Hypochlorite-responsive theranostic prodrug and its application in the detection of myeloperoxidase and drug release in acute myeloid leukemia

Abstract

The present invention discloses a fluorescent diagnostic and therapeutic prodrug and its application in the selective detection and drug release of acute myeloid leukemia (AML) cells with high expression of myeloperoxidase (MPO). The fluorescent diagnostic and therapeutic prodrug FNC constructed by the present invention can selectively detect ClO in AML cells with high MPO expression ‑ . This fluorescent prodrug can be used to identify AML cells with high MPO expression and perform selective drug release. FNC can also be used as a cell differentiation indicator.

Description

Technical Field

[0001] The present invention belongs to the field of biological detection, and particularly relates to a fluorescent diagnostic and therapeutic prodrug and its application in selectively detecting acute myeloid leukemia (AML) with high expression of myeloperoxidase (MPO) and drug release. Background Art

[0002] Acute myeloid leukemia (AML) is a heterogeneous hematological disease with poor prognosis. Immature bone marrow cells proliferate without restriction and differentiation is blocked. Chemotherapy is the main treatment method for acute myeloid leukemia, but it has side effects, including infection, recurrence, and chemotherapy resistance. Diagnostic and therapeutic prodrugs are an important method for precision medicine, promising to improve drug delivery, reduce the toxicity of chemotherapy, and make drugs more targeted. Developing diagnostic and therapeutic drugs is an attractive strategy for treating AML.

[0003] Myeloperoxidase (MPO) is a marker enzyme of myeloid cells, and the level of cellular MPO affects the choice of chemotherapy drugs and treatment duration. The percentage of MPO-positive blasts is an important diagnostic and prognostic factor for AML. Promoting differentiation is an effective treatment method for AML-M3 (accounting for 10-15% of all AML), which differentiates promyelocytes into mature granulocytes and is prone to apoptosis, during which the expression of MPO is significantly reduced. The commonly used methods for clinical diagnosis are cytochemistry (CC) and flow cytometry (FC) to detect MPO. Cytochemical staining is a rapid and inexpensive method, but the results may be interfered by other peroxidases and certain hemoproteins, and MPO staining will interfere with the identification of cell structure due to the coverage of positive granules. Flow cytometry has high specificity and sensitivity, but the technical steps are complex and the cost is expensive. Since MPO is the only enzyme in cells that catalyzes the reaction of H2O2 and Cl - to generate OCl - , the activity of MPO can be detected by detecting OCl - . Fluorescent probes have the characteristics of intuitive perception, no radiation, real-time analysis, low cost, and simple operation, and are powerful tools for detecting important bioactive substances, and fluorescence methods for detecting MPO can be developed.

[0004] Studies have shown that AML with high MPO expression is resistant to certain drugs such as cytarabine (AraC). To improve the treatment of AML, it is necessary to develop specific prodrugs for AML with high MPO expression. In recent years, some prodrugs and probes responsive to HOCl or ROS have been developed for detection and drug release in tumor or leukemia cells. Considering that activated immune cells can also produce a large amount of HOCl in inflammatory conditions, the key is to make the diagnostic and therapeutic prodrugs unresponsive to HOCl in inflammatory cells. The inflammatory cell environment is acidic, and prodrugs can thus be designed.

[0005] Differentiation is a method for the treatment of leukemia. After drug-induced granulocytic or macrophage differentiation of HL-60, the expression of MPO decreases. Detecting the differentiation of HL-60 can measure the efficacy of drugs. Currently, there is no fluorescent probe for detecting the differentiation of AML cells with high expression of MPO.

[0006] Therefore, it is necessary to design a hypochlorite-responsive fluorescent diagnosis and treatment prodrug in this application, and to propose a simple and rapid method to identify AML cells with high expression of MPO and perform selective drug release treatment. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide a new fluorescent diagnosis and treatment prodrug FNC to achieve efficient, rapid recognition and selective drug release of AML cells with high expression of MPO; and to develop a method for detecting the differentiation of HL-60.

[0008] The technical solution of the present invention is: a compound shown in formula (I), the compound FNC uses fluorescein as the fluorescent group, hydrazide as the ClO - recognition group, and chlorambucil as the therapeutic drug.

[0009]

[0010] A kit containing the above-mentioned compound.

[0011] Use of the above-mentioned compound in the preparation of detection and treatment reagents for AML cells with high expression of MPO.

[0012] Use of the above-mentioned compound in the preparation of an AML cell differentiation indicator.

[0013] Compared with the prior art, the present invention has the following beneficial effects:

[0014] The fluorescent diagnosis and treatment prodrug FNC constructed by the present invention can sensitively, specifically and rapidly detect ClO in AML cells with high expression of MPO - . This fluorescent prodrug can quickly distinguish AML cells with high MPO expression from other cells and perform selective drug release. FNC can also be used as a differentiation indicator. Brief Description of the Drawings

[0015] Figure 1 It is a schematic diagram of the synthesis route and response mechanism of FNC;

[0016] Figure 2 It is a schematic diagram of the response mechanism of FNC to AML with high MPO expression;

[0017] Figure 3 It is the spectral performance analysis of the response of FNC to ClO - response;

[0018] Figure 4 Selectivity analysis of FNC for ClO - ;

[0019] Figure 5 Effect of pH on the reaction of FNC with ClO - ;

[0020] Figure 6 Study on the reaction mechanism of FNC by liquid chromatography - mass spectrometry

[0021] Figure 7 Study on the drug release efficiency of FNC by liquid phase

[0022] Figure 8 Fluorescence imaging of ClO in HL - 60 cells by FNC - ;

[0023] Figure 9 Differentiation of AML cells with high MPO expression from other cells by FNC

[0024] Figure 10 Differentiation of AML cells with high MPO expression from other cells by H2O2 - assisted FNC

[0025] Figure 11 Detection of granulocytic or macrophage differentiation of HL - 60 by FNC

[0026] Figure 12 Cytotoxicity of FNC to various cells

[0027] Figure 13 Fluorescence imaging of FNC in various cells at high concentration for a long time

[0028] Figure 14 1H nuclear magnetic resonance spectrum for FNC structure confirmation ( 1 1H NMR);

[0029] Figure 15 13C nuclear magnetic resonance spectrum for FNC structure confirmation ( 13 13C NMR);

[0030] Figure 16 High - resolution mass spectrum for FNC structure confirmation (HRMS). Embodiments

[0031] In the following experimental methods of the embodiments, unless otherwise specified, they are all conventional methods. The test materials used in the following embodiments, unless otherwise specified, are all purchased from commercial channels.

[0032] Example 1 Synthesis of FNC

[0033] Synthesized through two - step organic reactions, and the synthesis route is asFigure 1 as shown in a of

[0034] (1)Synthesis of FluN

[0035] Fluorescein (1.241 g, 3.731 mmol) and hydrazine hydrate (2.10 mL, 43.3 mmol, 11.6 eq) were stirred in 60 mL of methanol overnight at 70 °C. After cooling to room temperature, the mixture was acidified with dilute hydrochloric acid. The yellow precipitate was collected and purified by silica gel column chromatography using dichloromethane / methanol (20 / 1, v / v) as the eluent to obtain the yellow solid FluN (0.8333 g, 2.408 mmol, yield 64.6%). 1 H NMR (400 MHz, DMSO- d 6 ) δ 9.84 (s, 2H), 7.81–7.73 (m, 1H),7.54–7.43 (m, 2H), 7.02–6.95 (m, 1H), 6.58 (d, J = 2.4 Hz, 2H), 6.48–6.35 (m,4H), 4.40 (s, 2H).

[0036] (2)Synthesis of FNC

[0037] Chlorambucil (0.7312 g, 2.405 mmol), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 0.5035 g, 2.626 mmol, 1.1 eq), 1-hydroxybenzotriazole (HOBt, 0.351 g, 2.60 mmol, 1.1 eq) and 4-dimethylaminopyridine (DIEA, 0.75 mL, 4.538 mmol, 1.9 eq) were dissolved in 30 mL of ultra-dry DMF and stirred at 0 °C under N2 for 1 h. Then FluN (0.8333 g, 2.408 mmol, 1eq) was added and the mixture was stirred overnight at room temperature. After removing the solvent in vacuo using a rotary evaporator, the residue was purified by silica gel column chromatography using dichloromethane / methanol (100 / 1, v / v) as the eluent to obtain the pale yellow solid FNC (0.435 g, 0.688 mmol, yield 28.6%). 1 H NMR (400 MHz, DMSO- d6): δ= 9.89 (s, 1H), 7.81 (dd, J = 5.8, 3.1 Hz, 1H), 7.51 (dd, J = 5.8, 3.1 Hz,2H), 7.11–6.97 (m, 4H), 6.77 (dd, J = 8.3, 2.3 Hz, 1H), 6.66 (dd, J = 15.2,6.6 Hz, 4H), 6.53–6.43 (m, 2H), 4.53 (s, 2H), 3.69 (s, 8H), 2.60–2.47 (m,4H), 1.88 (p, J = 7.5 Hz, 2H). 13 C NMR (101 MHz, DMSO- d 6): δ = 171.96, 166.30,158.95, 152.65, 152.45, 151.27, 145.11, 133.36, 129.98, 129.88, 129.63,129.26, 128.50, 123.97, 123.13, 118.04, 117.96, 113.01, 112.50, 110.70,102.97, 65.09, 52.79, 41.71, 33.76, 33.45, 26.92. MS ESI(N): m / z 630.1569 for[M - H] - , calc. for C 34 H 30 Cl2N3O5= 630.1568。

[0038] Example 2 Spectral performance analysis of FNC response to ClO -

[0039] FNC and ClO - were tested in PBS (0.01 M, pH = 7.4) buffer solution ( Figure 3 ). Figure 3 where a is the UV-visible absorption spectra of FNC before and 2 minutes after the reaction with ClO - . FNC itself has no UV absorption. When FNC (5 µ M) reacts with ClO - (500 µ M), an obvious UV absorption appears at 512 nm; Figure 3 where b is the change in fluorescence intensity of FNC reacting with different concentrations of ClO - for 2 minutes. The fluorescence of the FNC solution is very weak. When FNC (5​µ M) reacted with ClO at different concentrations - (2.5 μ M - 20 μ M), the probe solution emitted obvious green fluorescence, with the maximum fluorescence wavelength being 534 nm, and the fluorescence intensity increased continuously with the increase of the ClO - concentration. Compared with fluorescein, the ultraviolet absorption and fluorescence spectra were redshifted by about 22 - 23 nm, indicating that fluorescein had undergone a chlorination reaction; Figure 3 where c is the linear correlation analysis of the fluorescence intensity of FNC at 534 nm with the change of ClO - concentration. FNC (5 μ M) and ClO - showed a linear relationship in the concentration range of 2.5 - 15 μ M (R 2 = 0.9974), and the detection limit was calculated to be 23.6 nM (3σ / s, n = 11). Figure 3 where d is the kinetic experiment of the reaction between FNC and ClO - . The fluorescence change of the reaction between FNC (5 μ M) and ClO - (20 μ M) was monitored in real time. The fluorescence change was obvious after 30 seconds of reaction, and the fluorescence reached stability at 1 minute of reaction.

[0040] Example 3 Selectivity analysis of FNC for ClO -

[0041] By comparing the response performance of FNC to various reactive oxygen species, reactive nitrogen species, metal ions, Cys, and GSH, the selectivity of FNC for ClO - was analyzed. FNC (5 μ M) was reacted with ClO - (20 μ M) or 100 μ M of H2O2, ONOO − , ·NO, O2 ·− , OH, TBHP, Cys, GSH, Fe 3+ , Al 3+ , Cu 2+ respectively. Only the reaction between FNC and ClO - resulted in an obvious increase in the fluorescence signal, while there was no reaction with other substances ( Figure 4 ). Therefore, FNC has excellent selectivity for ClO - .

[0042] Example 4 Influence of pH on the reaction between FNC and ClO - ​​

[0043] FNC (5 μ M) and ClO - (20 μ M) were reacted in buffer solutions with different pH values (5.0, 6.0, 7.4, 8.0, 9.0) for 2 minutes, and the fluorescence signals at 534 nm were collected ( Figure 5 ). FNC and ClO - hardly reacted at pH 5. When the pH was 6, a weak reaction between FNC and ClO - began. When the pH was neutral or alkaline, the reaction between FNC and ClO - was good.

[0044] Example 5 Response mechanism and drug release efficiency of FNC and ClO - FNC (20 mM, 2.2 mL, DMF) was reacted with NaClO (20 mM, 13 mL, H2O) to isolate chlorambucil and the structure was confirmed by 1H NMR. The confirmed yield was 83.6%. The reaction of FNC (0.2 mM) and NaClO (4 mM) was carried out in ethanol, and dichlorofluorescein, tetrachlorofluorescein and chlorambucil (

[0045] ) were analyzed by liquid chromatography-high resolution mass spectrometry (LC-HRMS). A standard curve of chlorambucil standard was made by high performance liquid chromatography (HPLC). According to the peak area corresponding to chlorambucil in the reaction solution, the release efficiency of chlorambucil was calculated to be 91.6% ( Figure 6 ), indicating efficient drug release. Figure 7

[0046] Example 6 Intracellular endogenous OCl- imaging in HL-60 cells

[0047] We used AML cells HL-60 (AML-M3) with high expression of MPO as the object for confocal imaging. Incubating cells with FNC (10 μ M) for 30 minutes could detect the basal OCl - in HL-60 cells, generating green fluorescence. Pretreating cells with the MPO enzyme inhibitor flufenamic acid (FFA, 100 μ M, 4 h) could not observe fluorescence. Treating the cells pre-incubated with FNC with H2O2 (10 μ M) could observe obvious green fluorescence. Treating the cells pre-incubated with FNC and FFA with H2O2 (10 μ M) would not produce fluorescence either ( Figure 8 ). It was proved that FNC could detect the basal OCl - in AML cells with high MPO expression and its fluctuations.

[0048] Example 7. FNC differentiates AML cells with high MPO expression from other cells

[0049] Incubate cells with FNC (10 μ μM) for 30 minutes and perform confocal imaging. Green fluorescence can be seen in AML cells HL-60 and Kasumi-1 with high MPO expression (AML-M 2b ), while no fluorescence is observed in leukemia cells K562, TF-1, CCRF-CEM, RAW264.7 with low MPO expression, cancer cell HeLa, normal cells 293T, HCoEpiC, and the inflammatory cell model (RAW 264.7 stimulated with 1 μg / mL lipopolysaccharide for 16 h and 1 μg / mL phorbol ester for 1 h). Fluorescence quantitative statistics show that the fluorescence of HL-60 and Kasumi-1 is 4.46 - 13.7 times that of other cells (excluding inflammation) and more than 3 times that of inflammatory cells ( Figure 9 a, b). After adding H2O2 (20 μ μM) for 15 minutes, the fluorescence difference becomes more obvious, and the fluorescence of HL-60 and Kasumi-1 is 16.5 - 57.5 times that of other cells ( Figure 10 ). Co-culture K562 cells with Kasumi-1 cells. K562 cells are pre-stained with the DNA dye Hoechst-33342, and then co-cultured and treated with FNC together. The green fluorescence signal does not overlap with the blue fluorescence signal ( Figure 9 c). These results indicate that FNC can selectively image AML cells with high MPO expression through OCl - .

[0050] Example 8. FNC detects whether HL-60 differentiates into granulocytes or macrophages

[0051] Promoting granulocytic differentiation of cells with all-trans retinoic acid (ATRA) is an important method for treating AML-M3. After the granulocytic or macrophage differentiation of HL-60 cells, the MPO expression decreases. After promoting the granulocytic differentiation of HL-60 cells with ATRA (10 μ μM, 3 days) or promoting the macrophage differentiation of HL-60 cells with phorbol ester (16 nM, 2 days), and then incubating the cells with FNC (10 μ μM) for 30 minutes, no fluorescence can be detected ( Figure 11 ). Therefore, FNC can be used as a differentiation indicator for HL-60 cells to evaluate the drug efficacy. Given the potential role of directed cell differentiation in regeneration, this work may provide important information for clinical evaluation.

[0052] Example 9. Selective cytotoxicity generated by FNC drug release

[0053] The cytotoxicity study of FNC at different concentrations on various cells was carried out for 48 hours, and the cell viability was calculated by the CCK-8 method. Incubate various cells with 50 μM FNC for 48 hours, and the cell viabilities of HL-60 and Kasumi-1 cells are 35.4% and 34.2% respectively; the cell viabilities of K562, TF-1, RAW264.7, HeLa, 293T, and HCoEpiC cells are between 70.5% and 89.9% ( Figure 12 ). The results show that FNC has a highly selective prodrug effect in AML with high MPO expression, releasing drugs, while having less effect on other cells and reducing side effects. AML with high MPO expression is chemoresistant to AraC, while FNC has selective toxicity to such cells.

[0054] Example 10 Imaging of leukemia cells with high and low MPO expression by FNC at high concentration for a long time

[0055] Incubate HL-60, Kasumi-1, K562, and TF-1 cells with 50 μM FNC for 48 hours. Confocal imaging shows that obvious fluorescence can be observed in the first two cells, while no fluorescence is observed in the latter two cells ( Figure 13 ), indicating that leukemia cells with low MPO expression at high concentration for a long time still cannot interact with FNC to produce fluorescence.

Claims

1. The compound shown in formula (I): (I).

2. A kit containing the compound according to claim 1.

3. Use of the compound according to claim 1 in the preparation of a hypochlorous acid detection reagent.

4. Use of the compound according to claim 1 in the preparation of a reagent for detecting and treating AML cells with high MPO expression.

5. Use of the compound according to claim 1 in the preparation of an HL-60 cell differentiation indicator.

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