CN114773361B - N-heteronile erythronium salt compound and preparation method and application thereof - Google Patents
N-heteronile erythronium salt compound and preparation method and application thereof Download PDFInfo
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- -1 salt compound Chemical class 0.000 title claims abstract description 42
- 241000245089 Erythronium Species 0.000 title claims abstract description 27
- 238000002360 preparation method Methods 0.000 title claims abstract description 15
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- NXYYRSJPSPRDEO-UHFFFAOYSA-N 5-(diethylamino)-2-nitrosophenol Chemical compound CCN(CC)C1=CC=C(N=O)C(O)=C1 NXYYRSJPSPRDEO-UHFFFAOYSA-N 0.000 description 1
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- 125000001664 diethylamino group Chemical group [H]C([H])([H])C([H])([H])N(*)C([H])([H])C([H])([H])[H] 0.000 description 1
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- PYWVYCXTNDRMGF-UHFFFAOYSA-N rhodamine B Chemical compound [Cl-].C=12C=CC(=[N+](CC)CC)C=C2OC2=CC(N(CC)CC)=CC=C2C=1C1=CC=CC=C1C(O)=O PYWVYCXTNDRMGF-UHFFFAOYSA-N 0.000 description 1
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- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 1
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- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D498/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms
- C07D498/02—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms in which the condensed system contains two hetero rings
- C07D498/04—Ortho-condensed systems
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
- A61K41/0057—Photodynamic therapy with a photosensitizer, i.e. agent able to produce reactive oxygen species upon exposure to light or radiation, e.g. UV or visible light; photocleavage of nucleic acids with an agent
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
- A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
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- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
- C09K2211/10—Non-macromolecular compounds
- C09K2211/1018—Heterocyclic compounds
- C09K2211/1025—Heterocyclic compounds characterised by ligands
- C09K2211/1044—Heterocyclic compounds characterised by ligands containing two nitrogen atoms as heteroatoms
- C09K2211/1048—Heterocyclic compounds characterised by ligands containing two nitrogen atoms as heteroatoms with oxygen
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Abstract
The invention relates to the technical field of biological medicines, in particular to N-heteronile erythronium salt compounds and a preparation method and application thereof. Has a structure shown in formula I. The N-heteronile erythronium salt compound provided by the invention can be quickly positioned in mitochondria at low concentration, track the mitochondria in living cells in real time and for a long time, and dye the mitochondria without being influenced by mitochondrial membrane potential. The N-heteronile erythronium salt compound can generate superoxide anions under the irradiation of a red light LED lamp, has a strong tumor cell killing effect, and has a good application prospect in mitochondrial fluorescence imaging and photodynamic therapy.
Description
Technical Field
The invention relates to the technical field of biological medicines, in particular to N-heteronile erythronium salt compounds and a preparation method and application thereof.
Background
Mitochondria are a semi-autonomous organelle of most eukaryotic cells, performing a variety of intracellular functions, including energy supply, signal transduction, calcium homeostasis (calciumhomeostasis), cell differentiation, and the like. Mitochondrial metabolism is critical for tumor development and tumor growth, and alterations in mitochondrial metabolism can increase the production of mitochondrial Reactive Oxygen Species (ROS) and alter cellular redox states, thereby altering gene expression and stimulating cancer cell proliferation, increasing tumor cell plasticity and addressing adverse environmental conditions through a variety of mechanisms. Mitochondria play a key role in the regulation of intrinsic apoptosis and the process of non-apoptotic cell death (regulatory necrosis) in various forms, so that targeting mitochondrial metabolism and mitochondria is an effective treatment strategy, and a large number of experiments show that targeting mitochondria can effectively improve the anti-tumor effect and possibly overcome the tumor drug resistance.
Photodynamic therapy is a promising non-invasive local therapy, in which a photosensitizer excited by light of appropriate wavelength generates highly toxic Reactive Oxygen Species (ROS) by a photochemical mechanism of type i (electron transfer) or type ii (energy transfer). ROS attack various biomolecules, including DNA, proteins, lipids, etc., causing apoptosis or necrosis to further treat the disease. Increasing subcellular organelle targeting of photosensitizers is one of the effective methods to increase photodynamic therapy efficacy, and since mitochondria are susceptible to excessive amounts of ROS, many photosensitizers have been designed and demonstrated to improve photodynamic therapy (PDT) through mitochondrial targeting. However, mitochondrial potential varies greatly with mitochondrial status, and may adversely affect electrostatic interactions of photosensitizers and mitochondrial targeting ability, especially the loss of decreased mitochondrial membrane potential under the action of photosensitizers, which may lead to leakage of photosensitizers from mitochondria. This limits the residence time of the photosensitizer in the mitochondria and greatly affects the therapeutic effect. Therefore, there is a need to develop photosensitizers with higher mitochondrial targeting stability.
In view of the above, the invention develops and designs the N-heteronile erythronium salt compound photosensitizer with the mitochondrion targeting stability, and has great value for the fluorescence labeling of the mitochondrion and the photodynamic treatment of cancer.
Disclosure of Invention
Aiming at the problems, the invention provides an N-heteronile erythronium salt compound for mitochondrion targeting and photodynamic therapy, a preparation method and application thereof, and the N-heteronile erythronium salt compound has the biomedical application of targeting mitochondrion to carry out in vivo and in vitro fluorescence imaging diagnosis and exert photodynamic therapy effect, in particular to the fluorescence imaging diagnosis and/or therapy of in vivo and in vitro tumor cells and tissues.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
n-heteronile red onium salt compound shown in general formula (I):
general formula I
Wherein n =7-13.
Preferably, the N-heteronile erythronium salt compound has the following structural formula:
in the present application, it is preferred that,
the code number and the name of the compound represented by the code number are NRNC-8: 4-octyl-9- (diethylamino) -5-oxo-5H-pyrido [3,2-a]Phenoxazine-4-ium iodide;
Another purpose of the invention is to provide a preparation method of N-heteronile erythronium salt compounds, which comprises the following steps:
step 1): adding 5-diethylamino-2-nitrosophenol hydrochloride and 8-hydroxyquinoline into DMF or acetic acid or toluene, heating to 90-120 ℃, and reacting for 5-24H to obtain 9- (diethylamino) -5H-pyrido [3,2-a ] phenoxazin-5-one;
step 2): and (2) carrying out reflux reaction on the 9- (diethylamino) -5H-pyrido [3,2-a ] phenoxazin-5-one obtained in the step (1) and 1-iodoalkane in acetonitrile for 3-24H to obtain the alkyl-substituted N-heteronile erythronium salt compound shown in the general formula (I).
The reaction scheme is as follows:
the invention also aims to provide the application of the N-heteronile erythronium salt compound in preparing a fluorescence imaging agent targeting tumor cell mitochondria, wherein the N-heteronile erythronium salt compound has stable fluorescence characteristics targeting tumor cell mitochondria and can rapidly realize fluorescence imaging of tumor cells at low concentration. Wherein the tumor cells are breast cancer, liver cancer, lung cancer, prostatic cancer, head and neck cancer, colon cancer, esophageal cancer, melanoma, etc.
The invention also aims to provide application of the N-heteronile erythronium salt compounds in preparation of medicines for photodynamic tumor treatment, which can realize fluorescence imaging and/or treatment of tumor tissues or cells. Wherein, the N-heteronile erythronium salt compound can generate superoxide anions to kill tumor cells after being irradiated by laser or LED red light.
The invention has the following beneficial effects:
the invention introduces lipophilic cations on a rigid plane structure N-heteronile red ring to obtain a mitochondrion targeted N-heteronile erythronium salt compound, which can be quickly positioned in the mitochondrion at low concentration, track the mitochondrion in living cells in real time and for a long time, and dye the mitochondrion without being influenced by the mitochondrion membrane potential. The N-heteronile erythronium salt compound can generate superoxide anions under the irradiation of a red light LED lamp, has a strong tumor cell killing effect, and has a good application prospect in mitochondrial fluorescence imaging and photodynamic therapy.
Drawings
FIG. 1 shows the mass spectra of the compounds NRNC-8, NRNC-10 and NRNC-12 in the example;
FIG. 2 EPR spectrum of NRNC-8, compound of example 1;
FIG. 3 example viable cell mitochondrial co-localization and fixed cell imaging of compounds NRNC-8, NRNC-10, NRNC-12.
Detailed Description
The present application will be further described with reference to the accompanying drawings and detailed description so that those skilled in the art can more readily understand the present application, but these examples are only used for illustrating the present invention and are not used to limit the scope of the present invention, i.e., the described examples are only a part of the examples of the present invention, but not all of the examples.
Thus, the following detailed description of a portion of the embodiments of the present invention is not intended to limit the scope of the invention as claimed, but is merely a selected embodiment of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments of the present invention without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
Preparation of 4-octyl-9- (diethylamino) -5-oxo-5H-pyrido [3,2-a ] phenoxazin-4-ium iodide (NRNC-8)
5-diethylamino-2-nitrosophenolate (1.62g, 7 mmol) was dissolved in 15ml of DMF, and 8-hydroxyquinoline (1.02g, 7 mmol) was added under nitrogen protection to react at 105 ℃ overnight, after which the reaction was completed, and concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (dichloromethane: methanol =20: 1) to obtain 0.23g of the compound 9- (diethylamino) -5H-pyrido [3,2-a ] phenoxazin-5-one in a yield of 10.3%. The characterization data are as follows:
1 H NMR(400MHz,CDCl 3 )：δ8.97-9.02(m，2H)，7.63(d，J＝9.2Hz，1H)，7.64((dd，J＝8.4，4.4Hz，1H)，6.71(dd，J＝9.2，2.8Hz，1H)，6.59(s，1H)，6.51(d，J＝2.8Hz，1H)，3.50(q，J＝7.2Hz，4H)，1.28(t，J＝7.2Hz，6H)。
13 C NMR(100MHz，CDCl 3 )：δ182.1，151.8，151.7，151.4，147.0，146.7，138.1，132.3，131.4，128.5，125.4，125.2，110.2，107.0，96.3，45.2，12.6。
HR-MS：m/z：calcd for[C 19 H 17 N 3 O 2 ]：319.1321；found：320.1379[M+H] + ，342.1234[M+Na] + ，661.2576[2M+Na] + 。
the compound 9- (diethylamino) -5H-pyrido [3,2-a ] phenoxazin-5-one (64mg, 0.2mmol) is dissolved in 10mL acetonitrile, 1-iodo-n-octane (240mg, 1.0 mmol) is added for reflux reaction for 24H, the solvent is evaporated under reduced pressure after the reaction is finished, and the residue is treated by an alkaline alumina column (eluent: dichloromethane/methanol = 25/1) to obtain 46mg of the product 4-octyl-9- (diethylamino) -5-oxo-5H-pyrido [3,2-a ] phenoxazin-4-iodonium (NRNC-8) with the yield of 41.1%. The characterization data are as follows:
1 H NMR(400MHz，CDCl 3 )：δ10.16(dd，J＝5.6，1.2Hz，1H)，9.78(dd，J＝8.8，0.8Hz，1H)，8.55(dd，J＝8.4，6.0Hz，1H)，7.76(d，J＝9.2Hz，1H)，6.96(dd，J＝9.2，2.8Hz，1H)，6.66(s，1H)，6.62(d，J＝2.8Hz，1H)，5.55(t，J＝7.6Hz，2H)，3.62(q，J＝7.2Hz，4H)，2.04(m，2H)，1.53(m，2H)，1.23-1.40(m，14H)，0.87(t，J＝6.8Hz，3H)。
13 C NMR(100MHz，CDCl 3 )：δ174.5，154.1，151.3，151.0，148.1，141.5，135.9，133.5，133.1，131.2，128.2，128.0，113.3，108.6，96.2，62.1，46.1，32.9，31.7，29.1，26.4，22.6，14.1，12.7。
HR-MS：m/z[M-I] + ：calcd for[C 27 H 34 N 3 O 2 + ]：432.2646；found：432.2644。
example 2
Preparation of 9- (diethylamino) -4-decyl-5-oxo-5H-pyrido [3,2-a ] phenoxazin-4-ium iodide (NRNC-10)
Referring to the preparation method of example 1, 1-iodo-n-octane was replaced with 1-iodo-n-decane to obtain compound NRNC-10, whose characterization data are as follows:
HR-MS：m/z[M-I] + ：calcd for[C 29 H 38 N 3 O 2 + ]：460.2959；found：460.2959。
example 3
Preparation of 9- (diethylamino) -4-dodecyl-5-oxo-5H-pyrido [3,2-a ] phenoxazin-4-ium iodide (NRNC-12)
Referring to the preparation method of example 1, 1-iodo-n-octane was replaced with 1-iodo-n-dodecane to finally obtain compound NRNC-12, whose characterization data are as follows:
HR-MS：m/z[M-I] + ：calcdfor[C 31 H 42 N 3 O 2 + ]：488.3272；found：488.3271。
example 4: NRNC-8 Electron Paramagnetic Resonance (EPR) analysis of the compound prepared in example 1 to detect O 2 Production of
Using the NRNC-8 prepared in example 1 as an example, it was tested on a Bruker A300 EPR spectrometer using 5, 5-dimethyl-1-pyrroline N-oxide (DMPO) as spin trap. NRNC-8 was dissolved in 100. Mu. LDMSO at a concentration of 2X 10 -3 M, then 5. Mu.L MPO was added to the NRNC-8 solution above, and the solution was irradiated with a red LED lamp for 2 minutes (50 mW/cm) 2 ). Finally, the EPR signal was recorded at room temperature. As shown in FIG. 2, pure DMPO solution irradiated with red LED without EPR signal, DMPO + NRNC-8 in the dark with weaker EPR signal, consistent with the results reported previously (Biochemistry, 2003,42, 11924-11931.), DMPO + NRNC-8 irradiated with red LED for 2 minutes, observed a significantly enhanced strongly characteristic paramagnetic adduct with O 2 The signal remained well consistent, indicating that NRNC-8 is highly efficient in producing superoxide anions under light.
Example 5: example tumor mitochondrial localization of N-heteronile Renilium salts
MCF-7 cells which grow well and are inoculated in a confocal glass bottom dish are incubated for 20 minutes by using a mitochondrial commercial dye rhodamine 123 (Rh 123) (1 mu M,200 mu L), then the compounds NRNC-8, NRNC-10 and NRNC-12 (100nM, 200 mu L) prepared in the example are respectively used for staining for 20 minutes, then DPBS is washed for three times, and laser confocal imaging (green channel: 488nm excitation, 500-550nm collection; red channel: 640nm excitation, 665-705nm collection) is used for observing the overlapping condition of the red fluorescence signals of the compounds obtained in the example and the fluorescence signals of the rhodamine 123, and the result shows that the NRNC-8, NRNC-10 and NRNC-12 can be efficiently and rapidly absorbed by tumor cells under in-vitro culture conditions, strong red fluorescence signals can be well coincided with green fluorescence signals from the mitochondria, and the result proves that the main cells are distributed in mitochondria after the compounds enter the cells, the figures of the compounds NRNC-8, NRNC-10, NRNC-12 and rhodamine have the co-localization coefficients of more than 0.8 and more than 3.8. The obtained result shows that the N-heteronile erythronium salt compound can quickly target the mitochondria of tumor cells at low concentration (100 nM) in a short time, has obvious targeting effect and provides a feasible method for fluorescence labeling tracing and medical diagnosis of the mitochondria.
TABLE 1 table of co-localization coefficients of N-heteronile erythronium salt compounds and Rh123
| Serial number | Photosensitizers | Commercial mitochondrial dyes | Co-localization factor (Pr) |
| Example 1 | NRNC-8 | Rh123 | 0.91 |
| Example 2 | NRNC-10 | Rh123 | 0.92 |
| Example 3 | NRNC-12 | Rh123 | 0.84 |
Mitochondrial Membrane Potential (MMP) is a main index of mitochondrial function, disappearance of mitochondrial membrane potential causes cell dysfunction and even death, and disappearance of mitochondrial membrane potential in the photodynamic therapy process causes photosensitizer to escape from mitochondria, reducing the therapeutic effect. The invention further tests the targeting of the compounds NRNC-8, NRNC-10 and NRNC-12 in the examples to mitochondria when the mitochondrial membrane potential disappears. When the cells are fixed, MMPs disappear. We further investigated the staining ability of NRNC-8, NRNC-10, NRNC-12 in fixed cells to verify whether they could be fixed in mitochondria after MMP disappearance. Viable MCF-7 cells were incubated with Rh123 (1. Mu.M, 200. Mu.L) for 20 minutes, then stained with each of the compounds NRNC-8, NRNC-10, NRNC-12 (100nM, 200. Mu.L) prepared in the examples for 20 minutes, and finally fixed with a 4% formaldehyde fixing solution for 20 minutes, and observed by laser confocal imaging (green channel: 488nm excitation, 500-550nm collection; red channel: 640nm excitation, 665-705nm collection), as shown in FIG. 3, the fluorescence of Rh123 after fixation was almost not seen, while the red fluorescence of NRNC-8, NRNC-10, NRNC-12 remained significantly large, which indicates that NRNC-8, NRNC-10, NRNC-12 could be fixed in mitochondria while MMP disappeared, with good mitochondrial targeting stability, which could effectively reduce the escape of photosensitizers from mitochondria in photodynamic therapy, and improve photodynamic therapy effect.
Example 6: dark toxicity and phototoxicity assay for tumor cells
MCF-7 cells were plated at approximately 1X 10 per well 5 The amount was inoculated in 96-well plates and incubated overnight. After removal of the medium, 100. Mu.L of DPBS solutions of NRNC-8, NRNC-12 at different concentrations (0-5. Mu.M) were added to the wells, the cells were incubated for a further 1h at 37 ℃ and then the cells were placed in a red LED lamp (50 mW/cm) 2 ) Irradiating for 0 min or 10 min, then changing into DMEM medium, and incubating at 37 deg.CIncubate 24 hours, add CCK-8 (10. Mu.L, 5 mg/mL) per well and in CO 2 The culture was continued in the incubator for 2 hours. The absorbance at 450nm was then measured on a microplate reader. The results of their CCK-8 assays are shown in Table 2, respectively. The result shows that the photosensitizer targeted by the mitochondria NRNC-8, NRNC-10 and NRNC-12 has low dark toxicity and strong phototoxicity and can be used for photodynamic anti-tumor treatment.
TABLE 2 evaluation table of photodynamic effects of photosensitizers obtained in examples 1 to 3
L (-) indicates no light; l (+) denotes red LED lamp illumination.
Claims (7)
1. N-heteronile red onium salt compound shown in general formula (I),
general formula I
Wherein n =7-13.
2. The N-heteronile erythronium salt compound as claimed in claim 1, wherein the structural formula of the N-heteronile erythronium salt compound is as follows:
3. the method for preparing N-heteronile erythronium salts compound of claim 1, which comprises the following steps:
step 1): adding 5-diethylamino-2-nitrosophenol hydrochloride and 8-hydroxyquinoline into DMF or acetic acid or toluene, heating to 90-120 ℃, and reacting for 5-24H to obtain 9- (diethylamino) -5H-pyrido [3,2-a ] phenoxazin-5-one;
step 2): and (2) carrying out reflux reaction on the 9- (diethylamino) -5H-pyrido [3,2-a ] phenoxazin-5-one obtained in the step (1) and 1-iodoalkane in acetonitrile for 3-24H to obtain the alkyl substituted N-heteronile erythronium salt compound shown in the general formula (I).
The reaction scheme is as follows:
wherein n =7-13.
4. Use of a compound of the N-heteronile erythronium salt class described in any of claims 1 and 2 for the preparation of a fluorescence imaging agent targeting the mitochondria of tumor cells.
5. Use of a compound of the N-heteronile erythronium salt type as claimed in any of claims 1 and 2 for the preparation of a photosensitizer for photodynamic therapy.
6. The use of a N-heteronile erythronium salt compound as claimed in any of claims 1 and 2 for the preparation of a medicament having photodynamic tumour therapeutic effect.
7. The use of claim 6, wherein the tumor is breast cancer, liver cancer, lung cancer, prostate cancer, head and neck cancer, colon cancer, esophageal cancer, or melanoma.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH10182651A (en) * | 1996-12-26 | 1998-07-07 | Ricoh Co Ltd | Pyridophenoxazine-metal chelate compound |
| CN1524859A (en) * | 2003-09-17 | 2004-09-01 | 中山大学 | Phenoxazones, preparing process and pharmaceutical uses thereof |
| WO2013190127A1 (en) * | 2012-06-21 | 2013-12-27 | Immusmol Sas | Antagonist to an enzyme and/or a metabolite of the kynurenine pathway |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH10182651A (en) * | 1996-12-26 | 1998-07-07 | Ricoh Co Ltd | Pyridophenoxazine-metal chelate compound |
| CN1524859A (en) * | 2003-09-17 | 2004-09-01 | 中山大学 | Phenoxazones, preparing process and pharmaceutical uses thereof |
| WO2013190127A1 (en) * | 2012-06-21 | 2013-12-27 | Immusmol Sas | Antagonist to an enzyme and/or a metabolite of the kynurenine pathway |
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| Angelo Alberti et al..Antitumor Agents 4. Characterization of Free Radicals Produced during Reduction of the Antitumor Drug 5H-Pyridophenoxazin-5-one: An EPR Study.Biochemistry.2003,第42卷11924-11931. * |
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