), microalbuminuria (30–300 mg / L), and macroalbuminuria (> 300 mg / L), thus the results meet the requirements for rapid bedside detection of urinary protein.

Claims

1. A drug-resistant urinary protein fluorescent probe, characterized in that, It has the structure shown in equation (Ⅰ): ； Where R1 is an aromatic ring group, selected from the following structures: ； Where R2 is a nitrogen substituent, selected from the following structures: 。 2. The urinary protein fluorescent probe against drug interference as described in claim 1, characterized in that, Selected from at least one of the compounds having the chemical formula shown below: 。 3. The method for preparing the drug-resistant urinary protein fluorescent probe according to claim 1 or 2, characterized in that, The preparation method includes the following steps: (1) The compound having the structure shown in formula (II) and acrolein were dissolved in 1,4-dioxane, K2CO3 was added as a catalyst, and the reaction was carried out by heating. After the reaction was completed, the intermediate compound having the structure shown in formula (III) was obtained by separation: ； (2) The intermediate compound having the structure shown in formula (III) is dissolved in ethanol, and the compound having the structure shown in formula (IV) and the catalyst KOH are added to react. After the reaction is completed, dilute hydrochloric acid is added to adjust the pH to neutral, and the compound having the structure shown in formula (I) is separated, which is the drug-resistant urinary protein fluorescent probe: 。 4. The preparation method according to claim 3, characterized in that, The molar ratio of the compound having the structure shown in formula (II) in step (1), acrolein, and the catalyst K2CO3 is 1:1:

2.

5. The preparation method according to claim 3, characterized in that, The heating temperature in step (1) is 80°C and the heating time is 8 hours.

6. The preparation method according to claim 3, characterized in that, The separation step described in step (1) is as follows: after the reaction is completed, the reaction solution is naturally cooled to room temperature, a yellow solid is precipitated in the solution, the solid is filtered out and dried to obtain a compound intermediate with the structure shown in formula (Ⅲ).

7. The preparation method according to claim 3, characterized in that, The molar ratio of the compound having the structure shown in formula (III), the compound having the structure shown in formula (IV), and the catalyst KOH in step (2) is 1:1.2:

2.

8. The preparation method according to claim 3, characterized in that, The reaction time described in step (2) is 8 hours.

9. The preparation method according to claim 3, characterized in that, The separation step in step (2) is as follows: after the solution is adjusted to neutral, it is concentrated and extracted with dichloromethane to obtain an organic phase, and then separated by silica gel column chromatography to obtain a compound with the structure shown in formula (I), which is the anti-drug interference urinary protein fluorescent probe.

10. The use of the drug-resistant urinary protein fluorescent probe according to claim 1 or 2 in the preparation of a point-of-care rapid detection kit / test strip for urinary protein.

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