Stereoisomer pair with near-infrared two-region luminescence property, fluorescent probe as well as preparation method and application of stereoisomer pair and fluorescent probe

Abstract

The invention discloses a stereoisomer pair with near-infrared two-region luminescence property, a fluorescent probe as well as a preparation method and application of the stereoisomer pair. The structural general formula of the stereoisomer pair is as follows: # imgabs0 #, wherein X <-> comprises any one of ClO4 <->, BF4 <->, CF3COO <->, Cl <->, Br <->, I <-> and NO3 <->; and R1 and R2 independently comprise any one of # imgabs1 # and # imgabs2 #. The stereoisomer with the near-infrared second-region luminescence property shows obvious optical / physicochemical property difference in the aspects of absorption / fluorescence emission wavelength, fluorescence quantum efficiency and responsiveness, one of the stereoisomers shows high-sensitivity and high-specificity responsiveness in the physiological environment, and the other stereoisomer shows high-sensitivity and high-specificity responsiveness in the physiological environment. And the other isomer hardly has responsiveness. Meanwhile, the stereoisomer shows obviously different biomedical activities in the process of inducing pyroptosis of cells, and has wide application prospects in the fields of biological imaging, drug screening and development and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of near-infrared second-region fluorescent probes, and specifically relates to a stereoisomer pair of near-infrared second-region luminescent properties with significant differences in optical properties and biomedical activities, a fluorescent probe, and a preparation method and application thereof. Background Art

[0002] Compared with traditional imaging technologies such as CT imaging, magnetic resonance imaging (MRI), positron emission tomography (PET) and ultrasound imaging (US), fluorescence imaging has the advantages of high sensitivity, strong specificity, good real-time performance, low cost and no ionizing radiation, and has received increasing attention in the field of biomedical imaging. Among them, near-infrared II (NIR-II, 900-1700nm) fluorescence imaging can effectively avoid the interference of spontaneous fluorescence of biological tissues, reduce the absorption and scattering of light in tissues, and significantly improve the tissue penetration depth and imaging contrast of fluorescence imaging, showing great application prospects in the fields of tumor detection, intraoperative fluorescence navigation, disease diagnosis and treatment, etc.

[0003] The development of fluorescence imaging in the NIR-II region is inseparable from the development of high-quality contrast agents. At present, a variety of NIR-II region fluorescent probes have been developed, including single-walled carbon nanotubes, quantum dots, rare earth nanoparticles, and organic molecular probes. Among them, organic fluorescent probes in the NIR-II region have shown obvious advantages in clinical transformation due to their high biocompatibility, easy functional design, and biometabolism. At present, various types of NIR-II region molecular probes have been reported, which has promoted the development of NIR-II region fluorescence imaging in the biomedical field.

[0004] CN108997771A discloses a class of dyes with strong absorption and photothermal effect in the near-infrared region, and a preparation method and application thereof. The method uses the rhodamine structure as a fluorescent moiety and designs a variety of cyanine molecules with trimethine, pentamethine and heptamethine structures. This type of probe has strong absorption characteristics in the near-infrared region and can be used for photothermal therapy applications.

[0005] However, the prior art has not disclosed near-infrared second-zone fluorescent probe stereoisomers and their preparation methods and applications.

[0006] Molecular stereoisomers usually exhibit different biological activities under physiological conditions. The most famous "Thalidomide" is such a molecule. One of its isomers is non-toxic and has a sedative effect, while the other isomer has pharmacological effects, but its metabolites have strong teratogenic effects. Therefore, it is of great significance to design, synthesize and separate stereoisomers of near-infrared zone II fluorescent probes to study their differences in photophysical and chemical properties and biomedical effects. However, due to the extremely small difference in polarity between stereoisomers, separation and purification are difficult, and the near-infrared zone II fluorescent probes currently used in research are still mixtures of stereoisomers. Summary of the invention

[0007] The main purpose of the present invention is to provide a stable stereoisomer pair with near-infrared second-zone luminescence properties that is easy to separate at low cost and high efficiency, and a preparation method thereof, so as to overcome the shortcomings of the prior art.

[0008] Another object of the present invention is to provide a near-infrared second-region fluorescent probe.

[0009] Another object of the present invention is to provide the application of the stereoisomer pair having near-infrared second-region luminescence properties or the near-infrared second-region fluorescent probe.

[0010] In order to achieve the above-mentioned invention object, the technical solution adopted by the present invention includes:

[0011] The embodiment of the present invention provides a pair of stereoisomers having near-infrared second-zone luminescence properties, and the general structural formula of the stereoisomers includes formula (I) and formula (II):

[0012]

[0013] Among them, X - Including ClO4 - 、BF4 - CF3COO - , Cl - Br - ,I - 、NO3 - Any of the following:

[0014] R1 includes Any of the following:

[0015] R2 includes Any of the following:

[0016] m is any integer from 0 to 2, n is any integer from 0 to 2, and z is 1 or 2.

[0017] The embodiment of the present invention also provides a method for preparing a stereoisomer pair having near-infrared second-region luminescence properties, which comprises:

[0018] allowing the first compound containing the oxonium ion to undergo a condensation reaction with a condensation agent to obtain an intermediate;

[0019] The intermediate is further subjected to a condensation reaction with a second compound containing an oxonium ion to obtain a stereoisomer pair having near-infrared second-region luminescence properties;

[0020] Wherein, the first compound containing oxonium ions has a structure as shown in formula (III):

[0021]

[0022] Among them, X - Including ClO4 - 、BF4 - CF3COO - , Cl - Br - ,I - 、NO3 - Any of the following:

[0023] R1 includes Any one of; m is any integer from 0 to 2;

[0024] The condensing agent has a structure as shown in formula (IV):

[0025]

[0026] Where z is 1 or 2;

[0027] The second compound containing an oxonium ion has a structure as shown in formula (V):

[0028]

[0029] Among them, X - Including ClO4 - 、BF4 - CF3COO - , Cl - Br - ,I - 、NO3 - Any of the following:

[0030] R2 includes Any one of; n is any integer from 0 to 2.

[0031] The embodiment of the present invention also provides a stereoisomer pair having near-infrared second-zone luminescence properties obtained by the aforementioned preparation method.

[0032] The embodiment of the present invention also provides a method for purifying a stereoisomer pair having near-infrared second-region luminescence properties.

[0033] The embodiment of the present invention further provides a near-infrared second-region fluorescent probe, which includes the aforementioned stereoisomer pair having near-infrared second-region luminescent properties.

[0034] The embodiments of the present invention also provide the use of the aforementioned stereoisomer pair having near-infrared second-region luminescence properties or near-infrared second-region fluorescent probes in biological imaging or visualization drug screening and development.

[0035] Compared with the prior art, the beneficial effects of the present invention include:

[0036] (1) The present invention provides a series of near-infrared second-zone fluorescent probe stereoisomer pairs, and the raw materials used in the preparation method are cheap and easily available, the synthesis conditions are relatively mild, and the synthesis efficiency is high; and the separation and purification of these stereoisomer molecules are simple, and they can be separated using silica gel columns or ordinary HPLC columns, which is easy to scale up production;

[0037] (2) The stereoisomer pair of near-infrared region II fluorescent probes provided by the present invention has obvious differences in photophysical and chemical properties and biomedical activities, and can be used for in vivo near-infrared region II fluorescent imaging or disease detection;

[0038] (3) The near-infrared second-region fluorescent probe stereoisomers provided by the present invention have significantly different cellular effects and can be used for drug development in addition to being used as imaging tools. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0040] Figure 1 is the H NMR spectrum of the stereoisomer R5-55 prepared in Example 1 of the present invention;

[0041] Figure 2 is the NMR carbon spectrum of the stereoisomer R5-55 prepared in Example 1 of the present invention;

[0042] Figure 3 is a high-resolution mass spectrum of the stereoisomer R5-55 prepared in Example 1 of the present invention;

[0043] Figure 4 is the H NMR spectrum of the stereoisomer S5-55 prepared in Example 1 of the present invention;

[0044] Figure 5 is the NMR carbon spectrum of the stereoisomer S5-55 prepared in Example 1 of the present invention;

[0045] Figure 6 is a high-resolution mass spectrum of the stereoisomer S5-55 prepared in Example 1 of the present invention;

[0046] Figure 7 is the H NMR spectrum of the stereoisomer R5-66 prepared in Example 2 of the present invention;

[0047] Figure 8 is the NMR carbon spectrum of the stereoisomer R5-66 prepared in Example 2 of the present invention;

[0048] Fig. 9 is a high-resolution mass spectrum of the stereoisomer R5-66 prepared in Example 2 of the present invention;

[0049] Fig.10 is the H NMR spectrum of the stereoisomer S5-66 prepared in Example 2 of the present invention;

[0050] Fig.11 is the NMR carbon spectrum of the stereoisomer S5-66 prepared in Example 2 of the present invention;

[0051] Fig.12 is a high-resolution mass spectrum of the stereoisomer S5-66 prepared in Example 2 of the present invention;

[0052] Fig.13 is the H NMR spectrum of the stereoisomer R5-77 prepared in Example 3 of the present invention;

[0053] Fig.14 is a high-resolution mass spectrum of the stereoisomer R5-77 prepared in Example 3 of the present invention;

[0054] Fig.15 is the H NMR spectrum of the stereoisomer S5-77 prepared in Example 3 of the present invention;

[0055] Fig.16 is a high-resolution mass spectrum of the stereoisomer S5-77 prepared in Example 3 of the present invention;

[0056] Figure 17a-Figure 17d They are respectively the absorption and fluorescence spectra of the stereoisomer R / S5-55 in Example 13 of the present invention in different solvents;

[0057] Figure 18a-18d They are respectively the absorption and fluorescence spectra of the stereoisomer R / S5-66 in Example 13 of the present invention in different solvents;

[0058] Figure 19a-19d They are respectively the absorption and fluorescence spectra of the stereoisomer R / S5-77 in Example 13 of the present invention in different solvents;

[0059] Figure 20a-Figure 20f They are respectively the absorption and fluorescence spectra of the stereoisomer R / S5-55 in Example 14 of the present invention at different pH values;

[0060] Figure 21a-Figure 21f They are respectively the absorption and fluorescence spectra of the stereoisomer R / S5-66 in Example 14 of the present invention at different pH values;

[0061] Figure 22a-22f They are respectively the absorption and fluorescence spectra of the stereoisomer R / S5-77 in Example 14 of the present invention at different pH values;

[0062] Figure 23a-23f They are respectively the absorption and fluorescence spectra of the stereoisomer R / S5-55 in Example 15 of the present invention after adding different active oxygen species;

[0063] Figure 24a-Figure 24f They are respectively the absorption and fluorescence spectrum changes of the stereoisomer R / S5-55 in Example 15 of the present invention after adding ONOO-;

[0064] Fig.25 The diagram is the mechanism diagram of the response of stereoisomer R / S5-55 to ONOO-;

[0065] Fig.26 HPLC chart of probe R / S5-55 after stereoisomer separation in Example 16 of the present invention;

[0066] Fig. 27 HPLC chart of probe R / S5-66 after stereoisomer separation in Example 16 of the present invention;

[0067] Fig.28 HPLC chart of probe R / S5-77 after stereoisomer separation in Example 16 of the present invention;

[0068] Fig.29 This is a graph showing the results of a ratiometric fluorescence diagnostic test of the probe R / S5-55 stereoisomer in Example 17 of the present invention on a mouse enteritis model;

[0069] Fig.30 The fluorescence spectrum of the mixed stereoisomers R7-66 and S7-66 in Example 17 of the present invention;

[0070] Fig.31 This is a diagram of the cell status of 4T1 cells in Example 18 of the present invention after being incubated with two stereoisomers R / S5-55 for 12 hours respectively. DETAILED DESCRIPTION

[0071] In view of the problems existing in the above-mentioned prior art, after long-term research and a large number of experiments, the inventors of this case proposed this technical solution, which mainly provides a stable near-infrared second-zone fluorescent stereoisomer pair that is easy to separate with low cost and high efficiency, and based on the differences in the photophysical and chemical properties and biomedical activities of the stereoisomers, provides its application in biomedical diagnosis and drug development.

[0072] The technical solution, its implementation process and principle will be further explained as follows.

[0073] As one aspect of the technical solution of the present invention, the general structural formula of a stereoisomer pair having near-infrared second-zone luminescence properties is shown in formula (I) and formula (II):

[0074]

[0075] Among them, X - Including ClO4 - 、BF4 - CF3COO - , Cl - Br - ,I - 、NO3 - Any one of the above;

[0076] R1 includes Any one of the above;

[0077] R2 includes Any one of the above;

[0078] m is any integer between 0 and 2 (such as 0, 1, 2), n is any integer between 0 and 2 (such as 0, 1, 2), and z is 1 or 2.

[0079] The stereoisomer pair with near-infrared second-zone luminescence properties of the present invention exhibits obvious differences in optical / physicochemical properties in terms of absorption / fluorescence emission wavelength, fluorescence quantum efficiency and responsiveness. One isomer in the stereoisomer pair of the present invention exhibits high sensitivity and high specific responsiveness in a physiological environment, while the other isomer has almost no responsiveness. At the same time, the stereoisomer pair exhibits significantly different biomedical activities in the process of inducing cell pyroptosis.

[0080] As another aspect of the technical solution of the present invention, a method for preparing a stereoisomer pair having near-infrared second-zone luminescence properties comprises:

[0081] allowing the first compound containing the oxonium ion to undergo a condensation reaction with a condensation agent to obtain an intermediate;

[0082] The intermediate is further subjected to condensation reaction with a second compound containing an oxonium ion to obtain a stereoisomer having near-infrared second-region luminescence properties.

[0083] In some embodiments, the first compound containing an oxonium ion (hereinafter referred to as C1) used in the present invention has a structure as shown in formula (III):

[0084]

[0085] Among them, X - Including ClO4 - 、BF4 - CF3COO - , Cl - Br - ,I - 、NO3 - Any one of the above;

[0086] R1 includes Any one of the above; m is any integer from 0 to 2 (such as 0, 1, 2).

[0087] In some embodiments, the condensing agent (referred to as S1) used in the present invention has a structure as shown in formula (IV):

[0088]

[0089] Where z is 1 or 2.

[0090] In some embodiments, the second compound containing an oxonium ion (hereinafter referred to as C2) used in the present invention has a structure as shown in Formula (V):

[0091]

[0092] Among them, X - Including ClO4 - 、BF4 - CF3COO - , Cl - Br - ,I - 、NO3 - Any one of the above;

[0093] R2 includes Any one of the above; n is any integer from 0 to 2 (such as 0, 1, 2).

[0094] In some specific embodiments, the preparation method includes: mixing a first compound containing an oxonium ion (abbreviated as C1), a condensation agent and a first reaction solvent, and performing a condensation reaction in a protective atmosphere to obtain an intermediate (abbreviated as M1).

[0095] In some specific embodiments, the preparation method includes: mixing the intermediate, a second compound containing an oxonium ion (which can be referred to as C2), a base and a second reaction solvent, and continuing a condensation reaction in a protective atmosphere to obtain a stereoisomer pair having near-infrared second-zone luminescence properties.

[0096] The reaction equation of the preparation method of the present invention is as follows:

[0097]

[0098] The preparation mechanism of the present invention is: firstly, oxonium ions and condensation agents are used for condensation reaction to obtain intermediate M1, and then intermediate M1 and oxonium ions are used for further condensation reaction to obtain a mixed stereoisomer pair with near-infrared second-zone luminescence properties.

[0099] In some more preferred embodiments, the method for preparing the stereoisomer pair having near-infrared second-region luminescence properties of the present invention comprises the following steps:

[0100] (1) Synthesis of intermediate M1: dissolving a first compound C1 containing an oxonium ion and a corresponding condensing agent S1 in a first reaction solvent, and then heating the mixture in a protective gas atmosphere. After the reaction, heating is stopped and the mixture is returned to room temperature to obtain intermediate M1.

[0101] (2) Synthesis of near-infrared zone II stereoisomers: Add a second compound C2 containing an oxonium ion and a base to the solution of the intermediate M1 obtained in step (1), and continue the reaction under a protective gas to obtain near-infrared zone II molecular stereoisomers, which is a mixture of two stereoisomers.

[0102] The present invention can prepare symmetrical and asymmetrical molecular stereoisomers by adjusting the reaction conditions. When the first compounds C1 and C2 of the oxonium ion are consistent, it is considered to be a symmetrical molecule, and when they are inconsistent, it is an asymmetrical molecule. Whether it is a symmetrical or asymmetrical molecule, it contains stereoisomers with different structures.

[0103] In some preferred embodiments, in the above-mentioned preparation method step (1), the first reaction solvent may include at least any one of acetic anhydride, toluene / n-butanol, benzene / n-butanol, ethanol, methanol, etc., preferably acetic anhydride.

[0104] In some preferred embodiments, in the above-mentioned preparation method step (1), the molar ratio of the first compound containing oxonium ions to the condensing agent may be 1:(0.1-2).

[0105] In some preferred embodiments, in step (1) of the above-mentioned preparation method, the temperature of the condensation reaction is 50-150°C, preferably 90-110°C.

[0106] In some preferred embodiments, in step (1) of the above-mentioned preparation method, the condensation reaction time is 0.5 to 12 hours, preferably 2 to 3 hours.

[0107] In some preferred embodiments, in step (2) of the above-mentioned preparation method, the base may include at least any one of sodium acetate, potassium acetate, potassium carbonate, sodium carbonate, triethylamine, etc., preferably sodium acetate or potassium acetate.

[0108] In some preferred embodiments, in step (2) of the above-mentioned preparation method, the second reaction solvent may include at least any one of acetic anhydride, toluene / n-butanol, benzene / n-butanol, etc., preferably acetic anhydride.

[0109] In some preferred embodiments, in step (2) of the above-mentioned preparation method, the molar ratio of the intermediate to the second compound containing the oxonium ion is 1:(0.5-10), preferably 1:(0.8-1.5).

[0110] In some preferred embodiments, in step (2) of the above-mentioned preparation method, the molar ratio of the intermediate to the base is 1:(1-30).

[0111] In some preferred embodiments, in step (2) of the above-mentioned preparation method, the temperature for continuing the condensation reaction is 0 to 150°C, preferably 20 to 60°C.

[0112] In some preferred embodiments, in step (2) of the above-mentioned preparation method, the condensation reaction is continued for 0.5 to 24 hours, preferably 6 to 12 hours.

[0113] In some preferred embodiments, the preparation method further comprises: using a silica gel column to separate and purify the product obtained by the continued reaction (ie, the crude product) to obtain stereoisomers of mixed structure.

[0114] Furthermore, the preparation method further comprises: using high performance liquid chromatography to separate and purify again the stereoisomers of the mixed structure after the silica gel column purification.

[0115] In summary, the preparation method of a series of near-infrared second-zone stereoisomer pairs provided by the present invention uses cheap and easily available raw materials, relatively mild synthesis conditions, and high synthesis efficiency; and these stereoisomer molecules are simple to separate and purify, and can be separated using silica gel columns or ordinary HPLC columns, which is easy to scale up production.

[0116] As another aspect of the technical solution of the present invention, it also relates to a stereoisomer pair having near-infrared second-zone luminescence properties obtained by the aforementioned preparation method.

[0117] In the stereoisomer pair with near-infrared second-zone luminescence properties provided by the present invention, one stereoisomer can cause significant cell pyroptosis, while the activity of the other stereoisomer is significantly weaker.

[0118] As another aspect of the technical solution of the present invention, it also relates to a method for purifying a stereoisomer pair having near-infrared second-region luminescence properties, which comprises: passing the stereoisomer pair having near-infrared second-region luminescence properties through a silica gel column for separation and purification.

[0119] In some embodiments, the crude product can be separated and purified using a silica gel column to directly obtain a stereoisomer pair.

[0120] Furthermore, the purification method further comprises: the mixed stereoisomer pair after the silica gel column purification needs to be further separated and purified by high performance liquid chromatography.

[0121] As another aspect of the technical solution of the present invention, it also relates to a near-infrared second-region fluorescent probe, which includes the aforementioned stereoisomer pair having near-infrared second-region luminescent properties.

[0122] Furthermore, the near-infrared second-region fluorescent probe exhibits different responsiveness to reactive oxygen species. More specifically, one stereoisomer exhibits a significant ratio fluorescence change to ONOO- under physiological conditions, while the other stereoisomer does not show a significant change.

[0123] The near-infrared second-zone fluorescent probe of the present invention is simple to synthesize, has a high yield and is easy to purify and separate, and has broad application prospects in the fields of biological imaging, drug screening and development, disease diagnosis and treatment, etc.

[0124] As another aspect of the technical solution of the present invention, it also relates to the application of the aforementioned stereoisomer pair having near-infrared second-region luminescence properties or near-infrared second-region fluorescent probe.

[0125] Furthermore, the stereoisomer pair or near-infrared region II fluorescent probe having near-infrared region II luminescence properties has obvious differences in photophysical and chemical properties and biomedical activity, and can be used for in vivo near-infrared region II fluorescence imaging or disease detection, such as in vivo imaging of mice.

[0126] Furthermore, the stereoisomer pair or near-infrared region II fluorescent probe having near-infrared region II luminescence properties has significantly different cellular effects and can be used in drug development in addition to being an imaging tool, specifically in visual drug screening and development.

[0127] Furthermore, the stereoisomer pair or near-infrared region II fluorescent probe having near-infrared region II luminescence properties can also be used in the preparation of products that induce cell death.

[0128] The specific implementation of the present invention will be described in more detail below in conjunction with the embodiments and drawings, but the embodiments do not limit the present invention. All variations associated with or derived from the contents disclosed in the present invention are considered to be within the protection scope of the present invention.

[0129] Example 1

[0130] The oxonium ion C5 (0.1mmol) and the condensing agent S5 (0.1mmol) were dissolved in 20mL acetic anhydride at room temperature, and then the raw materials were completely dissolved by ultrasound. Under a nitrogen atmosphere, the reaction system was heated to 110°C and the reaction was continued for 3 hours to obtain the intermediate M1. When the reaction solution was cooled to room temperature, it was poured into a large amount of water for precipitation, and the precipitate was washed with water for 2-3 times and then vacuum dried. The above precipitate (0.1mmol), oxonium ion C5 (0.1mmol) and sodium acetate (0.3mmol) were dissolved in acetic anhydride, reacted at room temperature for 12 hours under a nitrogen atmosphere, and the obtained reaction solution was poured into a large amount of water for precipitation to obtain a mixed stereoisomer probe. In order to obtain a single stereoisomer, the above precipitate was separated and purified by preparative high performance liquid chromatography (HPLC), and the mobile phase was acetone and water.

[0131] The H NMR spectrum of the stereoisomer R5-55 obtained in this example is as follows Figure 1 As shown, the NMR carbon spectrum of R5-55 is as follows Figure 2 As shown, the high-resolution mass spectrum of R5-55 is as follows Figure 3 As shown, its structural characterization data is:

[0132] R5-55: 1H NMR (400MHz, DMSO-d6) 68.10 (d, J=7.8Hz, 2H), 7.79 (m, 3H), 7.69-7.61 (m, 3H), 7.39 (dd, J=7.6, 3.5Hz, 2H), 6.82-6.71 (m, 5H), 6.5 7-6.50 (m, 1H), 6.32 (t, J=13.4Hz, 1H), 3.52-3.47 (m, 8H), 2.78 (d, J=6.6Hz, 4H), 2.64 (m, 2H), 2.45 (m, 2H), 1.17 (t, J=7.1Hz, 12H).

[0133] 13 C NMR (101MHz, DMSO-d6) 6167.50, 164.24, 155.96, 151.23, 145.52, 138.57, 138.07, 136.06, 135.03, 133.17, 131.71, 131.12, 130.92, 130 .11, 129.85, 129.15, 127.36, 125.43, 124.31, 114.07, 112.29, 110.48, 97.99, 97.40, 44.95, 44.30, 33.13, 25.26, 24.65, 19.72, 12.87.

[0134] HR-MS m / z calculation for C 49 H 47 N2O6 + , [M-ClO4] + :759.34,found:759.34.

[0135] The H NMR spectrum of the stereoisomer S5-55 obtained in this example is as follows Figure 4 As shown, the NMR carbon spectrum of S5-55 is as follows Figure 5 As shown, the high-resolution mass spectrum of S5-55 is as follows Figure 6 As shown, its structural characterization data is:

[0136] S5-55: 1H NMR (400MHz, DMSO-d6) δ8.09 (d, J=7.8Hz, 2H), 7.76 (t, J=7.4Hz, 2H), 7.69-7.59 (m, 4H), 7.33 (d, J=7.5Hz, 2H), 6.85-6.69 (m , 6H), 6.33 (t, J=13.2Hz, 1H), 3.49 (q, J=7.1Hz, 8H), 2.80 (t, J=6.5Hz, 4H), 2.61 (m, 2H), 2.45 (m, 2H), 1.16 (t, J=7.0Hz, 12H).

[0137] 13 C NMR (101MHz, DMSO-d6) δ167.48, 164.22, 155.95, 151.24, 145.50, 138.67, 134.99, 133.14, 131.6 7, 131.12, 130.08, 129.85, 127.36, 124.30, 114.05, 112.30, 97.38, 44.95, 25.27, 24.64, 12.88.

[0138] HR-MS m / z calculation for C 49 H 47 N2O6 + , [M-ClO4] + :759.34,found:759.34.

[0139]

[0140] Example 2

[0141] The oxonium ion C6 (0.1mmol) and the condensing agent S5 (0.1mmol) were dissolved in 20mL acetic anhydride at room temperature, and then the raw materials were completely dissolved by ultrasound. Under a nitrogen atmosphere, the reaction system was heated to 90°C and the reaction was continued for 3 hours to obtain the intermediate M2. When the reaction solution was cooled to room temperature, it was poured into a large amount of water for precipitation, and the precipitate was washed with water for 2-3 times and then vacuum dried. The above precipitate (0.1mmol), oxonium ion C6 (0.1mmo1) and sodium acetate (0.3mmol) were dissolved in acetic anhydride, reacted at room temperature for 12 hours under a nitrogen atmosphere, and the obtained reaction solution was poured into a large amount of water for precipitation to obtain a mixed stereoisomer probe. In order to obtain a single stereoisomer, the above precipitate was separated and purified using a silica gel column, and the eluent was dichloromethane and methanol.

[0142] The H NMR spectrum of the stereoisomer R5-66 obtained in this example is as follows Figure 7 As shown, the NMR carbon spectrum of R5-66 is as follows Figure 8 As shown, the high-resolution mass spectrum of R5-66 is as follows Fig. 9 As shown, its structural characterization data is:

[0143] R5-66: 1 H NMR (400MHz, DMSO-d6) 68.18-8.06 (m, 4H), 7.82-7.75 (m, 2H), 7.67 (td, J=7. 7, 1.3Hz, 2H), 7.32 (d, J=7.4Hz, 2H), 6.91 (d, J=2.4Hz, 2H), 6.77 (dd, J=9.2, 2.5Hz, 2H), 6.69 (d, J=13.4Hz, 1H), 6.57 (d, J=9.1Hz, 2H), 3.51 (m, 8H), 2.69 -2.59(m, 4H), 2.29-2.14(m, 4H), 1.76-1.62(m, 4H), 1.18(t, J=7.0Hz, 12H).

[0144] 13 C NMR (101MHz, DMSO-d6) δ167.26, 159.49, 155.87, 151.88, 142.93, 135.78, 133.19, 131.05, 130. 10, 129.69, 127.93, 121.54, 117.98, 113.86, 112.41, 96.49, 44.91, 26.83, 24.87, 20.81, 12.99.

[0145] HR-MS m / z calculation for C 51 H 51 N2O6 + , [M-ClO4] + :787.37,found:787.37.

[0146] The H NMR spectrum of the stereoisomer SS-66 obtained in this example is as follows: Fig.10 As shown, the NMR carbon spectrum of S5-66 is as follows Fig.11 As shown, the high-resolution mass spectrum of S5-66 is as follows Fig.12 As shown, its structural characterization data is:

[0147] S5-66: 1H NMR (400MHz, DMSO-d6) δ8.17 (d, J=7.9Hz, 4H), 7.84 (t, J=7.6Hz, 2H), 7.72 (t , J=7.7Hz, 2H), 7.37 (d, J=7.5Hz, 2H), 6.96 (d, J=2.4Hz, 2H), 6.81 (t, J=10.6 Hz, 2H), 6.73 (q, J=9.9, 7.9Hz, 1H), 6.63 (d, J=9.1Hz, 2H), 3.60-3.55 (m, 8H) , 2.69(m, 4H), 2.39-2.19(m, 4H), 1.84-1.64(m, 4H), 1.24(t, J=6.8Hz, 12H).

[0148] 13 C NMR (101MHz, DMSO-d6) 6167.26, 159.49, 155.88, 151.89, 149.50, 142.95, 135.77, 133.19, 131.10, 1 30.10, 129.69, 127.94, 121.54, 117.98, 113.86, 112.43, 96.48, 44.90, 26.84, 24.86, 20.79.13.00.

[0149] HR-MS m / z calculation for C 51 H 51 N2O6 + , [M-ClO4] + :787.37,found:787.37.

[0150]

[0151] Example 3

[0152] Oxonium ion C7 (0.1mmol) and condensing agent S5 (0.1mmol) were dissolved in 20mL acetic anhydride at room temperature, and then the raw materials were completely dissolved by ultrasound. Under a nitrogen atmosphere, the reaction system was heated to 110°C and the reaction was continued for 3 hours to obtain an intermediate M3. When the reaction solution was cooled to room temperature, it was poured into a large amount of water for precipitation, and the precipitation was continued to be washed with water 2-3 times and then vacuum dried. The above precipitate (0.1mmol), oxonium ion C7 (0.1mmol) and sodium acetate (0.5mmol) were dissolved in acetic anhydride, reacted at 60°C for 5 hours under a nitrogen atmosphere, and the obtained reaction solution was poured into a large amount of water for precipitation to obtain a mixed stereoisomer probe. In order to obtain a single stereoisomer, the above precipitate was crudely purified using a silica gel column, and further separated and purified using preparative high performance liquid chromatography (HPLC) to obtain a stereoisomer pair.

[0153] The H NMR spectrum of the stereoisomer R5-77 obtained in this example is as follows Fig.13 As shown, the high-resolution mass spectrum of R5-77 is as follows Fig.14 As shown, its structural characterization data is:

[0154] R5-77: 1 H NMR (400MHz, DMSO-d6) 68.13 (dd, J=7.9, 1.3Hz, 2H), 7.79 (m, 3H), 7.68 (m, 3H), 7.32 (dd, J=7.6, 1.3Hz, 2H), 6.92 (d, J=2.5Hz, 2H), 6. 77 (d, J=8.6Hz, 2H), 6.52 (d, J=8.9Hz, 3H), 3.51 (m, 8H), 2.77 (m, 4H), 2.34 (m, 4H), 1.80 (m, 4H), 1.62 (m, 4H), 1.17 (t, J=7.0Hz, 12H).

[0155] HR-MS m / z calculation for C 53 H 55 N2O6 + , [M-ClO4] + :815.41, found:815.40.

[0156] The H NMR spectrum of the stereoisomer S5-77 obtained in this example is as follows Fig.15 As shown, the high-resolution mass spectrum of S5-77 is as follows Fig.16 As shown, its structural characterization data is:

[0157] S5-77: 1 H NMR (400MHz, DMSO-d6) δ8.13 (dd, J=7.9, 1.3Hz, 2H), 7.79 (td, J=7.5, 1.4Hz, 3H), 7.68 (td, J=7.7, 1.4Hz, 3H), 7.32 (dd, J=7.7, 1.3Hz, 2H), 6.92(m, 2H), 6.76(m, 2H), 6.52(m, 3H), 3.51(m, 8H), 2.77(m, 4H), 2.34(m, 4H), 1.80(m, 4H), 1.61(m, 4H), 1.18(t, J=7.0Hz, 12H).

[0158] HR-MS m / z calculation for C 53 H 55 N2O6 + , [M-ClO4] +:815.41, found:815.40.

[0159]

[0160] Example 4

[0161] Oxonium ion C5 (0.1mmol) and condensing agent S7 (0.1mmol) were dissolved in 20mL acetic anhydride at room temperature, and then the raw materials were completely dissolved by ultrasound. Under a nitrogen atmosphere, the reaction system was heated to 110°C and the reaction was continued for 3 hours to obtain intermediate M4. When the reaction solution was cooled to room temperature, it was poured into a large amount of water for precipitation, and the precipitation was continued to be washed with water 2-3 times and then vacuum dried. The above-mentioned precipitate (0.1mmol), oxonium ion C5 (0.1mmol) and sodium acetate (0.3mmol) were dissolved in acetic anhydride, reacted at room temperature for 5 hours under a nitrogen atmosphere, and the resulting reaction solution was poured into a large amount of water for precipitation to obtain a mixed stereoisomer probe. In order to obtain a single stereoisomer, preparative high performance liquid chromatography (HPLC) was used for separation and purification.

[0162]

[0163] Example 5

[0164] Oxonium ion C6 (0.1mmol) and condensing agent S5 (0.1mmol) were dissolved in 20mL acetic anhydride at room temperature, and then the raw materials were completely dissolved by ultrasound. Under a nitrogen atmosphere, the reaction system was heated to 110°C and the reaction was continued for 3 hours to obtain intermediate M5. When the reaction solution was cooled to room temperature, it was poured into a large amount of water for precipitation, and the precipitation was continued to be washed with water for 2-3 times and then vacuum dried. The above-mentioned precipitate (0.1mmol), oxonium ion C6 (0.1mmol) and sodium acetate (0.3mmol) were dissolved in acetic anhydride, reacted at room temperature for 12 hours under a nitrogen atmosphere, and the obtained reaction solution was poured into a large amount of water for precipitation to obtain a mixed stereoisomer probe. In order to obtain a single stereoisomer, preparative high performance liquid chromatography (HPLC) was used for separation and purification.

[0165]

[0166] Example 6

[0167] The oxonium ion C7 (0.1mmol) and the condensing agent S7 (0.1mmol) were dissolved in 20mL acetic anhydride at room temperature, and then the raw materials were completely dissolved by ultrasound. Under a nitrogen atmosphere, the reaction system was heated to 110°C and the reaction was continued for 3 hours to obtain the intermediate M6. When the reaction solution was cooled to room temperature, it was poured into a large amount of water for precipitation, and the precipitate was continued to be washed with water 2-3 times and then vacuum dried. The above precipitate (0.1mmol), oxonium ion C7 (0.1mmol) and sodium acetate (0.3mmol) were dissolved in acetic anhydride, reacted at 60°C for 3 hours under a nitrogen atmosphere, and the resulting reaction solution was poured into a large amount of water for precipitation to obtain a mixed stereoisomer probe. In order to obtain a single stereoisomer, preparative high performance liquid chromatography (HPLC) was used for separation and purification.

[0168]

[0169] Example 7

[0170] The oxonium ion B5 (0.1 mmol) and the condensation agent S5 (0.1 mmol) were dissolved in 20 mL of ethanol at room temperature, and then the raw materials were completely dissolved by ultrasound. Under a nitrogen atmosphere, the reaction system was heated to 80 ° C and the reaction was continued for 3 hours to obtain the intermediate M7. Then the obtained M7 (0.1 mmol), oxonium ion C6 (0.1 mmol) and potassium acetate (3 mmol) were dissolved in 20 mL of toluene / n-butanol (volume ratio 1: 1), and reacted at 130 ° C for 3 hours under a nitrogen atmosphere. The obtained reaction solution was evaporated to remove the solvent to obtain a mixed stereoisomer probe. In order to obtain a single stereoisomer, preparative high performance liquid chromatography (HPLC) was used for separation and purification.

[0171]

[0172] Example 8

[0173] The oxonium ion B6 (0.1mmol) and the condensation agent S5 (0.2mmol) were dissolved in 20mL of acetic anhydride at room temperature, and then the raw materials were completely dissolved by ultrasound. Under a nitrogen atmosphere, the reaction system was heated to 110°C and the reaction was continued for 3 hours. When the reaction solution cooled to room temperature, it was poured into a large amount of water for precipitation. The precipitate was washed with water for 2-3 times and then vacuum dried to obtain the intermediate M8. The above precipitate (0.1mmol), oxonium ion C7 (0.05mmo1) and sodium carbonate (0.3mmol) were dissolved in toluene / n-butanol (volume ratio 3:1), reacted at 100°C for 0.5 hours under a nitrogen atmosphere, and the obtained reaction solution was evaporated to remove the solvent to obtain a mixed stereoisomer probe. In order to obtain a single stereoisomer, preparative high performance liquid chromatography (HPLC) was used for separation and purification.

[0174]

[0175] Example 9

[0176] Oxonium ion C7 (0.1mmol) and condensing agent S5 (0.05mmol) were dissolved in 20mL acetic anhydride at room temperature, and then the raw materials were completely dissolved by ultrasound. Under a nitrogen atmosphere, the reaction system was heated to 50°C and the reaction was continued for 3 hours. When the reaction solution was cooled to room temperature, it was poured into a large amount of water for precipitation. The precipitate was washed with water for 2-3 times and then vacuum dried to obtain intermediate M9. The above precipitate (0.05mmol), oxonium ion C7 (0.1mmol) and triethylamine (1mmol) were dissolved in acetic anhydride, reacted at room temperature for 4 hours under a nitrogen atmosphere, and the resulting reaction solution was poured into a large amount of water for precipitation to obtain a mixed stereoisomer probe. In order to obtain a single stereoisomer, preparative high performance liquid chromatography (HPLC) was used for separation and purification.

[0177]

[0178] Example 10

[0179] The oxonium ion B6 (0.1mmol) and the condensing agent S7 (0.1mmol) were dissolved in 20mL of ethanol at room temperature, and then the raw materials were completely dissolved by ultrasound. Under a nitrogen atmosphere, the reaction system was heated to 80°C and the reaction was continued for 3 hours. When the reaction solution was cooled to room temperature, it was poured into a large amount of water for precipitation. The precipitate was washed with water for 2-3 times and then vacuum dried to obtain the intermediate M10. The above precipitate (0.1mmol), oxonium ion C7 (1mmol) and triethylamine (3mmol) were dissolved in acetic anhydride, reacted at room temperature of 0°C for 24 hours under a nitrogen atmosphere, and the obtained reaction solution was poured into a large amount of water for precipitation to obtain a mixed stereoisomer probe. In order to obtain a single stereoisomer, preparative high performance liquid chromatography (HPLC) was used for separation and purification.

[0180]

[0181] Embodiment 11

[0182] The oxonium ion C5 (0.1mmol) and the condensing agent S7 (0.05mmol) were dissolved in 20mL methanol at room temperature, and then the raw materials were completely dissolved by ultrasound. Under a nitrogen atmosphere, the reaction system was heated to 150°C and the reaction was continued for 0.5 hours. When the reaction solution was cooled to room temperature, it was poured into a large amount of water for precipitation. The precipitate was washed with water for 2-3 times and then vacuum dried to obtain the intermediate M11. The above precipitate (0.1mmol), oxonium ion C7 (0.08mmol) and triethylamine (3mmol) were dissolved in acetic anhydride, reacted at room temperature of 120°C for 1 hour under a nitrogen atmosphere, and the obtained reaction solution was poured into a large amount of water for precipitation to obtain a mixed stereoisomer probe. In order to obtain a single stereoisomer, preparative high performance liquid chromatography (HPLC) was used for separation and purification.

[0183]

[0184] Example 12

[0185] The oxonium ion C5 (0.1 mmol) and the condensation agent S7 (0.01 mmol) were dissolved in 20 mL of benzene / n-butanol (volume ratio 2:1) at room temperature, and then the raw materials were completely dissolved by ultrasound. Under a nitrogen atmosphere, the reaction system was heated to 50°C and the reaction was continued for 12 hours. When the reaction solution cooled to room temperature, it was poured into a large amount of water for precipitation. The precipitate was washed with water for 2-3 times and then vacuum dried to obtain the intermediate M12. The above precipitate (0.1 mmol), the oxonium ion C7 (0.15 mmol) and sodium carbonate (0.1 mmol) were dissolved in acetic anhydride, reacted at room temperature of 150°C for 1 hour under a nitrogen atmosphere, and the resulting reaction solution was poured into a large amount of water for precipitation to obtain a mixed stereoisomer probe. In order to obtain a single stereoisomer, preparative high performance liquid chromatography (HPLC) was used for separation and purification.

[0186]

[0187] Example 13

[0188] The inventors of this case also performed spectral characterization on the stereoisomer probes obtained in Examples 1-3 above: the single stereoisomers were dissolved in methanol, acetone, dichloromethane, chloroform, DMSO, DMF and tetrahydrofuran, respectively, and the final probe concentration was 10μμM. The absorption and fluorescence spectra of the probes in the corresponding solvents were tested respectively. The results showed that Figure 17a-Figure 17d The absorption and fluorescence spectra of stereoisomer R / S5-55 in different solvents are shown respectively. Figure 18a-18d The absorption and fluorescence spectra of stereoisomer R / S5-66 in different solvents are shown respectively. Figure 19a-19d The absorption and fluorescence spectra of stereoisomer R / S5-77 in different solvents are shown respectively.

[0189] Embodiment 14

[0190] The inventors of this case also conducted pH stability tests on the stereoisomer probes obtained in Examples 1-3 above: the single stereoisomers were dissolved in PBS buffer containing 50% acetonitrile, and the pH of the buffer was adjusted using dilute hydrochloric acid and dilute sodium hydroxide solution. The final probe concentration was 10μμM. After incubation in the corresponding pH buffer for 2 hours, the spectra of the solutions were tested to evaluate the stability of the single stereoisomer molecules. The results showed that Figure 20a-Figure 20f The absorption and fluorescence spectra of stereoisomer R / S5-55 at different pH are shown respectively. Figure 21a-Figure 21f The absorption and fluorescence spectra of the stereoisomer R / S5-66 at different pH are shown respectively. Figure 22a-22f The absorption and fluorescence spectra of stereoisomer R / S5-77 at different pH values ​​are shown respectively.

[0191] Embodiment 15

[0192] The inventors of this case also tested the responsiveness of the stereoisomer probes R5-55 and S5-55 obtained in Example 1 above to active oxygen: the two stereoisomers were dissolved in PBS buffer containing 50% acetonitrile, and then common active oxygen species were added to the solution respectively, and the addition continued for 2 hours. The absorption and near-infrared fluorescence spectra of the probes were tested to evaluate the responsiveness of the probes to ONOO-. The results showed that the absorption and fluorescence spectra of the stereoisomer R / S5-55 after adding different active oxygen species were as follows: Figure 23a-23f As shown, the absorption and fluorescence spectra of stereoisomer R / S5-55 after adding ONOO- and the fluorescence imaging changes in the near-infrared first region and near-infrared second region are shown in Figures 1 and 2. Figure 24a-Figure 24f As shown, the mechanism diagram of the stereoisomer R / S5-55 response to ONOO- is shown in Fig.25 shown.

[0193] Example 16

[0194] HPLC purification and separation of mixed stereoisomers: Preparative HPLC was used to purify and separate mixed stereoisomers, where the chromatographic column did not need to use a chiral separation column, and the mobile phase was acetone and water. The results showed that the HPLC graph of probe R / S5-55 after stereoisomer separation was as follows Fig.26 As shown, the HPLC diagram of probe R / S5-66 after stereoisomer separation is as follows Fig. 27 As shown, the HPLC diagram of probe R / S5-77 after stereoisomer separation is as follows Fig.28 shown.

[0195] Embodiment 17

[0196] In vivo detection: Construct a colitis mouse model. Use R5-55 and S5-55 to diagnose colitis mice. Gavage the probe, then image the mice at a specific time, detect the mouse fluorescence images under the 1000nm long-pass channel and 820nm bandpass channel, and perform mouse ratio imaging. The results of the ratio fluorescence diagnosis test of the mouse colitis model with probe R / S5-55 stereoisomers are as follows Fig.29 As shown, the fluorescence spectra of mixed stereoisomers R7-66 and S7-66 are shown in Fig.30 The experimental results show that the two stereoisomers exhibit significant differences in properties in vivo, indicating that the stereoisomer probes have different physiological activities.

[0197] Embodiment 18

[0198] Stereoisomers induce cell pyroptosis: Cells were cultured with R5-55 and S5-55 probes, and then the cell status was monitored. After 12 hours of culture, the cell status was monitored. The results show that the cell status of 4T1 cells after incubation with two stereoisomers R / S5-55 for 12 hours is shown in the figure below. Fig.31 shown.

[0199] The above are only preferred embodiments of the present invention, which do not limit the protection scope of the present invention. For those skilled in the art, the present invention may have various modifications and changes. Within the spirit and principle of the present invention, changes, modifications, replacements, integrations and parameter changes to these embodiments by conventional substitutions or by being able to achieve the same functions without departing from the principles and spirit of the present invention all fall within the protection scope of the present invention.

Claims

1. A stereoisomer pair having near-infrared second-zone luminescence properties, characterized in that: The general structural formula of the stereoisomers includes formula (I) and formula (II): Among them, X - Including ClO4 - 、BF4 - CF3COO - , Cl - Br - ,I - 、NO3 - Any of the following; R1 includes Any of the following; R2 includes Any of the following; m is any integer from 0 to 2, n is any integer from 0 to 2, and z is 1 or 2.

2. A method for preparing a stereoisomer pair having near-infrared second-zone luminescence properties, characterized in that: include: allowing the first compound containing the oxonium ion to undergo a condensation reaction with a condensation agent to obtain an intermediate; The intermediate is further subjected to a condensation reaction with a second compound containing an oxonium ion to obtain a stereoisomer pair having near-infrared second-region luminescence properties; Wherein, the first compound containing oxonium ions has a structure as shown in formula (III): Among them, X - Including ClO4 - 、BF4 - CF3COO - , Cl - Br - ,I - 、NO3 - Any of the following; R1 includes Any one of; m is any integer from 0 to 2; The condensing agent has a structure as shown in formula (IV): Where z is 1 or 2; The second compound containing an oxonium ion has a structure as shown in formula (V): Among them, X - Including ClO4 - 、BF4 - CF3COO - , Cl - Br - ,I - 、NO3 - Any of the following; R2 includes Any one of; n is any integer from 0 to 2.

3. The preparation method according to claim 2, characterized in that: include: Mixing a first compound containing an oxonium ion, a condensation agent and a first reaction solvent, and performing a condensation reaction in a protective atmosphere to obtain an intermediate; Preferably, the first reaction solvent comprises at least any one of acetic anhydride, toluene / n-butanol, benzene / n-butanol, ethanol, and methanol, and acetic anhydride is particularly preferred; and / or, the molar ratio of the first compound containing oxonium ions to the condensing agent is 1:(0.1-2); and / or, the condensation reaction temperature is 50 to 150° C., preferably 90 to 110° C.; And / or, the condensation reaction time is 0.5 to 12 hours, preferably 2 to 3 hours.

4. The preparation method according to claim 2, characterized in that: include: The intermediate, the second compound containing an oxonium ion, a base and a second reaction solvent are mixed, and a condensation reaction is continued in a protective atmosphere to obtain a stereoisomer pair having near-infrared second-region luminescence properties; Preferably, the base includes at least any one of sodium acetate, potassium acetate, potassium carbonate, sodium carbonate, and triethylamine, preferably sodium acetate or potassium acetate; Preferably, the second reaction solvent includes at least any one of acetic anhydride, toluene / n-butanol, benzene / n-butanol, and acetic anhydride is particularly preferred.

5. The preparation method according to claim 2, characterized in that: The molar ratio of the intermediate to the second compound containing an oxonium ion is 1:(0.5-10), preferably 1:(0.8-1.5); and / or, the molar ratio of the intermediate to the base is 1:(1-30); and / or, the temperature for continuing the condensation reaction is 0 to 150° C., preferably 20 to 60° C.; And / or, the condensation reaction is continued for 0.5 to 24 hours, preferably 6 to 12 hours.

6. The preparation method according to claim 4, characterized in that: Also includes: The product obtained by the continued reaction is separated and purified by a silica gel column to obtain a stereoisomer pair of mixed structure; Preferably, the preparation method further comprises: using high performance liquid chromatography to separate and purify the stereoisomer pair of mixed structure after silica gel column purification again to obtain stereoisomers of single structure.

7. A stereoisomer pair having near-infrared second-region luminescence properties obtained by the preparation method according to any one of claims 2 to 6.

8. The method for purifying a stereoisomer pair having near-infrared second-region luminescence properties according to claim 7, characterized in that: include: Passing the stereoisomer having near-infrared second-region luminescence properties through a silica gel column for separation and purification; Preferably, the purification method further comprises: using high performance liquid chromatography to separate and purify the stereoisomer pair again after purification by silica gel column.

9. A near-infrared second-region fluorescent probe, characterized in that: The near-infrared second-region fluorescent probe comprises the stereoisomer pair having near-infrared second-region luminescence properties as described in claim 1 or 7.

10. Use of the stereoisomer pair with near-infrared second-region luminescence properties as described in claim 1 or 7 or the near-infrared second-region fluorescent probe as described in claim 9 in the screening and development of anti-tumor drugs for biological imaging or visualization.

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