Small molecule probe as well as preparation method and application thereof

Abstract

The invention relates to a small molecule probe as well as a preparation method and application thereof, the chemical structural formula of the probe is as follows: # imgabs0 #, in which R1 is 1-propanesulfonic acid or 1-butanesulfonic acid; r2 is independently selected from trifluoromethyl or sulfenyl pentafluoride, and the substitution position of R2 is ortho-position, para-position or meta-position of hydroxyl on a benzene ring; the preparation method comprises the following steps: directly introducing a fluorine substituent group into a spiropyrane molecular structure with pH response through condensation reaction of salicylaldehyde containing the fluorine substituent group and indoline, and preparing the pH response type bimodal imaging small molecular probe with both < 19 > F MRI (Magnetic Resonance Imaging) and fluorescence imaging. Compared with the prior art, the small molecule probe shows remarkably different < 19 > F NMR chemical shifts and fluorescence intensities in different pH environments, high-resolution < 19 > F MRI imaging and fluorescence imaging in different pH environments can be realized, the imaging sensitivity and accuracy are greatly improved, and the small molecule probe has important application prospects in the fields of biosensing, cell imaging, medical diagnosis and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of organic small molecule probes, and relates to a small molecule probe and a preparation method and application thereof, and specifically relates to a pH-responsive 19 F magnetic resonance imaging and fluorescence imaging small molecule probes and their preparation methods and applications. Background Art

[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 with water protons, endogenous fluorine atoms in organisms exist only in trace amounts (<10- 6 M), and is mainly accumulated in teeth and bones. Due to its extremely short transverse relaxation time (T2), it exhibits negligible magnetic resonance signals. 19 F MRI ( 19 F MRI) is considered to be an imaging technique with zero background interference. 19 The linear relationship between F content and signal intensity allows for quantitative imaging. 19 F MRI has brought new opportunities in molecular and cellular imaging, and has shown broad application prospects in the fields of cell tracking, biological target imaging, and related disease diagnosis.

[0003] 19 The main challenge facing F MRI in practical applications is its low sensitivity. This is mainly due to the low dose 19 The longitudinal relaxation effect of FMRI probes is poor, and a longer acquisition time is required to obtain high-resolution images. 19 F MRI imaging agents can achieve good imaging effects at low concentrations and short acquisition times. Typical agents include perfluorocarbons, perfluoropolyethers, fluorinated polymers, fluorinated inorganic compounds, etc. These imaging agents are characterized by high fluorine content, chemical inertness, and difficulty in decomposition. However, they also have problems such as difficulty in metabolism, poor water solubility, and difficulty in functionalization.

[0004] pH stimulus-responsive small molecules 19 F MRI probes provide a new solution to the above problems. The pH-stimuli-responsive small molecule probes constructed by molecular engineering can not only obtain good water solubility, biocompatibility, and rapid metabolism, but also emit signals only after the pH changes, which can significantly improve the specificity and sensitivity of imaging. 19 There are two main types of F MRI probes. The first type is the stimulus-responsive type based on the paramagnetic relaxation enhancement (PRE) effect. 19F MRI molecular probes, but the probes constructed in this way 19 F MRI signals only have a single change from weak to strong, and have low anti-interference ability. Another type of probe is based on the change of the chemical environment within the molecule caused by external stimuli, which leads to 19 FNMR chemical shift changes produce distinct 19 F MRI signal, achieving high-sensitivity detection, but the maximum sensitivity that this type of probe molecule can achieve is 19 The chemical shift variation of F NMR is limited to 2 ppm, which greatly restricts its application in practice. Summary of the invention

[0005] The purpose of the present invention is to provide a pH responsive 19 F magnetic resonance imaging and fluorescence imaging small molecule probes and preparation methods and applications thereof. The present invention introduces fluorine-containing substituents into the structure of spiropyrans that respond to pH stimulation, and introduces fluorine-containing substituents into the spiropyrans that respond to pH stimulation through the condensation reaction of salicylaldehyde containing fluorine substituents and indoline, thereby successfully constructing a spiropyrans that responds to pH stimulation. 19 F NMR chemical shift changes and significant fluorescence intensity changes 19 F MRI imaging and fluorescence imaging dual-modality small molecule probe. The probe exhibits significantly different 19 F NMR chemical shift and fluorescence intensity, using this remarkable 19 FNMR chemical shift changes and fluorescence changes can achieve high resolution in different pH environments 19 F MRI imaging and fluorescence imaging greatly improve the sensitivity and accuracy of imaging.

[0006] The purpose of the present invention can be achieved by the following technical solutions:

[0007] The first aspect of the present invention provides a small molecule probe, the chemical structure of the probe is as follows:

[0008]

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

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

[0011] 1) adding 2,3,3-trimethylindole and alkyl sulfonate into an organic solvent, and heating the mixture to obtain an intermediate product;

[0012] 2) adding the intermediate product and fluorine-containing substituted salicylaldehyde into an organic solvent, and heating the resulting product to obtain a small molecule probe.

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

[0014] Furthermore, in step 1), the alkyl sultone is selected from 1,3-propane sultone or 1,4-butane sultone.

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

[0016] Furthermore, in step 2), the molar ratio of the intermediate product to the fluorine-containing substituted salicylaldehyde is 1:1.

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

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

[0019] Furthermore, in step 1), the heating reaction is carried out for a heating time of 5-48 h and a heating temperature of 70-120°C; in step 2), the heating reaction is carried out for a heating time of 24-72 h and a heating temperature of 70-120°C.

[0020] The 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 by the present invention mainly exists in the form of spiropyran open-ring isomers in an acidic environment. When the pH value of the environment becomes alkaline, the open-ring isomers are quickly converted into closed-ring isomers. The significant chemical structural difference between the open-ring isomers and the closed-ring isomers of spiropyran greatly changes the chemical environment of the fluorinated substituents, resulting in the electron density of the fluorinated substituents in the open-ring isomers being significantly lower than that in the closed-ring isomers. Therefore, the two can generate up to 13 ppm. 19F NMR chemical shift changes. In addition, since the probe exists mainly in the form of open-ring isomers in acidic solutions, the indole unit and the chromene structural unit are conjugated with each other, and there is an obvious intramolecular charge transfer process (ICT), which can produce strong fluorescence. After the open-ring isomer is converted into a closed-ring body, this conjugated structure is cut off, resulting in fluorescence quenching. Using this significant 19 F NMR chemical shift changes and fluorescence changes can achieve high resolution in different pH environments 19 F MRI imaging 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) The present invention introduces a fluorine-containing substituent into the pH-responsive spiropyran molecular structure through a condensation reaction of salicylaldehyde containing a fluorine substituent and indoline, thereby preparing a spiropyran having both 19 A pH-responsive dual-modality imaging small molecule probe for F MRI and fluorescence imaging, which has important application prospects in the fields of biosensing, cell imaging, and medical diagnosis;

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

[0025] 3) The small molecule probe prepared by the present invention 19 F NMR chemical shifts can vary by up to 13 ppm, enabling high-resolution analysis of the 19 F MRI imaging resolves common pH stimulus responses 19 F MRI probes only have 19 F NMR / 19 F MRI signal strength changes and 19 F NMR / 19 The problem of small change trend of F MRI signal has significantly improved 19 F MRI imaging sensitivity;

[0026] 4) The small molecule probe prepared by the present invention can also cause changes in the intensity of the probe fluorescence, thereby achieving fluorescence imaging in different pH environments and further improving the accuracy of imaging. BRIEF DESCRIPTION OF THE DRAWINGS

[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-visible 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 F MRI images;

[0031] Figure 5 These are confocal imaging images of the small molecule probe 1a prepared in Example 1 in Staphylococcus: (A) dark field image under acidic pH conditions, (B) bright field image under acidic pH conditions, (C) dark field image under alkaline pH conditions, and (D) bright field image under alkaline pH conditions. DETAILED DESCRIPTION

[0032] The present invention is described in detail below in conjunction with the accompanying drawings and specific embodiments. This embodiment is implemented based on the technical solution of the present invention, and provides a detailed implementation method and specific operation process, but the protection scope of the present invention is not limited to the following embodiments.

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

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

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

[0036] Embodiment 1:

[0037] A small molecule probe having the following molecular structure:

[0038]

[0039] The preparation method comprises the following steps:

[0040] S1: 2,3,3-trimethylindole (1.59 g, 1 mmol) and 1,3-propane sultone (1.22 g, 1 mmol) were added to a 100 mL single-necked flask, and 50 mL of ethanol was added for reflux heating reaction at 85 °C for 5 hours. After the reaction was completed, the reaction solution was poured into water and cooled to room temperature. The coolant was vacuum filtered to obtain the intermediate product a (4-(2,3,3-trimethyl-3H-indol-1-ium-1-yl)propane-1-sulfonic acid inner salt)

[0041] S2: Add the intermediate product a (1.41 g, 0.5 mmol) and 2-hydroxy-5-trifluoromethylbenzaldehyde (0.95 g, 0.5 mmol) into a 150 mL single-necked flask, add 100 mL of toluene, and reflux and react at a temperature of 115 °C for 24 hours. After the reaction, pour the reaction solution into water, cool to room temperature, and vacuum filter the coolant to obtain a small molecule probe, which is recorded as small molecule probe 1a.

[0042] The NMR data of the small molecule probe 1a are: 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 small molecule probe 1a was tested:

[0044] Phosphate buffer solution was used to prepare probe solutions of different pH solutions, and the nuclear magnetic resonance (NMR) of probe 1a under different pH conditions was measured by NMR spectrometer. 19 F spectrum( 19 F NMR spectrum) to monitor the pH-induced 19 F NMR chemical shift changes; fluorescence intensity changes of probe 1a under different pH conditions were monitored by fluorescence spectrometer; absorption spectrum changes of probe 1a under different pH conditions were monitored by UV-visible spectrophotometer; probe 1a under acidic and alkaline pH conditions were measured under different pulse excitations of small animal nuclear magnetic resonance imaging system. 19 F Magnetic resonance imaging effect; Fluorescence imaging of Staphylococcus aureus incubated with probe molecules under acid-base conditions was tested by laser confocal fluorescence microscopy.

[0045] The results are as follows:

[0046] Figure 1 The small molecule probe 1a is expressed under different pH conditions. 19 F NMR spectrum, from which it can be seen that the small molecule probe 1a changes by 13 ppm with the increase of pH, proving that the small molecule probe 1a has great 19 F NMR chemical shift changes.

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

[0048] Figure 3 The UV-visible absorption spectra of the small molecule probe 1a under different pH conditions. It can be seen from the figure that the small molecule probe 1a has significant absorption changes at different pH values, proving that the 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) 19F MRI images show that the probe can achieve high-resolution imaging under different pH environments.

[0050] Figure 5 This is a confocal imaging picture of the small molecule probe 1a in Staphylococcus. From the picture, it can be seen that the small molecule probe 1a exhibits strong fluorescence in acidic conditions and no fluorescence in alkaline conditions, proving that the small molecule probe 1a can image bacteria under acidic conditions.

[0051] Embodiment 2:

[0052] A small molecule probe, the preparation method of which is different from that of Example 1 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). The remaining conditions were the same as those in Example 1. The prepared small molecule probe was recorded as small molecule probe 1b.

[0054] Embodiment 3:

[0055] A small molecule probe, the preparation method of which is different from that of Example 1 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). The remaining conditions were the same as those in Example 1. The prepared small molecule probe was recorded as small molecule probe 1c.

[0057] Embodiment 4:

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

[0059] S1: 2,3,3-trimethylindole (1.59 g, 1 mmol) and 1,4-butanesulfonate (1.36 g, 1 mmol) were added to a 100 mL single-necked flask, and 50 mL of ethanol was added for reflux heating reaction at 85 °C for 24 hours. After the reaction was completed, the reaction solution was poured into water and cooled to room temperature. The coolant was vacuum filtered to obtain the intermediate product b (4-(2,3,3-trimethyl-3H-indol-1-ium-1-yl)butane-1-sulfonic acid inner salt);

[0060] S2: The 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 were added for reflux heating reaction. The heating temperature was 115 °C and the heating time was 36 hours. After the reaction was completed, the reaction solution was poured into water and cooled to room temperature. After vacuum filtering the coolant, a small molecule probe was obtained, which was recorded as small molecule probe 2a.

[0061] Embodiment 5:

[0062] A small molecule probe, the preparation method of which is different from that of Example 4 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). The remaining conditions were the same as those in Example 4. The prepared small molecule probe was designated as small molecule probe 2b.

[0064] Embodiment 6:

[0065] A small molecule probe, the preparation method of which is different from that of Example 4 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). The remaining conditions were the same as those in Example 4. The prepared small molecule probe was recorded as small molecule probe 2c.

[0067] Embodiment 7:

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

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

[0070] Embodiment 8:

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

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

[0073] Embodiment 9:

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

[0075] In step S2, the reflux time is 72 hours. The other conditions are the same as those in Example 4. The prepared small molecule probe is recorded as small molecule probe 4a.

[0076] Embodiment 10:

[0077] A small molecule probe, the preparation method of which is different from that of Example 4 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. The remaining conditions were the same as those in Example 4. A small molecule probe was prepared, which was recorded as small molecule probe 4b.

[0079] Embodiment 11:

[0080] A small molecule probe, the preparation method of which is different from that of Example 4 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. The remaining conditions were the same as those in Example 4. A small molecule probe was prepared, which was recorded as small molecule probe 4c.

[0082] The small molecule probe prepared in the above embodiment has pH stimulus response characteristics. At acidic and neutral pH, the spiropyran small molecule probe is mainly an open-ring isomer. When the pH value becomes alkaline, the open-ring isomer is rapidly converted into a closed-ring isomer. Based on this pH-induced structural transformation of the open-ring and closed-ring isomers, the probe 19 F NMR chemical shifts can vary by up to 13 ppm, enabling high-resolution analysis of the 19 F MRI imaging resolves common pH stimulus responses 19 F MRI probes only have 19 F NMR / 19F MRI signal strength changes and 19 F NMR / 19 The problem of small change trend of F MRI signal has significantly improved 19 At the same time, the structural transformation of open- and closed-ring isomers can also cause changes in the intensity of probe fluorescence, enabling fluorescence imaging in different pH environments and further improving the accuracy of imaging.

[0083] The above description of the embodiments is to facilitate the understanding and use of the invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments, and 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 present invention should be within the scope of protection of the present invention.

Claims

1. 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 pentafluorosulfur, and the substitution position of R2 is the ortho, para or meta position of the hydroxyl group on the benzene ring.

2. The method for preparing a small molecule probe according to claim 1, characterized in that: The method comprises the following steps: 1) adding 2,3,3-trimethylindole and alkyl sulfonate into an organic solvent, and heating the mixture to obtain an intermediate product; 2) adding the intermediate product and fluorine-containing substituted salicylaldehyde into an organic solvent, and heating the resulting product to obtain a small molecule probe.

3. The method for preparing a small molecule probe according to claim 2, characterized in that: In step 1), the molar ratio of 2,3,3-trimethylindole to alkyl sulfonate is 1:

1.

4. The method for preparing a small molecule probe according to claim 2, characterized in that: In step 1), the alkyl sultone is selected from 1,3-propane sultone or 1,4-butane sultone.

5. The method for preparing a small molecule probe according to claim 2, characterized in that: In step 1), the organic solvent is selected from one or more of ethanol, acetonitrile or toluene.

6. The method for preparing a small molecule probe according to claim 2, characterized in that: In step 2), the molar ratio of the intermediate product to the fluorine-containing substituted salicylaldehyde is 1:

1.

7. The method for preparing a small molecule probe according to claim 2, characterized in that: In step 2), the fluorine-containing substituted salicylaldehyde is selected from any one of 2-hydroxy-5-trifluoromethylbenzaldehyde, 2-hydroxy-4-trifluoromethylbenzaldehyde, 2-hydroxy-3-trifluoromethylbenzaldehyde, 2-hydroxy-4-pentafluorosulfenylbenzaldehyde or 2-hydroxy-5-pentafluorosulfenylbenzaldehyde.

8. The method for preparing a small molecule probe according to claim 2, characterized in that: In step 2), the organic solvent is selected from one or more of ethanol, acetonitrile or toluene.

9. The method for preparing a small molecule probe according to claim 2, characterized in that: In step 1), the heating time of the heating reaction is 5-48 hours and the heating temperature is 70-120°C; in step 2), the heating time of the heating reaction is 24-72 hours and the heating temperature is 70-120°C.

10. The use of a small molecule probe as claimed in claim 1, characterized in that: The small molecule probe responds to pH stimulation 19 Applications in F MRI and fluorescence imaging.

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