A preparation method and application of Nile red and its derivatives

Abstract

The present application relates to a preparation method and application of Nile red and its derivatives. The method comprises: S1, reacting the compound represented by the formula A with nitrite in the presence of an acid solution, and then drying the product after the reaction to obtain the intermediate represented by the formula B; S2, reacting the intermediate with the compound represented by the formula D in the presence of an organic solvent to obtain the crude product of Nile red and its derivatives represented by the formula C. It also comprises: S3, crystallizing and purifying the crude product of Nile red and its derivatives to obtain the purified Nile red and its derivatives. The preparation method of Nile red and its derivatives described in the present application is a simple chemical synthesis method, which is simple in preparation method and high in synthesis efficiency, and realizes the rapid and large-scale synthesis of Nile red and its derivatives. At the same time, the obtained product is purified by crystallization, and finally the purity of Nile red and its derivatives reaches more than 97%, which can be well used in fluorescent dyes.

Description

Technical Field

[0001] The present application relates to the technical field of chemical organic synthesis, and in particular to a preparation method and application of Nile red and its derivatives. Background Art

[0002] The full name of Nile Red is 9-(diethylamino)benzo[a]phenoxazin-5(5H)-one (structural formula is Figure 1 As shown). In recent years, Nile Red has become a cell membrane and lipid stain due to its unparalleled fluorescence properties in lipophilic environments. Nile Red itself is electrically neutral, and the 5-position is a carbonyl group that can act as an electron acceptor. Therefore, Nile Red is a fluorophore with strong electron push and pull capabilities, and has obvious solvent-induced color changes and the ability to image living cells. In addition, Nile Red is widely used in biological fluorescence labeling and made into fluorescent probes because of its lipophilicity, near-infrared (NIR) emission, and large Stokes shift.

[0003] However, the existing preparation methods of Nile red or its derivatives are relatively complicated, the preparation process is time-consuming, and it is impossible to achieve rapid mass production of Nile red or its derivatives. At the same time, the existing methods for purifying crude Nile red are not effective, and the impurity content in the purified Nile red is still high.

[0004] Therefore, in order to reduce the difficulty of operation and improve the purity of the product, it is necessary to provide a simple and efficient method for preparing Nile red and its derivatives. Summary of the invention

[0005] In order to solve the deficiencies of the prior art, the present application provides a new method for preparing Nile red and its derivatives, which is a simple chemical synthesis method, and subsequent purification by crystallization can make the purity of the prepared Nile red and its derivatives reach more than 97%.

[0006] To this end, the first aspect of the present application provides a method for preparing Nile red and its derivatives, the method comprising: firstly modifying a nitroso group on a compound represented by formula A to obtain an intermediate represented by formula B; then directly annealing the intermediate to obtain Nile red and its derivatives represented by formula C;

[0007]

[0008] Wherein, R1 and R2 in Formula A, Formula B and Formula C are independently selected from -H, an alkane group having 1 to 5 carbon atoms and Any one of; R in the formula C 1 , R 2 , R 3 and R 4Each is independently selected from any one of -H, -OH, -NO2, -CN, -CF3, -NH2 and halogen.

[0009] In the present application, the Nile red and its derivatives adopt a synthesis strategy of first adding nitroso groups to obtain intermediates, and then directly cyclizing the intermediates after drying without purification. The synthesis method is simple and efficient, and can achieve rapid synthesis of large quantities of Nile red and its derivatives.

[0010] In some preferred embodiments, the alkane group is selected from any one of -CH3, -C2H5 and -C3H7; and the halogen group is -Br or -F.

[0011] In some specific embodiments, R1 and R2 are the same and are any one of -H, -CH3, -C2H5 and -C3H7; 1 , R 2 , R 3 and R 4 Each is independently selected from any one of -H, -OH and -NH2.

[0012] In some embodiments, the method specifically comprises the following steps:

[0013] S1, reacting the compound represented by formula A with nitrite in the presence of an acid solution, and then drying the reaction product to obtain an intermediate represented by formula B;

[0014] S2, reacting the intermediate with a compound represented by formula D in the presence of an organic solvent to obtain a crude product of Nile red and its derivatives represented by formula C;

[0015]

[0016] Wherein, R in the formula D 1 , R 2 , R 3 and R 4 respectively with R in the formula C 1 , R 2 , R 3 and R 4 The definition is the same.

[0017] In some embodiments, in step S1, the molar ratio of the compound represented by formula A to nitrite is 1:(1-1.5).

[0018] In the present application, the molar amount of nitrite is generally the same as or slightly higher than the molar amount of the compound shown in Formula A. Since the cost of the compound shown in Formula A is relatively high, in order to ensure that the compound shown in Formula A reacts fully, the molar amount of nitrite is generally slightly higher than the molar amount of the compound shown in Formula A.

[0019] In some preferred embodiments, the molar ratio of the compound represented by formula A to nitrite is 1:(1.1-1.5). For example, in some embodiments, the molar ratio of the compound represented by formula A to nitrite is 1:1.1, 1:1.2, 1:1.3, 1:1.4 or 1.5, etc.

[0020] In some embodiments, the nitrite is sodium nitrite.

[0021] In some embodiments, the acid solution is selected from any one of a hydrochloric acid aqueous solution, a sulfuric acid aqueous solution, a phosphoric acid aqueous solution and an acetic acid aqueous solution. For example, the acid solution is a hydrochloric acid aqueous solution, a sulfuric acid aqueous solution, and the like.

[0022] In some embodiments, in step S1, the reaction temperature is -5 to 10°C, and the reaction time is 5 to 10 hours.

[0023] In the present application, under the above reaction conditions, the compound represented by formula A can be fully reacted with nitrite to obtain the intermediate represented by formula B. If the reaction temperature is too high (such as below -5°C), the reaction system is easy to freeze into a solid, thereby hindering the reaction; if the reaction temperature is too high (such as above 10°C), the nitrite is easily destroyed, causing adverse effects on the reaction, such as reducing the yield of the intermediate.

[0024] In some preferred embodiments, the reaction temperature is 0-5° C. Carrying out the reaction at this temperature can ensure that the reaction proceeds quickly and efficiently, and the yield of the intermediate represented by formula B in the reaction product is high.

[0025] In some embodiments, in step S1, the drying temperature is 40 to 50° C. and the drying time is 5 to 10 hours.

[0026] In the present application, the drying temperature needs to be controlled at 40-50° C. If the drying temperature is too high (such as exceeding 50° C.), the nitroso part in the intermediate shown in formula B will be oxidized; if the drying temperature is too low (such as below 40° C.), the drying time will be too long, which is not conducive to the efficient reaction.

[0027] In some specific embodiments, the drying temperature is 50° C. and the drying time is 8 hours.

[0028] In some embodiments, in step S2, the molar ratio of the intermediate to the compound represented by formula D is (1 to 1.5):(1 to 1.5).

[0029] In some preferred embodiments, the molar ratio of the intermediate to the compound represented by formula D is 1:1.

[0030] In some embodiments, the organic solvent is dimethylformamide (DMF) or N-methylpyrrolidone (NMP).

[0031] In the present application, the presence of the above organic solvent is conducive to the efficient reaction of the intermediate with the compound shown in formula D. At the same time, after the reaction, the above organic solution is also conducive to being removed by reduced pressure evaporation to obtain the crude product of Nile red and its derivatives shown in formula C.

[0032] In some embodiments, in step S2, the reaction temperature is 140-160° C., and the reaction time is 4-5 hours.

[0033] In the present application, under the above reaction conditions, the intermediate can be fully reacted with the compound shown in formula D to obtain Nile red and its derivatives shown in formula C. If the reaction temperature is too low (such as below 140°C), the ring-binding reaction is not complete, resulting in too many by-products in the reaction product; if the reaction temperature is too high (such as above 160°C), the reaction becomes complicated, and in addition to the ring-binding reaction required by the present application, side reactions will also occur, which will also result in too many by-products in the reaction product.

[0034] In some preferred embodiments, the reaction temperature is 145-150° C. Carrying out the reaction at this temperature can ensure that the reaction proceeds quickly and efficiently, and the yield of the Nile red and its derivatives represented by the reaction product formula C is high.

[0035] In some specific embodiments, the reaction temperature is 150° C., and the reaction time is 4 hours.

[0036] In some embodiments, the method further comprises the following steps:

[0037] S3, purifying the crude product of Nile red and its derivatives by crystallization to obtain the purified Nile red and its derivatives.

[0038] In the present application, the crude product of Nile red and its derivatives is purified by crystallization. Compared with the purification method of column chromatography in the prior art, the purification by crystallization can make the purity of the purified product reach more than 97%. However, the highest purity of the purified product can only reach 95% by column chromatography. It can be seen that the purification by crystallization can make the purity of the purified Nile red and its derivatives better.

[0039] In some embodiments, the solvent used in the crystallization purification is a mixed solvent of dimethylformamide and ethanol, and the volume ratio of dimethylformamide (DMF) to ethanol in the mixed solvent is (1-2):(1-2).

[0040] In the present application, when the Nile red and its derivatives are crystallized and purified by the mixed solvent, the purified product can have a high yield and good purity, and the solvent residue can be easily removed.

[0041] In some preferred embodiments, the volume ratio of dimethylformamide (DMF) to ethanol in the mixed solvent is 1: 1. When the volume ratio of dimethylformamide (DMF) to ethanol in the mixed solvent is 1: 1, the yield and purity of the purified product can be optimized.

[0042] In some specific embodiments, the method for preparing Nile red and its derivatives comprises the following steps:

[0043] (1) adding an acid solution into a reaction device, then slowly adding a compound represented by formula A, and then slowly adding an aqueous solution of nitrite after dissolution to obtain a reaction system; reacting the reaction system at -5 to 10° C. (preferably 0 to 5° C.) for 5 to 10 hours (preferably 8 hours) to obtain a reaction product; filtering and drying the reaction product to obtain an intermediate represented by formula B;

[0044] (2) adding an intermediate represented by formula B, a compound represented by formula D and an organic solvent into a reaction apparatus to obtain a reaction system; reacting the reaction system at 140 to 160° C. (preferably 145 to 150° C.) for 4 to 5 hours (preferably 5 hours) to obtain a reaction product; removing the solvent in the reaction product by reduced pressure evaporation to obtain a crude product of Nile red and its derivatives represented by formula C;

[0045] (3) Using a mixed solvent of dimethylformamide and ethanol (the volume ratio of dimethylformamide to ethanol in the mixed solvent is (1-2):(1-2)) to crystallize and purify the crude product of Nile red and its derivatives, thereby obtaining purified Nile red and its derivatives.

[0046] The second aspect of the present application provides a use of Nile red and its derivatives prepared by the method described in the first aspect of the present application as fluorescent dyes.

[0047] The method described in the present application can prepare Nile red and its derivatives quickly and efficiently, realize the rapid large-scale synthesis of Nile red and its derivatives, and the prepared Nile red and its derivatives are of high purity, so they can be well used in fluorescent dyes.

[0048] Beneficial technical effects of the present application: The preparation method of Nile red and its derivatives described in the present application is a simple chemical synthesis method. In the preparation process, a nitroso group is first added to obtain an intermediate, and then the intermediate is dried to directly synthesize Nile red and its derivatives. The preparation method is simple and has high synthesis efficiency, and the rapid mass synthesis of Nile red and its derivatives is achieved. At the same time, the obtained product is purified by crystallization, and the purity of the obtained Nile red and its derivatives finally reaches more than 97%, which can be well used in fluorescent dyes. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 The hydrogen nuclear magnetic resonance spectrum of the intermediate M1 prepared in Example 1 and Example 2 is shown in FIG.

[0050] Figure 2 This is the hydrogen nuclear magnetic resonance spectrum of Nile Red prepared in Example 1.

[0051] Figure 3 This is the hydrogen nuclear magnetic resonance spectrum of the Nile red derivative prepared in Example 2. DETAILED DESCRIPTION

[0052] In order to make the present application easier to understand, the present application will be further described in detail below in conjunction with the examples, which are merely illustrative and are not intended to limit the scope of application of the present application. The raw materials or components used in the present application can be obtained through commercial routes or conventional methods unless otherwise specified.

[0053] Example 1: Preparation of Nile Red

[0054] Step 1: Synthesis of intermediate M1

[0055] The synthetic route is as follows:

[0056]

[0057] Add 1025.0mL concentrated hydrochloric acid and 700mL water to the reaction device, cool the ice water to 0°C while stirring, slowly add 500g (3.03mol) of 3-hydroxy-N,N-diethylaniline, stir in an ice bath until dissolved, control the temperature at 0°C, and slowly drop sodium nitrite aqueous solution [NaNO2 (235.92gg, 3.42mol), H2O (200mL)] into the system. After 30 minutes of dropwise addition, react at 4°C for 8 hours and filter to obtain a yellow product. Among them, the molar ratio of the added 3-hydroxy-N,N-diethylaniline to NaNO2 is 1:1.3. The yellow product is dried at 50°C for 8 hours until the product is constant weight to obtain a yellow-green solid-intermediate M1 (mass: 530g, yield: 90%). The hydrogen nuclear magnetic resonance spectrum of the obtained intermediate M1 is as follows Figure 1 shown.

[0058] Step 2: Synthesize Nile Red

[0059] The synthetic route is as follows:

[0060]

[0061] Add 500 g (2.57 mol) of intermediate M1, 371 g (2.57 mol) of 1-naphthol and anhydrous dry DMF (2000 mL) into a reaction kettle, stir evenly, and prepare a reaction system. Heat the reaction system to 150° C., react for 4 hours, and then evaporate DMF under reduced pressure to prepare a crude Nile red product. The molar ratio of the added intermediate M1 to 1-naphthol is 1:1.

[0062] The crude Nile Red was crystallized and purified using 1500 mL of a mixed solvent of DMF and ethanol in a volume ratio of 1:1 to obtain 750 g of pure Nile Red with a yield of 91%. The purity of the Nile Red in the pure product was 97% as determined by high performance liquid chromatography (HPLC). The hydrogen nuclear magnetic resonance spectrum of the obtained Nile Red was as follows: Figure 2 shown.

[0063] Example 2: Preparation of Nile Red Derivatives

[0064] Step 1: Synthesis of intermediate M1

[0065] The synthetic route is as follows:

[0066]

[0067] 1368mL of water was added to the reaction device, and 684mL of concentrated sulfuric acid was slowly added under stirring. Then, the ice water was cooled to 0°C, and 500g (3.03mol) of 3-hydroxy-N,N-diethylaniline was slowly added. Stirring was carried out in an ice bath until dissolved. The temperature was controlled at 0°C, and an aqueous sodium nitrite solution [NaNO2 (235.92gg, 3.42mol), H2O (200mL)] was slowly added dropwise to the system. After 30 minutes of addition, the mixture was reacted at 4°C for 8 hours, and a yellow product was obtained by filtration. Among them, the molar ratio of the added 3-hydroxy-N,N-diethylaniline to NaNO2 was 1:1.3. The yellow product was dried by air at 50°C for 8 hours until the product had a constant weight, and a yellow-green solid-intermediate M1 (mass: 530g, yield: 90%) was obtained. The hydrogen nuclear magnetic resonance spectrum of the obtained intermediate M1 is as follows: Figure 1 shown.

[0068] Step 2: Synthesis of Nile Red Derivatives

[0069] The synthetic route is as follows:

[0070]

[0071] Add 500 g (2.57 mol) of intermediate M1, 411.6 g (2.57 mol) of 1,7-dihydroxynaphthalene and anhydrous dry DMF (2000 mL) into a reaction kettle, stir evenly, and prepare a reaction system. Heat the reaction system to 150° C., react for 4 hours, and then evaporate DMF under reduced pressure to prepare a crude Nile red derivative. The molar ratio of the added intermediate M1 to 1,7-dihydroxynaphthalene is 1:1.

[0072] The crude Nile red derivative was crystallized and purified using 1500 mL of a mixed solvent of DMF and ethanol in a volume ratio of 1:1 to obtain 731 g of pure Nile red derivative with a yield of 85%. The purity of the Nile red derivative in the pure product was 98% as determined by high performance liquid chromatography (HPLC). The hydrogen nuclear magnetic resonance spectrum of the obtained Nile red derivative was as follows: Figure 3 shown.

[0073] Example 3: Preparation of Nile Red

[0074] The preparation process of step 1 is basically the same as that of Example 1, except that the reaction temperature in step 1 is 15° C. The mass of the intermediate M1 finally obtained is 486 g, and the yield is 83%.

[0075] The preparation process of step 2 is the same as that of embodiment 1.

[0076] Example 4: Preparation of Nile Red

[0077] The preparation process of step 1 is the same as that of Example 1.

[0078] The preparation process of step 2 is basically the same as that of Example 1, except that the organic solvent added is dry NMP (2000 mL). Finally, 742 g of pure Nile Red was obtained through purification, with a yield of 90%. The purity of Nile Red in the pure product was 97% as determined by high performance liquid chromatography (HPLC).

[0079] Example 5: Preparation of Nile Red

[0080] The preparation process of step 1 is the same as that of Example 1.

[0081] The preparation process of step 2 is basically the same as that of Example 1, except that the reaction temperature is 140° C. Finally, 735 g of pure Nile Red is obtained through purification, with a yield of 89%. The purity of Nile Red in the pure product is 96% as determined by high performance liquid chromatography (HPLC).

[0082] Example 6: Preparation of Nile Red

[0083] The preparation process of step 1 is the same as that of Example 1.

[0084] The preparation process of step 2 is basically the same as that of Example 1, except that the reaction temperature is 160° C. Finally, 729 g of pure Nile Red is obtained through purification, with a yield of 88%. The purity of Nile Red in the pure product is 96% as determined by high performance liquid chromatography (HPLC).

[0085] Example 7: Preparation of Nile Red

[0086] The preparation process of step 1 is the same as that of Example 1.

[0087] The preparation process of step 2 is basically the same as that of Example 1, except that the mixed solvent used for crystallization and purification is 1500 mL of DMF and propanol in a volume ratio of 1:1. Finally, 738 g of pure Nile Red was obtained through purification, with a yield of 89%. The purity of Nile Red in the pure product was 91% as detected by high performance liquid chromatography (HPLC).

[0088] Example 8: Preparation of Nile Red

[0089] The preparation process of step 1 is the same as that of Example 1.

[0090] The preparation process of step 2 is basically the same as that of Example 1, except that the mixed solvent used for crystallization and purification is 1500 mL of DMF and ethanol in a volume ratio of 1:2. Finally, 741 g of pure Nile Red was obtained through purification, with a yield of 90%. The purity of Nile Red in the pure product was 94% as detected by high performance liquid chromatography (HPLC).

[0091] Example 9: Preparation of Nile Red

[0092] The preparation process of step 1 is the same as that of Example 1.

[0093] The preparation process of step 2 is basically the same as that of Example 1, except that the mixed solvent used for crystallization and purification is 1500 mL of DMF and ethanol in a volume ratio of 2:1. Finally, 745 g of pure Nile Red was obtained through purification, with a yield of 90%. The purity of Nile Red in the pure product was 95% as detected by high performance liquid chromatography (HPLC).

[0094] It should be noted that the embodiments described above are only used to explain the present application and do not constitute any limitation to the present application. The present application is described with reference to typical embodiments, but it should be understood that the words used therein are descriptive and explanatory words, rather than restrictive words. The present application may be modified as specified within the scope of the claims of the present application, and the present invention may be revised without departing from the scope and spirit of the present application. Although the present application described therein relates to specific methods, materials and embodiments, it does not mean that the present application is limited to the specific examples disclosed therein. On the contrary, the present application can be extended to all other methods and applications with the same functions.

Claims

1. A method for preparing Nile red and its derivatives, characterized in that: The method comprises: firstly modifying a nitroso group on a compound shown in formula A to obtain an intermediate shown in formula B; then directly performing a ring-bonding on the intermediate to obtain Nile red and its derivatives shown in formula C; Wherein, R1 and R2 in Formula A, Formula B and Formula C are independently selected from -H, an alkane group having 1 to 5 carbon atoms and Any one of; R in the formula C 1 , R 2 , R 3 and R 4 Each is independently selected from any one of -H, -OH, -NO2, -CN, -CF3, -NH2 and halogen; The method specifically comprises the following steps: S1, reacting the compound represented by formula A with nitrite in the presence of an acid solution, and then drying the reaction product to obtain an intermediate represented by formula B; S2, reacting the intermediate with a compound represented by formula D in the presence of an organic solvent to obtain a crude product of Nile red and its derivatives represented by formula C; Wherein, R in the formula D 1 , R 2 , R 3 and R 4 respectively with R in the formula C 1 , R 2 , R 3 and R 4 The definition of is the same as; S3, crystallizing and purifying the crude product of Nile red and its derivatives to obtain purified Nile red and its derivatives; the solvent used in the crystallization and purification is a mixed solvent of dimethylformamide and ethanol, and the volume ratio of dimethylformamide to ethanol in the mixed solvent is 1:

1.

2. The method according to claim 1, characterized in that The alkane group is selected from any one of -CH3, -C2H5 and -C3H7; the halogen group is -Br or -F.

3. The method according to claim 1, characterized in that In step S1, the molar ratio of the compound represented by formula A to the nitrite is 1:(1-1.5); and / or The acid solution is selected from any one of a hydrochloric acid aqueous solution, a sulfuric acid aqueous solution, a phosphoric acid aqueous solution and an acetic acid aqueous solution.

4. The method according to claim 3, characterized in that The nitrite is sodium nitrite.

5. The method according to any one of claims 1 to 4, characterized in that: In step S1, the reaction temperature is -5 to 10°C; the reaction time is 5 to 10 hours.

6. The method according to claim 5, characterized in that The reaction temperature is 0-5°C.

7. The method according to any one of claims 1 to 4, characterized in that: In step S1, the drying temperature is 40-50°C and the drying time is 5-10 hours.

8. The method according to any one of claims 1 to 4, characterized in that: In step S2, the molar ratio of the intermediate to the compound represented by formula D is (1 to 1.5): (1 to 1.5); and / or The organic solvent is dimethylformamide or N-methylpyrrolidone.

9. The method according to claim 8, characterized in that The molar ratio of the intermediate to the compound represented by formula D is 1:

1.

10. The method according to any one of claims 1 to 4, characterized in that: In step S2, the reaction temperature is 140-160° C. and the reaction time is 4-5 hours.

11. The method according to claim 10, characterized in that The reaction temperature is 145-150°C.

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