Small molecule probe based on imidazo [1, 5-a] pyridine skeleton, preparation method and application

Abstract

The invention discloses a small molecular probe based on an imidazo [1, 5-a] pyridine skeleton, a preparation method and application, and belongs to the field of activatable small molecular fluorescent probes. The preparation method of the small molecular probe based on the imidazo [1, 5-a] pyridine skeleton comprises the following steps: dissolving 2-benzoylpyridine and ammonium acetate in acetic acid, adding salicylaldehyde, heating and stirring to generate a compound 3; and dissolving the compound 3 in dichloromethane, adding pyridine, stirring, adding 4-bromobutyryl chloride, and continuously stirring to obtain a compound 1, namely the small molecule probe. The small molecule probe can be used for detecting hydrazine in a biological system or drinking water.

Description

Technical Field

[0001] The present invention belongs to the field of activatable small molecule fluorescent probes, and particularly relates to a small molecule probe based on an imidazo[1,5-a]pyridine skeleton, a preparation method and an application thereof. Background Art

[0002] As a highly toxic and carcinogenic substance, hydrazine can cause acute damage to many organs of the human body, such as the kidneys, lungs and liver, and may cause cancer, seriously endangering human health. At the same time, hydrazine is also an important industrial chemical and is widely used in many fields such as pharmaceuticals, chemicals, catalysis, and agriculture. It is easy to cause environmental pollution during industrial production, disposal and other processes, thus making people exposed to this environment at risk of poisoning. Therefore, it is particularly important to establish a detection method for hydrazine to monitor the concentration of hydrazine in biological systems and drinking water. Existing hydrazine detection methods include titrimetric analysis, spectrophotometry, electrochemistry, chromatography-mass spectrometry, Raman spectroscopy, and so on. However, these methods have defects such as high cost, cumbersome operation or long analysis time, which hinder their application in practice.

[0003] Fluorescent probes have the advantages of high sensitivity, good selectivity and good biocompatibility, and have been widely developed in the detection of small molecule targets in recent years. Although many hydrazine detection fluorescent probe molecules have been reported at present, most of them have the disadvantages of strong background signal, poor selectivity, high cytotoxicity and complex synthesis process. Research shows that imidazo[1,5-a]pyridine and its derivatives have been widely used in organic light-emitting diodes and organic field-effect transistors. Importantly, these compounds also have excellent optical properties, such as a large Stokes shift and high photostability, which effectively improve the detection sensitivity of the probe molecule. Therefore, how to construct a novel fluorescent probe based on the imidazo[1,5-a]pyridine skeleton structure, which has simple synthesis, high detection sensitivity and high selectivity for hydrazine recognition, has become a technical problem to be solved urgently at present. Summary of the Invention

[0004] Aiming at the deficiencies of the prior art, the purpose of the present invention is to provide a small molecule probe based on an imidazo[1,5-a]pyridine skeleton, a preparation method and an application thereof, which solves the problems in the prior art.

[0005] The purpose of the present invention can be achieved by the following technical solutions:

[0006] A small molecule probe based on an imidazo[1,5-a]pyridine skeleton, the structural formula of the small molecule probe is:

[0007]

[0008] Preparation method of small molecule probe based on imidazo[1,5-a]pyridine skeleton, comprising the following steps:

[0009] S1, Dissolve 2-benzoylpyridine and ammonium acetate in acetic acid, add salicylaldehyde, heat and stir to generate compound 3;

[0010] S2, Dissolve compound 3 in dichloromethane, add pyridine, stir and then add 4-bromobutyryl chloride, continue to stir to obtain compound 1, which is the small molecule probe.

[0011] Further, in S1, the temperature of heating and stirring is 110 °C.

[0012] Further, in S1, the molar ratio of 2-benzoylpyridine, ammonium acetate to salicylaldehyde is 1:5:1.5.

[0013] Further, in S2, the molar ratio of compound 3, pyridine to 4-bromobutyryl chloride is 1:80:1.2.

[0014] Further, the production route of compound 3 is:

[0015]

[0016] Further, the synthesis route of the said compound 1 is:

[0017]

[0018] Application of the above-mentioned small molecule probe based on imidazo[1,5-a]pyridine skeleton in detecting hydrazine.

[0019] A hydrazine detection reagent, comprising the above-mentioned small molecule probe based on imidazo[1,5-a]pyridine skeleton.

[0020] A biological system and drinking water detection reagent, comprising the above-mentioned hydrazine detection reagent.

[0021] Advantages of the present invention:

[0022] 1. The synthesis route of the small molecule probe provided by the present invention is simple, the raw materials are easily available and the cost is low.

[0023] 2. The small molecule probe provided by the present invention can recognize hydrazine, has high selectivity and sensitivity to hydrazine, and has potential application value in the field of hydrazine detection.

[0024] 3. The small molecule probe provided by the present invention can detect low-concentration hydrazine in biological systems and drinking water, has good biocompatibility, and has potential application value in the field of toxic and harmful substance detection. Description of the drawings

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the accompanying drawings required in the description of the embodiments or the prior art. Obviously, for those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0026] Figure 1 is the hydrogen nuclear magnetic resonance spectrum of Compound 3 of the present invention;

[0027] Figure 2 is the high-resolution mass spectrum of Compound 3 of the present invention;

[0028] Figure 3 is the hydrogen nuclear magnetic resonance spectrum of Compound 1 of the present invention;

[0029] Figure 4 is the carbon nuclear magnetic resonance spectrum of Compound 1 of the present invention;

[0030] Figure 5 is the high-resolution mass spectrum of Compound 1 of the present invention;

[0031] Figure 6 is the response spectrum of Compound 1 of the present invention to hydrazine;

[0032] Figure 7 is the live cell imaging image of Compound 1 of the present invention. Detailed Embodiments

[0033] The following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

[0034] The small molecule probe based on the imidazo[1,5-a]pyridine skeleton has the following structural formula:

[0035]

[0036] The preparation process of the small molecule probe based on the imidazo[1,5-a]pyridine skeleton is as follows:

[0037] Step 1: Dissolve 2-benzoylpyridine and ammonium acetate in acetic acid, add salicylaldehyde, heat and stir to generate Compound 3.

[0038] Step 2: Dissolve Compound 3 in dichloromethane, add pyridine, stir at room temperature for 30 minutes, then add 4-bromobutyryl chloride, and continue to stir at room temperature for 24 hours to generate Compound 1, that is, the small molecule probe.

[0039] In Step 1, the temperature for heating and stirring is 110 °C, and the reaction time is 6 hours.

[0040] In Step 1, the molar ratio of 2-benzoylpyridine, ammonium acetate, and salicylaldehyde is 1:5:1.5.

[0041] In Step 2, the molar ratio of Compound 3, pyridine, and 4-bromobutyryl chloride is 1:80:1.2.

[0042] The following examples illustrate the specific preparation process and application of the small molecule probe.

[0043] Example 1

[0044] A method for preparing a small molecule probe based on an imidazo[1,5-a]pyridine skeleton, comprising the following steps:

[0045] S1. Preparation of Compound 3. The preparation route is as follows:

[0046]

[0047] The specific preparation process of Compound 3 is as follows:

[0048] Dissolve 2-benzoylpyridine (2.0 g, 10.9 mmol) and ammonium acetate (4.2 g, 54.5 mmol) in acetic acid (30 mL), and then add salicylaldehyde (2.0 g, 16.35 mmol). Stir the reaction solution at 110 °C for 6 hours. Cool the reaction solution to room temperature, add water (50 mL), and extract with ethyl acetate (3 × 15 mL). The combined organic phases are washed with water (2 × 15 mL) and saturated brine (2 × 15 mL) respectively, and dried over anhydrous sodium sulfate. Evaporate the solvent under reduced pressure and separate by silica gel column chromatography (PE:EA = 3:1) to obtain a light green solid, namely Compound 3 (2.3 g, 75%);

[0049] As Figure 1 shown, the results of the hydrogen nuclear magnetic resonance spectrum of Compound 3 are as follows: 1 H NMR (400 MHz, CDCl 3 ) δ11.99 (s, 1H), 8.54 (d, J = 7.6 Hz, 1H), 7.92 - 7.89 (m, 3H), 7.79 (dd, J = 8.0, 1.6 Hz, 1H), 7.50 (t, J = 7.6 Hz, 2H), 7.37 - 7.31 (m, 2H), 7.19 (dd, J = 8.0, 1.2 Hz, 1H), 7.02 (t, J = 7.6 Hz, 1H), 6.90 - 6.86 (m, 1H), 6.73 - 6.69 (m, 1H).

[0050] As Figure 2As shown, the high-resolution mass spectrometry result of compound 3 is: HRMS(ESI) calcd for C 19 H 15 N 2 O[M+H] + , 287.1184; found, 287.1179.

[0051] S2. Using compound 3 to synthesize a small molecule probe (compound 1) based on the imidazo[1,5-a]pyridine skeleton, the synthetic route is as follows:

[0052]

[0053] The specific synthesis process is as follows: Dissolve compound 3 (200 mg, 0.70 mmol) in dichloromethane (10 mL), add pyridine (7.8 mL, 56.0 mmol), and stir the reaction solution at room temperature for 30 minutes. Then add 4-bromobutyryl chloride (155 mg, 0.84 mmol) to the reaction solution and continue to stir at room temperature for 24 hours. Add water (20 mL) to the reaction solution, and extract with dichloromethane (3×10 mL). The combined organic phases are washed with water (2×15 mL) and saturated brine (2×15 mL) respectively, and dried over anhydrous sodium sulfate. The solvent is removed by rotary evaporation under vacuum, and separated by silica gel column chromatography (PE:EA = 5:1) to obtain a green solid, namely compound 1 (249 mg, 82%).

[0054] As Figure 3 shown, the hydrogen nuclear magnetic resonance spectrum result of compound 1 is: 1 H NMR(400 MHz, CDCl 3 ) δ 7.93 - 7.90 (m, 2H), 7.87 - 7.80 (m, 2H), 7.71 (dd, J = 7.6, 2.0 Hz, 1H), 7.54 - 7.50 (m, 1H), 7.46 - 7.39 (m, 3H), 7.31 - 7.26 (m, 2H), 6.84 - 6.80 (m, 1H),

[0055] 6.59 - 6.56 (m, 1H), 3.16 (t, J = 6.4 Hz, 2H), 2.46 (t, J = 7.2 Hz, 2H), 1.97 - 1.90 (m, 2H).

[0056] As Figure 4 shown, the carbon nuclear magnetic resonance spectrum result of compound 1 is: 13 C NMR(150 MHz, CDCl 3)δ170.70,149.10,131.25,130.60,128.73,127.29,126.65,126.42,123.37,122.17,120.09,118.95,113.16,77.24,77.03,76.82,32.39,32.19,27.34.

[0057] As Figure 5 shown, the high-resolution mass spectrometry results of Compound 1 are: HRMS(ESI) calcd for C 19 H 15 N 2 O[M+H] + , 435.0708; found, 435.0703.

[0058] Example 2

[0059] In this example, the small molecule probe (Compound 1) prepared in Example 1 was used to test the response of the small molecule probe to hydrazine. The response spectrogram of Compound 1 to hydrazine is as Figure 6 shown;

[0060] Figure 6 (A) in reflects the absorption change of Compound 1 (10 μM) after adding 200 μM hydrazine hydrate; Figure 6 (B) in reflects the fluorescence spectrogram change of Compound 1 (10 μM) after incubation for 4 minutes in PBS buffer (10 mM, pH 7.4, 1% DMSO) after adding hydrazine at different concentrations (1 - 100 eq); Figure 6 (C) in reflects the standard curve of the fluorescence intensity of Compound 1 (10 μM) at a wavelength of 475 nm after adding hydrazine at different concentrations (1 - 10 eq); Figure 6 (D) in reflects the fluorescence intensity response of Compound 1 (10 μM) to different ions and amino acids at 475 nm.

[0061] From Figure 6It can be seen that in the absence of hydrazine hydrate, Compound 1 has a weak absorption at 360 nm; when 200 μM hydrazine hydrate is added, a strong absorption peak (A) appears at 300 nm, indicating that Compound 1 has a good optical response to hydrazine hydrate. Secondly, in the absence of hydrazine hydrate, Compound 1 has no fluorescence signal; when hydrazine hydrate is added, an obvious fluorescence effect (B) appears at 470 nm, showing a large Stokes shift. The fluorescence response of Compound 1 to hydrazine hydrate at different concentrations (1 - 100 μM, corresponding to 1 - 100 eq) shows a significant dose-dependence. As the concentration of hydrazine hydrate increases from 1 μM to 100 μM, the fluorescence signal of Compound 1 increases by 90 times (B). Between 1 μM - 10 μM, the fluorescence intensity of Compound 1 shows a good linear correlation with the concentration of hydrazine hydrate (C), and from this, the detection limit of Compound 1 for hydrazine hydrate is calculated to be 0.15 μM. These results indicate that Compound 1 has good sensitivity as a fluorescence detection probe for hydrazine hydrate. In addition to hydrazine hydrate, the fluorescence response value of Compound 1 to other species is weak (D), indicating that Compound 1 has good selectivity as a fluorescence detection probe for hydrazine hydrate.

[0062] Example 3

[0063] In this example, the small molecule probe (Compound 1) prepared in Example 1 was used for cell experiments to illustrate the detection ability of this small molecule probe for hydrazine. The live cell imaging images of Compound 1 are as Figure 7 shown;

[0064] Figure 7 In (a)-(c), the HeLa cells were co-incubated with Compound 1 (10 μM) at 37 °C for 30 minutes as the control group; Figure 7 In (d)-(f), the HeLa cells were co-incubated with isoniazid (100 μM) at 37 °C for 30 minutes. Isoniazid can release hydrazine in live cells, and then they were incubated with Compound 1 (10 μM) for 30 minutes; Figure 7 In (g)-(i), the HeLa cells were co-incubated with hydrazine (100 μM) at 37 °C for 30 minutes, and then they were incubated with Compound 1 (10 μM) for 30 minutes; Figure 7 In (j)-(l), the HeLa cells were co-incubated with hydrazine (200 μM) at 37 °C for 30 minutes, and then they were incubated with Compound 1 (10 μM) for 30 minutes.

[0065] From Figure 7It can be seen that Compound 1 did not show fluorescence when incubated alone with cells; when isoniazid, a precursor of hydrazine, was added to the incubation system, the cells showed obvious blue fluorescence; similarly, when hydrazine was added to the incubation system, the cells showed obvious blue fluorescence, and the fluorescence intensity had a good correlation with the concentration of hydrazine. The above results indicate that Compound 1, as a fluorescence detection probe, is suitable for detecting hydrazine in living cells, and its advantages are mainly reflected in low cytotoxicity and good biocompatibility.

[0066] In the description of this specification, the description with reference to terms such as "one embodiment", "example", "specific example", etc. means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner.

[0067] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above embodiments, and what is described in the above embodiments and the specification only illustrates the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention will have various changes and improvements, and these changes and improvements all fall within the scope of the present invention claimed.

Claims

1. A small molecule probe based on an imidazo[1,5-a]pyridine skeleton, characterized in that: The structural formula of the small molecule probe is:

2. A method for preparing a small molecule probe based on an imidazo[1,5-a]pyridine skeleton, characterized in that: The following steps are involved: S1, dissolving 2-benzoylpyridine and ammonium acetate in acetic acid, adding salicylaldehyde, heating and stirring to generate compound 3; S2, dissolving compound 3 in dichloromethane, adding pyridine, stirring, then adding 4-bromobutyryl chloride, and continuing to stir to obtain compound 1, which is the small molecule probe.

3. The method for preparing a small molecule probe based on an imidazo[1,5-a]pyridine skeleton according to claim 2, characterized in that: In S1, the temperature of heating and stirring was 110°C.

4. The method for preparing a small molecule probe based on an imidazo[1,5-a]pyridine skeleton according to claim 2, characterized in that: The molar ratio of 2-benzoylpyridine, ammonium acetate and salicylaldehyde in S1 is 1:5:1.

5.

5. The method for preparing a small molecule probe based on an imidazo[1,5-a]pyridine skeleton according to claim 2, characterized in that: In S2, the molar ratio of compound 3, pyridine and 4-bromobutyryl chloride is 1:80:1.

2.

6. The method for preparing a small molecule probe based on an imidazo[1,5-a]pyridine skeleton according to claim 2, characterized in that: The production route of compound 3 is:

7. The method for preparing a small molecule probe based on an imidazo[1,5-a]pyridine skeleton according to claim 2, characterized in that: The synthetic route of compound 1 is:

8. Use of the small molecule probe based on the imidazo[1,5-a]pyridine skeleton according to claim 1 in detecting hydrazine.

9. A hydrazine detection reagent, characterized in that It includes the small molecule probe based on the imidazo[1,5-a]pyridine skeleton as described in claim 1.

10. A biological system and drinking water detection reagent, characterized in that: The invention comprises a hydrazine detection reagent as claimed in claim 9.

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