CN114790165B

CN114790165B - Synthesis method of near-infrared two-region fluorescent dye FD-1080

Info

Publication number: CN114790165B
Application number: CN202210476562.8A
Authority: CN
Inventors: 杨原
Original assignee: Xi'an Kangfunuo Biotechnology Co ltd
Current assignee: Xi'an Kangfunuo Biotechnology Co ltd
Priority date: 2022-04-30
Filing date: 2022-04-30
Publication date: 2023-04-04
Anticipated expiration: 2042-04-30
Also published as: CN114790165A

Abstract

The invention discloses a synthesis method of a near-infrared two-zone fluorescent dye FD-1080, which takes benzo [ cd ] indole-2 (1H) -ketone as a starting raw material, firstly introduces tert-butylcarbonyl (Boc) on an amino group to protect the amino group, then introduces methyl by utilizing Grignard reagent ring opening, then completes Boc protecting group removal and intramolecular cyclization reaction by using an ethyl acetate solution of hydrogen chloride, then introduces sulfonic acid by nucleophilic substitution, and finally reacts with N- [ (3- (anilinomethylene) -2-chloro-1-cyclohexene-1-yl) methylene ] anilino hydrochloride to synthesize the FD-1080.

Description

Synthesis method of near-infrared two-region fluorescent dye FD-1080

Technical Field

The invention belongs to the technical field of synthesis of fluorescent dyes, and particularly relates to a synthesis method of a near-infrared two-region fluorescent dye.

Background

Fluorescence biological imaging utilizes a fluorescence signal emitted by a fluorescence probe to optically image cells, tissues or organisms, and compared with imaging technologies such as nuclear magnetic resonance imaging, the fluorescence biological imaging has the characteristics of high safety, high time resolution, high spatial resolution, no radiation, low cost, nondestructive detection and the like, and becomes a popular research direction in the field of biomedical imaging. Fluorescence imaging can be divided into three regions according to their emission wavelengths: visible light region (400-700 nm), near infrared region I (NIR-I, 700-900 nm) and near infrared region II (NIR-II, 1000-1700 nm). NIR-II can also be subdivided into three sub-windows NIR-IIa' (1000 to 1300 nm), NIR-IIa (1300 to 1400 nm) and NIR-IIb (1500 to 1700 nm).

There are only two organic near-infrared dyes currently in clinical approval: methylene Blue (MB) and indocyanine green (ICG). Both dyes are small molecules that are rapidly excreted from the body, however, their fluorescence emission is in the NIR-I region and has limited penetration depth for in vivo imaging. The photon penetration depth is mainly determined by the scattering and absorption of tissue components, and the noise and background level are generated by the autofluorescence and scattered photons of the tissue, so compared with NIR-I fluorescence imaging, NIR-II fluorescence imaging has the characteristics of lower tissue absorption, scattering, autofluorescence and the like, has higher signal-to-noise ratio, and can realize deep penetration and high-quality imaging in vivo. More and more researchers have been invested in the research field of near-infrared two-zone fluorescence imaging, including the development of related new instruments and the research of novel fluorescent materials, and it is very meaningful to develop a new near-infrared two-zone fluorescent dye with better luminous effect and optimize the production and synthesis route thereof.

The dyes currently used in NIR-II fluorescence imaging are mainly of the following classes: rare earth element doped nano material, carbon nano tube, quantum dot, organic molecular polymer and organic small molecular compound. And after the carbon nanotube material, the rare earth doped nanomaterial, the quantum dot and other inorganic nanomaterials enter a living body as a near-infrared two-region probe, the inorganic nanomaterials are easily accumulated in a reticuloendothelial system (such as a liver and a spleen) due to non-biodegradability, have potential long-term cytotoxicity and limit clinical application of the inorganic nanomaterials, and organic micromolecules have a definite molecular structure, a definite molecular weight and an excellent metabolic capability, so the organic micromolecules have a wider application prospect in the aspect of NIR-II imaging.

In 2015, the Dai task group reports for the first time the application of the organic small molecular probe CH1055 in near-infrared two-region fluorescence imaging, the organic small molecular probe CH1055 is metabolized rapidly in vivo, 90% of the organic small molecular probe CH1055 can be discharged with urine through the kidney within 24 hours after being injected into a mouse as a developer, and the NIR-II imaging quality of the derivative CH1055-PEG is far better than ICG in the aspect of imaging of blood, lymph vessels, tumors and lymph nodes of the mouse through the experiment of the task group. Noninvasive identification of tumors in the brain of mice can be achieved through the intact scalp and skull at a depth of about 4 mm. Its NIR-II imaging provides a 5-fold higher tumor-to-normal tissue ratio than traditional NIR-I imaging, and can be used for accurate image-guided tumor ablation. CH1055 was the first small molecule probe reported for NIR-II fluorescence imaging, and the study of the Dai topic group opened the sequence for the near infrared two-domain organic small molecule probe.

In 2018, zhang task group reports for the first time that a new NIR-II region dye FD-1080 is synthesized by introducing a sulfonate group into the structure of a commercialized NIR-II dye IR-1048, has an absorption wavelength of 1046nm and an emission wavelength of 1080nm, and can be used for in-vivo imaging. The subject group, which is the design of heptamethylamine structures, shifts absorption and emission into the NIR-II region. The water solubility and the stability of the sulfonated group and the cyclohexene group are improved. The quantum yield of FD-1080 is 0.31%, and can be even improved to 5.94% after being combined with Fetal Bovine Serum (FBS), and through mouse experiments, FD-1080 can not only realize noninvasive high-resolution deep tissue hindlimb blood vessel and cerebrovascular biological imaging, but also can quantify the respiratory rate based on the dynamic imaging of respiratory craniofacial motion of conscious and anesthetized mouse liver, and has a good application prospect. The synthetic route reported by this group of subjects is as follows:

when the FD-1080 is synthesized by the above route, the product needs to be purified by a crystallization method for multiple times, and the product needs to be purified by column chromatography in the last step, so that the overall yield is low, and the separation of experimental products is difficult, which finally causes the high price of FD-1080 and further influences the use and popularization of FD-1080 in various biological experiments.

Disclosure of Invention

In order to solve the problems of high production cost and high price of FD-1080, the invention provides a method for synthesizing FD-1080 at low cost.

The synthesis method of FD-1080 provided by the invention comprises the following steps:

1. dissolving benzo [ cd ] indole-2 (1H) -ketone in acetonitrile, adding 4-dimethylamino pyridine and di-tert-butyl dicarbonate under stirring, stirring at room temperature for 1-2 hours, carrying out reduced pressure distillation to remove acetonitrile, washing unreacted 4-dimethylamino pyridine by hydrochloric acid, extracting by ethyl acetate, and concentrating an ethyl acetate phase to obtain an intermediate 1.

2. Dissolving the intermediate 1 with dry tetrahydrofuran, cooling to below-40 ℃ in nitrogen atmosphere, dropwise adding a tetrahydrofuran solution of methyl magnesium chloride under stirring, naturally heating to 0-5 ℃ after dropwise adding, stirring for reacting for 30-60 minutes, pouring the reaction solution into a mixed solution of ethyl acetate and water in a volume ratio of 1-2 for quenching reaction, extracting with ethyl acetate, and concentrating an ethyl acetate phase to obtain an intermediate 2.

3. And adding the intermediate 2 into an ethyl acetate solution of HCl, stirring at room temperature for 4-6 hours to remove the Boc protecting group and complete intramolecular cyclization, after the reaction is completed, removing a supernatant, ultrasonically washing hydrochloric acid away by using ethyl acetate, and performing rotary evaporation to remove the residual ethyl acetate to obtain an intermediate 3.

4. Dissolving 1, 4-butane sultone in acetonitrile, adding the intermediate 3 and sodium acetate, sealing a tube at 100-110 ℃, heating for reaction for 6-8 hours, naturally cooling to room temperature after reaction, removing supernatant, ultrasonically dispersing the solid in acetonitrile, and filtering to obtain an intermediate 4.

5. Adding the intermediate 4 and sodium acetate into a mixed solvent of acetic anhydride and acetic acid in a volume ratio of 4-5, heating to 50-60 ℃, keeping the temperature for 10-30 minutes, adding N- [ (3- (anilinomethylene) -2-chloro-1-cyclohexene-1-yl) methylene ] aniline hydrochloride under a stirring state, reacting for 30-40 minutes, then adding the same amount of N- [ (3- (anilinomethylene) -2-chloro-1-cyclohexene-1-yl) methylene ] aniline hydrochloride, continuing to react for 4-6 hours at 50-60 ℃, pouring the reaction solution into methyl tert-butyl ether for precipitation after the reaction is finished, filtering to obtain black precipitate, then adding the precipitate into acetonitrile, performing ultrasonic washing, and filtering again to obtain FD-1080.

In the step 1, the mole ratio of the benzo [ cd ] indol-2 (1H) -one to the 4-dimethylaminopyridine to the di-tert-butyl dicarbonate is preferably 1.2-1.5.

In the step 1, the concentration of the hydrochloric acid is preferably 0.5 to 1mol/L.

In the step 2, the molar ratio of the intermediate 1 to the methyl magnesium chloride is preferably 1.2 to 1.5.

In the step 2, the concentration of the methyl magnesium chloride in the tetrahydrofuran solution of the methyl magnesium chloride is preferably 3mol/L.

In the step 3, the mass-volume ratio of the intermediate 2 to the ethyl acetate solution of HCl is preferably 1g to 5mL, and the concentration of HCl in the ethyl acetate solution of HCl is preferably 2mol/L.

In the step 4, the molar ratio of the intermediate 3 to the 1, 4-butane sultone and the sodium acetate is preferably 1.65 to 1.8.

In the step 5, the molar ratio of the intermediate 4 to sodium acetate and N- [ (3- (anilinomethylene) -2-chloro-1-cyclohexen-1-yl) methylene ] aniline hydrochloride is preferably 2 to 2.1, and the mass-volume ratio of the intermediate 4 to acetic anhydride is 1 g.

The invention has the following beneficial effects:

the method comprises the steps of taking benzo [ cd ] indole-2 (1H) -ketone as a starting material, introducing tert-butylcarbonyl (Boc) on an amino group to protect the amino group, then introducing methyl by utilizing Grignard reagent ring opening, then completing Boc protecting group removal and intramolecular cyclization reaction by using an ethyl acetate solution of hydrogen chloride, introducing sulfonic acid by nucleophilic substitution, and finally reacting with N- [ (3- (anilinomethylene) -2-chloro-1-cyclohexene-1-yl) methylene ] aniline hydrochloride to synthesize FD-1080. According to the invention, tertiary butyl carbonyl (Boc) is introduced to the amino group to protect the amino group, so that the attack rate of a Grignard reagent on a reaction intermediate in the subsequent steps of the reaction is increased, the reaction yield is increased, the post-treatment of each step of the experiment is simple, the whole synthesis process is safe and pollution-free, a product with high purity can be synthesized without column chromatography purification, and the method can be used for large-scale production of FD-1080.

Drawings

FIG. 1 is a nuclear magnetic hydrogen spectrum of intermediate 1 synthesized in example 1.

FIG. 2 is a nuclear magnetic hydrogen spectrum of intermediate 2 synthesized in example 1.

FIG. 3 is a nuclear magnetic hydrogen spectrum of intermediate 3 obtained by carrying out the synthesis of example 1.

FIG. 4 is a nuclear magnetic hydrogen spectrum of intermediate 4 synthesized in example 1.

FIG. 5 is a nuclear magnetic hydrogen spectrum of FD-1080 synthesized in example 1.

FIG. 6 is a high resolution mass spectrum of FD-1080 synthesized in example 1.

Fig. 7 shows the results of hplc-ms coupled testing of FD-1080 synthesized in example 1.

Detailed Description

The present invention will be described in further detail with reference to the drawings and examples, but the scope of the present invention is not limited to these examples.

Example 1

1. 10g (0.059 mol) of benzo [ cd]Indol-2 (1H) -one was added to a 500mL round-bottom flask, followed by 100mL acetonitrile and stirred until benzo[cd]Dissolving indole-2 (1H) -ketone completely, adding 10.83g (0.088 mol) of 4-dimethylamino pyridine, adding 19.35g (0.088 mol) of di-tert-butyl dicarbonate into a round-bottom flask in portions by one spoon under a stirring state, generating a large amount of bubbles in the flask but not obviously heating, obtaining turbid liquid in the flask, ultrasonically stirring the solid in the flask for 2min after the materials are added to uniformly disperse the solid, stirring the mixture at normal temperature for 2H, removing acetonitrile by reduced pressure distillation, adding 0.5mol/L hydrochloric acid to wash away unreacted 4-dimethylamino pyridine, extracting the mixture twice by ethyl acetate, drying the mixture by organic phase anhydrous sodium sulfate, concentrating the mixture, and obtaining 13.5g of intermediate 1 after the concentration is finished, wherein the yield is 84.9%. The nuclear magnetic hydrogen spectrum of the intermediate 1 is shown in figure 1: ¹ HNMR(600MHz,CDCl ₃ )δ8.12(t,J＝8.0Hz,2H),7.82-7.72(m,2H),7.68(d,J＝8.4Hz,1H),7.57(t,J＝6.0Hz,1H),1.71(s,9H)。

2. adding 10g (0.037 mol) of the intermediate 1 into a 500mL round-bottom flask, adding 100mL of dry tetrahydrofuran, stirring until the mixture is completely dissolved, pumping nitrogen in the flask, placing the flask on a low-temperature reactor, cooling to-40 ℃, slowly and dropwise adding 18.5mL of 3mol/L tetrahydrofuran solution of methyl magnesium chloride into the flask by using a syringe, naturally heating to 0 ℃ after dropwise adding, reacting for 30min, slowly pouring the reaction solution into 500mL of a mixed solution of ethyl acetate and water with the volume ratio of 1 to quench the reaction, extracting the reaction solution twice by using ethyl acetate, adding the ethyl acetate into anhydrous sodium sulfate, drying, and concentrating to obtain 9.2g of light yellow powdery intermediate 2 with the yield of 87.1%. The nuclear magnetic hydrogen spectrum of the intermediate 2 is shown in figure 2: ¹ H NMR(400MHz,CDCl ₃ )δ7.66(d,J＝6.5Hz,1H),7.54-7.49(m,1H),7.45-7.36(m,3H),7.23(s,1H),3.11(s,3H),2.80(s,9H)。

3. adding 8g (0.028 mol) of intermediate 2 into a 250mL round-bottom flask, adding 80mL of 2mol/LHCl ethyl acetate solution, stirring at room temperature for 4 hours, separating out a solid attached to the wall of the flask after the reaction is finished, pouring off a supernatant, repeatedly adding ethyl acetate into the flask, ultrasonically washing off HCl, and distilling under reduced pressure to remove the residual ethyl acetate to obtain 3.8g of intermediate 3 with the yield of 81.1%. The nuclear magnetic hydrogen spectrum of the intermediate 3 is shown in figure 3: ¹ HNMR(400MHz,CDCl ₃ )δ8.48(dd,J＝7.2,5.5Hz,2H),8.31(d,J＝7.2Hz,1H),8.12(d,J＝8.3Hz,1H),7.98(t,J＝7.6Hz,1H),7.79(t,J＝7.8Hz,1H),3.19(s,3H)。

4. dissolving 7.32g (0.054 mol) of 1, 4-butane sultone in 30mL of acetonitrile, filling the solution into a 100mL sealed tube, adding 2.4g (0.030 mol) of sodium acetate and 3g (0.018 mol) of intermediate 3, heating the solution to 110 ℃, reacting for 8h, changing the state of a reaction system in the bottle from a uniform liquid phase to a solid at the bottom after the reaction is finished, turning off the heating, naturally cooling the solution to room temperature, pouring out a supernatant, adding acetonitrile into the solid, performing ultrasonic dispersion, and filtering to obtain 4.5g of intermediate 4, wherein the yield is 82.4%. The nuclear magnetic hydrogen spectrum of the intermediate 4 is shown in figure 4: ¹ H NMR(600MHz,MeOD)δ8.87(d,J＝7.2Hz,1H),8.73(d,J＝8.1Hz,1H),8.47(d,J＝7.4Hz,1H),8.39(d,J＝8.2Hz,1H),8.14(t,J＝7.8Hz,1H),7.98(t,J＝7.8Hz,1H),4.75(t,J＝7.8Hz,2H),2.91(t,J＝7.2Hz,2H),2.24(dt,J＝15.4,7.7Hz,2H),1.98(dt,J＝14.9,7.3Hz,2H)。

5. adding 2g (0.0066 mol) of intermediate 4 and 0.54g (0.0066 mol) of sodium acetate into a 250mL round-bottom flask, adding 40mL of acetic anhydride and 10mL of acetic acid, placing the mixture into an oil bath pot after ultrasonic homogenization, heating the mixture to 50 ℃, preserving the temperature for 10min, and adding 0.56g (0.0015 mol) of N- [ (3- (anilinomethylene) -2-chloro-1-cyclohexene-1-yl) methylene chloride]Aniline hydrochloride, stirring at 50 deg.C for 30min, adding 0.56g (0.0015 mol) N- [ (3- (anilinomethylene) -2-chloro-1-cyclohexen-1-yl) methylene]And (2) after aniline hydrochloride reacts for 4 hours at 50 ℃, the reaction liquid is poured into 200mL of methyl tert-butyl ether for precipitation, then the precipitate is filtered, 100mL of acetonitrile is added into a filter cake for 10min of ultrasonic treatment, the filtration is carried out again, the acetonitrile is added to wash the filter cake until the effluent liquid under a funnel is clear and transparent, the solid is transferred into a round-bottom flask, the solvent is pumped by an oil pump, and 1.9g of black powdery FD-1080 is obtained, wherein the yield is 85.3%. The nuclear magnetic hydrogen spectrum of FD-1080 is shown in figure 5: ¹ HNMR (400mhz, meod) δ 8.37 (d, J =12.6hz, 2h), 7.97 (m, 2H), 7.86 (m, 2H), 7.71 (m, 2H), 7.29 (d, J =20.2hz, 2h), 7.06 (m, 1H), 6.32 (d, J =13.1hz, 2h), 4.60 (m, 4H), 3.74 (m, 4H), 2.84 (m, 4H), 2.75 (m, 4H), 1.83 (m, 6H). The high resolution mass spectrum of FD-1080 is shown in FIG. 6. The purity of the product can reach 98.5% by high pressure liquid chromatography-mass spectrometer, as shown in figure 7.

Claims

1. A synthesis method of a near-infrared two-region fluorescent dye FD-1080, wherein the structural formula of the FD-1080 is shown as follows:

the method is characterized by comprising the following steps:

(1) Dissolving benzo [ cd ] indole-2 (1H) -ketone in acetonitrile, adding 4-dimethylamino pyridine and di-tert-butyl dicarbonate under the stirring state, stirring at room temperature for 1-2 hours, carrying out reduced pressure distillation to remove acetonitrile, washing unreacted 4-dimethylamino pyridine by hydrochloric acid, extracting by ethyl acetate, and concentrating an ethyl acetate phase to obtain an intermediate 1;

(2) Dissolving the intermediate 1 with dry tetrahydrofuran, cooling to below-40 ℃ in a nitrogen atmosphere, dropwise adding a tetrahydrofuran solution of methyl magnesium chloride under a stirring state, naturally heating to 0-5 ℃ after dropwise adding, stirring for reacting for 30-60 minutes, pouring a reaction solution into a mixed solution of ethyl acetate and water in a volume ratio of 1-2 for quenching reaction, extracting with ethyl acetate, and concentrating an ethyl acetate phase to obtain an intermediate 2;

(3) Adding the intermediate 2 into an ethyl acetate solution of HCl, stirring at room temperature for 4-6 hours to remove a Boc protecting group and complete intra-molecular cyclization, after the reaction is completed, removing a supernatant, ultrasonically washing hydrochloric acid away by using ethyl acetate, and performing rotary evaporation to remove the residual ethyl acetate to obtain an intermediate 3;

(4) Dissolving 1, 4-butane sultone in acetonitrile, adding an intermediate 3 and sodium acetate, sealing a tube at 100-110 ℃, heating for reaction for 6-8 hours, naturally cooling to room temperature after reaction, removing supernatant, ultrasonically dispersing the solid with acetonitrile, and filtering to obtain an intermediate 4;

(5) Adding the intermediate 4 and sodium acetate into a mixed solvent of acetic anhydride and acetic acid in a volume ratio of 4-5, heating to 50-60 ℃, keeping the temperature for 10-30 minutes, adding N- [ (3- (anilinomethylene) -2-chloro-1-cyclohexene-1-yl) methylene ] aniline hydrochloride under a stirring state, reacting for 30-40 minutes, then adding the same amount of N- [ (3- (anilinomethylene) -2-chloro-1-cyclohexene-1-yl) methylene ] aniline hydrochloride, continuing to react for 4-6 hours at 50-60 ℃, pouring a reaction solution into methyl tert-butyl ether for precipitation after the reaction is finished, filtering to obtain black precipitate, then adding the precipitate into acetonitrile, performing ultrasonic washing, and filtering again to obtain FD-1080;

2. the method of claim 1, wherein the synthesis of the near-infrared two-region fluorescent dye FD-1080 is as follows: in the step (1), the molar ratio of the benzo [ cd ] indol-2 (1H) -one to the 4-dimethylaminopyridine to the di-tert-butyl dicarbonate is 1.2-1.5.

3. The method for synthesizing the near-infrared two-region fluorescent dye FD-1080 as claimed in claim 1, wherein: in the step (1), the concentration of the hydrochloric acid is 0.5-1 mol/L.

4. The method of claim 1, wherein the synthesis of the near-infrared two-region fluorescent dye FD-1080 is as follows: in the step (2), the molar ratio of the intermediate 1 to the methyl magnesium chloride is 1.2-1.5.

5. The method for synthesizing the near-infrared two-region fluorescent dye FD-1080 as claimed in claim 1, wherein: in the step (2), the concentration of the methyl magnesium chloride in the tetrahydrofuran solution of the methyl magnesium chloride is 3mol/L.

6. The method for synthesizing the near-infrared two-region fluorescent dye FD-1080 as claimed in claim 1, wherein: in the step (3), the mass-volume ratio of the intermediate 2 to the HCl ethyl acetate solution is 1g.

7. The method for synthesizing the near-infrared two-region fluorescent dye FD-1080 as claimed in claim 1, wherein: in the step (4), the molar ratio of the intermediate 3 to the 1, 4-butane sultone and the sodium acetate is 1.65-1.8.

8. The method for synthesizing the near-infrared two-region fluorescent dye FD-1080 as claimed in claim 1, wherein: in the step (5), the molar ratio of the intermediate 4 to sodium acetate and N- [ (3- (anilinomethylene) -2-chloro-1-cyclohexene-1-yl) methylene ] aniline hydrochloride is 2-2.1.

9. The method for synthesizing the near-infrared two-region fluorescent dye FD-1080 as claimed in claim 1, wherein: in the step (5), the mass-volume ratio of the intermediate 4 to acetic anhydride is 1g.