Near-infrared fluorescent small molecule probe as well as preparation method and application thereof

Abstract

The invention discloses a near-infrared fluorescent small-molecule probe as well as a preparation method and application thereof. The fluorescent small-molecule probe is shown as a formula I in the specification. Excitation wavelengths of the fluorescent small-molecule probe are all in a near-infrared region, and the fluorescent small-molecule probe can be effectively combined with a myelin sheath part to remarkably enhance the imaging effect of the myelin sheath part and can be applied to myelin sheath fluorescence imaging, so that a novel molecular imaging reagent # imgabs0 # is provided for diagnosis and curative effect evaluation of central nervous system diseases such as multiple sclerosis.

Description

Technical Field

[0001] The present invention relates to the field of fluorescent molecular imaging, and in particular to a class of near-infrared fluorescent small molecule probes and a preparation method and application thereof. Background Art

[0002] Myelin is a membrane wrapped around the axons of nerve cells, composed of myelin, and generally appears in the central nervous system of vertebrates. Myelination refers to the process of myelin development, which speeds up the conduction of nerve excitation along nerve fibers and ensures its directional conduction to avoid mutual interference of information. At the same time, it can guide the regeneration of axons when some axons are damaged. Demyelination refers to the damage of myelin sheath after myelination, which may cause a variety of central nervous system diseases, the most common of which is multiple sclerosis (MS). In the diagnosis and efficacy evaluation of MS, seeking methods to visualize myelin sheaths qualitatively or quantitatively in vivo or in vitro has always been the goal of researchers and clinicians.

[0003] To achieve this goal, the key is to develop molecular probes that can specifically bind to myelin through the blood-brain barrier. In the current clinical diagnosis technology of MS, it mainly relies on nuclear magnetic resonance imaging technology, which does not have target specificity, and has other disadvantages such as complex operation and high cost. With the continuous advancement of near-infrared imaging technology, it will be possible to use near-infrared imaging technology for the diagnosis and efficacy evaluation of MS in the future, and researchers have also developed a variety of myelin-targeted near-infrared fluorescent molecular probes. Among them, two aminophenoxazinone structural compounds ADMPO and ADEPO were found to have application potential as myelin-targeted near-infrared fluorescent imaging small molecule probes (Tetrahedron, 2023, 136: 133358). However, they have disadvantages such as low lipid-water partition coefficient (LogP) and short fluorescence emission wavelength, which affect the blood-brain barrier pass rate and imaging depth. Summary of the invention

[0004] The purpose of the present invention is to provide a class of near-infrared fluorescent small molecule probes and their preparation method and application, which can specifically bind to the myelin sheath through the blood-brain barrier, thereby realizing in vivo visualization imaging of the myelin sheath, especially visualization imaging under near-infrared conditions, and providing an intuitive, simple, fast and inexpensive imaging method and imaging reagent for the diagnosis and efficacy evaluation of central nervous system diseases such as multiple sclerosis and the corresponding drug efficacy evaluation.

[0005] The present invention provides a type of near-infrared fluorescent small molecule probe, the probe structural formula I is:

[0006]

[0007] The R1 is a straight chain or branched (C1-C8) alkyl group;

[0008] The R2 is a linear or branched (C1-C8) alkyl group.

[0009] Preferably,

[0010] The R1 is a straight chain or branched (C1-C4) alkyl group;

[0011] The R2 is a linear or branched (C1-C4) alkyl group.

[0012] Preferably,

[0013] The R1 is methyl or ethyl;

[0014] The R2 is methyl, ethyl, propyl, butyl, isopropyl or isobutyl.

[0015] Preferably, the near-infrared fluorescent small molecule probe is as follows:

[0016]

[0017] The preparation method of the near-infrared fluorescent small molecule probe of the present invention comprises the following steps:

[0018]

[0019] The raw material II is dissolved in a solvent, an acid binding agent is added, and iodinated alkane III is slowly added dropwise at 0°C. The reaction is carried out at a certain temperature for a period of time. After the reaction is completed, an appropriate amount of water is added, and then extracted with an organic solvent. The organic phase is collected, washed with salt, washed with water, and dried. The organic solvent is evaporated under reduced pressure to obtain a crude product, which is then subjected to silica gel column chromatography to obtain compound I.

[0020] Preferably, the molar ratio of the raw material II to the compound III is 1:1 to 1:1.5.

[0021] Preferably, the dissolving solvent is methanol, N,N-dimethylformamide (DMF) or dimethyl sulfoxide (DMSO), the acid binding agent is sodium carbonate, potassium carbonate, sodium bicarbonate or triethylamine, the certain temperature is 0 to 80°C, the period of time is 4 to 24 hours, and the extraction solvent is ethyl acetate or dichloromethane or toluene.

[0022] Preferably, the compound III is methyl iodide, ethyl iodide, propyl iodide, butyl iodide, isopropyl iodide or isobutyl iodide.

[0023] The present invention also provides use of the above-mentioned near-infrared fluorescent small molecule probe or the near-infrared fluorescent small molecule probe prepared by the above-mentioned preparation method in myelin sheath imaging.

[0024] Beneficial effects:

[0025] (1) The near-infrared fluorescent small molecule probes described in the present invention have emission wavelengths in the near-infrared region (>650nm), and belong to near-infrared fluorescent small molecule probes;

[0026] (2) A type of near-infrared fluorescent small molecule probe described in the present invention has a lipid-water partition coefficient (LogP) between 2.5 and 4.0, can smoothly pass through the blood-brain barrier and specifically bind to the myelin sheath;

[0027] (3) The near-infrared fluorescent small molecule probe described in the present invention can realize visualization imaging of the myelin sheath in vivo, especially visualization imaging under near-infrared conditions, thereby providing an intuitive, simple, fast and inexpensive imaging method and imaging reagent for the diagnosis and efficacy evaluation of central nervous system diseases such as multiple sclerosis and the screening of corresponding therapeutic drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the technical solution of the present application, the drawings required for use in the embodiments are briefly introduced below. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0029] Figure 1 The mass spectrum of the target product A prepared in Example 1 of the present invention;

[0030] Figure 2 The mass spectrum of the target product B prepared in Example 2 of the present invention;

[0031] Figure 3 The mass spectrum of the target product C prepared in Example 3 of the present invention;

[0032] Figure 4 The mass spectrum of the target product D prepared in Example 4 of the present invention;

[0033] Figure 5 This is the mass spectrum of the target product E prepared in Example 5 of the present invention;

[0034] Figure 6 This is the mass spectrum of the target product F prepared in Example 6 of the present invention;

[0035] Figure 7 This is the mass spectrum of the target product G prepared in Example 7 of the present invention;

[0036] Figure 8 This is the mass spectrum of the target product H prepared in Example 8 of the present invention;

[0037] Fig. 9 The mass spectrum of the target product I prepared in Example 9 of the present invention;

[0038] Fig.10 This is the mass spectrum of the target product J prepared in Example 10 of the present invention;

[0039] Fig.11 This is the mass spectrum of the target product K prepared in Example 11 of the present invention;

[0040] Fig.12 This is the mass spectrum of the target product L prepared in Example 12 of the present invention;

[0041] Fig.13 This is an in vitro staining image of mouse brain using the near-infrared fluorescent small molecule probe prepared by the present invention. DETAILED DESCRIPTION

[0042] In order to enable those skilled in the art to understand the characteristics and effects of the present invention, the following is a general description and definition of the terms and expressions mentioned in the specification and claims. Unless otherwise specified, all technical and scientific terms used in the text are the common meanings understood by those skilled in the art for the present invention. In the event of a conflict, the definition in this specification shall prevail.

[0043] The theories or mechanisms described and disclosed herein, whether correct or incorrect, should not limit the scope of the present invention in any way, that is, the present invention can be implemented without being limited by any specific theory or mechanism.

[0044] Herein, all features such as values, quantities, contents and concentrations defined in the form of numerical ranges or percentage ranges are for simplicity and convenience only. Accordingly, the description of numerical ranges or percentage ranges should be considered to have included and specifically disclosed all possible secondary ranges and individual values ​​within the range (including integers and fractions).

[0045] In this document, in order to make the description concise, not all possible combinations of various technical features in various embodiments or examples are described. Therefore, as long as there is no contradiction in the combination of these technical features, the various technical features in various embodiments or examples can be combined arbitrarily, and all possible combinations should be considered to be within the scope of this specification.

[0046] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall within the scope limited by the appended claims of the application equally.

[0047] The following examples use conventional instruments and equipment in the art. The experimental methods in the following examples where specific conditions are not specified are usually carried out under conventional conditions or under conditions recommended by the manufacturer. The various raw materials used in the following examples are conventional commercial products unless otherwise specified, and their specifications are conventional specifications in the art. In the specification of the present invention and the following examples, unless otherwise specified, "%" means weight percentage, "part" means weight part, and ratio means weight.

[0048] The following is a further description of a class of near-infrared fluorescent small molecule probes provided in the present application and their preparation methods and applications in conjunction with specific embodiments.

[0049] 1. Synthesis of target product AL

[0050] Example 1: Synthesis of target product A

[0051] A magnetic bar was added to a 50 mL round-bottom flask, and 2 mmol of sodium carbonate and 1 mmol of raw material ADMPO were added. The flask was sealed and nitrogen was passed through for protection. Then 10 mL of dry DMF was added. After stirring for 30 min, 1.0 mmol of iodomethane was slowly added dropwise at 0°C. The reaction was carried out at 0°C for 24 h. The reaction process was monitored by TLC (developing solvent: ethyl acetate: n-hexane = 1:1). After the reaction was completed, ethyl acetate was extracted (30 mL × 3). The organic phases were combined, washed with saturated NaCl solution, dried over anhydrous Na2SO4, and ethyl acetate was removed by vacuum distillation to obtain a crude product. Solid product A was obtained by silica gel column chromatography. HR-MS (ESI) m / z calculated for (C 15 H 15 N3O2)[M+H] + 270.1237, found 270.1238. Figure 1 shown.

[0052] Example 2: Synthesis of target product B

[0053] A magnetic bar was added to a 50 mL round-bottom flask, and 2 mmol of sodium bicarbonate and 1 mmol of raw material ADMPO were added. The flask was sealed and nitrogen was passed through for protection. Then 10 mL of dry DMF was added. After stirring for 30 min, 1.3 mmol of iodoethane was slowly added dropwise at 0 °C. The reaction was carried out at 0 °C for 24 h. The reaction process was monitored by TLC (developing solvent: ethyl acetate: n-hexane = 1:1). After the reaction was completed, ethyl acetate was extracted (30 mL × 3). The organic phases were combined, washed with saturated NaCl solution, dried over anhydrous Na2SO4, and ethyl acetate was removed by vacuum distillation to obtain a crude product. Solid product B was obtained by silica gel column chromatography. HR-MS (ESI) m / z calculated for (C 16 H 17 N3O2)[M+H]+ 284.1394, found 284.1391. Figure 2 shown.

[0054] Example 3: Synthesis of target product C

[0055] A magnetic bar was added to a 50 mL round-bottom flask, and 2 mmol of potassium carbonate and 1 mmol of raw material ADMPO were added. The flask was sealed and nitrogen was passed through for protection. Then 10 mL of anhydrous methanol was added. After stirring for 30 min, 1.5 mmol of iodopropane was slowly added dropwise at 0°C. The reaction was carried out at 50°C for 12 h. The reaction process was monitored by TLC (developing solvent: ethyl acetate: n-hexane = 1:1). After the reaction was completed, dichloromethane was extracted (30 mL × 3). The organic phases were combined, washed with saturated NaCl solution, dried with anhydrous Na2SO4, and dichloromethane was removed by vacuum distillation to obtain a crude product. Solid product C was obtained by silica gel column chromatography. HR-MS (ESI) m / z calculated for (C 17 H 19 N3O2)[M+Na] + 320.1369, found 320.1372. Figure 3 shown.

[0056] Example 4: Synthesis of target product D

[0057] A magnetic bar was added to a 50 mL round-bottom flask, and 2 mmol of potassium carbonate and 1 mmol of raw material ADMPO were added. The flask was sealed and nitrogen was passed through. Then 10 mL of anhydrous methanol was added. After stirring for 30 min, 1.5 mmol of iodobutane was slowly added dropwise at 0°C. The reaction was carried out at 50°C for 12 h. The reaction process was monitored by TLC (developing solvent: ethyl acetate: n-hexane = 1:1). After the reaction was completed, dichloromethane was extracted (30 mL × 3). The organic phases were combined, washed with saturated NaCl solution, dried with anhydrous Na2SO4, and dichloromethane was removed by vacuum distillation to obtain a crude product. Solid product D was obtained by silica gel column chromatography. HR-MS (ESI) m / z calculated for (C 18 H 21 N3O2)[M+H] + 312.1633, found 312.1706. Figure 4 shown.

[0058] Example 5: Synthesis of target product E

[0059] A magnetic bar was added to a 50 mL round-bottom flask, and 2 mmol of triethylamine and 1 mmol of raw material ADMPO were added. The flask was sealed and nitrogen was passed through for protection. Then 10 mL of dry DMSO was added. After stirring for 30 min, 1.5 mmol of isopropyl iodide was slowly added dropwise at 0°C. The reaction was carried out at 80°C for 4 h. The reaction process was monitored by TLC (developing solvent: ethyl acetate: n-hexane = 1:1). After the reaction was completed, toluene was extracted (30 mL × 3). The organic phases were combined, washed with saturated NaCl solution, dried with anhydrous Na2SO4, and toluene was removed by vacuum distillation to obtain a crude product. Solid product E was obtained by silica gel column chromatography. HR-MS (ESI) m / z calculated for (C 17 H 19 N3O2)[M+H] + 298.1550, found 298.1549. Figure 5 shown.

[0060] Example 6: Synthesis of target product F

[0061] A magnetic bar was added to a 50 mL round-bottom flask, and 2 mmol of triethylamine and 1 mmol of raw material ADMPO were added. The flask was sealed and nitrogen was passed through for protection. Then 10 mL of dry DMSO was added. After stirring for 30 min, 1.5 mmol of isobutyl iodide was slowly added dropwise at 0°C. The reaction was carried out at 80°C for 4 h. The reaction process was monitored by TLC (developing solvent: ethyl acetate: n-hexane = 1:1). After the reaction was completed, toluene was extracted (30 mL × 3). The organic phases were combined, washed with saturated NaCl solution, dried with anhydrous Na2SO4, and toluene was removed by vacuum distillation to obtain a crude product. Solid product F was obtained by silica gel column chromatography. HR-MS (ESI) m / z calculated for (C 18 H 21 N3O2)[M+H] + 312.1712, found 312.1647. Figure 6 shown.

[0062] Example 7: Synthesis of target product G

[0063] A magnetic bar was added to a 50 mL round-bottom flask, and 2 mmol of sodium carbonate and 1 mmol of raw material ADEPO were added. The flask was sealed and nitrogen was passed through. Then 10 mL of anhydrous methanol was added. After stirring for 30 min, 1.1 mmol of iodomethane was slowly added dropwise at 0°C. The reaction was carried out at 0°C for 24 h. The reaction process was monitored by TLC (developing solvent: ethyl acetate: n-hexane = 1:1). After the reaction was completed, ethyl acetate was extracted (30 mL × 3). The organic phases were combined, washed with saturated NaCl solution, dried with anhydrous Na2SO4, and ethyl acetate was removed by vacuum distillation to obtain a crude product. Solid product G was obtained by silica gel column chromatography. HR-MS (ESI) m / z calculated for (C 17 H 19 N3O2)[M+H] + 298.1556, found 298.1550. Figure 7 shown.

[0064] Example 8: Synthesis of target product H

[0065] A magnetic bar was added to a 50 mL round-bottom flask, and 2 mmol of sodium bicarbonate and 1 mmol of raw material ADEPO were added. The flask was sealed and nitrogen was passed through for protection. Then 10 mL of anhydrous methanol was added. After stirring for 30 min, 1.2 mmol of iodoethane was slowly added dropwise at 0°C. The reaction was carried out at 0°C for 24 h. The reaction process was monitored by TLC (developing solvent: ethyl acetate: n-hexane = 1:1). After the reaction was completed, ethyl acetate was extracted (30 mL × 3). The organic phases were combined, washed with saturated NaCl solution, dried with anhydrous Na2SO4, and ethyl acetate was removed by vacuum distillation to obtain a crude product. The solid product H was obtained by silica gel column chromatography. HR-MS (ESI) m / z calculated for (C 18 H 21 N3O2)[M+H] + 312.1706, found 312.1700. Figure 8 shown.

[0066] Example 9: Synthesis of target product I

[0067] A magnetic bar was added to a 50 mL round-bottom flask, and 2 mmol of potassium carbonate and 1 mmol of raw material ADEPO were added. The flask was sealed and nitrogen was passed through for protection. Then 10 mL of dry DMF was added. After stirring for 30 min, 1.3 mmol of iodopropane was slowly added dropwise at 0 °C and reacted at 60 °C for 12 h. The reaction process was monitored by TLC (developing solvent: ethyl acetate: n-hexane = 1:1). After the reaction was completed, dichloromethane was extracted (30 mL × 3), and the organic phases were combined, washed with saturated NaCl solution, dried over anhydrous Na2SO4, and dichloromethane was removed by vacuum distillation to obtain a crude product. Solid product I was obtained by silica gel column chromatography. HR-MS (ESI) m / z calculated for (C 19 H 23 N3O2)[M+H] + 326.1863, found 326.1862. Fig. 9 shown.

[0068] Example 10: Synthesis of target product J

[0069] A magnetic bar was added to a 50 mL round-bottom flask, and 2 mmol of potassium carbonate and 1 mmol of raw material ADEPO were added. The flask was sealed and nitrogen was passed through for protection. Then 10 mL of dry DMF was added. After stirring for 30 min, 1.3 mmol of iodobutane was slowly added dropwise at 0 °C and reacted at 60 °C for 12 h. The reaction process was monitored by TLC (developing solvent: ethyl acetate: n-hexane = 1:1). After the reaction was completed, dichloromethane was extracted (30 mL × 3), and the organic phases were combined, washed with saturated NaCl solution, dried over anhydrous Na2SO4, and dichloromethane was removed by vacuum distillation to obtain a crude product. Solid product J was obtained by silica gel column chromatography. HR-MS (ESI) m / z calculated for (C 20 H 25 N3O2)[M+H] + 340.2019, found 340.2019. Fig.10 shown.

[0070] Example 11: Synthesis of target product K

[0071] A magnetic bar was added to a 50 mL round-bottom flask, and 2 mmol of triethylamine and 1 mmol of raw material ADEPO were added. The flask was sealed and nitrogen was passed through for protection. Then 10 mL of dry DMSO was added. After stirring for 30 min, 1.5 mmol of iodomethane was slowly added dropwise at 0°C. The reaction was carried out at 80°C for 4 h. The reaction process was monitored by TLC (developing solvent: ethyl acetate: n-hexane = 1:1). After the reaction was completed, toluene was extracted (30 mL × 3). The organic phases were combined, washed with saturated NaCl solution, dried with anhydrous Na2SO4, and toluene was removed by vacuum distillation to obtain a crude product. Solid product K was obtained by silica gel column chromatography. HR-MS (ESI) m / z calculated for (C 19 H 23 N3O2)[M+H] + 326.1863, found 326.1858. Fig.11 shown.

[0072] Example 12: Synthesis of target product L

[0073] A magnetic bar was added to a 50 mL round-bottom flask, and 2 mmol of triethylamine and 1 mmol of raw material ADEPO were added. The flask was sealed and nitrogen was passed through for protection. Then 10 mL of dry DMSO was added. After stirring for 30 min, 1.5 mmol of iodomethane was slowly added dropwise at 0 °C. The reaction was carried out at 80 °C for 4 h. The reaction process was monitored by TLC (developing solvent: ethyl acetate: n-hexane = 1:1). After the reaction was completed, toluene was extracted (30 mL × 3). The organic phases were combined, washed with saturated NaCl solution, dried with anhydrous Na2SO4, and toluene was removed by vacuum distillation to obtain a crude product. Solid product L was obtained by silica gel column chromatography. HR-MS (ESI) m / z calculated for (C 20 H 25 N3O2)[M+H] + 340.2025, found 340.1957. Fig.12 shown.

[0074] 2. Fluorescence performance test

[0075] The product was dissolved in dimethyl sulfoxide (DMSO) to prepare a 1mM solution, and its excitation wavelength and emission wavelength were tested using a fluorescence spectrophotometer. The results showed that its emission wavelength was greater than 650nm, which is in the near-infrared region.

[0076] The emission wavelengths and LogP of some near-infrared fluorescent small molecule probes are shown in Table 1.

[0077] Table 1 Emission wavelength and LogP of some near-infrared fluorescent small molecule probes

[0078]

[0079] 3. In vitro staining experiment

[0080] In vitro staining experiments are an effective method for detecting the specific binding and staining performance of compounds with myelin. The compound to be stained is prepared into a 1mM solution with dimethyl sulfoxide solvent, and then the corpus callosum, striatum and spinal cord of the mouse brain in paraffin sections are stained in vitro. After 25 minutes of exposure and imaging, it can be clearly observed that after in vitro staining, the white matter areas (richer myelin distribution) of the three parts are significantly brighter than the gray matter areas (less myelin), which means that the tested compound can specifically bind to the myelin and can be well stained and imaged. Combined with the previous fluorescence performance test results, it shows that this type of structural compound is suitable for myelin targeted imaging under near-infrared conditions. Fig.13 This is an in vitro staining image of the mouse brain of Example 1-12. The results show that the white matter portion of Example 1-12 is significantly brighter than the gray matter region, indicating good myelin-specific binding and potential for application as a myelin-targeted near-infrared fluorescent small molecule probe.

[0081] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A type of near-infrared fluorescent small molecule probe, characterized in that: The probe structural formula I is: The R1 is a straight chain or branched (C1-C8) alkyl group; The R2 is a linear or branched (C1-C8) alkyl group.

2. The near-infrared fluorescent small molecule probe according to claim 1, characterized in that: The R1 is a straight chain or branched (C1-C4) alkyl group; The R2 is a linear or branched (C1-C4) alkyl group.

3. The near-infrared fluorescent small molecule probe according to claim 1, characterized in that: The R1 is methyl or ethyl; The R2 is methyl, ethyl, propyl, butyl, isopropyl or isobutyl.

4. The near-infrared fluorescent small molecule probe according to claim 1, characterized in that: The probe is as follows:

5. A method for preparing a near-infrared fluorescent small molecule probe, characterized in that: The steps are: The raw material II is dissolved in a solvent, an acid binding agent is added, and iodinated alkane III is slowly added dropwise at 0°C. The reaction is carried out at a certain temperature for a period of time. After the reaction is completed, an appropriate amount of water is added, and then extracted with an organic solvent. The organic phase is collected, washed with salt, washed with water, and dried. The organic solvent is evaporated under reduced pressure to obtain a crude product, which is then subjected to silica gel column chromatography to obtain compound I.

6. The method for preparing the near-infrared fluorescent small molecule probe according to claim 5, characterized in that: The molar ratio of the raw material II to the compound III is 1:1 to 1:1.

5.

7. The method for preparing the near-infrared fluorescent small molecule probe according to claim 5, characterized in that: The dissolving solvent is methanol, N,N-dimethylformamide or dimethyl sulfoxide, the acid binding agent is sodium carbonate, potassium carbonate, sodium bicarbonate or triethylamine, the certain temperature is 0 to 80° C., the period of time is 4 to 24 hours, and the extraction solvent is ethyl acetate, dichloromethane or toluene.

8. The method for preparing the near-infrared fluorescent small molecule probe according to claim 5, characterized in that: The compound III is methyl iodide, ethyl iodide, propyl iodide, butyl iodide, isopropyl iodide or isobutyl iodide.

9. Use of the near-infrared fluorescent small molecule probe as claimed in any one of claims 1 to 4 or the near-infrared fluorescent small molecule probe prepared by the preparation method as claimed in any one of claims 5 to 8 in myelin imaging.

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