Heterocyclic compound and application thereof in nano-protein fluorescent probe

Abstract

The application discloses a heterocyclic compound and application thereof in a nano-protein fluorescent probe, and the structure of the probe is as shown in the following figure: wherein the substituent R is selected from any one of N,N-dimethylaminophenyl and p-cyanophenyl. The probe is prepared from pyrrozine fused BOPYIN as raw material, coupled with phenylboronic acid through tetra-triphenylphosphine palladium catalysis, and has the advantages of simple synthesis method, convenient separation and purification and high yield. The aromatic ring is introduced on the parent structure, so that the absorption spectrum is red-shifted and has strong stability, and the probe can be used for glioma tumor targeting color development.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of fine chemical industry, and more particularly, to a heterocyclic compound and application thereof in nano-protein fluorescent probe. BACKGROUND

[0002] The boron fluoride dipyrrin fluorescent dye has higher selectivity, high sensitivity, good optical stability and other advantages compared with fluorescein, rhodamine or cyanine dye. The BOPYIN of the present application introduces different electronic effect substituents at the position of bromine atom, so that the dye has larger molar absorption coefficient and fluorescence quantum yield, and the absorption and emission spectrum is red-shifted.

[0003] Hydrophobin is a kind of small molecule protein with special physicochemical properties produced by fungi at a specific period, which can self-assemble into a film at the interface of two phases, so as to enhance the hydrophilicity of the hydrophobic surface and the hydrophobicity of the hydrophilic surface, and has great application value. At present, it has been applied in the medical field. Hydrophobin can specifically recognize some specific expression materials, so as to have targeting to some tissues. However, the current tumor targeting probes generally have low water solubility and poor biocompatibility.

[0004] In view of the above problems, the present application provides a heterocyclic compound and application thereof in nano-protein fluorescent probe. The heterocyclic compound has good targeting to glioma tumor and can be used for biological imaging. SUMMARY

[0005] The main purpose of the present application is to provide a heterocyclic compound and application thereof in nano-protein fluorescent probe. The technical scheme of the present application is as follows:

[0006] The heterocyclic compound and application thereof in nano-protein fluorescent probe, the chemical structural formula of the compound is:

[0007] ;

[0008] Wherein, the substituent R is any one selected from N,N-dimethylamino phenyl and p-cyanophenyl. As a preferred scheme, the chemical structural formula of the compound is:

[0009]

[0010] Any one of the two.

[0011] The synthesis method of the heterocyclic compound and application thereof in nano-protein fluorescent probe includes the following synthesis path:

[0012]

[0013] The method comprises the following steps:

[0014] (1) adding compound I-1, 1,4-dioxane, compound 2, magnetically stirring to dissolve, and then adding tetrakis triphenylphosphine palladium and potassium hydroxide aqueous solution to obtain a reaction solution;

[0015] (2) purifying the reaction solution in step (1) after washing with water, extraction, drying, and concentration to obtain product I-1, a new fluorescent probe.

[0016] In step (1), the feeding ratio of compound I-1, compound 2 and tetrakis triphenylphosphine palladium is 1:1-10:0.01-1.

[0017] In step (1), the concentration of compound potassium hydroxide in the potassium hydroxide aqueous solution is 0.1 mol / mL-10 mol / mL.

[0018] The feeding sequence of step (1) is compound I, 1,4-dioxane, compound 2, tetrakis triphenylphosphine palladium and potassium hydroxide aqueous solution.

[0019] The heating reflux reaction temperature of step (1) is 30-90 DEG C, and the heating time is 2-18 hours.

[0020] In step (1), the feeding ratio of compound I-1 and potassium hydroxide is 1:1-10.

[0021] The present application has the following beneficial effects:

[0022] (1) The compound of the present application is a heterocyclic compound, and the hydrophobic protein modified by RGD peptide is prepared by genetic engineering method and specifically combined with α v β3, and the heterocyclic compound is wrapped in the hydrophobic protein to form a nano-protein fluorescent probe, the nano-protein shell not only retains the fluorescence emission intensity of the heterocyclic compound, but also increases the water solubility and biocompatibility. The probe has targeting property for glioma tumor and can be used for biological imaging.

[0023] (2) The heterocyclic compound preparation method is simple, has good absorption and emission intensity, and is small in volume and easy to be wrapped in nano-protein. DETAILED DESCRIPTION

[0024] Figure 1 is the ultraviolet spectrum of compound I-2 obtained in Example 4.

[0025] Figure 2 is the ultraviolet spectrum of compound I-3 obtained in Example 8.

[0026] Figure 3is a fluorescence spectrum of compound I-2 obtained in Example 4.

[0027] Figure 4 is a fluorescence spectrum of compound I-3 obtained in Example 8.

[0028] Figure 5 is a hydrogen spectrum of compound I-2 obtained in Example 4.

[0029] Figure 6 is a hydrogen spectrum of compound I-3 obtained in Example 8.

[0030] Figure 7 is a luminous efficiency graph of compound I-2 obtained in Example 9.

[0031] Figure 8 is a luminous efficiency graph of compound I-3 obtained in Example 10. DETAILED DESCRIPTION

[0032] The present application is further illustrated by the following examples, but the scope of the present application is not limited to the scope of the examples.

[0033] Example 1

[0034]

[0035] Compound I-1 (532 mg, 1 mmol) was weighed, and 30.00 mL of 1,4-dioxane was added to dissolve it, and then 4-diphenylaminophenylboronic acid (289 mg, 1 mmol), tetrakis triphenylphosphine palladium (11.6 mg, 0.001 mmol), and an aqueous solution of potassium hydroxide (1 mmol) were sequentially added. It was stirred in a water bath and heated to 60°C, and after 8 hours of reaction, the reaction was complete, and the yield was 45.1%.

[0036] Example 2

[0037]

[0038] Compound I-1 (532 mg, 1 mmol) was weighed, and 30.00 mL of 1,4-dioxane was added to dissolve it, and then 4-diphenylaminophenylboronic acid (578 mg, 2 mmol), tetrakis triphenylphosphine palladium (11.6 mg, 0.001 mmol), and an aqueous solution of potassium hydroxide (1 mmol) were sequentially added. It was stirred in a water bath and heated to 60°C, and after 8 hours of reaction, the reaction was complete. When the amount of 4-diphenylaminophenylboronic acid was increased by 1 time compared to Example 1, the yield was increased by 18.9%.

[0039] Example 3

[0040]

[0041] Compound I-1 (532 mg, 1 mmol) was weighed, and 30.00 mL of 1,4-dioxane was added to dissolve it. Then, 4-diphenylaminophenylboronic acid (578 mg, 2 mmol), tetrakis(triphenylphosphine)palladium (11.6 mg, 0.001 mmol), and an aqueous solution of potassium hydroxide (1 mmol) were sequentially added. The reaction was stirred in a water bath and heated to 90°C. The reaction was complete after 6 hours. When the reaction temperature was increased by 30°C compared to Example 2, the reaction time was reduced by 2 hours, and the yield was increased by 21.6%.

[0042] Example 4

[0043]

[0044] Compound I-1 (532 mg, 1 mmol) was weighed, and 30.00 mL of 1,4-dioxane was added to dissolve it. Then, 4-diphenylaminophenylboronic acid (578 mg, 2 mmol), tetrakis(triphenylphosphine)palladium (23.2 mg, 0.002 mmol), and an aqueous solution of potassium hydroxide (1 mmol) were sequentially added. The reaction was stirred in a water bath and heated to 90°C. The reaction was complete after 6 hours. When the amount of tetrakis(triphenylphosphine)palladium was increased by 1-fold compared to Example 3, the yield was increased by 4.4%.

[0045] Example 5

[0046]

[0047] Compound I-1 (532 mg, 1 mmol) was weighed, and 30.00 mL of 1,4-dioxane was added to dissolve it. Then, 4-diphenylaminophenylboronic acid (578 mg, 2 mmol), tetrakis(triphenylphosphine)palladium (23.2 mg, 0.002 mmol), and an aqueous solution of potassium hydroxide (2 mmol) were sequentially added. The reaction was stirred in a water bath and heated to 90°C. The reaction was complete after 6 hours. When the amount of potassium hydroxide was increased by 1-fold compared to Example 4, i.e., the concentration of the aqueous solution of potassium hydroxide was increased by 1-fold compared to Example 1, the yield was decreased by 15.6%.

[0048] Example 6

[0049]

[0050] Compound I-1 (532 mg, 1 mmol) was weighed, and 60.00 mL of 1,4-dioxane was added to dissolve it, followed by the addition of 4-diphenylaminophenylboronic acid (578 mg, 2 mmol), tetrakis triphenylphosphine palladium (23.2 mg, 0.002 mmol), and an aqueous solution of potassium hydroxide (1 mmol). Stirring was performed in a water bath, and heating was performed to 90°C. After 6 hours of reaction, the reaction was complete. When the amount of 1,4-dioxane was increased by 1-fold relative to Example 4, there was no significant change in the yield.

[0051] Example 7

[0052]

[0053] Compound I-1 (532 mg, 1 mmol) was weighed, and 30.00 mL of 1,4-dioxane was added to dissolve it, followed by the addition of 4-diphenylaminophenylboronic acid (578 mg, 2 mmol), tetrakis triphenylphosphine palladium (23.2 mg, 0.002 mmol), and an aqueous solution of potassium hydroxide (1 mmol). Stirring was performed in a water bath, and heating was performed to 30°C. After 14 hours of reaction, the reaction was complete. When the reaction temperature was reduced by 60°C relative to Example 4, and the reaction time was increased by 8 hours, the yield was reduced by 31.5%.

[0054] A mother liquor was prepared by dissolving I-2 (6.82 mg, 0.01 mmol) in 1 mL of ethyl acetate, and 6 μL of the mother liquor was added to 3 mL of toluene, dichloromethane, acetone, acetonitrile, and dimethyl sulfoxide, respectively, to measure the absorption wavelength of the compound (λmax). Figure 1 The compound has a long absorption wavelength, and has absorption double peaks between 392 nm and 482 nm. The fluorescence quantum yield of the compound in toluene and dimethyl sulfoxide is greater than 99% (ΦF). Figure 3

[0055] Example 8

[0056]

[0057] Compound I-1 (532 mg, 1 mmol) was weighed, and 30.00 mL of 1,4-dioxane was added to dissolve it, followed by the addition of 4-diphenylaminophenylboronic acid (578 mg, 2 mmol), tetrakis triphenylphosphine palladium (23.2 mg, 0.002 mmol), and an aqueous solution of potassium hydroxide (1 mmol). Stirring was performed in a water bath, and heating was performed to 90°C. After 6 hours of reaction, the reaction was complete, and compound I-3 was obtained, with a yield of 84.7%.

[0058] ​The mother liquor was prepared by dissolving I-3 (5.40 mg, 0.01 mmol) in 1 mL of ethyl acetate, and 6 μL of the mother liquor was added to 3 mL of toluene, dichloromethane, acetone, acetonitrile, dimethyl sulfoxide, respectively, to measure the absorption wavelength of the compound (λmax) Figure 2 . The compound has a long absorption wavelength, with a double absorption peak between 354 nm and 471 nm, and the fluorescence quantum yield in toluene and dimethyl sulfoxide is greater than 99% Figure 4 , which can be used for fluorescence development in vivo.

[0059] Example 9 Luminescence efficiency of compound I-2 probe

[0060] Preparation of a mixed solution of nanoprobes and N-containing seven-membered heterocyclic compounds: I-2 was dissolved in dichloromethane (2 x 10 -5 M). Different concentrations of nanoprobes were prepared using phosphate buffer (pH = 7.4) (50-250 mg / mL). 30 mL of a dichloromethane solution of N-containing seven-membered heterocyclic compounds was added to nanoprobes of a certain concentration (200 mL). The solution was assembled using ultrasonic waves and stirred at less than 25°C for 1 hour. A transparent solution was obtained in the upper layer. The nanoprobes were injected intravenously into mice carrying glioma tumor cells, and it was observed that 2 hours later, weak fluorescence was observed at the tumor site (no fluorescence at the non-tumor site), indicating that the nanoprobes had combined with the compound and could specifically recognize glioma. The fluorescence intensity reached a peak value after 24 hours, and fluorescence disappeared after 78 hours Figure 7 .

[0061] Example 10 Luminescence efficiency of compound I-3 probe

[0062] Preparation of a mixed solution of nanoprobes and N-containing seven-membered heterocyclic compounds: I-3 was dissolved in dichloromethane (2 x 10 -5 M). Different concentrations of nanoprobes were prepared using phosphate buffer (pH = 7.4) (50-250 mg / mL). 30 mL of a dichloromethane solution of N-containing seven-membered heterocyclic compounds was added to nanoprobes of a certain concentration (200 mL). The solution was assembled using ultrasonic waves and stirred at less than 25°C for 1 hour. A transparent solution was obtained in the upper layer. The nanoprobes were injected intravenously into mice carrying glioma tumor cells, and it was observed that 2 hours later, weak fluorescence was observed at the tumor site (no fluorescence at the non-tumor site), indicating that the nanoprobes had combined with the compound and could specifically recognize glioma. The fluorescence intensity reached a peak value after 22 hours, and fluorescence disappeared after 65 hours Figure 8 .

Claims

1. Use of a heterocyclic compound for the manufacture of a pharmaceutical for a nanoprotein fluorescent probe, characterized in that, The chemical structural formula of the heterocyclic compound is: I The substituent R is selected from any one of 4-diphenylamine phenyl and p-cyanophenyl.

2. Use according to claim 1, characterized in that, The application comprises the following synthesis path: Compound 2 The substituent R is selected from any one of 4-diphenylamine phenyl and p-cyanophenyl; (1) At room temperature, compound I-1, 1,4-dioxane, compound 2, and tetraphenylphosphonium palladium are added to a reaction bottle and magnetically stirred to dissolve, and then potassium hydroxide aqueous solution is added, and heating is performed to obtain a reaction solution; (2) The reaction solution in step (1) is washed with water, extracted, dried, concentrated, and purified to obtain product I, namely a nano-protein fluorescent probe.

3. Use according to claim 2, characterized in that, In step (1), the feeding ratio of compound I-1, compound 2, and tetraphenylphosphonium palladium is 1:1-10:0.01-1.

4. Use according to claim 2, characterized in that, In step (1), the concentration of potassium hydroxide in the potassium hydroxide aqueous solution is 0.1 mol / mL-10 mol / mL.

5. Use according to claim 2, characterized in that, The feeding sequence of step (1) is compound I-1, 1,4-dioxane, compound 2, tetraphenylphosphonium palladium, and potassium hydroxide aqueous solution.

6. Use according to claim 2, characterized in that, The heating reflux reaction temperature of step (1) is 30-90 DEG C, and the heating time is 2-18 hours.

7. Use according to claim 2, characterized in that, In step (1), the feeding ratio of compound I-1 and potassium hydroxide is 1:1-10.

8. The use according to claim 1, characterized in that, The nano-protein fluorescent probe is used for preparing a glioma tumor targeting drug.

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