A near-infrared fluorescent dye, preparation method and application thereof

Abstract

The present invention discloses a compound having a structural formula as shown in formula (a) or formula (b): #imgabs0# wherein R is an alkyl group or a phenyl group, and R1-R5 are each independently selected from one of hydrogen, an alkyl group, a cycloalkyl group, an ether bond, a halogen, an alkoxy group, a hydroxyl group or a sulfonate group. The present invention also discloses a method for preparing the compound shown in formula (a) or formula (b). The compound obtained by the present invention has a novel structure, excellent anti-photobleaching performance, and exhibits the characteristic of fluorescence enhancement in blood as a dye. The synthesis steps are simple and the synthesis yield is high.

Description

Technical Field

[0001] The invention relates to a near-infrared fluorescent dye, a preparation method and application thereof, and belongs to the field of dye chemistry. Background Art

[0002] In recent years, near-infrared fluorescent dyes have been widely used in the biomedical field. In particular, the use of tail fluorescence in the second region of the near-infrared region of organic small molecules to achieve direct visualization of living tissues and the application of fluorescent surgical navigation has made the development of near-infrared fluorescent dyes a key scientific hotspot. In the past few decades, many near-infrared fluorescent dye core structures have been reported in the field, such as fluoroborane, squaric acid, and cyanine.

[0003] The rigidified core structure with the introduction of germanium atoms has not been reported yet. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a new compound, which, as a near-infrared fluorescent dye, has the characteristics of strong photobleaching resistance and high imaging resolution. The present invention also discloses a preparation method of the compound, which has simple steps and high synthesis yield.

[0005] In order to solve the above technical problems, the present invention first discloses a compound with a structural formula as shown in formula (a) or formula (b):

[0006]

[0007] Wherein, R is an alkyl group or a phenyl group, R 1 -R 5 Each is independently selected from one of hydrogen, alkyl, cycloalkyl, ether bond, halogen, alkoxy, hydroxyl or sulfonate.

[0008] Furthermore, the R is methyl or phenyl, the R 1 -R 5 Each is independently selected from hydrogen, methyl, ethyl or alkoxy.

[0009] The present invention also discloses a method for preparing the above compound, and the preparation reaction formula is as follows:

[0010]

[0011] in,

[0012]

[0013] Specifically:

[0014] Step 1, the compound represented by formula (1) is reacted with an amino compound to obtain a compound represented by formula (2);

[0015] Step 2: The compound represented by formula (2) is subjected to an alkylation reaction with an alkylating agent to obtain a compound represented by formula (3) or formula (7);

[0016] Step 3: Under the catalysis of protonic acid or Lewis acid, the compound represented by formula (3) or the compound represented by formula (7) is subjected to condensation reaction with formaldehyde or paraformaldehyde to obtain the compound represented by formula (4) or the compound represented by formula (8);

[0017] Step 4: The compound represented by formula (4) or the compound represented by formula (8) is subjected to lithium halogen exchange with a lithium reagent under an inert gas atmosphere to obtain a dilithium reagent, and the dilithium reagent obtained is then reacted with dialkylgermanium dichloride or diphenylgermanium dichloride to obtain a compound represented by formula (5) or a compound represented by formula (9), respectively; the dialkylgermanium dichloride is preferably dimethylgermanium dichloride.

[0018] Step 5: The compound represented by formula (5) or the compound represented by formula (9) is oxidized with an oxidant to obtain the compound represented by formula (6) or the compound represented by formula (10);

[0019] Step 6: The compound represented by formula (6) or the compound represented by formula (10) is subjected to nucleophilic addition reaction in the presence of a phenyllithium reagent or a phenyl Grignard reagent, and then acid-catalyzed to obtain the compound represented by formula (a) or the compound represented by formula (b).

[0020] Furthermore, in step 1, the amination reaction is carried out in a closed high-pressure container, the reaction temperature is 135-155° C., and the amino compound is aqueous ammonia.

[0021] Furthermore, the alkylation reaction in step 2 is carried out in a solvent DMF (N,N-dimethylformamide), the alkylation agent is 1-bromo-3-chloropropane, and potassium carbonate is also added to the reaction system to consume the hydrobromic acid produced during the reaction and promote the reaction. In this step 2, the solvent is used to dissolve the intermediate product to promote the smooth reaction and increase the yield of the product. In this reaction step 2, when the reaction temperature is 130°C-145°C, the alkylation reaction obtains the compound shown in formula (3); when the reaction temperature is 155°C-175°C, the alkylation reaction obtains the compound shown in formula (7).

[0022] Furthermore, the condensation reaction in step three is carried out in acetic acid as a solvent, refluxed at 80-95° C., the protonic acid is concentrated sulfuric acid or hydrochloric acid, and the Lewis acid is boron trifluoride etherate or ferric chloride.

[0023] Furthermore, in step 4, anhydrous tetrahydrofuran or diethyl ether is used as solvent, the lithium reagent used is n-butyl lithium or sec-butyl lithium, and the lithium halogen exchange temperature is -78°C.

[0024] Furthermore, the oxidant used in step 5 is chlorobenzoquinone, potassium permanganate or hydrogen peroxide, the solvent used is ethyl acetate or acetone, and the reaction temperature is -15°C to room temperature.

[0025] Furthermore, in step six, the nucleophilic addition uses ethers as the reaction solvent, and the phenyllithium reagent used is a 2,6-dimethylphenyllithium reagent, an o-methylphenyllithium reagent or a 2,6-dimethoxyphenyllithium reagent. These phenyllithium reagents can be prepared by themselves, and the phenyl Grignard reagent used can be a Grignard reagent corresponding to the phenyllithium reagent, which can be purchased directly. The phenyllithium reagent can be prepared according to the following method: weigh the corresponding bromobenzene reagent in a dried Slack reaction bottle, place the reaction system in a low-temperature reactor at -78°C for about 0.5h, and measure 2.5mol / L of n-butyllithium (1.2 molar equivalents) and quickly add it to the low-temperature reaction system and stir for 0.5h. The prepared phenyllithium reagent is ready for use, and it should be ready for use under low temperature conditions.

[0026] The phenyl Grignard reagent used is the Grignard reagent corresponding to the phenyllithium reagent, the ether solvent is tetrahydrofuran or diethyl ether; the acid catalyst used in the acid catalysis is methanesulfonic acid, the reaction is carried out at room temperature, and the halogenating reagent is used as the solvent. The halogenating reagent is dichloromethane or 1,2-dichloroethane, preferably dichloromethane, which is easy to post-process.

[0027] The compound of the invention is used as a near-infrared fluorescent dye and is applied to in vivo imaging in the field of biological hospitals.

[0028] The compounds shown in formula (a) and formula (b) of the present invention have novel structures, excellent resistance to photobleaching, and strong advantages in in vivo imaging. Its synthesis steps are simple (six steps are completed), the synthesis yield is high, and the synthesis cost is low. Specifically, by introducing germanium atoms into the parent core structure, that is, rigidifying the parent core structure, and formula (b) closing the end group alkyl chain in a ring, that is, increasing the electron supply capacity of the end group, two types of near-infrared fluorescent dyes (a) and (b) are constructed. As infrared dyes, they absorb / emit 880 / 930nm, and the tail fluorescence can reach the near-infrared IIa+IIx (1300-1500nm) band or even tail to the near-infrared IIb band (1500-1700nm), and still maintain a high brightness, that is, this type of dye shows the characteristics of fluorescence enhancement in blood. Based on its excellent brightness in the near-infrared IIa+IIx band, high-resolution imaging of systemic blood vessels and cerebral blood vessels of mice can be achieved. In the present invention, the amino compound used in step 1 can be amine compounds such as methylamine, diethylamine, etc. in addition to ammonia water, to obtain the corresponding naphthylamine, and then enter the next step of reaction; the alkylating agent used in step 2 can be alkyl bromides or alkyl iodides containing bromine or iodine at other end groups, such as iodomethane, iodoethane, or 3-iodopropanol in addition to 1-bromo-3-chloropropane. When different amino compounds or different alkylating agents are used, different substituents can be obtained at both ends of formula (a) or formula (b) or on the germanium atom, but because they have the same parent core structure, they still have similar properties. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The absorption and emission spectra of the dye of compound (b) obtained in Example 2 in chloroform show that the test results are absorption emission at 865 / 890nm, and the fluorescence emission tails to 1200nm;

[0030] Figure 2 The absorption and emission of the dye of compound (b) obtained in Example 2 in DMSO solvent are 891 / 930 nm, respectively, and the fluorescence emission is tailed to 1200 nm;

[0031] Figure 3 The absorption and emission of the dye of compound (b) obtained in Example 2 in methanol solvent are 866 / 895 nm, respectively, and the fluorescence emission is tailed to 1200 nm;

[0032] Figure 4 The absorption and emission of the dye of compound (b) obtained in Example 2 in serum solvent are 888 / 920 nm, respectively, and the fluorescence emission is tailed to 1200 nm;

[0033] Figure 5 Schematic diagram showing the comparison of the photobleaching resistance of the dye of compound (b) obtained in Example 2 and the commercial dye ICG;

[0034] Figure 6 This is a graph showing the test results of in vitro fluorescence of the dye of compound (b) obtained in Example 2;

[0035] Figure 7 This is a graph showing the in vivo living imaging test results of the dye of compound (b) obtained in Example 2;

[0036] Figure 8 This is a diagram showing the verification result of the in vivo circulation time of the dye of compound (a) obtained in Example 1;

[0037] Fig. 9 This is a diagram showing the verification results of the in vivo circulation time of the dye of compound (b) obtained in Example 2. DETAILED DESCRIPTION

[0038] The present invention is further explained below in conjunction with the examples. The following examples are only used to illustrate the present invention, but are not intended to limit the scope of the present invention.

[0039] Example 1

[0040] Preparation of a compound represented by formula (a), wherein R, R 1 and R 5 All are methyl, R 2 , R 3 and R 4 When it is hydrogen, its preparation reaction formula is as follows:

[0041]

[0042] Specifically:

[0043] The compound 7-bromo-2-hydroxynaphthalene represented by formula (1) was directly purchased domestically.

[0044] Step 1: Compound (2) is obtained by amination reaction: 7-bromo-2-hydroxynaphthalene (40.0 g, 179.0 mmol) and sodium pyrosulfite (40.9 g, 215.0 mmol) are weighed in a 350 mL pressure bottle, 120 mL of water is added to the pressure bottle to fully dissolve, and ammonia water (35 mL, 900 mmol) is also added to the pressure bottle. The reaction system is placed in a 155°C oil bath and heated for about 48 hours until the raw materials react completely. The reaction system is cooled to room temperature and filtered to obtain compound (2) (40 g, yield 98%). 1 H NMR (400 MHz, CDCl 3)δ7.74(d,J=1.6Hz,1H),7.61(d,J=8.7Hz,1H),7.54(d,J=8.7Hz,1H),7.29(d,J=1.9Hz,1H),6.93(dd,J=8.7,2.3Hz,1H),6.87(d,J=2.1Hz,1H). 13 CNMR (151MHz, CDCl 3 )δ145.00,136.13,129.34,129.20,127.67,126.21,125.64,120.51,118.46,107.35.HRMS(ESI)m / zChemical formula:C 10 H 8 BrN[M] + Calculated value: 221.9913; Found: 221.9915. In this step, sodium metabisulfite will combine with the naphthalene ring to form an intermediate to obtain the product smoothly.

[0045] Step 2, compound (3) is obtained by alkylation reaction: Compound (2) (10 g, 45 mmol) is weighed into a 250 mL round-bottom flask, and an appropriate amount of N,N-dimethylformamide (DMF) is added to completely dissolve the reactant. 1-Bromo-3-chloropropane (6.7 mL, 68 mmol) is also weighed into the reaction system, and an appropriate amount of potassium carbonate is added into the reaction system to fully mix the reaction system. The reaction system is placed in an oil bath at 135°C, heated to reflux, and stirred with a magnetic stirrer until compound (2) is completely reacted (about 24 h). The reaction system is cooled to room temperature, extracted with dichloromethane, concentrated, and finally separated by column chromatography to obtain compound (3) (13 g, yield 93%) (ethyl acetate: petroleum ether = 1:8 separation). 1 H NMR (400 MHz, CDCl 3 )δ7.85(d,J=1.7Hz,1H),7.49(t,J=9.0Hz,2H),7.28–7.18(m,1H),7.04(d,J=9.1Hz,1H),3.61(t,J=6.1Hz,2H),3.55(t,J=8Hz 2H),3.32–3.27(m,2H),2.94(t,J=6.5Hz,2H),2.10–2.00(m,4H). 13 C NMR (101 MHz, CDCl 3)δ143.52,134.58,129.87,127.27,125.02,124.54,123.81,120.89,115.25,112.00,49.46,49.27,42.86,30.16,23.64,21.76.HRMS(ESI)m / zChemical formula:C 16 H 17 BrClN, calculated value [M] + :338.0306 Measured value:338.0303.

[0046] Step 3, acid-catalyzed condensation reaction to obtain compound (4): 20 mL of acetic acid was measured and added to the reaction system. Formaldehyde aqueous solution (37% saturated aqueous solution) (5 mL, 60 mmol) was measured and added to the reaction system, and finally a catalytic equivalent of concentrated sulfuric acid was added dropwise to the reaction system and stirred for 0.5 h. Compound (3) (1 g, 5.9 mmol) was weighed into a 100 mL pressure bottle, the mixed reaction solution was sealed in the pressure bottle, heated to 80 ° C, and stirred with a magnetic stirrer for about 24 h. The reaction system was cooled to room temperature, extracted with dichloromethane, concentrated, and separated by column chromatography to obtain compound (4) (0.4 g, yield 80%) (ethyl acetate: petroleum ether = 1:6 separation) Note that the remaining 0.5 g of the reaction raw material can be recycled and used again. The formaldehyde aqueous solution in the reaction system can be replaced with paraformaldehyde. 1 H NMR (400 MHz, CDCl 3 )δ8.00(s,1H),7.36(d,J=9.1Hz,1H),7.23(s,1H),7.00(d,J=9.1Hz,1H),4.32(s,1H),3.61(t,J=6. 1Hz, 2H), 3.56–3.39 (m, 3H), 3.41–3.21 (m, 2H), 2.98 (t, J = 6.4Hz, 2H), 2.05 (dd, J = 14.2, 7.6Hz, 4H). 13 C NMR (101 MHz, CDCl 3 )δ143.03,133.30,131.89,129.62,126.91,125.77,125.27,124.38,115.39,111.78,49.44,49.25,42.79,41.78,30.15,23.62,21.76.HRMS(ESI)m / zChemical formula:C 33 H 35 Br 2 Cl 2 N 2 [M+H] + , calculated value 687.0539, measured value: 687.0542.

[0047] Step 4, lithium halogen exchange and introduction of germanium element to obtain compound (5): Weigh compound (4) (0.56 mg, 0.81 mmol) in a dried Slack reaction bottle, blow argon into the reaction system to replace the air, and then add an argon balloon to seal the mouth of the reaction bottle. In addition, 10 mL of anhydrous tetrahydrofuran is added to the reaction system to completely dissolve the raw materials, and the reaction system is placed in a low-temperature reactor at -78°C for about 0.5 h to ensure that the temperature in the system reaches -78°C. In addition, 2.5 mol / L of n-butyl lithium (0.97 mL, 2.4 mmol) is quickly added to the low-temperature reaction system, and the reaction system is heated to 0°C and stirred for 20 min after continuing to stir for 30 min. Dichlorodimethylgermanium (0.11 mL, 0.98 mmol) is quickly injected into the reaction system and stirred for 4-6 h (6 h in this embodiment). Saturated aqueous ammonium chloride is added to quench the reaction system, extracted with ethyl acetate, concentrated, and separated by column chromatography to obtain compound (5).

[0048] Step 5: Compound (6) can be obtained by oxidation of the compound (5) separated as above, or directly oxidized from the system after the reaction in step 4. In this embodiment, the system after the reaction in step 4 is directly oxidized:

[0049] Saturated aqueous ammonium chloride solution (for quenching unreacted n-butyl lithium) was added to the reaction system obtained in step 4, and then ethyl acetate was added to the reaction system, and 2 molar equivalents of tetrachlorobenzoquinone as an oxidant were directly added, and the reaction system was fully stirred for 24 hours. The reaction system was extracted with ethyl acetate, and compound (6) (0.3 mg, yield 57%) was separated by column chromatography (ethyl acetate: petroleum ether = 1:5 for separation).

[0050] Alternatively, add saturated aqueous ammonium chloride solution to the reaction system obtained in step 4, extract with ethyl acetate, concentrate, dissolve the reaction system with acetone, add 1 molar equivalent of potassium permanganate at 0°C for oxidation, filter, concentrate, and separate by column chromatography to obtain compound 6 (0.23 mg, yield 44%). 1 H NMRδ8.86(s,2H),7.89(s,2H),7.78(d,J=9.1Hz,2H),7.13(d,J=9.1Hz,2H),3.7 5–3.48(m,8H),3.42(t,J=5.5Hz,4H),3.14(t,J=6.5Hz,4H),2.49–1.75(m,8H). 13 C NMR (101 MHz, CDCl 3)δ188.84,145.42,138.63,134.90,134.67,132.56,130.70,127.35,126.96,116.29,112.48,78.02,77.70,77.38,50.34,49.91,43.39,31.86,30.82,24.26,22.43,0.67,-0.01.HRMS(ESI)m / zChemical formula:C 35 H 38 Cl 2 N 2 O[M+H] + , measured value: 647.1306.

[0051] Step 6: Obtain compound (a) through nucleophilic addition reaction:

[0052] First, prepare the phenyllithium reagent, weigh 2,6-dimethyl bromobenzene reagent into a dried Slack reaction bottle, place the reaction system in a low-temperature reactor at -78°C for about 0.5h, and quickly add 2.5mol / L n-butyllithium (1.2 molar equivalents) into the low-temperature reaction system and stir for 0.5h. The prepared phenyllithium reagent is ready for use, and it should be kept ready under low temperature conditions.

[0053] Weigh compound (6) (100 mg, 0.15 mmol) in a dried Slack reactor, add the above-prepared 2,6-dimethylbenzene lithium reagent (1.5 molar equivalents) to the reaction system and stir for 0.5 h, slowly warm the reaction system to room temperature, and stir for 4-6 h (6 h in this embodiment). Extract with ethyl acetate to obtain an organic layer, concentrate to obtain a mixed product, add dichloromethane to dissolve, add catalytic equivalents of methanesulfonic acid dropwise at room temperature and stir for 4-6 h (6 h in this embodiment). TLC plate until the raw material is completely reacted, neutralize the reaction system with sodium bicarbonate aqueous solution to pH = 7 (note, it cannot be adjusted to alkaline), extract with dichloromethane to obtain an organic phase, concentrate, and separate by column chromatography to obtain compound (a) (45 mg, yield 39%). 1 H NMRδ7.85(s,2H),7.51(s,2H),7.47–7.35(m,2H),7.26(d,J=6.4Hz,6H),7.18(d,J=9.4Hz,2H),3 .91–3.63(m,14H),3.12(t,J=6.4Hz,4H),2.18(dt,J=12.9,6.5Hz,9H),2.00(s,6H),0.78(s,6H). 13 C NMR (101 MHz, CDCl 3)δ151.86,143.03,140.97,136.39,135.46,135.13,132.42,130.38,129.14,128.02,127.61,116.24,115.64,50.88,50.20,42.60,31.45,30.47,29.91,23.24,21.20,20.05,0.20.HRMS(ESI)m / zChemical formula: C 43 H 47 Cl 2 N 2 + [M+H] + , measured value: 735.2326.

[0054] Example 2

[0055] Preparation of a compound represented by formula (b), wherein R, R 1 and R 5 All are methyl, R 2 , R 3 and R 4 When it is hydrogen, its preparation reaction formula is as follows:

[0056]

[0057] Specifically:

[0058] Step 1: Same as Example 1.

[0059] Step 2, compound (7) is obtained by alkylation reaction: Compound 2 (10 g, 45 mmol) is weighed into a 250 ml round-bottom flask, and an appropriate amount of N,N-dimethylformamide (DMF) is added to completely dissolve the reactant. 1-Bromo-3-chloropropane (9 mL, 90 mmol) is also weighed into the reaction system, and a catalytic equivalent of potassium carbonate is added to the reaction system to fully mix the reaction system. The reaction system is placed in an oil bath at 160° C., heated to reflux, and stirred with a magnetic stirrer until the raw material 2 is completely reacted (approximately 36 h). The reaction system is cooled to room temperature, extracted with dichloromethane, concentrated, and finally separated by column chromatography to obtain compound 8 (10 g, yield 73%) (ethyl acetate: petroleum ether = 1:8 separation). 1 H NMRδ7.79(d,J=1.9Hz,1H),7.40(d,J=8.5Hz,1H),7.23(s,1H),7.18(dd,J=8.5,1.9Hz,1H),3.22 (q,J=5.6Hz,5H),2.97(t,J=6.7Hz,2H),2.92–2.88(m,2H),2.14–2.06(m,2H),2.05–1.97(m,2H).13 C NMR (101 MHz, CDCl 3 )δ142.09,133.53,129.13,125.71,125.39,124.93,124.18,123.46,119.71,110.15,50.50,49.86,28.69,23.40,22.00,21.67.HRMS(ESI)m / zChemical formula:C 16 H 16 BrN[M+H] + , measured value: 302.0539.

[0060] Step 3, acid-catalyzed condensation reaction to obtain compound (8): Weigh compound (7) (1 g, 3.31 mmol) in a 100 mL pressure bottle, measure 15 mL of acetic acid and add it to the reaction system. In addition, measure formaldehyde aqueous solution (37% saturated aqueous solution) (2.5 mL, 33 mmol) and add it to the reaction system, and finally drop a catalytic equivalent of concentrated sulfuric acid into the reaction system. The mixed reaction solution is sealed in a pressure bottle, heated to 80°C, and stirred with a magnetic stirrer for about 24 hours. The reaction system is cooled to room temperature, extracted with dichloromethane, concentrated, and separated by column chromatography to obtain compound 8 (0.62 g, yield 62%) (ethyl acetate: petroleum ether = 1:6 separation). The formaldehyde aqueous solution in the reaction system can be replaced with paraformaldehyde. 1 H NMRδ7.92(s,1H),7.12(s,1H),7.08(s,1H),4.30(d,J=7.5Hz,1H),3.25–3.16(m,5H), 2.99(t,J=6.7Hz,2H),2.83(t,J=6.5Hz,2H),2.10(p,J=6.3Hz,3H),2.02–1.92(m,2H). 13 C NMR (101 MHz, CDCl 3 )δ141.59,132.25,131.70,129.05,128.98,125.81,125.77,125.12,124.92,123.26,110.02,50.52,49.91,41.81,28.63,23.42,22.04,21.74.HRMS(ESI)m / zChemical formula:C 33 H 32 Br 2 N 2 [M+H] + , measured value: 617.0986.

[0061] Step 4, lithium halogen exchange and introduction of germanium element to obtain compound (9): Weigh compound (8) (0.6 mg, 0.83 mmol) in a dried Slack reaction bottle, blow argon into the reaction system to replace the air, and then add an argon balloon to seal the mouth of the reaction bottle. In addition, measure 10 mL of anhydrous tetrahydrofuran and add it to the reaction system to completely dissolve the raw materials. Place the reaction system in a low-temperature reactor at -78°C for about 0.5 h to ensure that the temperature in the system reaches -78°C. In addition, measure 2.5 mol / L of n-butyl lithium (1.1 mL, 2.6 mmol) and quickly add it to the low-temperature reaction system. Continue stirring for 30 min and then heat the reaction system to 0°C and stir for 20 min. Measure dichlorodimethylgermanium (0.12 mL, 1 mmol) and quickly inject it into the reaction system and stir for 4-6 h (6 h in this embodiment). Add saturated aqueous ammonium chloride to quench the reaction system.

[0062] Step 5, oxidation reaction to obtain compound (10): saturated aqueous ammonium chloride solution and ethyl acetate were added to the system obtained in step 4, and then chloranil as an oxidant was added (the reaction system was stirred for 24 h. The reaction system was extracted with ethyl acetate and separated by column chromatography to obtain compound (10) (0.35 mg, yield 60%) (ethyl acetate: petroleum ether = 1:5 separation). 1 H NMR(400MHz,Chloroform-d)δ8.76(s,2H),7.79(s,2H),7.44(s,2H),3.27(t,J=5.7Hz,8H),3.12(d, J=7.1Hz,4H),2.92(t,J=6.5Hz,4H),2.12(t,J=6.0Hz,4H),2.03–1.96(m,4H),0.68(d,J=5.4Hz,6H). 13 C NMR (101 MHz, CDCl 3 )δ188.99,144.10,137.53,134.35,134.04,131.82,128.48,127.13,126.79,126.45,119.70,110.29,51.23,50.53,29.39,24.00,22.56,22.25,-2.89.HRMS(ESI)m / zChemical formula:C 35 H 36 N 2 O[M+H] + , measured value: 573.1828. When dichlorodimethylgermanium is replaced by dichlorodiphenylgermanium, R is phenyl.

[0063] Step 6: Obtaining compound (b) by nucleophilic addition reaction: First prepare a phenyllithium reagent, which is the same as in Example 1. Alternatively, the phenylGenzel reagent can be purchased directly.

[0064] Weigh compound (10) (100 mg, 0.15 mmol) in a dried Slack reactor, add the prepared 2,6-dimethylbenzene lithium reagent (1.5 molar equivalents) to the reaction system and stir for 0.5 h, slowly warm the reaction system to room temperature and stir for 4-6 h (6 h in this embodiment). Extract with ethyl acetate to obtain an organic layer, concentrate to obtain a mixed product, add dichloromethane to dissolve, add catalytic equivalents of methanesulfonic acid dropwise at room temperature and stir for 4-6 h (6 h in this embodiment). TLC plate until the raw material is completely reacted, extract with dichloromethane to obtain an organic phase, concentrate, and separate by column chromatography to obtain compound 11 (45 mg, yield 39%). 1 H NMR(400MHz,Chloroform+Methanol-d)δ7.77(d,J=6.2Hz,2H),7.45–7.37(m,3H),7.25(q,J=8.6,7.0Hz,3H) ,7.04(s,2H),3.55(t,J=5.9Hz,8H),2.78(s,15H),2.15(p,J=6.2Hz,7H),2.04(s,9H),0.77(d,J=5.3Hz,7H). 13 C NMR (101 MHz, CDCl 3 )δ150.30,141.91,140.52,139.44,136.52,134.71,132.37,132.14,130.00,129.05,128.02,127.44,126.90,113.99,52.03,51.13,39.56,28.34,23.07,21.23,20.87,20.07,-0.01.HRMS(ESI)m / zChemical formula:C 43 H 45 N 2 + [M+H] + , measured value: 666.2796.

[0065] In step 6 of Example 1 and Example 2, different bromobenzene reagents can be used to prepare the corresponding phenyllithium reagents. Different R 1 -R 5 Substituents, when using o-methylbenzene lithium reagent, R 1 is methyl, R 2 -R 5 is hydrogen; when 2,6-dimethoxyphenyllithium reagent is used, R 1 ,R 5 is methoxy, R 2 -R 4 For hydrogen.

[0066] The basic spectral data of the compound (b) dye obtained in Example 2 was tested. Figure 1-Figure 4 , Figure 1-Figure 4 The absorption test data were collected from the Shimadzu UV3600 UV-visible spectrometer, and the fluorescence test data were collected from the Edinburgh FLS980.

[0067] from Figure 1-Figure 4 It can be seen that in different solvents, it has excellent absorption and emission properties, and its fluorescence emission tails to 1200nm.

[0068] Figure 5 Comparison of the photobleaching resistance of the compound (b) dye obtained in Test Example 2 with the commercial dye ICG; Compound (b) was formulated to have an OD value of about 1 at 888nm; similarly, ICG dye was formulated to have an OD value of about 1 at 795nm. Irradiation conditions: 880 laser for compound (b) and 808 laser for ICG. Power density: 0.35W laser was continuously irradiated to test the attenuation of the OD values ​​at 888 and 795nm. After 30 minutes of irradiation, it was found that the OD value of the ICG dye at 795nm was almost reduced to 0, while the germanium material (b) dye remained stable.

[0069] Figure 6 The test results of the in vitro fluorescence of the dye compound (b) obtained in Example 2 are shown in Figure 2: The fluorescence of the test compound (b) in a centrifuge tube, using an InGaAs camera, the laser is 880nm, (a) the brightness of the filter material with a 1000LP, (b) the brightness of the filter compound (b) with a 1300LP. The test structure shows that the germanium material has a brighter tail fluorescence at 1300nm, which is suitable for in vivo imaging in this band.

[0070] Figure 7 The in vivo imaging test results of the compound (b) dye obtained in Example 2 are shown in the figure: Experimental conditions: Compound (b) was prepared into a 1 mg / mL PBS buffer solution (containing 2% DMSO), and 200 μL of the solution was injected into the tail vein of mice. Experimental equipment: InGaAs camera was selected, and the laser was 793 nm. Power density: 41 mW / cm 2 The 1300LP filter was selected and the exposure time was 30ms. This indicated that under this condition, compound (b) could be bright in the mouse body, and the blood vessels of the mouse system could be clearly seen.

[0071] Figure 8 , 9The results of the in vivo circulation time of compound (a) obtained in Example 1 and compound (b) obtained in Example 2 are shown in the figure: The experimental conditions are to prepare compound (a) and (b) into 1 mg / mL PBS buffer solution (containing 2% DMSO), and inject 200 μL of the solution into the tail vein of mice. Experimental equipment: InGaAs camera, laser is 880nm. Power density: 30mW / cm 2 Select 1300LP filter and exposure time 50ms. Figure 8 The corresponding (a) shows that the blood vessels can still be clearly seen after 30 min, indicating that the dye is still in the blood vessels. Fig. 9 The corresponding figure is (b). After 30 minutes, the blood vessel fluorescence is very weak, and it quickly accumulates in the liver and the brightness increases in the intestine. This shows that compound (b) circulates faster in the blood. A shorter circulation time corresponds to a shorter metabolic time and less biological toxicity in clinical practice.

Claims

1. A compound having a structural formula as shown in formula (a) or formula (b): in, R, R1 and R5 are all methyl, and R2, R3 and R4 are all hydrogen.

2. A method for preparing the compound as claimed in claim 1, characterized in that: The preparation reaction is as follows: Wherein, formula (3) is further reacted as follows: Formula (7) is further reacted as follows: Specifically: Step 1, the compound represented by formula (1) is reacted with an amino compound to obtain a compound represented by formula (2); Step 2: The compound represented by formula (2) is subjected to an alkylation reaction with an alkylating agent to obtain a compound represented by formula (3) or formula (7); Step 3: Under the catalysis of protonic acid or Lewis acid, the compound represented by formula (3) or the compound represented by formula (7) is subjected to condensation reaction with formaldehyde or paraformaldehyde to obtain the compound represented by formula (4) or the compound represented by formula (8); Step 4: The compound represented by formula (4) or the compound represented by formula (8) is subjected to lithium halogen exchange with a lithium reagent under an inert gas atmosphere to obtain a dilithium reagent, and the dilithium reagent obtained is then reacted with dialkylgermanium dichloride or diphenylgermanium dichloride to obtain the compound represented by formula (5) or the compound represented by formula (9); Step 5: The compound represented by formula (5) or the compound represented by formula (9) is oxidized with an oxidant to obtain the compound represented by formula (6) or the compound represented by formula (10); Step 6: The compound represented by formula (6) or the compound represented by formula (10) is subjected to nucleophilic addition reaction in the presence of a phenyllithium reagent or a phenyl Grignard reagent, and then acid-catalyzed to obtain the compound represented by formula (a) or the compound represented by formula (b).

3. The preparation method according to claim 2, characterized in that: In step 1, the amination reaction is carried out in a closed high-pressure container at a reaction temperature of 135-155° C., and the amino compound is aqueous ammonia.

4. The preparation method according to claim 2, characterized in that: The alkylation reaction in step 2 is carried out in a solvent DMF, and the alkylation agent is 1-bromo-3-chloropropane; when the reaction temperature is 130°C-145°C, the alkylation reaction obtains the compound represented by formula (3); when the reaction temperature is 155°C-175°C, the alkylation reaction obtains the compound represented by formula (7).

5. The preparation method according to claim 2, characterized in that: The condensation reaction in step three is carried out in acetic acid as a solvent, refluxed at 80° C.-95° C., the protonic acid is concentrated sulfuric acid or hydrochloric acid, and the Lewis acid is boron trifluoride etherate or ferric chloride.

6. The preparation method according to claim 2, characterized in that: Step 4: Anhydrous tetrahydrofuran or diethyl ether is used as solvent, the lithium reagent used is n-butyl lithium or sec-butyl lithium, and the lithium halogen exchange temperature is -78°C.

7. The preparation method according to claim 2, characterized in that: The oxidant used in step 5 is chlorobenzoquinone, potassium permanganate or hydrogen peroxide, the solvent used is ethyl acetate or acetone, and the reaction temperature is -15°C to room temperature.

8. The preparation method according to claim 2, characterized in that: In step 6, an ether is used as a reaction solvent for the nucleophilic addition reaction. The phenyllithium reagent used is a 2,6-dimethylphenyllithium reagent, an o-methylphenyllithium reagent or a 2,6-dimethoxyphenyllithium reagent. The phenyl Grignard reagent used is a Grignard reagent corresponding to the phenyllithium reagent. The ether solvent is tetrahydrofuran or diethyl ether. When acid catalysis is used, the acid catalyst used is methanesulfonic acid. The reaction is carried out at room temperature, and a halogenated reagent is used as a solvent.

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