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Fluorescent substrate for detecting human cytochrome P450 3A4 as well as preparation method and application of fluorescent substrate

A cytochrome and fluorescent substrate technology, applied in the field of medicine, can solve problems such as complex methods, and achieve the effects of high detection throughput, efficient and rapid real-time detection, and high-throughput detection.

Pending Publication Date: 2022-05-13
SHANGHAI UNIV OF T C M
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  • Description
  • Claims
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Problems solved by technology

[0005] Aiming at the above-mentioned technical problems in the prior art, the present invention provides a fluorescent substrate for detecting human cytochrome P450 3A4 (Cytochrome P450 3A4) and its preparation method and application. The fluorescent substrate of P450 3A4 (Cytochrome P450 3A4) and its preparation method and application need to solve the complicated technical problem of the method for detecting CYP3A4 enzyme biopsy in the prior art

Method used

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  • Fluorescent substrate for detecting human cytochrome P450 3A4 as well as preparation method and application of fluorescent substrate
  • Fluorescent substrate for detecting human cytochrome P450 3A4 as well as preparation method and application of fluorescent substrate
  • Fluorescent substrate for detecting human cytochrome P450 3A4 as well as preparation method and application of fluorescent substrate

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Embodiment 1 The chemical synthesis of N-fluorobenzyl-1,8-naphthalimide compounds

[0038] (1) Dissolve one equivalent of 1,8-naphthalene dicarboxylic anhydride in 20 mL of absolute ethanol, add 1.2 equivalents of 2-fluorobenzylamine / 3-fluorobenzylamine / 4-fluorobenzylamine, stir and slowly heat to Reflux, and continue for 3h;

[0039] (2) The reaction solution was naturally cooled to room temperature, filtered to obtain a white filter cake as a crude product, and purified by silica gel chromatography (eluent: dichloromethane) to obtain pure N-(2'-fluoro Benzyl)-1,8-naphthalimide (B-2, yield: 89.8%, see figure 2 a).

[0040] 1 H NMR (600MHz, CDCl 3 )δ8.63(d, J=7.3Hz, 2H), 8.23(d, J=8.2Hz, 2H), 7.77(t, J=7.7Hz, 2H), 7.30(t, J=7.5Hz, 1H) ,7.24–7.18(m,1H),7.10–6.99(m,2H),5.48(s, 2H). 13 C NMR (150MHz, CDCl 3 )δ 164.13, 161.60, 159.96, 134.17, 131.66, 131.58, 129.25, 129.22, 128.92, 128.86, 128.29, 127.01, 124.19, 124.09, 124.07, 124.05, 122.534, 137.5

[0041] Obtain...

Embodiment 2

[0045] Example 2 Metabolic phenotype analysis of the specificity of N-fluorobenzyl-1,8-naphthalimide substrates

[0046] (1) Prepare 180 μl phase I metabolic reaction system, including PBS buffer (100 mM) at pH 7.4, commercial human P450 enzyme (10 nM), B-1 (1 μM), B-2 (1 μM), B-3 ( 1 μM) at 37°C for 3 minutes with shaking;

[0047] (2) Add 20 μl of NADPH with a final concentration of 1 mM to the reaction system to initiate the reaction;

[0048] (3) After 30 minutes, add 100 μl of glacial acetonitrile, shake vigorously, terminate the reaction, centrifuge at 20,000 g for 30 minutes, and take the supernatant for testing;

[0049] (4) Detection of the fluorescent signal of the product (E x =450nm,E m =555nm); Obtain the fluorescence intensity of the product in each sample, B-2, B-3, the subtype selectivity of the same family of B-1 are respectively 3.6,7.9,72.6 times ( Figure 4 a, 4b, 4c).

[0050] Conclusion: Among the three fluorescent substrates, the B-1 product was for...

Embodiment 3

[0051] The enzymatic kinetic analysis of embodiment 3 substrate B-1

[0052] (1) Prepare 180 μl phase I metabolic reaction system, including PBS buffer (100mM) at pH 7.4, commercialized human CYP 3A4 (0.025nM) / commercialized human liver microsomes (0.005mg / ml), different concentrations of B -1 Pre-incubate with shaking at 37°C for 3 minutes;

[0053] (2) Add 20 μl of NADPH with a final concentration of 1 mM to the reaction system to initiate the reaction;

[0054] (3) After 20 minutes, add 100 μl of glacial acetonitrile, shake vigorously, terminate the reaction, centrifuge at 20,000 g for 30 minutes, and take the supernatant for testing;

[0055] (4) Detection of the fluorescent signal of the product (E x =450nm,E m =555nm); the fluorescence intensity of the product in each sample was obtained, and the kinetic curve of the enzymatic reaction was fitted. In both single enzyme and commercial human liver microsomes, Mie kinetics were exhibited, and K m Very close, respective...

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Abstract

The fluorescent substrate for detecting the human cytochrome P450 3A4 is an N-fluorobenzyl-1, 8-naphthalimide compound, and the structural general formula of the fluorescent substrate is as shown in the formula (1). The invention also provides a preparation method of the fluorescent substrate. The substrate disclosed by the invention is specifically catalyzed by CYP3A4 to generate C-4 hydroxylation reaction and generate a single 4-hydroxylation product, and the activity or residual activity of CYP3A4 in a biological sample can be quantitatively detected by detecting elimination of the substrate in unit time or the generation amount of the 4-hydroxylation product. A CYP3A4 activity detection method constructed by virtue of the fluorescent substrate can be used for evaluating the activity of CYP3A4 in products such as recombinant CYP enzyme and commercial cell / tissue preparations so as to carry out quality control on the products, and also can be used for screening and evaluating the regulation effect of compounds on CYP3A4.

Description

technical field [0001] The invention belongs to the technical field of medicine, and relates to a fluorescent substrate for detecting human cytochrome P450 3A4 (Cytochrome P450 3A4), a preparation method and application thereof. Background technique [0002] Cytochrome P450 3A (cytochrome P450 3A enzymes, CYP3A) is an important phase I oxidative metabolic enzyme in the body, which catalyzes the oxidative metabolism of various endogenous and exogenous compounds, and plays an important role in the process of drug metabolism clearance and metabolic activation. effect. According to statistics, about 50% of marketed drugs (such as paclitaxel, cisplatin, midazolam, etc.) can be metabolized by CYP3A. The CYP3A subfamily includes four members, namely CYP3A4, CYP3A5, CYP3A7 and CYP3A43. Among them, CYP3A4 is the most important. It is not only highly expressed in multiple organs, but also participates in the metabolic clearance and metabolic activation of many drugs, traditional Chi...

Claims

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Application Information

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IPC IPC(8): C07D221/14C09K11/06G01N21/64
CPCC07D221/14C09K11/06G01N21/6428
Inventor 葛广波何荣景田镇豪张凤
Owner SHANGHAI UNIV OF T C M
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