A small molecule probe, and a preparation method and application thereof

Abstract

The present application relates to a kind of small molecule probes and its preparation method and application, the chemical structure of the probe is as shown in the following formula: Wherein, R1 is 1-propyl sulfonic acid or 1-butyl sulfonic acid;R2 independently selected from trifluoromethyl or sulfur pentafluoride group, the substitution position of R2 is the ortho, para or meta of hydroxyl on benzene ring;Preparation method is by the condensation reaction of salicylaldehyde containing fluorine substituent group and indoline, directly introduce containing fluorine substituent group into the spiro-pyran molecule structure with pH response, prepared with 19 F MRI and fluorescence imaging pH-responsive bimodal imaging small molecule probe.Compared with the prior art, the small molecule probe of the present application exhibits significantly different 19 F NMR chemical shift and fluorescence intensity, high-resolution 19 F MRI imaging and fluorescence imaging, greatly improve the sensitivity and accuracy of imaging, has important application prospect in the field of biosensing, cell imaging, medical diagnosis etc.

Description

Technical Field

[0001] This invention belongs to the field of organic small molecule probe technology, and relates to a small molecule probe, its preparation method and application, specifically a pH-responsive probe. 19 Small molecule probes for magnetic resonance imaging and fluorescence imaging, their preparation methods, and applications. Background Technology

[0002] As a non-invasive, radiation-free, and high-resolution imaging technique, magnetic resonance imaging (MRI) is one of the most widely used tools in clinical diagnosis. Compared to water protons, endogenous fluorine atoms in living organisms exist in trace amounts (<10⁻¹⁰). 6 M), and mainly accumulates in teeth and bones. Due to its extremely short transverse relaxation time (T2), it exhibits negligible magnetic resonance signal. Therefore, pH-responsive... 19 F magnetic resonance imaging (F magnetic resonance imaging) 19 fMRI is considered an imaging technique with zero background interference. It utilizes exogenous... 19 The linear relationship between F content and signal intensity allows for quantitative imaging. The aforementioned significant advantages are... 19 fMRI has brought new opportunities to molecular and cellular imaging, showing broad application prospects in cell tracking, biological target imaging and related disease diagnosis.

[0003] 19 The main challenge facing fMRI in practical applications is its low sensitivity. This is primarily due to the low dose... 19 The longitudinal relaxation effect of fMRI probes is poor, requiring a long acquisition time to obtain high-resolution images. A material with high fluorine atomic density is used as... 19 fMRI imaging reagents can achieve good imaging results at low concentrations and short acquisition times. Typical reagents include perfluorinated carbon, perfluorinated polyethers, fluorinated polymers, and fluorinated inorganic compounds. These imaging reagents are characterized by high fluorine content, chemical inertness, and difficulty in decomposition, but they also have problems such as being difficult to metabolize, poor water solubility, and difficulty in functionalization.

[0004] pH-responsive small molecules 19 fMRI probes offer a new solution to the aforementioned problems. pH-responsive small molecule probes constructed using molecular engineering not only possess excellent water solubility, biocompatibility, and rapid metabolic capacity, but also emit signals only upon pH change, significantly improving imaging specificity and sensitivity. Currently reported pH-responsive probes... 19 fMRI probes are mainly divided into two categories. The first category is stimulus-responsive probes constructed based on the paramagnetic relaxation enhancement (PRE) effect. 19fMRI molecular probes, but probes constructed in this way... 19 fMRI signals only show a single change from weak to strong, resulting in low interference resistance. Another type of probe is based on changes in the intramolecular chemical environment induced by external stimuli, leading to... 19 FNMR chemical shift changes produce significantly different 19 fMRI signals can be used to achieve highly sensitive detection; however, the maximum reach of these probe molecules is currently limited. 19 The chemical shift variation of F NMR is limited to 2 ppm, which greatly restricts its practical application. Summary of the Invention

[0005] The purpose of this invention is to provide a pH-responsive type 19 This invention relates to small molecule probes for magnetic resonance imaging and fluorescence imaging, their preparation methods, and applications. It introduces fluorinated substituents into the structure of pH-responsive spiropyran. The fluorinated substituents are introduced into the pH-responsive spiropyran core structure through a condensation reaction between fluorinated salicylaldehyde and indoline, successfully constructing probes with extremely high fluorine content. 19 F NMR chemical shift changes and significant fluorescence intensity changes 19 A dual-modality small molecule probe for both fMRI and fluorescence imaging. This probe exhibits significantly different characteristics in different pH environments. 19 F NMR chemical shift and fluorescence intensity, utilizing this significant 19 FNMR chemical shift and fluorescence changes can achieve high resolution for different pH environments. 19 fMRI and fluorescence imaging greatly improve the sensitivity and accuracy of imaging.

[0006] The objective of this invention can be achieved through the following technical solutions:

[0007] The first aspect of this invention provides a small molecule probe, the chemical structure of which is shown below:

[0008]

[0009] Wherein, R1 is either 1-propanesulfonic acid or 1-butanesulfonic acid; R2 is independently selected from trifluoromethyl or sulfopentafluoride, and the substitution position of R2 is ortho, para or meta of the hydroxyl group on the benzene ring.

[0010] A second aspect of this invention provides a method for preparing a small molecule probe, the method comprising the following steps:

[0011] 1) 2,3,3-trimethylindole and alkyl sulfonate lactone were added to an organic solvent and reacted by heating to obtain an intermediate product;

[0012] 2) The intermediate product and fluorine-substituted salicylaldehyde were added to an organic solvent and heated to prepare a small molecule probe.

[0013] Further, in step 1), the molar ratio of 2,3,3-trimethylindole to alkyl sulfonate lactone is 1:1.

[0014] Further, in step 1), the alkyl sulfonate lactone is selected from 1,3-propanesulfonate lactone or 1,4-butanesulfonate lactone.

[0015] Further, in step 1), the organic solvent is selected from one or more of ethanol, acetonitrile, or toluene. Preferably, the organic solvent is ethanol.

[0016] Further, in step 2), the molar ratio of the intermediate product to the fluorinated salicylaldehyde is 1:1.

[0017] Further, in step 2), the fluorinated salicylaldehyde is selected from any one of 2-hydroxy-5-trifluoromethylbenzaldehyde, 2-hydroxy-4-trifluoromethylbenzaldehyde, 2-hydroxy-3-trifluoromethylbenzaldehyde, 2-hydroxy-4-pentafluoridesulfobenzaldehyde, or 2-hydroxy-5-pentafluoridesulfobenzaldehyde.

[0018] Further, in step 2), the organic solvent is selected from one or more of ethanol, acetonitrile, or toluene. Preferably, the organic solvent is toluene.

[0019] Further, in step 1), the heating reaction is carried out for 5-48 h and the heating temperature is 70-120℃; in step 2), the heating reaction is carried out for 24-72 h and the heating temperature is 70-120℃.

[0020] A third aspect of the present invention provides an application of a small molecule probe, wherein the small molecule probe responds to pH stimulation. 19 Applications in fMRI and fluorescence imaging.

[0021] The small molecule probe prepared in this invention exists primarily as the spiropyran open-ring isomer in acidic environments. When the pH changes to alkaline, the open-ring isomer rapidly transforms into the closed-ring isomer. The significant chemical structural difference between the open-ring and closed-ring isomers drastically alters the chemical environment of the fluorinated substituents, resulting in a significantly lower electron density of the fluorinated substituents in the open-ring isomer compared to that in the closed-ring isomer. Therefore, a reaction can occur between the two, reaching up to 13 ppm. 19F NMR chemical shift changes. Furthermore, since the probe exists primarily as an open-ring isomer in acidic solution, the indole and chromene structural units are conjugated, resulting in a significant intramolecular charge transfer (ICT) process, thus producing strong fluorescence. Upon conversion from the open-ring isomer to the closed-ring isomer, this conjugated structure is cleaved, leading to fluorescence quenching. This significant [further details are needed for accurate translation]. 19 F NMR chemical shift and fluorescence changes can achieve high resolution for different pH environments. 19 fMRI and fluorescence imaging greatly improve the sensitivity and accuracy of imaging.

[0022] Compared with the prior art, the present invention has the following characteristics:

[0023] 1) This invention introduces fluorine-substituented compounds into the pH-responsive spiropyran molecule structure through the condensation reaction of fluorine-substituented salicylaldehyde with indoline, thus preparing a compound that possesses both... 19 A pH-responsive dual-modal imaging small molecule probe for fMRI and fluorescence imaging, which has important application prospects in biosensing, cell imaging, and medical diagnostics.

[0024] 2) The preparation method of this invention is simple to operate and has high synthesis efficiency, and can realize the preparation of probes at the g scale or above;

[0025] 3) The small molecule probes prepared in this invention 19 F NMR chemical shifts can produce changes of up to 13 ppm, enabling high-resolution NMR under different pH conditions. 19 fMRI imaging resolved common pH stimulation responses 19 Only present in fMRI probes 19 F NMR / 19 fMRI signal intensity changes and 19 F NMR / 19 The problem of small trend changes in fMRI signal was significantly improved. 19 Sensitivity of fMRI imaging;

[0026] 4) The small molecule probes prepared by this invention can also cause changes in the intensity of probe fluorescence, enabling fluorescence imaging in different pH environments and further improving the accuracy of imaging. Attached Figure Description

[0027] Figure 1 The small molecule probe 1a prepared in Example 1 under different pH conditions 19 F NMR spectrum;

[0028] Figure 2 The fluorescence spectra of the small molecule probe 1a prepared in Example 1 under different pH conditions;

[0029] Figure 3 The UV-Vis absorption spectra of the small molecule probe 1a prepared in Example 1 under different pH conditions;

[0030] Figure 4 The small molecule probe 1a prepared in Example 1 under acidic pH conditions (A) and alkaline pH conditions (B) 19 fMRI images;

[0031] Figure 5 The following are confocal images of the small molecule probe 1a prepared in Example 1 in Staphylococcus aureus: (A) Dark field image under acidic pH conditions, (B) Bright field image under acidic pH conditions, (C) Dark field image under alkaline pH conditions, (D) Bright field image under alkaline pH conditions. Detailed Implementation

[0032] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. These embodiments are based on the technical solution of the present invention and provide detailed implementation methods and specific operating procedures. However, the scope of protection of the present invention is not limited to the following embodiments.

[0033] The following are more detailed implementation examples, which further illustrate the technical solution of the present invention and the technical effects that can be obtained.

[0034] In the following embodiments, unless otherwise specified, the raw materials, reagents or processing techniques are all conventional commercial products or conventional processing techniques in the art.

[0035] In the following embodiments, 2-hydroxy-5-trifluoromethylbenzaldehyde (CAS No.: 210039-65-9, catalog No.: BD257156), 2-hydroxy-4-trifluoromethylbenzaldehyde (CAS No.: 58914-34-4, catalog No.: BD228764), 2-hydroxy-3-trifluoromethylbenzaldehyde (CAS No.: 336628-67-2, catalog No.: BD75596), 2-hydroxy-4-pentafluorinated sulfobenzaldehyde (CAS: 1807636-87-8, catalog No.: BD01215151), and 2-hydroxy-5-pentafluorinated sulfobenzaldehyde (CAS: 1159512-31-8, catalog No.: BD00775089) were all from Shanghai Bid Pharmaceutical Technology Co., Ltd.

[0036] Example 1:

[0037] A small molecule probe has the following molecular structural formula:

[0038]

[0039] Its preparation method includes the following steps:

[0040] S1: 2,3,3-Trimethylindole (1.59 g, 1 mmol) and 1,3-propanesulfonic acid lactone (1.22 g, 1 mmol) were added to a 100 mL single-necked flask, along with 50 mL of ethanol. The mixture was refluxed and heated to 85 °C for 5 hours. After the reaction was complete, the reaction solution was poured into water and cooled to room temperature. The cooled solution was then filtered under vacuum to obtain the intermediate product a (4-(2,3,3-trimethyl-3H-indole-1-onthium-1-yl)propane-1-sulfonic acid lactone).

[0041] S2: Intermediate product a (1.41 g, 0.5 mmol) and 2-hydroxy-5-trifluoromethylbenzaldehyde (0.95 g, 0.5 mmol) were added to a 150 mL single-necked flask, and 100 mL of toluene was added. The mixture was refluxed and heated to 115 °C for 24 hours. After the reaction was completed, the reaction solution was poured into water and cooled to room temperature. The cooled solution was then filtered under vacuum to obtain a small molecule probe, denoted as small molecule probe 1a.

[0042] The NMR data for this small molecule probe 1a are as follows: 1 H NMR (400 MHz, DMSO-d6) δ:11.95 (s, 1H,O H ), 8.62 (d, J = 8.0 Hz, 1H, Ar H ), 8.53-8.51 (d, J = 16.4 Hz, 1H, C H =C), 8.12 – 8.06(m, 1H, Ar) H ), 8.04-8.02 (d, J = 16.4 Hz, 1 H , CH=C), 7.90– 7.86(m, 1H, Ar H ),7.77-7.75 (dd, J = 8.0, 1H, Ar H ), 7.65 – 7.61 (m, 2H, Ar H ),7.22-7.20 (d, J = 8.0 Hz, 1H,Ar H ), 4.82 (t, J = 7.6 Hz, 2H, NC H2 ), 2.64 (t,J = 6.5 Hz, 2H, SC H2 ), 2.26-2.18 (m, 2H,C H2), 1.82 (s, 6H, C(C H 3)2) ppm; HRMS(ESI, m / z): [M + H]+calcd for C 22 H 23 F3NO4S,454.1300; found 454.1302.

[0043] The performance of the above-mentioned small molecule probe 1a was tested:

[0044] Probe solutions with different pH values ​​were prepared using phosphate buffer solution, and the nuclear magnetic resonance (NMR) values ​​of probe 1a under different pH conditions were measured using a nuclear magnetic resonance (NMR) spectrometer. 19 F-map ( 19 (F NMR spectrum), monitoring pH-induced changes in probe 1a 19 F NMR chemical shift changes; fluorescence intensity changes of probe 1a under different pH conditions monitored by fluorescence spectroscopy; absorption spectral changes of probe 1a under different pH conditions monitored by UV-Vis spectrophotometer; and the absorption spectrum of probe 1a under acidic and alkaline pH conditions determined by a small animal NMR imaging system under different pulse excitations. 19 F-magnetic resonance imaging effect; fluorescence imaging of Staphylococcus aureus incubated with probe molecules under acid-base conditions using laser confocal fluorescence microscopy.

[0045] The results are as follows:

[0046] Figure 1 For small molecule probe 1a under different pH conditions 19 The F NMR spectrum shows that the small molecule probe 1a changes by 13 ppm with increasing pH, demonstrating that the small molecule probe 1a has extremely high pH sensitivity. 19 F NMR chemical shift changes.

[0047] Figure 2 The figure shows the fluorescence spectra of small molecule probe 1a under different pH conditions. It can be seen from the figure that the fluorescence intensity of small molecule probe 1a decreases with increasing pH, which proves that small molecule probe 1a has strong fluorescence under acidic conditions and fluorescence quenching under alkaline conditions.

[0048] Figure 3 The figure shows the UV-Vis absorption spectra of small molecule probe 1a under different pH conditions. As can be seen from the figure, small molecule probe 1a has significant absorption changes under different pH conditions, which proves that small molecule probe 1a has significant pH responsiveness.

[0049] Figure 4 The small molecule probe 1a under acidic pH conditions (A) and alkaline pH conditions (B) 19The fMRI images demonstrate that the probe can achieve high-resolution imaging under different pH conditions.

[0050] Figure 5 This is a confocal imaging image of small molecule probe 1a in Staphylococcus aureus. The image shows that small molecule probe 1a exhibits strong fluorescence under acidic conditions but no fluorescence under alkaline conditions, proving that small molecule probe 1a can image bacteria under acidic conditions.

[0051] Example 2:

[0052] A small molecule probe, the preparation method of which differs from that in Example 1 only in that:

[0053] In step S2, 2-hydroxy-5-trifluoromethylbenzaldehyde (0.95 g, 0.5 mmol) was replaced with 2-hydroxy-4-trifluoromethylbenzaldehyde (0.95 g, 0.5 mmol). All other conditions were the same as in Example 1. The prepared small molecule probe was designated as small molecule probe 1b.

[0054] Example 3:

[0055] A small molecule probe, the preparation method of which differs from that in Example 1 only in that:

[0056] In step S2, 2-hydroxy-5-trifluoromethylbenzaldehyde (0.95 g, 0.5 mmol) was replaced with 2-hydroxy-3-trifluoromethylbenzaldehyde (0.95 g, 0.5 mmol). All other conditions were the same as in Example 1. The prepared small molecule probe was designated as small molecule probe 1c.

[0057] Example 4:

[0058] A small molecule probe, the preparation method of which includes the following steps:

[0059] S1: 2,3,3-trimethylindole (1.59 g, 1 mmol) and 1,4-butyryl lactone (1.36 g, 1 mmol) were added to a 100 mL single-necked flask, and 50 mL of ethanol was added. The mixture was refluxed and heated at 85 °C for 24 hours. After the reaction was completed, the reaction solution was poured into water and cooled to room temperature. The cooled solution was then filtered under vacuum to obtain intermediate product b (4-(2,3,3-trimethyl-3H-indole-1-onthium-1-yl)but-1-sulfonic acid lactone).

[0060] S2: Intermediate product b (1.47 g, 0.5 mmol) and 2-hydroxy-5-trifluoromethylbenzaldehyde (0.95 g, 0.5 mmol) were added to a 150 mL single-necked flask, and 100 mL of toluene was added. The mixture was refluxed and heated at 115 °C for 36 hours. After the reaction was completed, the reaction solution was poured into water and cooled to room temperature. The cooled solution was then filtered under vacuum to obtain a small molecule probe, denoted as small molecule probe 2a.

[0061] Example 5:

[0062] A small molecule probe, the preparation method of which differs from that in Example 4 only in that:

[0063] In step S2, 2-hydroxy-5-trifluoromethylbenzaldehyde (0.95 g, 0.5 mmol) was replaced with 2-hydroxy-4-trifluoromethylbenzaldehyde (0.95 g, 0.5 mmol). All other conditions were the same as in Example 4. The prepared small molecule probe was designated as small molecule probe 2b.

[0064] Example 6:

[0065] A small molecule probe, the preparation method of which differs from that in Example 4 only in that:

[0066] In step S2, 2-hydroxy-5-trifluoromethylbenzaldehyde (0.95 g, 0.5 mmol) was replaced with 2-hydroxy-3-trifluoromethylbenzaldehyde (0.95 g, 0.5 mmol). All other conditions were the same as in Example 4. The prepared small molecule probe was designated as small molecule probe 2c.

[0067] Example 7:

[0068] A small molecule probe, the preparation method of which differs from that in Example 1 only in that:

[0069] In step S2, 2-hydroxy-5-trifluoromethylbenzaldehyde (0.95 g, 0.5 mmol) was replaced with 2-hydroxy-5-pentafluorinated sulfobenzaldehyde (1.24 g, 0.5 mmol), and the reflux time was 48 hours. All other conditions were the same as in Example 1. The prepared small molecule probe was designated as small molecule probe 3a.

[0070] Example 8:

[0071] A small molecule probe, the preparation method of which differs from that in Example 1 only in that:

[0072] In step S2, 2-hydroxy-5-trifluoromethylbenzaldehyde (0.95 g, 0.5 mmol) was replaced with 2-hydroxy-4-pentafluorinated sulfobenzaldehyde (1.24 g, 0.5 mmol), and the reflux time was 48 hours. All other conditions were the same as in Example 1. The prepared small molecule probe was designated as small molecule probe 3b.

[0073] Example 9:

[0074] A small molecule probe, the preparation method of which differs from that in Example 4 only in that:

[0075] In step S2, the reflux time was 72 hours. All other conditions were the same as in Example 4. The prepared small molecule probe was designated as small molecule probe 4a.

[0076] Example 10:

[0077] A small molecule probe, the preparation method of which differs from that in Example 4 only in that:

[0078] In step S2, 2-hydroxy-5-trifluoromethylbenzaldehyde (0.95 g, 0.5 mmol) was replaced with 2-hydroxy-4-trifluoromethylbenzaldehyde (0.95 g, 0.5 mmol), and the reflux time was 72 hours. All other conditions were the same as in Example 4. The resulting small molecule probe was designated as small molecule probe 4b.

[0079] Example 11:

[0080] A small molecule probe, the preparation method of which differs from that in Example 4 only in that:

[0081] In step S2, 2-hydroxy-5-trifluoromethylbenzaldehyde (0.95 g, 0.5 mmol) was replaced with 2-hydroxy-3-trifluoromethylbenzaldehyde (0.95 g, 0.5 mmol), and the reflux time was 72 hours. All other conditions were the same as in Example 4. The resulting small molecule probe was designated as small molecule probe 4c.

[0082] The small molecule probes prepared in the above embodiments exhibit pH-responsive properties. Under acidic and neutral pH conditions, the spiropyran small molecule probes are predominantly open-ring isomers. When the pH changes to alkaline, the open-ring isomers rapidly convert to closed-ring isomers. Based on this pH-induced structural transformation between open and closed-ring isomers, the probes… 19 F NMR chemical shifts can produce changes of up to 13 ppm, enabling high-resolution NMR under different pH conditions. 19 fMRI imaging resolved common pH stimulation responses 19 Only present in fMRI probes 19 F NMR / 19fMRI signal intensity changes and 19 F NMR / 19 The problem of small trend changes in fMRI signal was significantly improved. 19 The sensitivity of fMRI imaging is improved. Furthermore, the structural transformation between open and closed-ring isomers can alter the fluorescence intensity of the probe, enabling fluorescence imaging under different pH conditions and further enhancing imaging accuracy.

[0083] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. An application of a small molecule probe, characterized in that, The chemical structure of the probe is shown below: Wherein, R1 is 1-propanesulfonic acid or 1-butanesulfonic acid; R2 is independently selected from trifluoromethyl or sulfopentafluoride, and the substitution position of R2 is ortho, para or meta of the hydroxyl group on the benzene ring; The small molecule probe responds to pH stimulation. 19 Applications in fMRI and fluorescence imaging, but not for the treatment or diagnosis of diseases.

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