Medical examination fluorescent molecular probe and preparation method thereof
The preparation of flavin compounds by modifying 3,4-difluorobenzaldehyde has solved the problems of low sensitivity and poor selectivity of existing molecular probes, and improved the sensitivity and selectivity of fluorescent molecular probes.
Patent Information
- Application Number
- CN202510731222.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-03
- Publication Date
- 2025-09-02
AI Technical Summary
Existing molecular probes have low sensitivity and poor selectivity.
3,4-difluorobenzaldehyde is prepared by benzylamine modification, 3,4-dibenzaldehyde is then reacted with 1,4-dimethylpyridine iodide and piperidine, and after ion exchange, it is bound to 1-isopropylpyrimidine-2,4,6(1H,3H,5H)-trione to form flavin compounds, which enhances the steric hindrance and selectivity of the molecular probe.
A fluorescent molecular probe with high sensitivity and anti-interference ability has been achieved, which significantly improves the selectivity of the target object.
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Figure CN120574232A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of medical testing technology, and specifically relates to a medical testing fluorescent molecular probe and a preparation method thereof. Background Art
[0002] Fluorescent probes are sensors that selectively identify specific target molecules because of their strong selectivity, high sensitivity, no need for complex instruments and operations, low experimental costs, ability to work in complex environments, and non-contact detection. Fluorescent probes react with target analytes to cause changes in the fluorescence spectrum, and determine the analyte substrate based on the interaction of the reaction and the resulting spectral changes. Ordinary molecular fluorescent probes contain three components: fluorescent groups, linkers, and recognition groups. Fluorescent groups are reporters that convert recognition information into fluorescent signals. Recognition groups are groups that can bind to specific objects or trigger special chemical reactions. Linkers connect recognition groups and fluorescent groups. The fluorescence inner filter effect refers to the phenomenon that a substance in the reaction system absorbs the specific excitation light or emission light of the fluorescent substance, resulting in a decrease in the fluorescence emission intensity.
[0003] The existing technologies currently have the following main problems: the molecular probes currently used have low sensitivity and poor selectivity. Summary of the Invention
[0004] In view of the above situation, in order to overcome the defects of the prior art, the present invention provides a medical test fluorescent molecular probe and a preparation method thereof. In order to solve the molecular probe problem, the present invention proposes to modify 3,4-difluorobenzaldehyde by benzylamine to achieve the technical effect of improving steric hindrance and thus improving selectivity. At the same time, 1-isopropylpyrimidine-2,4,6(1H,3H,5H)-trione is modified and flavin compounds are synthesized to obtain a highly sensitive anti-interference fluorescent molecular probe.
[0005] In order to achieve the above-mentioned purpose, the technical solution adopted by the present invention is as follows: The present invention proposes a medical test fluorescent molecular probe, which includes the following components in parts by weight: 17-19 parts of 3,4-difluorobenzaldehyde, 12-15 parts of benzylamine, 10-11 parts of piperidine, 24-26 parts of 1,4-lutidine iodide, 14-15 parts of 1-isopropylpyrimidine-2,4,6(1H,3H,5H)-trione and 6.2-7.1 parts of potassium fluoroborate.
[0006] The present invention provides a method for preparing a fluorescent molecular probe for medical testing, which specifically comprises the following steps:
[0007] S1. Add 3,4-difluorobenzaldehyde to acetonitrile, then add benzylamine, stir at room temperature for about 1 hour, stop the reaction, and remove the solvent under reduced pressure to obtain 3,4-dibenzylaminobenzaldehyde;
[0008] S2. Dissolve the 3,4-dibenzylaminobenzaldehyde obtained in S1 in anhydrous ethanol, then add 1,4-lutidine iodide and piperidine, and stir in an oil bath to obtain a reaction solution;
[0009] S3, the reaction solution obtained in S2 was cooled to room temperature, potassium fluoroborate was added and stirred for 2 h, ion exchange was performed, and then potassium fluoroborate was removed by passing through a diatomaceous earth column, recrystallized from ethanol, filtered, and dried to obtain an intermediate;
[0010] S4. 1,4-dioxane and deionized water were mixed at a volume ratio of 1:0.06 to obtain a mixed solution, selenium dioxide was added to the mixed solution in an amount of 0.22-0.26 g / mL, 1-isopropylpyrimidine-2,4,6(1H,3H,5H)-trione was added to the mixed solution in an amount of 0.34-0.38 g / mL, the mixture was heated at 75° C. with stirring for 4 hours, cooled, filtered, and the filtrate was evaporated to dryness to obtain a derivative;
[0011] S5. Add the derivative obtained in S4 to glacial acetic acid, and then add the intermediate obtained in S3 to obtain a mixed solution. Add boric acid to the mixed solution, stir in a water bath under argon protection, filter, and recrystallize with ethanol to obtain a fluorescent molecular probe for medical testing.
[0012] Preferably, in S1, the amount of 3,4-difluorobenzaldehyde added to acetonitrile is 0.05-0.06 g / mL.
[0013] Preferably, in S2, the amount of 3,4-dibenzylaminobenzaldehyde obtained in S1 added to anhydrous ethanol is 0.25-0.35 g / mL.
[0014] Preferably, in S2, the oil bath stirring temperature is 90-100°C, the speed is 40-50 rpm, and the time is 18-20 h.
[0015] Preferably, in S5, the amount of the derivative obtained in S4 added to glacial acetic acid is 0.01-0.015 g / mL.
[0016] Preferably, in S5, the amount of boric acid added to the mixed solution is 0.007-0.009 g / mL.
[0017] Preferably, in S5, the water bath stirring temperature is 40-50°C, the speed is 60-80 rpm, and the time is 10-12 h.
[0018] The beneficial effects achieved by the present invention are as follows: 3,4-difluorobenzaldehyde is modified using benzylamine to obtain 3,4-dibenzylaminobenzaldehyde, the steric hindrance of the molecular recognition site is improved by amino substitution, and a precursor with a benzylamino group is obtained; 3,4-dibenzylaminobenzaldehyde is then reacted with 1,4-dimethylpyridine iodide, and the iodide ion is removed by ion substitution to obtain an intermediate; 1-isopropylpyrimidine-2,4,6(1H,3H,5H)-trione is modified to obtain a four-oxopyrimidine compound, i.e., a derivative; and finally, the intermediate and the derivative are combined to obtain a flavin compound, i.e., a fluorescent molecular probe for medical testing, which has good fluorescence properties and selectivity. BRIEF DESCRIPTION OF THE DRAWINGS
[0019] Figure 1 The results of the interference test of Examples 1-3 and Comparative Examples 1-2 of the present invention are shown in FIG.
[0020] Figure 2 This is a flow chart of preparing 3,4-dibenzylaminobenzaldehyde in an embodiment of the present invention;
[0021] Figure 3 This is a flow chart of preparing intermediates in the embodiments of the present invention;
[0022] Figure 4 This is a flow chart of preparing fluorescent molecular probes for medical testing in an embodiment of the present invention.
[0023] The accompanying drawings are used to provide further understanding of the present invention and constitute a part of the specification. They are used to explain the present invention together with the embodiments of the present invention and do not constitute a limitation of the present invention. DETAILED DESCRIPTION
[0024] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments; based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.
[0025] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those familiar to those skilled in the art. Furthermore, any methods and materials similar or equivalent to those described herein can be applied to the present invention. The preferred embodiments and materials described herein are for illustrative purposes only and are not intended to limit the scope of this application.
[0026] The experimental methods in the following examples are conventional methods unless otherwise specified; the experimental materials used in the following examples are purchased from commercial channels unless otherwise specified.
[0027] Example 1
[0028] A fluorescent molecular probe for medical testing comprises the following components in parts by weight: 17 parts of 3,4-difluorobenzaldehyde, 12 parts of benzylamine, 10 parts of piperidine, 24 parts of 1,4-lutidine iodide, 14 parts of 1-isopropylpyrimidine-2,4,6(1H,3H,5H)-trione and 6.2 parts of potassium fluoroborate.
[0029] The present invention provides a method for preparing a fluorescent molecular probe for medical testing, which specifically comprises the following steps:
[0030] S1. Add 3,4-difluorobenzaldehyde in an amount of 0.05 g / mL to acetonitrile, then add benzylamine, stir at room temperature for about 1 hour, stop the reaction, and remove the solvent under reduced pressure to obtain 3,4-dibenzylaminobenzaldehyde;
[0031] S2. Dissolve the 3,4-dibenzylaminobenzaldehyde obtained in S1 in anhydrous ethanol at an addition amount of 0.25 g / mL, then add 1,4-lutidine iodide and piperidine, and stir in an oil bath at 90° C. and 40 rpm for 18 h to obtain a reaction solution;
[0032] S3, the reaction solution obtained in S2 was cooled to room temperature, potassium fluoroborate was added and stirred for 2 h, ion exchange was performed, and then potassium fluoroborate was removed by passing through a diatomaceous earth column, recrystallized from ethanol, filtered, and dried to obtain an intermediate;
[0033] S4. 1,4-dioxane and deionized water were mixed in a volume ratio of 1:0.06 to obtain a mixed solution, selenium dioxide was added to the mixed solution in an amount of 0.22 g / mL, 1-isopropylpyrimidine-2,4,6(1H,3H,5H)-trione was added to the mixed solution in an amount of 0.34 g / mL, and the mixture was heated at 75° C. with stirring for 4 hours. The mixture was cooled, filtered, and the filtrate was evaporated to dryness to obtain a derivative;
[0034] S5. Add the derivative obtained in S4 to glacial acetic acid at a dosage of 0.01 g / mL, then add the intermediate obtained in S3 to obtain a mixed solution, add boric acid at a dosage of 0.007 g / mL to the mixed solution, stir at 60 rpm in a 40°C water bath under argon protection for 10 h, filter, and recrystallize from ethanol to obtain a fluorescent molecular probe for medical testing.
[0035] Example 2
[0036] A fluorescent molecular probe for medical testing comprises the following components in parts by weight: 19 parts of 3,4-difluorobenzaldehyde, 15 parts of benzylamine, 11 parts of piperidine, 26 parts of 1,4-lutidine iodide, 15 parts of 1-isopropylpyrimidine-2,4,6(1H,3H,5H)-trione and 7.1 parts of potassium fluoroborate.
[0037] The present invention provides a method for preparing a fluorescent molecular probe for medical testing, which specifically comprises the following steps:
[0038] S1. Add 3,4-difluorobenzaldehyde in an amount of 0.06 g / mL to acetonitrile, then add benzylamine, stir at room temperature for about 1 hour, stop the reaction, and remove the solvent under reduced pressure to obtain 3,4-dibenzylaminobenzaldehyde;
[0039] S2. Dissolve 0.35 g / mL of 3,4-dibenzylaminobenzaldehyde obtained in S1 in anhydrous ethanol, then add 1,4-lutidine iodide and piperidine, and stir in an oil bath at 100° C. and 50 rpm for 20 h to obtain a reaction solution;
[0040] S3, the reaction solution obtained in S2 was cooled to room temperature, potassium fluoroborate was added and stirred for 2 h, ion exchange was performed, and then potassium fluoroborate was removed by passing through a diatomaceous earth column, recrystallized from ethanol, filtered, and dried to obtain an intermediate;
[0041] S4. 1,4-dioxane and deionized water were mixed in a volume ratio of 1:0.06 to obtain a mixed solution, selenium dioxide was added to the mixed solution in an amount of 0.26 g / mL, 1-isopropylpyrimidine-2,4,6(1H,3H,5H)-trione was added to the mixed solution in an amount of 0.38 g / mL, and the mixture was heated at 75° C. with stirring for 4 hours. The mixture was cooled, filtered, and the filtrate was evaporated to dryness to obtain a derivative;
[0042] S5. Add the derivative obtained in S4 to glacial acetic acid at a dosage of 0.015 g / mL, then add the intermediate obtained in S3 to obtain a mixed solution, add boric acid to the mixed solution at a dosage of 0.009 g / mL, and under argon protection, stir at 80 rpm in a 50°C water bath for 12 h, filter, and recrystallize from ethanol to obtain a fluorescent molecular probe for medical testing.
[0043] Example 3
[0044] A fluorescent molecular probe for medical testing comprises the following components in parts by weight: 18 parts of 3,4-difluorobenzaldehyde, 13.5 parts of benzylamine, 10.5 parts of piperidine, 25 parts of 1,4-dimethylpyridinium iodide, 14.5 parts of 1-isopropylpyrimidine-2,4,6(1H,3H,5H)-trione and 6.6 parts of potassium fluoroborate.
[0045] The present invention provides a method for preparing a fluorescent molecular probe for medical testing, which specifically comprises the following steps:
[0046] S1. Add 3,4-difluorobenzaldehyde in an amount of 0.055 g / mL to acetonitrile, then add benzylamine, stir at room temperature for about 1 hour, stop the reaction, and remove the solvent under reduced pressure to obtain 3,4-dibenzylaminobenzaldehyde;
[0047] S2. Dissolve the 3,4-dibenzylaminobenzaldehyde obtained in S1 in anhydrous ethanol at an addition amount of 0.3 g / mL, then add 1,4-lutidine iodide and piperidine, and stir in an oil bath at 95° C. and 45 rpm for 19 h to obtain a reaction solution;
[0048] S3, the reaction solution obtained in S2 was cooled to room temperature, potassium fluoroborate was added and stirred for 2 h, ion exchange was performed, and then potassium fluoroborate was removed by passing through a diatomaceous earth column, recrystallized from ethanol, filtered, and dried to obtain an intermediate;
[0049] S4. 1,4-dioxane and deionized water were mixed in a volume ratio of 1:0.06 to obtain a mixed solution, selenium dioxide was added to the mixed solution in an amount of 0.24 g / mL, 1-isopropylpyrimidine-2,4,6(1H,3H,5H)-trione was added to the mixed solution in an amount of 0.36 g / mL, and the mixture was heated at 75° C. with stirring for 4 hours. The mixture was cooled, filtered, and the filtrate was evaporated to dryness to obtain a derivative;
[0050] S5. Add the derivative obtained in S4 to glacial acetic acid at a dosage of 0.0125 g / mL, then add the intermediate obtained in S3 to obtain a mixed solution, add boric acid to the mixed solution at a dosage of 0.008 g / mL, under argon protection, stir at 70 rpm in a 45°C water bath for 11 h, filter, and recrystallize from ethanol to obtain a fluorescent molecular probe for medical testing.
[0051] Comparative Example 1
[0052] This comparative example provides a molecular probe, which differs from Example 1 only in that 3,4-difluorobenzaldehyde is modified with isopropylamine, and the remaining components and component contents are the same as those in Example 1.
[0053] Comparative Example 2
[0054] This comparative example provides a molecular probe, which differs from Example 1 only in that 3,4-diaminobenzaldehyde is used instead of 3,4-difluorobenzaldehyde, and the remaining components and component contents are the same as those in Example 1.
[0055] Experimental example
[0056] 1. Interference test
[0057] The fluorescent probes synthesized in Examples 1-3 and Comparative Examples 1-2 were dissolved in DMSO solvent to a concentration of 5 mmol / L, and the basic photophysical properties of the probe system were measured using a UV-visible spectrophotometer and a fluorescence spectrometer. VB1 was added to the system containing the probes of Examples 1-3 and Comparative Examples 1-2, followed by VB2, VB3, VB5, and VB6 as interfering substances. After shaking, the fluorescence spectra were immediately tested, and the maximum emission intensities before and after addition were recorded. The maximum occurrence intensity reduction rate was calculated using the following formula:
[0058] Maximum emission intensity reduction rate = (intensity before addition - maximum emission intensity after addition) / intensity before addition.
[0059] Figure 1 This is a graph showing the results of the interference test of Examples 1-3 and Comparative Examples 1-2 of the present invention; as shown in the figure, in the interference test, the maximum emission intensity reduction rates of Examples 1-3 and Comparative Examples 1-2 are 53.6%, 56.9%, 51.6% and 26.3%, 19.6%, respectively. The maximum emission intensity reduction rate of Example 1-3 is significantly higher than that of Comparative Example 1-2, indicating that the modification of the probe by benzylamine improves the anti-interference ability of the probe and improves the selectivity of the probe for VB1.
[0060] Figure 2 Flow chart of preparing 3,4-dibenzylaminobenzaldehyde in an embodiment of the present invention; as shown in the figure, 3,4-dibenzylaminobenzaldehyde is prepared from 3,4-difluorobenzaldehyde and benzylamine.
[0061] Figure 3 Flowchart for preparing an intermediate in an embodiment of the present invention; as shown in the figure, the intermediate is prepared from 3,4-dibenzylaminobenzaldehyde and 1,4-dimethylpyridine iodide.
[0062] Figure 4 This is a flow chart of preparing fluorescent molecular probes for medical testing according to an embodiment of the present invention. As shown in the figure, fluorescent molecular probes for medical testing are prepared from intermediates and derivatives.
[0063] While the embodiments of the present invention have been shown and described, it will be apparent to those skilled in the art that various changes, modifications, substitutions, and alterations can be made to the embodiments without departing from the principles and spirit of the invention.
[0064] The present invention and its embodiments are described above. Such description is not restrictive. The drawings show only one embodiment of the present invention, and actual applications are not limited thereto. In short, if a person skilled in the art is inspired by the above, and does not deviate from the purpose of the present invention, any method and embodiment similar to the technical solution without creative design shall fall within the scope of protection of the present invention.
Claims
1. A fluorescent molecular probe for medical testing, characterized in that , including the following components in parts by weight: 17-19 parts of 3,4-difluorobenzaldehyde, 12-15 parts of benzylamine, 10-11 parts of piperidine, 24-26 parts of 1,4-lutidine iodide, 14-15 parts of 1-isopropylpyrimidine-2,4,6(1H,3H,5H)-trione and 6.2-7.1 parts of potassium fluoroborate.
2. A method for preparing a fluorescent molecular probe for medical testing according to claim 1, characterized in that: The specific steps include: S1. Add 3,4-difluorobenzaldehyde to acetonitrile, then add benzylamine, stir at room temperature for about 1 hour, stop the reaction, and remove the solvent under reduced pressure to obtain 3,4-dibenzylaminobenzaldehyde; S2. Dissolve the 3,4-dibenzylaminobenzaldehyde obtained in S1 in anhydrous ethanol, then add 1,4-lutidine iodide and piperidine, and stir in an oil bath to obtain a reaction solution; S3, the reaction solution obtained in S2 was cooled to room temperature, potassium fluoroborate was added and stirred for 2 h, ion exchange was performed, and then potassium fluoroborate was removed by passing through a diatomaceous earth column, recrystallized from ethanol, filtered, and dried to obtain an intermediate; S4. 1,4-dioxane and deionized water were mixed at a volume ratio of 1:0.06 to obtain a mixed solution, selenium dioxide was added to the mixed solution in an amount of 0.22-0.26 g / mL, 1-isopropylpyrimidine-2,4,6(1H,3H,5H)-trione was added to the mixed solution in an amount of 0.34-0.38 g / mL, the mixture was heated at 75° C. with stirring for 4 hours, cooled, filtered, and the filtrate was evaporated to dryness to obtain a derivative; S5. Add the derivative obtained in S4 to glacial acetic acid, and then add the intermediate obtained in S3 to obtain a mixed solution. Add boric acid to the mixed solution, stir in a water bath under argon protection, filter, and recrystallize with ethanol to obtain a fluorescent molecular probe for medical testing.
3. The method for preparing a fluorescent molecular probe for medical testing according to claim 2, wherein: In S1, the amount of 3,4-difluorobenzaldehyde added to acetonitrile was 0.05-0.06 g / mL.
4. The method for preparing a fluorescent molecular probe for medical testing according to claim 3, wherein: In S2, the amount of 3,4-dibenzylaminobenzaldehyde obtained in S1 added to anhydrous ethanol is 0.25-0.35 g / mL.
5. The method for preparing a fluorescent molecular probe for medical testing according to claim 4, wherein: In S2, the oil bath stirring temperature is 90-100°C, the speed is 40-50 rpm, and the time is 18-20 h.
6. The method for preparing a fluorescent molecular probe for medical testing according to claim 5, wherein: In S5, the derivative obtained in S4 is added to glacial acetic acid in an amount of 0.01-0.015 g / mL.
7. The method for preparing a fluorescent molecular probe for medical testing according to claim 6, wherein: In S5, the amount of boric acid added to the mixed solution is 0.007-0.009 g / mL.
8. The method for preparing a fluorescent molecular probe for medical testing according to claim 7, wherein: In S5, the water bath stirring temperature is 40-50°C, the speed is 60-80 rpm, and the time is 10-12 h.