CN114656450A - Preparation method and application of N ^ N ^ N ligand with ultraviolet-visible absorption and fluorescence luminescence characteristics - Google Patents

Abstract

The invention belongs to the technical field of photodynamic therapy photosensitizers, and in particular relates to a preparation method and application of a N^N^N ligand with ultraviolet-visible absorption and fluorescence emission characteristics. The N^N^N ligand disclosed in the invention is composed of 2 ,2′:6′,2″-terpyridine-4′-carboxaldehyde and 1,2,3,3-tetramethyl-3H-indolium iodide or 1-ethyl-2,3,3-tri Methyl-3H-indole-1-onium iodide or 1-ethyl iodide 2,3,3-trimethylbenzindole is prepared after reaction. Compared with traditional N^N^N ligands , has better ultraviolet-visible absorption and better fluorescence luminescence ability, and is used for the synthesis of metal complex photosensitizers, which can effectively improve the optical properties of photosensitizers, thereby improving its photodynamic therapy effect; at the same time, the N^N of the present invention ^N ligand has strong growth inhibitory ability for common human cervical cancer cell lines, which is helpful for the development of highly effective anticancer drugs.

Description

A kind of preparation method and application of N^N^N ligand with ultraviolet-visible absorption and fluorescence emission characteristics

technical field

The invention belongs to the technical field of photodynamic therapy photosensitizers, and in particular relates to a preparation method and application of a N^N^N ligand with ultraviolet-visible absorption and fluorescence emission characteristics.

Background technique

Cancer is one of the deadliest diseases in the world. The current traditional treatment methods for cancer mainly include surgical resection, chemotherapy and radiation therapy. Compared with traditional methods, photodynamic therapy (PDT), as a new type of treatment, has the advantages of non-invasiveness, accurate spatiotemporal manipulation, etc., which has attracted widespread attention. Ideally, photosensitizers (PS) have negligible toxicity in the dark, but can be activated by light irradiation of specific wavelengths to generate cytotoxic reactive oxygen species (ROS). ROS can rapidly destroy nearby biomolecules (proteins, lipids, or nucleic acids, etc.), eventually leading to cell death. At present, although a variety of different PSs have been reported, such as porphyrin, BODIPY and cyanine dyes, etc. However, most PSs suffer from poor water solubility and easy photobleaching, which impair their efficacy in cancer PDT therapy. As an alternative, metal complexes such as ruthenium (Ru) and iridium (Ir) complexes are considered as promising therapeutic agents, showing very attractive photophysical properties such as high water solubility, good photostability and high ROS production capacity. Therefore, the use of metal complexes for PDT has attracted great interest. It is worth mentioning that the complex TLD-1433 as PS for the treatment of bladder cancer with PDT has completed a phase II clinical trial, which has greatly increased the enthusiasm for the research and development of new metal complexes in PDT.

An ideal PS must possess strong visible light absorption, efficient intersystem crossover (ISC) efficiency, high ROS production, deep tissue permeability, and long-wavelength excitation performance. Among them, long-wavelength excitation can minimize the light damage to normal tissues, and obtain better tissue penetration and excellent spatial resolution. Therefore, long-wavelength excitation of PS is crucial to ensure effective therapeutic effects. Currently, a variety of metal complexes have been reported for photodynamic therapy, but most photosensitizers still require irradiation with short-wavelength excitation of ultraviolet or visible light to generate ROS, which is due to the depth of tissue penetration. problems, thus limiting the application of PS in deep tissue and solid tumors. To address the issue of tissue penetration depth, near-infrared (NIR) metal complex photosensitizers activated by a two-photon process have been developed, but their excitation efficiency is low and activation occurs only at the focus of high-intensity pulsed laser light. Therefore, two-photon absorption metal complex photosensitizers have certain drawbacks for PDT. It can be seen that the development of metal complexes that are directly activated at long wavelengths is promising for PDT.

Currently, conventional N^N^N ligands have poor photophysical and chemical properties (such as poor UV-vis absorption and fluorescence emission), which limit the application of N^N^N in PDT. The conjugated structure can effectively improve its photophysical and chemical properties; at the same time, by introducing transition metal atoms and ligands to form complexes, the ISC ability of the metal complex photosensitizer can be improved, and the separation of HOMO and LUMO can be effectively enhanced. Therefore, metal complex photosensitizers formed by rationally structurally modified N^N^N ligands and transition metals can clearly combine the advantages of transition metal complexes with the long-lived triple heavy metal ligand charge transfer ( ³ MLCT) states and The combination of the strong π-π* transitions of fluorescent molecules at long wavelengths, resulting in high ROS generation capacity and long-wavelength excitation, can significantly improve their PDT therapeutic effects. Therefore, it is necessary to develop new N^N^N ligands to improve the UV-vis absorption and fluorescence emission capabilities of N^N^N ligands to improve their effects in photodynamic therapy.

SUMMARY OF THE INVENTION

In order to overcome the above-mentioned deficiencies of the prior art, the primary purpose of the present invention is to provide a N^N^N ligand with ultraviolet-visible absorption and fluorescence emission characteristics.

The second object of the present invention is to provide a method for preparing the above-mentioned N^N^N ligands with ultraviolet-visible absorption and fluorescence emission characteristics.

The third object of the present invention is to provide the application of the above-mentioned N^N^N ligands with UV-Vis absorption and fluorescence emission characteristics.

The above-mentioned first purpose of the present invention is achieved through the following technical solutions:

A N^N^N ligand with ultraviolet-visible absorption and fluorescence emission characteristics, the structure of the N^N^N ligand is shown in formula I and/or formula II and/or formula III (formula I and/or formula III). Or the ligands shown in formula II and/or formula III are abbreviated as tpyCN1, tpyCN2, tpyPCN):

The above-mentioned second purpose of the present invention is achieved through the following technical solutions:

The preparation method of the above-mentioned N^N^N ligand with ultraviolet-visible absorption and fluorescence emission characteristics is specifically: by 2,2′:6′,2″-terpyridine-4′-carbaldehyde and 1,2,3, 3-Tetramethyl-3H-indolium iodide or 1-ethyl-2,3,3-trimethyl-3H-indole-1-onium iodide or 1-ethyl iodide 2,3, 3-trimethylbenzindole is prepared by reaction; the 2,2′:6′,2″-terpyridine-4′-carbaldehyde, 1,2,3,3-tetramethyl-3H- Indolium iodide, 1-ethyl-2,3,3-trimethyl-3H-indole-1-onium iodide, 1-ethyl 2,3,3-trimethylbenzidinium iodide The structures of inole are shown in formula (1), formula (2), formula (3), and formula (4) respectively:

Preferably, the reaction is a heating and refluxing reaction, the reaction time is 18-20 hours, and the reaction temperature is 70-90°C. Specifically, the reaction time was 19 hours, and the reaction temperature was 80°C.

Preferably, the reaction is carried out under an inert gas range.

Preferably, the 2,2':6',2"-terpyridine-4'-carbaldehyde is combined with 1,2,3,3-tetramethyl-3H-indolium iodide or 1-ethyl-2 , 3,3-trimethyl-3H-indole-1-onium iodide or 1-ethyl iodide 2,3,3-trimethylbenzindole in a molar ratio of 1:1.

Preferably, the solvent used in the reaction includes, but is not limited to, ethanol.

Preferably, the subsequent ethanol recrystallization purification and drying steps are also included.

The above-mentioned third purpose of the present invention is achieved through the following technical solutions:

Application of the above N^N^N ligands with UV-Vis absorption and fluorescence emission properties in the preparation of photodynamic therapy photosensitizers.

The application of the above-mentioned N^N^N ligands with UV-vis absorption and fluorescence emission properties in the preparation of anti-cervical cancer drugs.

Compared with the traditional N^N^N ligand, the N^N^N ligand of the present invention has better ultraviolet-visible absorption and fluorescence emission capabilities, and at the same time, the N^N^N ligand itself is applied to the treatment of cervical cancer and has high performance. It is of great significance for the study of high-efficiency metal complex photosensitizer anti-tumor drugs, which lays an experimental and theoretical foundation for the clinical development of new metal anti-tumor drugs.

The application of the above N^N^N ligand with UV-vis absorption and fluorescence emission properties in the preparation of a medicine for inhibiting the proliferation of cervical cancer cells.

Preferably, the cervical cancer cells include, but are not limited to, HeLa cells.

Studies have shown that the ligand has cytotoxicity to cervical cancer cell lines (HeLa cells) and is a promising and highly effective anticancer drug.

Compared with the prior art, the beneficial effects of the present invention are:

The invention discloses an N^N^N ligand with ultraviolet-visible absorption and fluorescence emission characteristics, which is composed of 2,2′:6′,2″-terpyridine-4′-carbaldehyde and 1,2,3,3 -Tetramethyl-3H-indolium iodide or 1-ethyl-2,3,3-trimethyl-3H-indole-1-onium iodide or 1-ethyl iodide 2,3,3 -Trimethylbenzindole is prepared after reaction. Compared with traditional N^N^N ligands, the N^N^N ligand pair of the present invention has better UV-vis absorption and better fluorescence emission It can be used in the synthesis of metal complex photosensitizers, which can effectively improve the optical properties of photosensitizers, thereby enhancing its photodynamic therapy effect. Cancer cell lines have strong growth-inhibiting ability, which contributes to the development of highly effective anticancer drugs.

Description of drawings

Figure 1 shows the UV-Vis absorption spectra of N^N^N ligands (tpyCN1 ligand, tpyCN2 ligand and tpyPCN ligand) in eight different solvents;

Figure 2 shows the fluorescence spectra of N^N^N ligands (tpyCN1 ligand, tpyCN2 ligand and tpyPCN ligand) in eight different solvents;

Figure 3 shows the cytotoxicity of N^N^N ligands (tpyCN1 ligand, tpyCN2 ligand and tpyPCN ligand) on cervical cancer cell lines (HeLa).

Detailed ways

The specific embodiments of the present invention will be further described below. It should be noted here that the descriptions of these embodiments are used to help the understanding of the present invention, but do not constitute a limitation of the present invention. In addition, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

The experimental methods in the following examples are conventional methods unless otherwise specified, and the experimental materials used in the following examples can be purchased through conventional commercial channels unless otherwise specified.

Example 1 Preparation of novel N^N^N ligands

(1) Synthesis method of tpyCN1 ligand

The structure of the tpyCN1 ligand is shown in formula I:

The synthesis of tpyCN1 ligands can be carried out according to the following reaction formula:

The specific synthesis method is: according to the above reaction formula, compound (1) 2,2':6',2"-terpyridine-4'-carbaldehyde (0.523g, 2mmol) and compound (2)1,2,3, The mixture of 3-tetramethyl-3H-indolium iodide (0.602 g, 2 mmol) was added to absolute ethanol (30 mL) and heated to 80°C, and the reaction was reduced to 19 hours in the dark under argon atmosphere. At room temperature, the precipitate was filtered and washed with ether. The obtained crude product was recrystallized from ethanol to obtain orange-red crystals. The obtained crystals were further dried to obtain 0.98 g of orange-red powder, namely tpyCN1 ligand. The yield of the ligand was 92%.

By characterization, the molecular formula of this ligand is C ₂₈ H ₂₅ IN ₄ , ESIm/z (C ₂₈ H ₂₅ N ₄ ⁺ ), calc: 417.54; found: 417. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 9. 09(s,2H),8.81(dt,J＝4.6,1.5Hz,2H),8.70(d,J＝1.1Hz,1H),8.68(d,J＝1.1Hz,1H),8.65(d,J = 16.6Hz, 1H), 8.13–8.04 (m, 3H), 8.06–7.98 (m, 1H), 7.98–7.90 (m, 1H), 7.75–7.64 (m, 2H), 7.59 (ddd, J=7.5 , 4.7, 1.2Hz, 2H), 4.31(s, 3H), 1.86(s, 6H).

(2) Synthesis method of tpyCN2 ligand

The structure of the tpyCN2 ligand is shown in formula II:

The synthesis of tpyCN2 ligands can be carried out according to the following reaction formula:

The specific synthesis method is: according to the above reaction formula, compound (1) 2,2':6',2"-terpyridine-4'-carbaldehyde (0.523g, 2mmol) and compound (3) 1-ethyl-2 , 3,3-trimethyl-3H-indole-1-onium iodide (0.630 g, 2 mmol) was added to anhydrous ethanol (30 mL) and heated to 80 ° C, and the reaction was carried out under argon atmosphere in the dark and refluxed for 19 After an hour, the reaction was lowered to room temperature, and the precipitate was filtered and washed with diethyl ether. The obtained crude product was recrystallized from ethanol to obtain orange-red crystals, and the obtained crystals were further dried to obtain 1.11 g of orange-red powder, namely tpyCN2 ligand. The rate was 95.3%.

By characterization, the molecular formula of this ligand is C ₂₉ H ₂₇ IN ₄ , ESIm/z (C ₂₉ H ₂₇ N ₄ ⁺ ), calc: 31.57; found: 431. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 9. 10(s, 2H), 8.82(d, J＝4.7Hz, 2H), 8.74-8.65(m, 3H), 8.09(dd, J＝18.4, 11.5Hz, 4H), 7.96(d, J＝6.4Hz ,1H),7.73–7.67(m,2H),7.58(t,J=6.2Hz,2H),4.90(q,J=7.5Hz,2H),1.88(s,6H),1.54(t,J= 7.2Hz, 3H).

(3) Synthesis method of tpyPCN ligand

The structure of the tpyPCN ligand is shown in formula III:

The synthesis of tpyPCN ligands can be carried out according to the following reaction formula:

The specific synthesis method is: according to the above reaction formula, compound (1) 2,2':6',2"-terpyridine-4'-carbaldehyde (0.523g, 2.5mmol) and compound (4) 1-ethyl iodide The mixture of 2,3,3-trimethylbenzindole (0.730 g, 2.5 mmol) was added to absolute ethanol (30 mL) and heated to 80°C, and the reaction was carried out under argon atmosphere in the dark under reflux for 19 hours. It was cooled to room temperature, and the precipitate was filtered and washed with ether. The obtained crude product was recrystallized from ethanol to obtain reddish-brown crystals. The obtained crystals were further dried to obtain 0.7040 g of reddish-brown powder, namely tpyPCN ligand, with a yield of 46%. .

By characterization, the molecular formula of this ligand is C ₃₃ H ₂₉ IN ₄ , ESIm/z (C ₃₃ H ₂₉ N ₄ ⁺ ), calc: 481.24; found: 481. ¹ H NMR (400MHz, DMSO-d ₆ ) δ 9. 14(s, 2H), 8.85(d, J＝4.8Hz, 2H), 8.77(d, J＝16.0Hz, 2H), 8.73(s, 1H), 8.50(d, J＝8.5Hz, 1H), 8.36(d,J＝8.9Hz,1H),8.25(dd,J＝15.1,8.6Hz,2H),8.19-8.11(m,3H),7.86(t,J＝7.6Hz,1H),7.79(t , J=7.5Hz, 1H), 7.68–7.60 (m, 2H), 5.02 (q, J=7.3Hz, 2H), 2.11 (s, 6H), 1.61 (t, J=7.2Hz, 3H).

Example 2 UV-Vis Absorption Spectroscopy Measurement Experiment of Novel N^N^N Ligands in Eight Different Solvents

The N^N^N ligands (tpyCN1 ligand, tpyCN2 ligand and tpyPCN ligand) of the present invention were analyzed by UV-Vis spectrophotometer in eight different solvents (DMSO, DMF, H ₂ O, CH ₃ OH, CH ₃ CN, EG, EA, CH ₂ Cl ₂ ) in UV-Vis absorption spectra. By comparing with the UV-Vis absorption spectra of the reference substance 2,2':6',2"-terpyridine (tpy) in eight different solvents, the absorption capacity of the ligand in the UV-visible region was analyzed.

Among them, the experimental steps for measuring the UV-Vis absorption spectrum are as follows:

1 Prepare 10mM tpy, tpyCN1, tpyCN2, tpyPCN solutions with DMSO solvent;

② The tpy, tpyCN1, tpyCN2, tpyPCN (10 mM) solutions were diluted into 10 μM solutions with eight different solvents (DMSO, DMF, H ₂ O, CH ₃ OH, CH ₃ CN, EG, EA, CH ₂ Cl ₂ ), Then, the absorption intensity in the ultraviolet-visible region was measured in the wavelength range of 250-700 nm on an ultraviolet spectrometer.

As shown in Figure 1, comparing the ultraviolet spectra of tpyCN1, tpyCN2, and tpyPCN ligands with the spectra of control tpy, it can be seen that compared with conventional tpyN^N^N ligands (tpy), the N of the present invention The ^N^N ligand has a wider absorption range and higher intensity in the ultraviolet-visible light region, which is more conducive to the synthesis of a complex with a wider absorption range as a photosensitizer and improves the absorption efficiency of the photosensitizer.

Example 3 Fluorescence spectrum measurement experiment of novel N^N^N ligands in eight different solvents

The N^N^N ligands (tpyCN1 ligand, tpyCN2 ligand and tpyPCN ligand) of the present invention were analyzed by spectrofluorophotometer in eight different solvents (DMSO, DMF, H ₂ O, CH ₃ OH, CH ₃ Fluorescence emission spectra in CN, EG, EA, _{CH2Cl2 )} . The fluorescence emission ability of the ligand was analyzed by comparing the fluorescence spectra of the reference substance 2,2′:6′,2″-terpyridine (tpy) in eight different solvents.

Among them, the steps of the fluorescence spectrum measurement experiment are as follows:

1 Prepare 10mM tpy, tpyCN1, tpyCN2, tpyPCN solutions with DMSO solvent;

② The tpy, tpyCN1, tpyCN2, tpyPCN (10 mM) solutions were diluted into 10 μM solutions with eight different solvents (DMSO, DMF, H ₂ O, CH ₃ OH, CH ₃ CN, EG, EA, CH ₂ Cl ₂ ), The fluorescence intensity was then measured on a fluorescence spectrometer in the wavelength range of 400-800 nm (λ _ex =365 nm).

As shown in Figure 2, comparing the fluorescence spectra of tpyCN1, tpyCN2, and tpyPCN ligands with the fluorescence spectra of control tpy, it can be seen that compared with the conventional N^N^N ligand (tpy), the N^N^N ligands have stronger fluorescence intensity, which is more conducive to the synthesis of complexes for use as photosensitizers.

Experimental Example 4 New N^N^N Ligand Pair in Chemotherapy of Human Cervical Cancer

The anti-proliferative effect of the N-N-N ligands (tpyCN1 ligand, tpyCN2 ligand and tpyPCN ligand) of the present invention on human cervical cancer (HeLa) cells was analyzed by MTT colorimetry, and 2,2': 6',2"-terpyridine (tpy) served as a control.

MTT, namely thiazole blue, is a tetrazolium salt. In living cells, succinate dehydrogenase in mitochondria can reduce MTT to form a blue-violet crystal - formazan (soluble in dimethyl sulfoxide). ), and the product has an absorption peak at 595nm, so an enzyme-linked immunosorbent assay can be used to analyze the proliferation of cells.

The steps of the MTT experiment are as follows:

①Cell recovery: Take the HeLa tumor cells cryopreserved in liquid nitrogen, wash the cryopreservation solution with PBS after rapid thawing, and then use fresh complete culture medium (DMEM medium + 10% fetal bovine serum + 1% penicillin-streptomycin mixture) ) were cultured and passaged, and MTT experiments were performed after 2 passages.

② Seed plate: When the cells reach the logarithmic growth phase, seed them into a 96-well plate at a cell density of 5,000 cells/well (100 μL of culture medium (fresh complete medium) is used in each well to culture cells), and then transfer to 37°C, Culture in a 5% CO ₂ incubator.

③Administration: After the cells adhered to the 96-well plate, absorb the DMSO to configure the ligands (tpyCN1 ligand, tpyCN2 ligand and tpyPCN ligand) stock solution, and dilute with medium to prepare the concentration gradient of 1000, 500, 100, 10, 01, 0.1 μM working solution, aspirate 10 μL of the original medium in the 96-well plate, then add 10 μL of the ligand working solution of 6 concentration gradients to the 96-well plate, and shake it gently to make the ligand. The final concentration gradients were 100, 50, 10, 1, 0.1, and 0.01 μM, respectively, followed by incubation in a carbon dioxide incubator for 48 h.

④ After 48 hours of incubation, add 10 μL MTT (5 mg/mL) to each well, continue to incubate for 4 hours in a constant temperature incubator at 37°C, then aspirate the supernatant, add 100 μL dimethyl sulfoxide (DMSO) to each well, and use enzyme The A _595nm was detected by an immunoassay instrument, and the cell proliferation inhibition rate was calculated to obtain the IC ₅₀ value.

As shown in Figure 3, compared with tpy ligands, the N^N^N ligands (tpyCN1 ligand, tpyCN2 ligand and tpyPCN ligand) of the present invention have better killing effect on human cervical cancer (HeLa cells), Among them, the chemotherapeutic effect of tpyCN1 (3 μM) is better than that of tpyCN2 (4.143 μM), and the chemotherapeutic effect of tpyCN2 (4.143 μM) is better than that of tpyPCN (12.9 μM), indicating that the ligand itself of the present invention has better effect on cervical cancer cells. The cytotoxic effect of this method has good application value for the development of new metal complex photosensitizers.

To sum up, the N^N^N ligands of the present invention (tpyCN1 ligand, tpyCN2 ligand and tpyPCN ligand) have better UV-vis absorption and fluorescence emission capabilities than traditional N^N^N ligands, At the same time, it has high efficacy in the treatment of cervical cancer (HeLa cells), which is of great significance for the study of high-efficiency metal complex photosensitizer anti-tumor drugs, and lays an experimental and theoretical basis for the clinical development of new metal anti-tumor drugs.

The embodiments of the present invention have been described above in detail, but the present invention is not limited to the described embodiments. For those skilled in the art, without departing from the principle and spirit of the present invention, various changes, modifications, substitutions and alterations to these embodiments still fall within the protection scope of the present invention.

Claims (10)

1. a N^N^N ligand with ultraviolet-visible absorption and fluorescence emission characteristics, it is characterized in that, the structure of described N^N^N ligand is as shown in formula I and/or formula II and/or formula III. Show:

2. the preparation method of the N^N^N ligand with ultraviolet-visible absorption and fluorescence emission characteristics according to claim 1, it is characterized in that, by 2,2':6',2"-terpyridine-4'- Formaldehyde with 1,2,3,3-tetramethyl-3H-indolium iodide or 1-ethyl-2,3,3-trimethyl-3H-indole-1-onium iodide or 1- Ethyl iodide 2,3,3-trimethylbenzindole is prepared by reaction; the 2,2′:6′,2″-terpyridine-4′-carbaldehyde, 1,2,3, 3-Tetramethyl-3H-indolium iodide, 1-ethyl-2,3,3-trimethyl-3H-indole-1-onium iodide, 1-ethyl iodide 2,3, The structures of 3-trimethylbenzindole are respectively shown in formula (1), formula (2), formula (3), and formula (4):

3. the preparation method of a kind of N^N^N ligand with ultraviolet-visible absorption and fluorescence emission characteristics according to claim 2, is characterized in that, described reaction is heating reflux reaction, and the time of reaction is 18～20 hours, the temperature of the reaction is 70-90°C. 4. the preparation method of a kind of N^N^N ligand with UV-Vis absorption and fluorescence emission characteristics according to claim 2, is characterized in that, described 2,2':6',2"-terpyridine -4′-Carboxaldehyde with 1,2,3,3-tetramethyl-3H-indolium iodide or 1-ethyl-2,3,3-trimethyl-3H-indole-1-onium iodide The molar ratio of 2,3,3-trimethylbenzindole iodide or 1-ethyl iodide was 1:1. 5. The preparation method of a N^N^N ligand with UV-Vis absorption and fluorescence emission characteristics according to claim 2, wherein the reaction is carried out in an inert gas range. 6. the preparation method of a kind of N^N^N ligand with UV-Vis absorption and fluorescence emission characteristics according to claim 2, is characterized in that, the solvent used in the reaction includes but is not limited to ethanol. 7. The application of the N^N^N ligand with ultraviolet-visible absorption and fluorescence emission characteristics according to claim 1 in the preparation of a photodynamic therapy photosensitizer. 8. The application of the N^N^N ligand with ultraviolet-visible absorption and fluorescence emission characteristics according to claim 1 in the preparation of an anti-cervical cancer drug. 9. The application of the N^N^N ligand with ultraviolet-visible absorption and fluorescence emission characteristics according to claim 1 in the preparation of a medicine for inhibiting the proliferation of cervical cancer cells. 10. The use according to claim 8, wherein the cervical cancer cells include but are not limited to HeLa cells.

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