A near-infrared fluorescent dye and a fluorescent composite material

Abstract

The present application relates to a kind of near-infrared fluorescent dyes and fluorescent composite material, near-infrared fluorescent dyes has the molecular structure of general formula (I), the structure formula is 3,5 position guroni modified Aza-BODIPY structure;Ar1 And Ar2 in the structure formula are independently selected from substituted or unsubstituted: benzene ring, thiophene ring, furan ring, pyrrole ring or pyridine ring.It has the beneficial effect, has more optimal spectral property and fluorescence imaging application potential: in NIR-II region imaging interval shows bright fluorescence, fluorescence quantum yield is generally in 1.50% -5.23% range, and molar extinction coefficient is higher, brightness is high;Fluorescence intensity is extremely sensitive to water in environment, ACQ effect is better, compared with NIR-I region ACQ probe, the present application NIR-II region ACQ probe is used to mark in vivo drug carrier, in sensitivity and accuracy aspect advantage is obvious.

Description

Technical Field

[0001] This invention relates to the technical field of organic fluorescent dyes, and more particularly to a near-infrared fluorescent dye and a fluorescent composite material. Background Technology

[0002] Compared to currently used technologies such as magnetic resonance imaging (MRI), computed tomography (CT), ultrasound imaging, positron emission tomography (PET), single-photon emission computed tomography (SPECT), and photoacoustic imaging (PAT), fluorescence imaging has received great attention in the medical field due to its advantages such as simple operation, low cost, rapid response, high signal-to-noise ratio, and high safety.

[0003] Because tissue absorption of light is primarily concentrated in the ultraviolet-visible region, and the intensity of light scattering and autofluorescence decreases significantly with increasing wavelength, near-infrared dyes penetrate deeper and are less affected by tissue scattering and background fluorescence compared to shorter-wavelength ultraviolet-visible fluorescent dyes. Based on wavelength range, near-infrared fluorescent dyes can be specifically divided into near-infrared region I (NIR-I, 700-900 nm) and near-infrared region II (NIR-II, 900-1700 nm) dyes. Currently, near-infrared region I fluorescence imaging is widely used clinically. Indocyanine green (ICG) has been approved by the U.S. Food and Drug Administration (FDA) and successfully used in cardiovascular angiography, lymphangiography, gastrointestinal angiography, and as an adjunct to tumor resection surgery. Although NIR-I imaging has achieved good results in clinical applications, its penetration depth remains limited.

[0004] Aza-BODIPY, an analogue of the fluoroboron dipyrrole (BODIPY) dye family in which the carbon atom at position 8 is replaced by a nitrogen atom, has been widely used in biomedical fields such as photodynamic therapy, fluorescence sensors, and near-infrared fluorescence imaging. However, most of the reported Aza-BODIPY dyes are in the visible and near-infrared I regions, with generally short wavelengths. In 2019, Fan Quli's research group introduced the electron-rich julonidine into the 1,7 positions of Aza-BODIPY and obtained the near-infrared II fluorescent probe NJ1060 with a maximum emission wavelength of 1070 nm through strong intramolecular charge transfer (ICT) from julonidine to Aza-BODIPY (Chem. Commun. 2019, 55, 10920-10923). However, the spectral properties of this probe still need to be improved.

[0005] In recent years, research on various drug delivery systems has continued to heat up, yet related products are few and far between. Analyzing the in vivo fate and mechanism of action of these drug carriers is crucial for their clinical translation, but it is also extremely challenging. Traditional research methods often employ fluorescent probes and radionuclides for labeling, but the accuracy is not ideal because they cannot distinguish between carrier signals and free probe signals. To address this key scientific problem, the applicant has developed a series of environmentally responsive near-infrared (NIR-I) fluorescent probes based on the aggregation-caused quenching (ACQ) effect and applied them to explore the in vivo fate of various drug carriers. Among these, the results of in vivo tracking of drug carriers using a typical NIR-I ACQ probe (P2) have been published in dozens of articles, such as *Nanomedicine* 2015, 11:1939-1948; *Nanoscale* 2016, 8:7024-7035; and *J Mater Chem B* 2016, 4:2864-2873. However, the maximum emission wavelength of this series of ACQ probes is generally below 750 nm, and the sensitivity and accuracy of in vivo drug carrier tracking are still significantly affected by tissue scattering and signal penetration depth.

[0006] Therefore, developing novel and efficient organic near-infrared ACQ fluorescent dyes is an urgent technical problem to be solved. Summary of the Invention

[0007] (a) Technical problems to be solved

[0008] In view of the above-mentioned shortcomings and deficiencies of the prior art, the present invention provides a near-infrared fluorescent dye and a fluorescent composite material, which solves the problems of low brightness of existing NIR-II region Aza-BODIPY dye and strong in vivo fluorescence reproduction and low penetration depth of NIR-I region ACQ fluorescent probe.

[0009] (II) Technical Solution

[0010] To achieve the above objectives, the main technical solutions adopted by the present invention include:

[0011] In a first aspect, embodiments of the present invention provide a near-infrared fluorescent dye, wherein the fluorescent dye is a compound having the general formula (I):

[0012]

[0013] Wherein, general formula (I) is an Aza-BODIPY structure modified with julonidine at positions 3 and 5; Ar1 ​​and Ar2 are independently selected from substituted or unsubstituted: benzene ring, thiophene ring, furan ring, pyrrole ring or pyridine ring.

[0014] According to a preferred embodiment of the present invention, the near-infrared fluorescent dye is any one of the following two structural formulas:

[0015]

[0016] Wherein, R1 and R2 are substituents on the benzene ring, with 1-5 substituents on each benzene ring; R3 and R4 are substituents on the thiophene ring, with 1-3 substituents on each thiophene ring; R1, R2, R3, and R4 are independently selected from the set of the following groups: H, C1-C8 alkoxy groups, NR... a R b -COR c Halogen, C1-C8 alkyl, C3-C6 cycloalkyl, hydroxyl, amino, nitro, aldehyde, -CF3, -CN, carboxyl; among which, R a R b R c Each of the following groups is independently selected: H, substituted or unsubstituted C1-C10 alkyl, substituted or unsubstituted C3-C10 cycloalkyl, substituted or unsubstituted C2-C10 alkenyl, substituted or unsubstituted C6-C20 aryl, and substituted or unsubstituted C5-C14 heteroaryl.

[0017] According to a preferred embodiment of the present invention, in the near-infrared fluorescent dye, R1 and R2 are independently H or C1-C6 alkoxy groups.

[0018] According to a preferred embodiment of the present invention, the near-infrared fluorescent dye wherein the C1-C6 alkoxy group is a straight-chain or branched alkoxy group.

[0019] According to a preferred embodiment of the present invention, the near-infrared fluorescent dye wherein the straight-chain alkoxy group is a straight-chain diekoxy group.

[0020] According to a preferred embodiment of the present invention, the near-infrared fluorescent dye has the following structural formula:

[0021]

[0022] R5, R6, R7 and R8 are independently selected from substituted or unsubstituted C1-C4 alkyl groups.

[0023] According to a preferred embodiment of the present invention, the near-infrared fluorescent dyes, R5 and / or R6, are fused to the benzene ring, and / or R7 and / or R8 are fused to the benzene ring.

[0024] According to a preferred embodiment of the present invention, in the near-infrared fluorescent dye, in the structural formula (II), R1 and / or R2 are NR. a R b At that time, R a and R bAnd onto the benzene ring, or, R a and R b Forming a ring; in the structural formula (Ⅲ), R3 and / or R4 are NR a R b At that time, R a and R b And onto the thiophene ring, or, R a and R b Forming a ring.

[0025] According to a preferred embodiment of the present invention, the near-infrared fluorescent dye is specifically one of the following compounds:

[0026]

[0027] In a second aspect, embodiments of the present invention provide a fluorescent composite material in which the near-infrared fluorescent dye described in the first aspect is encapsulated within a carrier, wherein the carrier has a hydrophobic matrix structure or a hydrophobic core-hydrophilic shell structure, and the near-infrared fluorescent dye is encapsulated within the hydrophobic core.

[0028] According to a preferred embodiment of the present invention, the fluorescent composite material has a carrier at the nanoscale, and the hydrophobic matrix structure is selected from at least one of polymer nanoparticles / microspheres, solid lipid microparticles / nanoparticles, microemulsions, nanoemulsions, and nanocrystals; the hydrophobic core-hydrophilic shell structure is a nanopolymer micelle.

[0029] According to a preferred embodiment of the present invention, the fluorescent composite material is used in fluorescence imaging, diagnosis, and in vivo fluorescence tracing of drug carriers.

[0030] (III) Beneficial Effects

[0031] The beneficial effects of this invention are as follows: The near-infrared fluorescent dye and fluorescent composite material of this invention, due to the juulonidine modification of the 3,5 positions of Aza-BODIPY by the near-infrared fluorescent dye, and because the aryl group at the 3,5 positions contributes more to the molecular orbitals of Aza-BODIPY than the aryl group at the 1,7 positions, Aza-BODIPY modified with juulonidine at the 3,5 positions exhibits superior spectral properties and greater potential for fluorescence imaging applications.

[0032] The maximum emission wavelengths of these near-infrared fluorescent dyes are all in the range of 880-1030 nm. They exhibit bright fluorescence in the NIR-II imaging region, with fluorescence quantum yields generally ranging from 1.50% to 5.23%, and high molar extinction coefficients (60000–83000 M). -1 cm -1Compared to the previously reported 1,7-position julonidin-modified Aza-BODIPY (NJ1060), the molar extinction coefficient and fluorescence quantum yield are significantly improved, and the brightness is mostly 2–8 times that of NJ1060.

[0033] The fluorescence intensity is extremely sensitive to water in the environment, and the ACQ effect is better. In the water / DMSO system, the ACQ effect is significantly stronger than that of P2 and NJ1060.

[0034] Compared to the earlier NIR-I region ACQ probe (P2), the near-infrared fluorescent dye of the present invention has a deeper signal penetration depth, a higher signal-to-noise ratio, more realistic imaging, and excellent in vivo anti-fluorescence reproducibility (all below 3%) when used to label in vivo drug carriers. Therefore, it has higher sensitivity and accuracy when used for in vivo fluorescence tracing of drug carriers.

[0035] Furthermore, the experiment revealed that the near-infrared fluorescent compound dye provided by this invention did not exhibit significant photoquenching or photobleaching phenomena under normal operating conditions and in an environment without light protection, indicating that its chemical and optical properties are highly stable. Attached Figure Description

[0036] Figure 1 The absorption and emission spectra (λ) of compound 1-14 (5 μM) in chloroform (CHCl3) of this invention are shown. ex =808nm);

[0037] Figure 2 The absorption and emission spectra (λ) of compound 1-14 (5 μM) in dimethyl sulfoxide (DMSO) of this invention are shown. ex =808nm);

[0038] Figure 3 The absorption and emission spectra (λ) of compound 1 (5 μM) and NJ1060 (5 μM) in CHCl3 and DMSO, respectively. ex =808nm);

[0039] Figure 4 The fluorescence spectra of compound 1 in DMSO / water mixed solvents with different water contents, and the curve of fluorescence intensity at the maximum emission wavelength as a function of water content;

[0040] Figure 5 The graph shows the fluorescence intensity at the maximum emission wavelength of compounds 2-14, NJ1060, and P2 as a function of water content in DMSO / water mixed solvents with different water contents.

[0041] Figure 6 A comparison chart of the minimum water content required for complete fluorescence quenching of P2, NJ1060, and compounds 1-14;

[0042] Figure 7 Figure showing the fluorescence quenching stability of P2 and compounds 1-14 in plasma;

[0043] Figure 8 The fluorescence reproduction results of P2 and compounds 1-14 in plasma are shown in the figure.

[0044] Figure 9 (a) shows fluorescence imaging of PM nano-tracer solutions containing compounds 1 and P2 in capillaries at different depths in 1% fat emulsions under different long-pass filters; (b) shows the full width at half maximum (FWHM) results at different depths.

[0045] Figure 10 (a, b) are fluorescence in vivo imaging and magnified images of blood vessel locations after intravenous administration of PM nano-tracer solutions containing compounds 1 and P2 to mice; (a) corresponds to compound 1, (b) corresponds to P2; (c) is a graph showing the changes in fluorescence signals of blood vessels and surrounding areas after the imaging of compounds 1 and P2; (d) is the signal-to-noise ratio of blood vessels imaging of compounds 1 and P2; (c) is the half-width at half-maximum (WHM) of blood vessels imaging of compounds 1 and P2.

[0046] Figure 11 (a) Fluorescence in vivo imaging at different time points after intravenous administration of PM nano-tracers loaded with compound 1 and pre-quenching solution of compound 1 to mice; (b) Fluorescence imaging of ex vivo organs 24 h after intravenous administration of PM nano-tracers loaded with compound 1 and pre-quenching solution of compound 1 to mice; (c) Fluorescence in vivo imaging at different time points after intravenous administration of PM nano-tracers loaded with P2 and pre-quenching solution of P2 to mice; (d) Fluorescence in vivo imaging of PM nano-tracers loaded with P2 loaded with P2. (e) Fluorescence imaging of ex vivo organs 24 h after intravenous administration of PM nanotracer and P2 prequenching solution to mice; (d) Percentage of fluorescence re-enhancing in mouse abdomen at different time points compared to PM nanotracer solutions containing compounds 1 and P2; (e) Percentage of fluorescence re-enhancing in ex vivo organs 24 h after intravenous administration of PM nanotracer solutions containing compounds 1 and P2. Detailed Implementation

[0047] To better explain and facilitate understanding of the present invention, the present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0048] The present invention provides a near-infrared fluorescent dye and a fluorescent composite material, which solves the problem that the low brightness of existing NIR-II region Aza-BODIPY dye and the high in vivo fluorescence reproducibility and low penetration depth of NIR-I region ACQ fluorescent dye fluorescent probe affect the application of near-infrared fluorescent dyes in medicine and other fields. Because this near-infrared fluorescent dye modifies the 3,5 positions of azirconidine in Aza-BODIPY, the 3,5-position julonidine-modified Aza-BODIPY exhibits superior spectral properties and fluorescence imaging potential. This near-infrared fluorescent dye displays bright fluorescence in the NIR-II imaging region, with significantly improved molar extinction coefficient and fluorescence quantum yield, and brightness that is 2–8 times that of NJ1060. It also exhibits better ACQ effects, showing significantly stronger ACQ effects than P2 and NJ1060 in the water / DMSO system. The fluorescence intensity is extremely sensitive to water in the environment. Compared to the earlier NIR-I ACQ probe (P2), the near-infrared fluorescent dye of this invention, when used to label in vivo drug carriers, demonstrates deeper signal penetration, higher signal-to-noise ratio, more realistic imaging, and excellent in vivo fluorescence reproducibility (all below 3%). Therefore, it offers higher sensitivity and accuracy in in vivo fluorescence tracing of drug carriers.

[0049] To better understand the above technical solutions, exemplary embodiments of the present invention will be described in more detail below with reference to the accompanying drawings. Although exemplary embodiments of the present invention are shown in the drawings, it should be understood that the present invention can be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that the present invention can be understood more clearly and thoroughly, and that the scope of the present invention can be fully conveyed to those skilled in the art.

[0050] Example 1

[0051] This embodiment provides a method for preparing compound 1, which consists of four steps, as detailed below:

[0052] (1) Synthesis of 9-acetyljulonidine

[0053]

[0054] A solution of N,N-dimethylacetamide (DMA) (0.81 mL, 8.8 mmol) in 1,2-dichloroethane (DCE) (40 mL) was cooled to 0 °C under nitrogen protection. Phosphorus oxychloride (POCl3) (0.68 mL, 7.3 mmol) was then added dropwise. After stirring at room temperature for 30 min, a solution of julonidine (1000 mg, 5.8 mmol) in 1,2-dichloroethane (10 mL) was added, and the mixture was stirred at 80 °C. After the starting material had completely disappeared, a saturated aqueous sodium carbonate solution was added, and the mixture was extracted with dichloromethane (DCM). The extract was dried over anhydrous sodium sulfate, evaporated to dryness, and subjected to column chromatography to obtain 200 mg of the product as a pale yellow solid, with a yield of 16%.

[0055] (2) Synthesis of 1-(2,3,6,7-tetrahydro-1H,5H-pyridyl[3,2,1-ij]quinolin-9-yl)-3-(4-methoxyphenyl)-prop-2-en-1-one

[0056]

[0057] A 5 mL (50%) aqueous solution of potassium hydroxide (KOH) was slowly added to a 20 mL (EtOH) solution of 9-acetyljulonidine (989 mg, 4.6 mmol) and 4-methoxybenzaldehyde (694 mg, 5.1 mmol), followed by stirring at room temperature. After complete conversion of the starting materials, ice water was added to precipitate the product. The precipitate was filtered, recrystallized, and yielded 1100 mg of the product as a yellow solid, with a yield of 72%.

[0058] (3) Synthesis of 1-(2,3,6,7-tetrahydro-1H,5H-pyridyl[3,2,1-ij]quinolin-9-yl)-3-(4-methoxyphenyl)-4-nitro-butane-1-one

[0059]

[0060] 2.1 mL of 1,8-diazabispyrocyclo[5.4.0]undec-7-ene (DBU) was added to a mixed solution of 1-(2,3,6,7-tetrahydro-1H,5H-pyridyl[3,2,1-ij]quinolin-9-yl)-3-(4-methoxyphenyl)-prop-2-en-1-one (932 mg, 2.8 mmol) in 10 mL of nitromethane (CH3NO2) and 50 mL of methanol (MeOH), and then refluxed for 7 h. After complete conversion of the starting material, the solution was diluted with water, extracted with dichloromethane, and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, evaporated to dryness, and subjected to silica gel column chromatography to give 1010 mg of the product as a pale yellow solid, with a yield of 92%.

[0061] (4) Synthesis of Compound 1

[0062]

[0063] Ammonium acetate (NH4OAc) (3126 mg, 40.6 mmol) was added to a solution of 1-(2,3,6,7-tetrahydro-1H,5H-pyridyl[3,2,1-ij]quinolin-9-yl)-3-(4-methoxyphenyl)-4-nitro-butane-1-one (591 mg, 1.5 mmol) in n-butanol (n-BuOH) (20 mL), and then refluxed for 12 h. After complete conversion of the starting material, the mixture was cooled, filtered, washed with a small amount of ethanol, dried, and evaporated to dryness to give the precursor of compound 1.

[0064] N,N-diisopropylethylamine (DIPEA) (1.2 mL) and boron trifluoride diethyl ether (BF3·OEt2) (1.2 mL) were added to a 1,2-dichloroethane (100 mL) solution of the precursor of compound 1, and the reaction was then heated to 80 °C. After the reaction was complete, the reaction was quenched with saturated sodium carbonate aqueous solution, followed by extraction with dichloromethane, drying with anhydrous sodium sulfate, and rotary evaporation. Silica gel column chromatography yielded 163 mg of the product as a black solid, giving compound 1 in 29% yield.

[0065] The proton and carbon NMR spectra of compound 1 are as follows:

[0066] 1HNMR(600MHz,DMSO-d6)δ8.14(d,J=8.8Hz,4H),7.81(s,4H),7.45(s,2H),7.08(d,J=8 .9Hz,4H),3.85(s,6H),3.33(d,J=7.9Hz,8H),2.75(t,J=6.2Hz,8H),1.96–1.90(m,8H);

[0067] 13CNMR(151MHz,DMSO)δ159.77,153.45,145.21,143.59,137.69,129.96,12 9.10,125.31,120.54,116.82,116.65,114.08,55.31,49.45,27.26,21.07.

[0068] Example 2

[0069] This embodiment provides a method for preparing compound 2, which consists of three steps, as follows:

[0070] (1) Synthesis of 1-(2,3,6,7-tetrahydro-1H,5H-pyridyl[3,2,1-ij]quinolin-9-yl)-3-(3,4-dimethoxyphenyl)-prop-2-en-1-one

[0071]

[0072] The synthesis method was the same as that in step (2) of Example 1, which describes the synthesis of 1-(2,3,6,7-tetrahydro-1H,5H-pyridyl[3,2,1-ij]quinolin-9-yl)-3-(4-methoxyphenyl)-prop-2-en-1-one. Using 9-acetyljulonidine (989 mg, 4.6 mmol) and 3,4-dimethoxybenzaldehyde (847 mg, 5.1 mmol) as starting materials, 1300 mg of the product was obtained as a yellow solid with a yield of 78%.

[0073] (2) Synthesis of 1-(2,3,6,7-tetrahydro-1H,5H-pyridyl[3,2,1-ij]quinolin-9-yl)-3-(3,4-dimethoxyphenyl)-4-nitro-butane-1-one

[0074]

[0075] The synthesis method was the same as that in step (3) of Example 1, which describes the synthesis of 1-(2,3,6,7-tetrahydro-1H,5H-pyridyl[3,2,1-ij]quinoline-9-yl)-3-(4-methoxyphenyl)-4-nitro-butane-1-one. Using 1-(2,3,6,7-tetrahydro-1H,5H-pyridyl[3,2,1-ij]quinoline-9-yl)-3-(3,4-dimethoxyphenyl)-prop-2-en-1-one (1016 mg, 2.8 mmol) as the starting material, 990 mg of the product was obtained as a yellow solid, with a yield of 83%.

[0076] (3) Synthesis of compound 2

[0077]

[0078] The synthesis method was the same as that of compound 1 in step (4) of Example 1. Using 1-(2,3,6,7-tetrahydro-1H,5H-pyridyl[3,2,1-ij]quinolin-9-yl)-3-(3,4-dimethoxyphenyl)-4-nitro-butane-1-one (636 mg, 1.5 mmol) as the starting material, 138 mg of product was obtained as a black solid, yielding compound 2 with a yield of 23%.

[0079] The proton and carbon NMR spectra of compound 2 are as follows:

[0080] 1HNMR(600MHz,DMSO-d6)δ7.81(s,4H),7.76(dd,J=8.4,1.7Hz,2H),7.62(d,J=1.6Hz,2H),7.45(s,2H),7. 04(d,J=8.5Hz,2H),3.84(s,6H),3.75(s,6H),3.36–3.32(m,8H),2.76(t,J=6.2Hz,8H),1.98–1.89(m,8H);

[0081] 13CNMR(151MHz,DMSO)δ153.30,149.54,148.72,145.19,143.66,138.22,129.11,125.6 8,121.81,120.52,117.18,116.84,112.25,111.70,55.62,55.59,49.45,27.28,21.07.

[0082] Example 3

[0083] This embodiment provides a method for preparing compound 3, which consists of three steps, as follows:

[0084] (1) Synthesis of 1-(2,3,6,7-tetrahydro-1H,5H-pyridyl[3,2,1-ij]quinolin-9-yl)-3-(3,4,5-trimethoxyphenyl)-prop-2-en-1-one

[0085]

[0086] The synthesis method was the same as that in step (2) of Example 1, which describes the synthesis of 1-(2,3,6,7-tetrahydro-1H,5H-pyridyl[3,2,1-ij]quinolin-9-yl)-3-(4-methoxyphenyl)-prop-2-en-1-one. Using 9-acetyljulonidine (989 mg, 4.6 mmol) and 3,4,5-trimethoxybenzaldehyde (1000 mg, 5.1 mmol) as starting materials, 1200 mg of the product was obtained as a yellow solid, with a yield of 66%.

[0087] (2) Synthesis of 1-(2,3,6,7-tetrahydro-1H,5H-pyridyl[3,2,1-ij]quinolin-9-yl)-3-(3,4,5-trimethoxyphenyl)-4-nitro-butane-1-one

[0088]

[0089] The synthesis method was the same as that in step (3) of Example 1, which describes the synthesis of 1-(2,3,6,7-tetrahydro-1H,5H-pyridyl[3,2,1-ij]quinoline-9-yl)-3-(4-methoxyphenyl)-4-nitro-butane-1-one. Using 1-(2,3,6,7-tetrahydro-1H,5H-pyridyl[3,2,1-ij]quinoline-9-yl)-3-(3,4,5-trimethoxyphenyl)-prop-2-en-1-one (1100 mg, 2.8 mmol) as the starting material, 920 mg of the product was obtained as a yellow solid, with a yield of 72%.

[0090] (3) Synthesis of compound 3

[0091]

[0092] The synthesis method was the same as that of compound 1 in step (4) of Example 1. Using 1-(2,3,6,7-tetrahydro-1H,5H-pyridyl[3,2,1-ij]quinolin-9-yl)-3-(3,4,5-trimethoxyphenyl)-4-nitro-butane-1-one (681 mg, 1.5 mmol) as the starting material, 162 mg of product was obtained as a black solid, yielding compound 3 with a yield of 25%.

[0093] The 1H and 1C NMR spectra of compound 3 are as follows:

[0094] 1HNMR(600MHz,Pyridine-d5)δ8.32(s,4H),7.80(s,2H),7.67(s,4H),4.02(s,6H), 3.80(d,J=6.7Hz,12H),3.07–3.01(m,8H),2.76(t,J=6.2Hz,8H),1.80–1.72(m,8H);

[0095] 13CNMR(151MHz,Pyridine-d5)δ155.82,154.41,146.31,145.85,140.62,140.00,1 30.72,130.14,121.85,119.29,119.12,107.86,61.21,56.52,50.46,28.46,22.16.

[0096] Example 4

[0097] This embodiment provides a method for preparing compound 4, which consists of three steps, as follows:

[0098] (1) Synthesis of 1,3-bis(2,3,6,7-tetrahydro-1H,5H-pyridyl[3,2,1-ij]quinolin-9-yl)-prop-2-en-1-one

[0099]

[0100] The synthesis method was the same as that in step (2) of Example 1, which describes the synthesis of 1-(2,3,6,7-tetrahydro-1H,5H-pyridyl[3,2,1-ij]quinolin-9-yl)-3-(4-methoxyphenyl)-prop-2-en-1-one. Using 9-acetyljulonidine (989 mg, 4.6 mmol) and 9-aldehydejulonidine (1025 mg, 5.1 mmol) as raw materials, 1300 mg of product was obtained as a yellow solid with a yield of 71%.

[0101] (2) Synthesis of 1,3-bis(2,3,6,7-tetrahydro-1H,5H-pyridyl[3,2,1-ij]quinolin-9-yl)-4-nitro-butane-1-one

[0102]

[0103] The synthesis method was the same as that in step (3) of Example 1, which describes the synthesis of 1-(2,3,6,7-tetrahydro-1H,5H-pyridyl[3,2,1-ij]quinoline-9-yl)-3-(4-methoxyphenyl)-4-nitro-butane-1-one. Using 1,3-bis(2,3,6,7-tetrahydro-1H,5H-pyridyl[3,2,1-ij]quinoline-9-yl)-prop-2-en-1-one (1113 mg, 2.8 mmol) as the starting material, 998 mg of the product was obtained as a yellow solid, with a yield of 78%.

[0104] (3) Synthesis of compound 4

[0105]

[0106] The synthesis method was the same as that of compound 1 in step (4) of Example 1. Using 1,3-bis(2,3,6,7-tetrahydro-1H,5H-pyridyl[3,2,1-ij]quinolin-9-yl)-4-nitro-butane-1-one (689 mg, 1.5 mmol) as the starting material, 100 mg of product was obtained as a black solid, yielding compound 4 with a yield of 15%.

[0107] The proton and carbon NMR spectra of compound 4 are as follows:

[0108] 1HNMR(600MHz,Pyridine-d5)δ8.21(s,4H),7.96(s,4H),7.44(s,2H),3.08(s,8H),3.00(s,8H),2.76(d,J=25.2Hz,16H),1.85(s,8H),1.76(s,8H);

[0109] 13CNMR(151MHz,Pyridine-d5)δ155.79,145.83,145.55,144.29,141.78,130.21,129. 24,122.17,121.88,121.47,120.06,115.46,50.51,50.37,28.73,28.53,22.60,22.33.

[0110] Example 5

[0111] This embodiment provides a method for preparing compound 5, which consists of three steps, as follows:

[0112] (1) Synthesis of 1-(2,3,6,7-tetrahydro-1H,5H-pyridyl[3,2,1-ij]quinolin-9-yl)-3-phenyl-prop-2-en-1-one

[0113]

[0114] The synthesis method was the same as that in step (2) of Example 1, which describes the synthesis of 1-(2,3,6,7-tetrahydro-1H,5H-pyridyl[3,2,1-ij]quinolin-9-yl)-3-(4-methoxyphenyl)-prop-2-en-1-one. Using 9-acetyljulonidine (989 mg, 4.6 mmol) and 4-methoxybenzaldehyde (541 mg, 5.1 mmol) as raw materials, 950 mg of a yellow solid was obtained, with a yield of 68%.

[0115] (2) Synthesis of 1-(2,3,6,7-tetrahydro-1H,5H-pyridyl[3,2,1-ij]quinolin-9-yl)-3-phenyl-4-nitro-butane-1-one

[0116]

[0117] The synthesis method was the same as that in step (3) of Example 1, which describes the synthesis of 1-(2,3,6,7-tetrahydro-1H,5H-pyridyl[3,2,1-ij]quinoline-9-yl)-3-(4-methoxyphenyl)-4-nitro-butane-1-one. Using 1-(2,3,6,7-tetrahydro-1H,5H-pyridyl[3,2,1-ij]quinoline-9-yl)-3-phenyl-prop-2-en-1-one (848 mg, 2.8 mmol) as the starting material, 780 mg of the product was obtained as a pale yellow solid with a yield of 77%.

[0118] (3) Synthesis of compound 5

[0119]

[0120] The synthesis method was the same as that of compound 1 in step (4) of Example 1. Using 1-(2,3,6,7-tetrahydro-1H,5H-pyridyl[3,2,1-ij]quinolin-9-yl)-3-phenyl-4-nitro-butane-1-one (546 mg, 1.5 mmol) as the starting material, 110 mg of product was obtained as a black solid, yielding compound 5 with a yield of 21%.

[0121] The proton and carbon NMR spectra of compound 5 are as follows:

[0122] 1HNMR (600MHz, Pyridine-d5) δ8.37 (d, J = 7.7Hz, 4H), 8.24 (s, 4H), 7.62 (s, 2H), 7.55 (t, J = 7. 7Hz,4H),7.45(t,J=7.3Hz,2H),3.08–2.98(m,8H),2.79(t,J=6.2Hz,8H),1.80–1.70(m,8H);

[0123] 13CNMR(151MHz,Pyridine-d5)δ156.03,146.37,145.90,140.04,134.40,13 0.67,130.00,129.33,129.25,121.86,119.28,119.07,50.47,28.47,22.14.

[0124] Example 6

[0125] This embodiment provides a method for preparing compound 6, which consists of three steps, as follows:

[0126] (1) Synthesis of 1-(2,3,6,7-tetrahydro-1H,5H-pyridyl[3,2,1-ij]quinolin-9-yl)-3-(thiophen-2-yl)-prop-2-en-1-one

[0127]

[0128] The synthesis method was the same as that in step (2) of Example 1, which describes the synthesis of 1-(2,3,6,7-tetrahydro-1H,5H-pyridyl[3,2,1-ij]quinolin-9-yl)-3-(4-methoxyphenyl)-prop-2-en-1-one. Using 9-acetyljulonidine (989 mg, 4.6 mmol) and thiophene-2-carboxaldehyde (571 mg, 5.1 mmol) as raw materials, 1200 mg of the product was obtained as a yellow solid, yielding 84%.

[0129] (2) Synthesis of 1-(2,3,6,7-tetrahydro-1H,5H-pyridyl[3,2,1-ij]quinolin-9-yl)-3-(thiophen-2-yl)-4-nitro-butane-1-one

[0130]

[0131] The synthesis method was the same as that in step (3) of Example 1, which describes the synthesis of 1-(2,3,6,7-tetrahydro-1H,5H-pyridyl[3,2,1-ij]quinoline-9-yl)-3-(4-methoxyphenyl)-4-nitro-butane-1-one. Using 1-(2,3,6,7-tetrahydro-1H,5H-pyridyl[3,2,1-ij]quinoline-9-yl)-3-(thiophen-2-yl)-prop-2-en-1-one (865 mg, 2.8 mmol) as the starting material, 770 mg of the product was obtained as a yellow solid with a yield of 74%.

[0132] (3) Synthesis of compound 6

[0133]

[0134] The synthesis method was the same as that of compound 1 in step (4) of Example 1. Using 1-(2,3,6,7-tetrahydro-1H,5H-pyridyl[3,2,1-ij]quinolin-9-yl)-3-(thiophen-2-yl)-4-nitro-butane-1-one (555 mg, 1.5 mmol) as the starting material, 50 mg of product was obtained as a black solid, yielding compound 6 with a yield of 10%.

[0135] The proton and carbon NMR spectra of compound 6 are as follows:

[0136] 1HNMR (600MHz, Pyridine-d5) δ8.23 (s, 2H), 8.17 (s, 4H), 7.66 (s, 2H), 7.27 (s, 4H), 3.03 (s, 8H), 2.75 (d, J = 5.4Hz, 8H), 1.74 (s, 8H);

[0137] 13CNMR(151MHz,Pyridine-d5)δ162.34,156.16,146.36,145.26,134.69,13 0.61,129.40,129.10,128.82,121.81,119.00,117.24,50.46,28.43,22.12.

[0138] Example 7

[0139] This embodiment provides a method for preparing compound 7, which consists of three steps, as follows:

[0140] (1) Synthesis of 1-(2,3,6,7-tetrahydro-1H,5H-pyridyl[3,2,1-ij]quinolin-9-yl)-3-(4-(dimethylamino)phenyl)-prop-2-en-1-one

[0141]

[0142] The synthesis method was the same as that in step (2) of Example 1, which describes the synthesis of 1-(2,3,6,7-tetrahydro-1H,5H-pyridyl[3,2,1-ij]quinolin-9-yl)-3-(4-methoxyphenyl)-prop-2-en-1-one. Using 9-acetyljulonidine (989 mg, 4.6 mmol) and 4-(dimethylamino)benzaldehyde (760 mg, 5.1 mmol) as raw materials, 1180 mg of the product was obtained as a yellow solid, yielding a 74% yield.

[0143] (2) Synthesis of 1-(2,3,6,7-tetrahydro-1H,5H-pyridyl[3,2,1-ij]quinolin-9-yl)-3-(4-(dimethylamino)phenyl)-4-nitro-butane-1-one

[0144]

[0145] The synthesis method was the same as that in step (3) of Example 1, which describes the synthesis of 1-(2,3,6,7-tetrahydro-1H,5H-pyridyl[3,2,1-ij]quinoline-9-yl)-3-(4-methoxyphenyl)-4-nitro-butane-1-one. Using 1-(2,3,6,7-tetrahydro-1H,5H-pyridyl[3,2,1-ij]quinoline-9-yl)-3-(4-(dimethylamino)phenyl)-prop-2-en-1-one (969 mg, 2.8 mmol) as the starting material, 788 mg of the product was obtained as a yellow solid, with a yield of 69%.

[0146] (3) Synthesis of compound 7

[0147]

[0148] The synthesis method was the same as that of compound 1 in step (4) of Example 1. Using 1-(2,3,6,7-tetrahydro-1H,5H-pyridyl[3,2,1-ij]quinolin-9-yl)-3-(4-(dimethylamino)phenyl)-4-nitro-butane-1-one (611 mg, 1.5 mmol) as the starting material, 110 mg of product was obtained as a black solid, yielding compound 7 with a yield of 19%.

[0149] The proton and carbon NMR spectra of compound 7 are as follows:

[0150] 1HNMR(600MHz,Pyridine-d5)δ8.48(d,J=8.1Hz,4H),8.20(s,4H),7.48(s,2H ),6.96(d,J=8.1Hz,4H),3.01(s,8H),2.88(s,12H),2.78(s,8H),1.76(s,8H);

[0151] 13CNMR(151MHz,Pyridine-d5)δ156.15,151.55,145.83,145.77,141.25,131.3 2,130.29,122.70,121.56,119.78,115.94,113.00,50.40,40.37,28.51,22.28.

[0152] Example 8

[0153] This embodiment provides a method for preparing compound 8, which consists of three steps, as follows:

[0154] (1) Synthesis of 1-(2,3,6,7-tetrahydro-1H,5H-pyridyl[3,2,1-ij]quinolin-9-yl)-3-(2,4-dimethoxyphenyl)-prop-2-en-1-one

[0155]

[0156] The synthesis method was the same as that in step (2) of Example 1, which was the synthesis of 1-(2,3,6,7-tetrahydro-1H,5H-pyridyl[3,2,1-ij]quinolin-9-yl)-3-(4-methoxyphenyl)-prop-2-en-1-one. Using 9-acetyljulonidine (989 mg, 4.6 mmol) and 2,4-dimethoxybenzaldehyde (847 mg, 5.1 mmol) as raw materials, 1250 mg of the product was obtained as a yellow solid with a yield of 75%.

[0157] (2) Synthesis of 1-(2,3,6,7-tetrahydro-1H,5H-pyridyl[3,2,1-ij]quinolin-9-yl)-3-(2,4-dimethoxyphenyl)-4-nitro-butane-1-one

[0158]

[0159] The synthesis method was the same as that in step (3) of Example 1, which describes the synthesis of 1-(2,3,6,7-tetrahydro-1H,5H-pyridyl[3,2,1-ij]quinoline-9-yl)-3-(4-methoxyphenyl)-4-nitro-butane-1-one. Using 1-(2,3,6,7-tetrahydro-1H,5H-pyridyl[3,2,1-ij]quinoline-9-yl)-3-(2,4-dimethoxyphenyl)-prop-2-en-1-one (1016 mg, 2.8 mmol) as the starting material, 1050 mg of the product was obtained as a yellow solid with a yield of 88%.

[0160] (3) Synthesis of compound 8

[0161]

[0162] The synthesis method was the same as that of compound 1 in step (4) of Example 1. Using 1-(2,3,6,7-tetrahydro-1H,5H-pyridyl[3,2,1-ij]quinolin-9-yl)-3-(2,4-dimethoxyphenyl)-4-nitro-butane-1-one (636 mg, 1.5 mmol) as the starting material, 205 mg of product was obtained as a black solid, yielding compound 8 with a yield of 34%.

[0163] The proton and carbon NMR spectra of compound 8 are as follows:

[0164] 1HNMR(600MHz,Pyridine-d5)δ8.60(d,J=8.6Hz,2H),8.20(s,4H),7.77(s,2H),6.87(d,J=8.6Hz,2H),6. 77(s,2H),3.81(d,J=22.6Hz,6H),3.79(s,6H),3.00(s,8H),2.74(t,J=5.7Hz,8H),1.74(d,J=5.2Hz,8H);

[0165] 13CNMR(151MHz,Pyridine-d5)δ162.32,160.53,156.02,146.48,145.91,137.11,135. 03,130.39,121.67,119.67,116.17,106.14,99.44,56.10,55.83,50.40,28.47,22.21.

[0166] Example 9

[0167] This embodiment provides a method for preparing compound 9, which consists of three steps, as follows:

[0168] (1) Synthesis of 1-(2,3,6,7-tetrahydro-1H,5H-pyridyl[3,2,1-ij]quinolin-9-yl)-3-(4-ethoxyphenyl)-prop-2-en-1-one

[0169]

[0170] The synthesis method was the same as that in step (2) of Example 1, which was the synthesis of 1-(2,3,6,7-tetrahydro-1H,5H-pyridyl[3,2,1-ij]quinolin-9-yl)-3-(4-methoxyphenyl)-prop-2-en-1-one. Using 9-acetyljulonidine (989 mg, 4.6 mmol) and 4-ethoxybenzaldehyde (765 mg, 5.1 mmol) as starting materials, the product was a yellow solid of 1300 mg, with a yield of 81%.

[0171] The 1H and 1C NMR spectra of the product obtained in this step are as follows:

[0172] 1HNMR (600MHz, CDCl3) δ7.72(d,J=15.5Hz,1H),7.58(d,J=8.7Hz,2H),7.57(s,2H),7.47–7.42(m,1H),6.91(d,J =8.7Hz,2H),4.12–3.99(m,2H),3.32–3.24(m,4H),2.80(t,J=6.3Hz,4H),2.03–1.92(m,4H),1.46–1.38(m,3H);

[0173] 13CNMR (151MHz, CDCl3) δ187.71,160.57,146.83,141.89,129.96,128.41,125.24,120.17,120.09,114.87,63.72,50.13,27.95,21.65,14.91.

[0174] (2) Synthesis of 1-(2,3,6,7-tetrahydro-1H,5H-pyridyl[3,2,1-ij]quinolin-9-yl)-3-(4-ethoxyphenyl)-4-nitro-butane-1-one

[0175]

[0176] The synthesis method was the same as that in step (3) of Example 1, which describes the synthesis of 1-(2,3,6,7-tetrahydro-1H,5H-pyridyl[3,2,1-ij]quinoline-9-yl)-3-(4-methoxyphenyl)-4-nitro-butane-1-one. Using 1-(2,3,6,7-tetrahydro-1H,5H-pyridyl[3,2,1-ij]quinoline-9-yl)-3-(4-ethoxyphenyl)-prop-2-en-1-one (972 mg, 2.8 mmol) as the starting material, 960 mg of the product was obtained as a yellow solid with a yield of 84%.

[0177] The 1H and 1C NMR spectra of the product obtained in this step are as follows:

[0178] 1HNMR (600MHz, CDCl3) δ7.38 (s, 2H), 7.18 (d, J = 8.6Hz, 2H), 6.83 (t, J = 5.8Hz, 2H), 4.81 (dd, J = 12.4, 6.0Hz, 1H), 4.61 (dd, J = 12.3, 8.7 Hz,1H),4.17–4.06(m,1H),3.99(q,J=7.0Hz,2H),3.30–3.15(m,6H),2.73(t,J=6.3Hz,4H),2.00–1.89(m,4H),1.39(t,J=7.0Hz,3H);

[0179] 13CNMR(151MHz,CDCl3)δ194.72,158.44,147.19,131.70,128.63,127.96,123 .32,120.02,114.97,80.18,63.55,50.08,40.87,39.26,27.89,21.51,14.98.

[0180] (3) Synthesis of compound 9

[0181]

[0182] The synthesis method was the same as that of compound 1 in step (4) of Example 1. Using 1-(2,3,6,7-tetrahydro-1H,5H-pyridyl[3,2,1-ij]quinolin-9-yl)-3-(4-ethoxyphenyl)-4-nitro-butane-1-one (612 mg, 1.5 mmol) as the starting material, 163 mg of product was obtained as a black solid, and the yield of compound 9 was 28%.

[0183] In addition, since compound 9 has poor solubility in common deuterated reagents such as CDCl3, CD2Cl2, DMSO-d6 (deuterated dimethyl sulfoxide) and Pyridine-d5 (deuterated pyridine), its proton and carbon spectra could not be obtained.

[0184] Example 10

[0185] This embodiment provides a method for preparing compound 10, which consists of three steps, as follows:

[0186] (1) Synthesis of 1-(2,3,6,7-tetrahydro-1H,5H-pyridyl[3,2,1-ij]quinolin-9-yl)-3-(4-propoxyphenyl)-prop-2-en-1-one

[0187]

[0188] The synthesis method was the same as that in step (2) of Example 1, which describes the synthesis of 1-(2,3,6,7-tetrahydro-1H,5H-pyridyl[3,2,1-ij]quinolin-9-yl)-3-(4-methoxyphenyl)-prop-2-en-1-one. Using 9-acetyljulonidine (989 mg, 4.6 mmol) and 4-propoxybenzaldehyde (836 mg, 5.1 mmol) as starting materials, 1240 mg of the product was obtained as a yellow solid, yielding 75%.

[0189] (2) Synthesis of 1-(2,3,6,7-tetrahydro-1H,5H-pyridyl[3,2,1-ij]quinolin-9-yl)-3-(4-propoxyphenyl)-4-nitro-butane-1-one

[0190]

[0191] The synthesis method was the same as that in step (3) of Example 1, which describes the synthesis of 1-(2,3,6,7-tetrahydro-1H,5H-pyridyl[3,2,1-ij]quinoline-9-yl)-3-(4-methoxyphenyl)-4-nitro-butane-1-one. Using 1-(2,3,6,7-tetrahydro-1H,5H-pyridyl[3,2,1-ij]quinoline-9-yl)-3-(4-propoxyphenyl)-prop-2-en-1-one (1011 mg, 2.8 mmol) as the starting material, 870 mg of the product was obtained as a yellow solid, with a yield of 74%.

[0192] (3) Synthesis of compound 10

[0193]

[0194] The synthesis method was the same as that of compound 1 in step (4) of Example 1. Using 1-(2,3,6,7-tetrahydro-1H,5H-pyridyl[3,2,1-ij]quinolin-9-yl)-3-(4-propoxyphenyl)-4-nitro-butane-1-one (633 mg, 1.5 mmol) as the starting material, 120 mg of product was obtained as a black solid, yielding compound 10 with a yield of 20%.

[0195] The proton and carbon NMR spectra of compound 10 are as follows:

[0196] 1HNMR(600MHz,Pyridine-d5)δ8.39(d,J=8.3Hz,4H),8.21(s,4H),7.52(s,2H),7.21(d,J=8.4Hz,4H),3.8 7(t,J=6.4Hz,4H),3.00(t,J=5.4Hz,8H),2.76(t,J=6.0Hz,8H),1.76–1.66(m,12H),0.92(t,J=7.4Hz,6H);

[0197] 13CNMR(151MHz,Pyridine-d5)δ160.73,156.15,146.15,145.77,140.25,131.45,130. 55,127.01,121.74,119.30,117.89,115.51,70.11,50.44,28.50,23.31,22.20,11.08.

[0198] Example 11

[0199] This embodiment provides a method for preparing compound 11, which consists of three steps, as follows:

[0200] (1) Synthesis of 1-(2,3,6,7-tetrahydro-1H,5H-pyridyl[3,2,1-ij]quinolin-9-yl)-3-(4-isopropoxyphenyl)-prop-2-en-1-one

[0201]

[0202] The synthesis method was the same as that in step (2) of Example 1, which describes the synthesis of 1-(2,3,6,7-tetrahydro-1H,5H-pyridyl[3,2,1-ij]quinolin-9-yl)-3-(4-methoxyphenyl)-prop-2-en-1-one. Using 9-acetyljulonidine (989 mg, 4.6 mmol) and 4-isopropoxybenzaldehyde (836 mg, 5.1 mmol) as starting materials, 1210 mg of the product was obtained as a yellow solid, yielding 73%.

[0203] (2) Synthesis of 1-(2,3,6,7-tetrahydro-1H,5H-pyridyl[3,2,1-ij]quinolin-9-yl)-3-(4-isopropoxyphenyl)-4-nitro-butane-1-one

[0204]

[0205] The synthesis method was the same as that in step (3) of Example 1, which describes the synthesis of 1-(2,3,6,7-tetrahydro-1H,5H-pyridyl[3,2,1-ij]quinoline-9-yl)-3-(4-methoxyphenyl)-4-nitro-butane-1-one. Using 1-(2,3,6,7-tetrahydro-1H,5H-pyridyl[3,2,1-ij]quinoline-9-yl)-3-(4-isopropoxyphenyl)-prop-2-en-1-one (1011 mg, 2.8 mmol) as the starting material, 920 mg of the product was obtained as a yellow solid, with a yield of 78%.

[0206] (3) Synthesis of compound 11

[0207]

[0208] The synthesis method was the same as that of compound 1 in step (4) of Example 1. Using 1-(2,3,6,7-tetrahydro-1H,5H-pyridyl[3,2,1-ij]quinolin-9-yl)-3-(4-isopropoxyphenyl)-4-nitro-butane-1-one (633 mg, 1.5 mmol) as the starting material, 162 mg of product was obtained as a black solid, yielding compound 11 with a yield of 27%.

[0209] The proton and carbon NMR spectra of compound 11 are as follows:

[0210] 1HNMR(600MHz,Pyridine-d5)δ8.41(d,J=8.1Hz,4H),8.24(s,4H),7.56(s,2H),7.23(d,J=8.8Hz,4H),4.62(d t,J=12.0,6.1Hz,2H),3.03(t,J=5.4Hz,8H),2.79(t,J=6.1Hz,8H),1.82–1.72(m,8H),1.29(d,J=5.9Hz,12H);

[0211] 13CNMR(151MHz,Pyridine-d5)δ158.20,154.84,144.82,144.45,138.95,130.23,1 29.21,125.52,120.41,117.98,116.52,115.19,68.99,49.11,27.17,21.15,20.87.

[0212] Example 12

[0213] This embodiment provides a method for preparing compound 12, which consists of three steps, as follows:

[0214] (1) Synthesis of 1-(2,3,6,7-tetrahydro-1H,5H-pyridyl[3,2,1-ij]quinolin-9-yl)-3-(4-pentoxyphenyl)-prop-2-en-1-one

[0215]

[0216] The synthesis method was the same as that in step (2) of Example 1, which was the synthesis of 1-(2,3,6,7-tetrahydro-1H,5H-pyridyl[3,2,1-ij]quinolin-9-yl)-3-(4-methoxyphenyl)-prop-2-en-1-one. Using 9-acetyljulonidine (989 mg, 4.6 mmol) and 4-pentoxybenzaldehyde (979 mg, 5.1 mmol) as raw materials, 1200 mg of the product was obtained as a yellow solid, with a yield of 67%.

[0217] (2) Synthesis of 1-(2,3,6,7-tetrahydro-1H,5H-pyridyl[3,2,1-ij]quinolin-9-yl)-3-(4-pentoxyphenyl)-4-nitro-butane-1-one

[0218]

[0219] The synthesis method was the same as that in step (3) of Example 1, which was the synthesis of 1-(2,3,6,7-tetrahydro-1H,5H-pyridyl[3,2,1-ij]quinoline-9-yl)-3-(4-methoxyphenyl)-4-nitro-butane-1-one. Using the synthesis of 1-(2,3,6,7-tetrahydro-1H,5H-pyridyl[3,2,1-ij]quinoline-9-yl)-3-(4-pentoxyphenyl)-prop-2-en-1-one (1089 mg, 2.8 mmol) as the starting material, 810 mg of the product was obtained as a yellow solid with a yield of 64%.

[0220] (3) Synthesis of compound 12

[0221]

[0222] The synthesis method was the same as that of compound 1 in step (4) of Example 1. Using 1-(2,3,6,7-tetrahydro-1H,5H-pyridyl[3,2,1-ij]quinolin-9-yl)-3-(4-pentoxyphenyl)-4-nitro-butane-1-one (675 mg, 1.5 mmol) as the starting material, 137 mg of product was obtained as a black solid, yielding compound 12 with a yield of 21%.

[0223] The proton and carbon NMR spectra of compound 12 are as follows:

[0224] 1HNMR (600MHz, Pyridine-d5) δ8.44(d,J=8.0Hz,4H),8.25(s,4H),7.57(s,2H),7.28(d,J=8.1Hz,4H),3.99(t,J=6.4Hz,4H),3.04(s ,8H),2.80(s,8H),1.75(dt,J=14.4,6.1Hz,12H),1.38(dt,J=12.6,6.3Hz,4H),1.28(dd,J=14.2,7.0Hz,4H),0.88(t,J=7.2Hz,6H);

[0225] 13CNMR(151MHz,Pyridine-d5)δ160.78,156.16,146.16,145.78,140.26,131.48,130.55,127 .02,121.75,119.31,117.89,115.53,68.67,50.44,29.68,28.86,28.50,23.13,22.20,14.61.

[0226] Example 13

[0227] This embodiment provides a method for preparing compound 13, which consists of three steps, as follows:

[0228] (1) Synthesis of 1-(2,3,6,7-tetrahydro-1H,5H-pyridyl[3,2,1-ij]quinolin-9-yl)-3-(4-tert-butoxyphenyl)-prop-2-en-1-one

[0229]

[0230] The synthesis method was the same as that in step (2) of Example 1, which describes the synthesis of 1-(2,3,6,7-tetrahydro-1H,5H-pyridyl[3,2,1-ij]quinolin-9-yl)-3-(4-methoxyphenyl)-prop-2-en-1-one. Using 9-acetyljulonidine (989 mg, 4.6 mmol) and 4-tert-butoxybenzaldehyde (908 mg, 5.1 mmol) as starting materials, 1020 mg of the product was obtained as a yellow solid, yielding a 59% yield.

[0231] (2) Synthesis of 1-(2,3,6,7-tetrahydro-1H,5H-pyridyl[3,2,1-ij]quinolin-9-yl)-3-(4-tert-butoxyphenyl)-4-nitro-butane-1-one

[0232]

[0233] The synthesis method was the same as that in step (3) of Example 1, which describes the synthesis of 1-(2,3,6,7-tetrahydro-1H,5H-pyridyl[3,2,1-ij]quinoline-9-yl)-3-(4-methoxyphenyl)-4-nitro-butane-1-one. Using 1-(2,3,6,7-tetrahydro-1H,5H-pyridyl[3,2,1-ij]quinoline-9-yl)-3-(4-tert-butoxyphenyl)-prop-2-en-1-one (1050 mg, 2.8 mmol) as the starting material, 720 mg of the product was obtained as a yellow solid, with a yield of 59%.

[0234] (3) Synthesis of compound 13

[0235]

[0236] The synthesis method was the same as that of compound 1 in step (4) of Example 1. Using 1-(2,3,6,7-tetrahydro-1H,5H-pyridyl[3,2,1-ij]quinolin-9-yl)-3-(4-tert-butoxyphenyl)-4-nitro-butane-1-one (654 mg, 1.5 mmol) as the starting material, 137 mg of product was obtained as a black solid, yielding compound 13 with a yield of 22%.

[0237] The proton and carbon NMR spectra of compound 13 are as follows:

[0238] 1HNMR(600MHz,Pyridine-d5)δ8.37(d,J=7.8Hz,4H),8.24(s,4H),7.58–7.56(m,2H),7.34 (d,J=7.8Hz,4H),3.04(s,8H),2.79(t,J=5.5Hz,8H),1.77(d,J=5.2Hz,8H),1.40(s,18H);

[0239] 13CNMR(151MHz,Pyridine-d5)δ157.34,156.14,146.25,145.84,140.03,130.9 1,130.60,129.25,121.80,119.20,118.41,79.33,50.45,29.34,28.48,22.17.

[0240] Example 14

[0241] This embodiment provides a method for preparing compound 14, which consists of three steps, as follows:

[0242] (1) Synthesis of 1-(2,3,6,7-tetrahydro-1H,5H-pyridyl[3,2,1-ij]quinolin-9-yl)-3-(4-(2-methoxyethoxy)phenyl)-prop-2-en-1-one

[0243]

[0244] The synthesis method was the same as that in step (2) of Example 1, which describes the synthesis of 1-(2,3,6,7-tetrahydro-1H,5H-pyridyl[3,2,1-ij]quinolin-9-yl)-3-(4-methoxyphenyl)-prop-2-en-1-one. Using 9-acetyljulonidine (989 mg, 4.6 mmol) and 4-(2-methoxyethoxy)benzaldehyde (918 mg, 5.11 mmol) as starting materials, 1320 mg of the product was obtained as a yellow solid, with a yield of 76%.

[0245] (2) Synthesis of 1-(2,3,6,7-tetrahydro-1H,5H-pyridyl[3,2,1-ij]quinolin-9-yl)-3-(4-(2-methoxyethoxy)phenyl)-4-nitro-butane-1-one

[0246]

[0247] The synthesis method was the same as that in step (3) of Example 1, which describes the synthesis of 1-(2,3,6,7-tetrahydro-1H,5H-pyridyl[3,2,1-ij]quinoline-9-yl)-3-(4-methoxyphenyl)-4-nitro-butane-1-one. Using 1-(2,3,6,7-tetrahydro-1H,5H-pyridyl[3,2,1-ij]quinoline-9-yl)-3-(4-(2-methoxyethoxy)phenyl)-prop-2-en-1-one (1055 mg, 2.8 mmol) as the starting material, 1060 mg of the product was obtained as a yellow solid with a yield of 87%.

[0248] (3) Synthesis of compound 14

[0249]

[0250] The synthesis method was the same as that of compound 1 in step (4) of Example 1. Using 1-(2,3,6,7-tetrahydro-1H,5H-pyridyl[3,2,1-ij]quinolin-9-yl)-3-(4-(2-methoxyethoxy)phenyl)-4-nitro-butane-1-one (657 mg, 1.5 mmol) as the starting material, 210 mg of product was obtained, yielding compound 14, a black solid with a yield of 34%.

[0251] The proton and carbon NMR spectra of compound 14 are as follows:

[0252] 1HNMR(600MHz,Pyridine-d5)δ8.39(d,J=6.4Hz,4H),8.24(s,4H),7.54(s,2H),7.26(d,J=6.7Hz,4H),4. 21(s,4H),3.74(s,4H),3.36(d,J=3.7Hz,6H),3.04(s,8H),2.80(d,J=4.4Hz,8H),1.77(d,J=4.3Hz,8H);

[0253] 13CNMR(151MHz,Pyridine-d5)δ160.48,156.13,146.17,145.75,140.10,131.44,130. 55,127.24,121.75,119.28,117.95,115.55,71.66,68.20,59.20,50.44,28.49,22.19.

[0254] Example 15

[0255] This embodiment uses the commercially available near-infrared fluorescent dye IR-26 as a reference to test the optical parameters of 14 compound dyes (hereinafter referred to as analytes) provided in Examples 1-14 and NJ1060 dye. The tests include:

[0256] (1) Ultraviolet absorption spectrum test of dyes

[0257] Prepare 0.5 mM solutions of the 14 analytes to be tested. The molar extinction coefficient can be obtained using the following formula: A = εbc. A represents the absorption intensity, ε is the molar extinction coefficient, c is the concentration of the compound, and b is the width of the quartz cell used for detection (usually 1 cm).

[0258] Specific steps: Weigh out dye M×5×10 respectively. -3 mg was transferred to a 10 mL volumetric flask and brought to a final volume as sample A (0.5 mM). 80 μL of this sample was then taken out and brought to a final volume of 4 mL with the same solvent. The sample was then tested using a double-sided transparent quartz cell. The maximum absorption wavelength and molar extinction coefficient were ultimately obtained.

[0259] (2) Fluorescence spectroscopy test of dyes

[0260] The fluorescence quantum yield is obtained using the following formula: Yu = YsFu / FsAs / Aunu 2 / ns 2Yu, Ys: Fluorescence quantum yields of the analyte and the reference standard; Fu, Fs: Integrated fluorescence intensities of the analyte and the reference standard; Au, As: Absorbance of the analyte and the reference standard at the incident light at this excitation wavelength (using the commercially available near-infrared fluorescent dye IR-26 as a reference); nu, ns: Refractive indices of the solvents of the analyte and the reference standard. The absorbance at the excitation wavelength (808 nm) was adjusted to between 0.04 and 0.07 for testing. The final fluorescence emission wavelength and fluorescence quantum yield were obtained.

[0261] The spectra of the 14 compound dyes tested were in Figure 1 and 2 The maximum absorption wavelength (λ) in chloroform (CHCl3) and dimethyl sulfoxide (DMSO) is shown in the figure. ab ), maximum fluorescence emission wavelength (λ) em The data for molar extinction coefficient (ε) and fluorescence quantum yield (Φ) are listed in Table 1.

[0262] Table 1

[0263]

[0264]

[0265] a Tested in CHCl3 / DMSO; b Tested in CHCl3; c The test in CHCl3 used IR-26 (Φ = 0.05%, 1,2-dichloroethane (DCE)) as a reference.

[0266] The data in Table 1 show that the 14 analytes exhibit strong absorption in the wavelength range of 700–900 nm. Figure 1 It has a high molar extinction coefficient (60000–83000 M). -1 cm -1 The maximum emission wavelengths are between 880-1030 nm, and the fluorescence quantum yields are generally in the range of 1.50%–5.23%. Compared with the previously reported 1,7-position julonidine-modified Aza-BODIPY (NJ1060), the molar extinction coefficients and fluorescence quantum yields of the 14 analytes are significantly improved, and the brightness is mostly 4–8 times that of NJ1060. Among them, compounds 5 and 6, which have the lowest quantum yields, still have an intensity 2–3 times that of NJ1060. Using a commercially available 808 nm laser as the best-matched light source, the absorption intensity of compound 1 and NJ1060 at 808 nm and the fluorescence intensity under the same excitation power were compared at the same concentration. Figure 3As shown, compound 1 at the same concentration exhibits stronger absorption at 808 nm than NJ1060, and compound 1 also shows significantly stronger NIR-II fluorescence than NJ1060 in both CHCl3 and DMSO. These studies indicate that the 14 analytes have better potential for fluorescence imaging applications than Aza-BODIPY modified with julodin at positions 1,7.

[0267] Example 16

[0268] This embodiment tests the ACQ (aggregation-induced quenching) effect of compound 1.

[0269] The experimental method was as follows: 14 samples of 20 μL, 5 mM of compound 1 dye were added to 4 mL of water-DMSO mixed solutions with water contents of 0%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 80%, 90%, 95%, and 100%. After vortexing for 5 s, the fluorescence emission spectrum was immediately scanned, and the fluorescence intensity at the maximum emission wavelength was measured.

[0270] like Figure 4 As shown, when the water content in the system reaches 10%, the fluorescence intensity of compound 1 decreases rapidly due to aggregation. When the water content reaches 40%, the fluorescence of compound 1 is almost completely quenched. This indicates that compound 1 exhibits a significant ACQ effect in the water / DMSO system, and its fluorescence intensity is extremely sensitive to water in the environment.

[0271] Examples 17-31

[0272] Examples 17-31 determined and compared the ACQ effect of compounds 2-14, 1,7-julonidine-modified Aza-BODIPY (NJ1060), and a typical NIR-I region ACQ probe (P2). The experimental methods were the same as in Example 16, except that compound 1 in Example 16 was replaced with compounds 2-14, NJ1060, and P2.

[0273] Figure 5 The graphs show the fluorescence changes of each dye in response to water content in the system for compounds 2-14. As the water content gradually increases, the fluorescence intensity of P2 decreases slowly; however, when the water content reaches 40%, its fluorescence decays rapidly. Similar to compound 1, the fluorescence of compounds 2-14 decays rapidly when the water content increases to 10% to 20%. This indicates that the ACQ effect of compounds 1-14 is stronger than that of P2. Although the fluorescence intensity of NJ1060 also decreases rapidly when the water content reaches 10% to 20%, NJ1060 retains some fluorescence as the water content continues to increase until it reaches 90%, at which point the fluorescence of NJ1060 is almost completely quenched.

[0274] Figure 6 The minimum water content required for complete fluorescence quenching of 16 dyes, including compounds 1-14, NJ1060, and P2, in Examples 16-31 was summarized. P2 and NJ1060 require water contents of 60% and 90%, respectively, for complete fluorescence quenching. The minimum water content required for complete fluorescence quenching of compounds 1-14 generally ranges from 40% to 55%, with only compound 6 reaching a minimum water content of 60%. The water quenching spectra of each dye are summarized... Figure 5 Therefore, it can be considered that the ACQ effect of the compound provided by the present invention is significantly stronger than that of P2 and NJ1060.

[0275] Example 32

[0276] This example tests and compares the fluorescence quenching stability of 15 dye compounds (compounds 1-14 and P2) in plasma. The experimental method is as follows: 1.34 mM DMSO solutions of each dye are mixed with rat plasma at a volume ratio of 1:100 and incubated at 37°C with a shaking speed of 120 rpm. Fluorescence intensity is measured after 24 hours, and quenching stability is calculated with the fluorescence of the DMSO solution at the same concentration of each dye as 100%.

[0277] like Figure 7 As shown, since P2 molecules can translocate into the hydrophobic waters of plasma proteins, the fluorescence retention rate of P2 in plasma is approximately 20%, which undoubtedly greatly reduces the accuracy of in vivo drug carrier tracing. In contrast, the fluorescence retention rates of compounds 1-14 provided in this invention are significantly lower than that of P2, all less than 8%, with compounds 1, 4, 7, 10, 11, and 12 all having fluorescence retention rates below 2%.

[0278] Example 33

[0279] This embodiment tests and compares the fluorescence reproducibility of water-quenched solutions of 15 dye compounds (compounds 1-14 and P2) in plasma. The specific procedure is as follows: 13.4 mM DMSO solutions of each dye were mixed with pure water at a volume ratio of 1:100 and vortexed to quench the fluorescence. The water-quenched solutions were then mixed with rat plasma at a volume ratio of 1:10 and incubated at 37°C with a shaking speed of 120 rpm. After 24 hours, samples were taken to measure the fluorescence intensity. The fluorescence reproducibility percentage was calculated with the fluorescence of the DMSO solution of each dye at the same concentration as 100%.

[0280] like Figure 8As shown, compared to the P2 water quenching solution, the water quenching solutions of compounds 1-14 exhibited excellent fluorescence reproducibility in plasma. After 24 hours of incubation, the fluorescence reproducibility of P2 was approximately 4.5%; while the fluorescence reproducibility of compounds 1-14 was all below 1.5%, with compounds 1, 4, 9, 10, 11, and 13 showing fluorescence reproducibility below 0.2%.

[0281] Example 34

[0282] In this embodiment, representative compounds 1, 4, 10, and 11 were selected to label two drug carriers: biodegradable polyethylene glycol-racemic polylactic acid (mPEG-PDLLA) polymer micelles (PM) and polycaprolactone (PCL) nanoparticles (PN), and the nanotracers were characterized. The experimental methods are as follows:

[0283] (1) Preparation of PM and PN nanotracers

[0284] Dye-labeled mPEG was prepared by thin-film dispersion method. 2k -PDLLA 2k Polymer micelles were prepared by mixing 0.535 μmol of each of the above compounds with 160 mg of mPEG. 2k -PDLLA 2k Dissolve in 4 mL of dichloromethane, and remove the solvent by rotary evaporation at 60 °C and 85 rpm. Add 5 mL of acetonitrile, redissolve under sonication in a water bath, and then remove the acetonitrile by rotary evaporation to obtain a uniform film. Add 4 mL of phosphate-buffered saline (PBS) preheated to 60 °C, and stir at 60 °C (500 rpm) for 30 min. Filter the aqueous solution through a 0.22 μm syringe filter to obtain PM.

[0285] Dye-labeled PCL PN was prepared by emulsification solvent evaporation method. 0.268 μmol of each of the above dye compounds and 68 mg of PCL were respectively used. 45k The organic phase was mixed with the probe and dissolved in 1 mL of dichloromethane. The organic phase was then slowly added to 4 mL of 1% PVA solution. The probe was sonicated (350 W, 3 min) under ice bath conditions to form a primary emulsion. The dichloromethane was removed by magnetic stirring (200 rpm, 6 h) at room temperature. The emulsion was then filtered through a 0.45 μm syringe filter to obtain PCL nanoparticles.

[0286] The formulation labeled with P2 dye was prepared according to the same procedure, keeping its molar concentration the same as that of the aforementioned compound dye.

[0287] (2) Characterization and testing of PM and PN nanotracers

[0288] The particle size and zeta potential of the prepared PM and PN nanotracers were measured at room temperature. All measurements were performed after diluting the formulations to 1-5 mg / mL (carrier material concentration) with deionized water. Encapsulation efficiency was determined by ultrafiltration.

[0289] Prepare a 16 μM probe DMSO stock solution, dilute it with DMSO to different concentrations, and measure the fluorescence intensity using an Edinburgh FS-5 (three copies) to obtain a standard curve of fluorescence intensity versus probe concentration.

[0290] The aforementioned diluted PM and PN nanotracer were placed in an ultrafiltration tube ( The filtrate was obtained by centrifugation (6000 rpm) for 20 min in an ultra-0.5 (100 kDa) solution. After lyophilizing the filtrate, it was dissolved in 0.5 mL of dimethyl sulfoxide (DMSO). The fluorescence intensity was measured, and the concentration of the encapsulated probe, c, was calculated by substituting the values ​​into the standard curve. The theoretical concentration is c0. Therefore, the encapsulation efficiency EE% = c / c0 × 100%.

[0291] The particle size, PDI, Zeta potential and encapsulation efficiency of the aforementioned PM nanotracers labeled with different compounds are listed in Table 2; the particle size, PDI, Zeta potential and encapsulation efficiency of the aforementioned PN nanotracers labeled with different compounds are listed in Table 3.

[0292] Table 2

[0293]

[0294] Table 3

[0295]

[0296]

[0297] The PM nanotracers labeled with the above compounds have a particle size of 20-23 nm and a zeta potential within ±7 mV; the PN nanotracers labeled with the above compounds have a particle size of 202-234 nm and a zeta potential within ±3 mV. Both the PM and PN nanotracers labeled with the above compounds are monodisperse and have an encapsulation efficiency higher than 90%. These results indicate that the 14 compound dyes provided by this invention can be effectively encapsulated into various drug carriers for in vivo tracking assays.

[0298] Example 35

[0299] This embodiment tests and compares the penetration depth of the PM nanotracer signals loaded with compounds 1 and P2 prepared in Example 34. The experimental method is as follows: capillaries containing PM nanotracer solutions loaded with compounds 1 and P2 are immersed in 1% Intralipid fat emulsions at different depths, and the fluorescence signals are then detected using an imaging system. The NIR-II region signal of compound 1 is measured using an InGaAs camera (Princeton Instruments), with an 808nm external laser as the excitation source and a power density set to 50mW / cm². 2 The exposure time was adjusted to obtain a sufficient signal value. The NIR-I region signal of P2 was measured using an IVISspectrum small animal in vivo imaging system (PerkinElmer), with the excitation wavelength set to 710 nm and the emission wavelength to 760 nm. The full width at half maximum (FWHM) of each dye signal was calculated, and the signal sharpness was measured as the ratio of FWHM to the true capillary width.

[0300] like Figure 9 As shown, the NIR-II fluorescence signal of compound 1 penetrates deeper than the near-infrared I signal of P2. The near-infrared I signal of P2 shows increasingly enhanced scattering with increasing fat emulsion thickness. When the fat emulsion thickness increases to 3 mm, the P2 signal gradually becomes blurred, and its full width at half maximum (FWHM) increases rapidly. However, when collecting the NIR-II fluorescence of compound 1 at depths beyond 1200 nm and 1300 nm, its FWHM remains constant even with a fat emulsion coverage depth of 7 mm, clearly revealing the capillary contours.

[0301] Example 36

[0302] This embodiment tests and compares the signal-to-noise ratio of PM nano-tracers loaded with compounds 1 and P2 prepared in Example 34 on nude mice. The experimental method was as follows: Male nude mice weighing between 20 ± 2 g were selected and randomly divided into two groups of three mice each. The first group of mice received a tail vein injection of 200 μL of PM nano-tracers loaded with compound 1 (probe concentration 0.134 mM for both groups), while the second group received a tail vein injection of 200 μL of PM nano-tracers loaded with P2 (probe concentration 0.134 mM for both groups). Imaging systems were then used to detect fluorescence signals. For the first group: An InGaAs camera (Princeton Instruments) was used to measure the NIR-II region signal of compound 1. The signal acquisition parameters were: an 808 nm external laser as the excitation source, a power density of 60 mW / cm², an exposure time of 500 ms, and a 1300 nm long-pass filter (OD>5) to assist signal acquisition. Group 2: The NIR-I region signal of P2 was measured using an IVISspectrum small animal in vivo imaging system (PerkinElmer). The excitation wavelength was set to 710 nm, the emission wavelength to 760 nm, and the exposure time to automatic mode. The fluorescence images were processed using ImageJ software, and fluorescence change curves near the blood vessels were plotted, and the signal-to-noise ratio (SNR) and full width at half maximum (FWHM) were calculated.

[0303] Figure 10 As shown, compound 1 clearly displays the outlines of blood vessels and the liver in live nude mice, while the P2 signal in live nude mice is extremely unclear due to scattering, failing to delineate the outlines of blood vessels and the liver. Calculations of the signal-to-noise ratio (SNR) and half-width at half-maximum (HWHM) of the blood vessels show that the SNR of blood vessels visualized by compound 1 is approximately 2.0, while that of P2 is only 1.1. Furthermore, the HWHM of blood vessels visualized by compound 1 is significantly smaller than that visualized by P2, and its HWHM is closer to the true radius of the blood vessels.

[0304] Example 37

[0305] This embodiment tests and compares the distribution of PM nanotracers containing compounds 1 and P2 prepared in Example 34 in animals and the maximum fluorescence reproduction of compound 1 at the animal level.

[0306] The experimental method was as follows: Male nude mice weighing between 20 and 2 g were randomly divided into four groups of three mice each. Group 1 mice were injected intravenously with 200 μL of PM nano-tracer containing compound 1; Group 2 mice were injected intravenously with 200 μL of pre-quenching solution containing compound 1; Group 3 mice were injected intravenously with 200 μL of PM nano-tracer containing P2; and Group 4 mice were injected intravenously with pre-quenching solution containing P2. The concentration of the dye compound in each group was 0.134 mM. The pre-quenching solutions for compound 1 and P2 were prepared as follows: 10 μL of a 13.4 mM DMSO solution containing the dye (compound 1 or P2) was added to 1 mL of PBS (pH = 7.4), and the mixture was vortexed to obtain the pre-quenching solutions for compound 1 and P2, respectively. The fluorescence signals of the compound dyes in each group were detected at 5 min, 15 min, 0.5 h, 1 h, 2 h, 4 h, 8 h, 12 h and 24 h after drug administration.

[0307] Meanwhile, nude mice in each group were sacrificed 24 hours after drug administration. After perfusion of the heart with 0.9% saline, organs such as brain, heart, liver, spleen, lung, kidney, muscle, skin, fat, and testis were harvested, and the fluorescence signals of each dye were detected.

[0308] For the first and second groups containing compound 1, the NIR-II fluorescence signals of the compound 1 nanotracer and the corresponding dye pre-quenching solution were detected in living and ex vivo tissues using an InGaAs camera (Princeton Instruments). The signal acquisition parameters were: an external 808nm excitation source with a power density of 60mW / cm². 2 The exposure time was 500 ms, and a 1300 nm long-pass filter (OD>5) was used to assist signal acquisition. ImageJ software was used to measure the average fluorescence intensity of the abdomens of nude mice in the first and second groups, and the in vivo reproducibility percentage was calculated. ImageJ software was also used to measure the average fluorescence intensity of each organ in the ex vivo organ images of nude mice in the first and second groups, and the reproducibility level of the pre-quenched solution of compound 1 in each organ in the second group was calculated (compared to the PM nano-tracer encapsulating compound 1 in the first group).

[0309] For groups three and four containing P2, the NIR-I region signal was measured using the IVISspectrum small animal in vivo imaging system (PerkinElmer), with the excitation wavelength set to 710 nm, the emission wavelength set to 760 nm, and the exposure time set to 0.1 s. The average fluorescence intensity in the abdomen of nude mice in groups three and four and the average fluorescence intensity in each isolated organ were measured using the system's built-in software, and the percentage of fluorescence reproduction in each tissue, both in vivo and in isolated, was calculated.

[0310] like Figure 11As shown, in live tissue, within 2 hours of intravenous administration, the fluorescence signal of the first group (PM nanotracers encapsulating compound 1) was mainly distributed in the blood vessels of mice. Subsequently, the signal of compound 1 in the blood vessels gradually weakened, while the signal in the liver and other tissues gradually increased. Among them, the signal in the liver was the highest and continued to increase until 8 hours, with a clear outline. This is mainly due to the recognition and uptake of PM by the reticuloendothelial system (RES). In contrast, after the corresponding dye prequenching solution entered the mice, the signal was almost unobservable, and only a very weak fluorescence signal could be found in the liver after adjusting the signal scale. Similar to the first group, the signal of the third group (PM nanotracers encapsulating P2) was rapidly distributed throughout the body and gradually accumulated in the abdomen where the liver is located; in the fourth group, the signal of the P2 prequenching solution gradually appeared in the abdomen 8 hours after entering the nude mice. Unlike the second group (compound 1 prequenching solution), the P2 prequenching solution showed a stronger in vivo signal and gradually appeared in sites outside the liver. This is mainly because the fluorescence-quenched P2 dye is easily redissolved by hydrophobic regions in the liver and other sites, resulting in obvious fluorescence reappearance.

[0311] Quantitative fluorescence analysis at the site of maximum fluorescence reproducibility (abdomen) showed that, compared to the third group (PM nano-tracers loaded with P2), the fourth group (P2 pre-quenching solution) achieved fluorescence of over 10% in the mouse abdomen; while the second group (compound 1 pre-quenching solution) showed fluorescence of only about 2% of the first group (PM nano-tracers loaded with compound 1) in the mouse abdomen. It can be concluded that the percentage of maximum in vivo fluorescence reproducibility produced by compound 1 is significantly lower than that of the P2 dye.

[0312] Twenty-four hours after drug administration, in ex vivo tissues, the fluorescence signal intensity accumulation order of the first group (PM nano-tracers encapsulating compound 1) in the tested organs / tissues was liver > lung > adipose tissue ≈ kidney > heart, consistent with the results of in vivo tissue imaging. The signal of the second group (pre-quenched solution of compound 1) was extremely weak in all organs and tissues, almost unobservable; after adjusting the signal scale, only an extremely weak fluorescence signal could be found in the liver. The signal of the fourth group (pre-quenched solution of P2) was more obvious in the liver and lungs.

[0313] Quantitative fluorescence analysis at the site of maximum fluorescence reproducibility (abdomen) showed that, compared to the first group (PM nano-tracers encapsulating compound 1), the fluorescence reproducibility percentage of the second group (pre-quenched solution of compound 1) in all organs was below 3%; while the fluorescence reproducibility percentage of the third group (PM nano-tracers encapsulating P2) in all organs was significantly higher than that of the first group (PM nano-tracers encapsulating compound 1), with reproducibility percentages in the liver and lungs reaching over 12% and 8%, respectively. This further confirms that the maximum in vivo fluorescence reproducibility produced by compound 1 is significantly lower than that of P2.

[0314] Compounds 1-14, represented by compound 1, have significant advantages in accurately tracing drug carriers at both in vitro and in vivo levels.

[0315] In summary, the 3,5-position juulonidine-modified Aza-BODIPY dyes provided by this invention exhibit excellent near-infrared II imaging capabilities, ACQ effect, and in vivo anti-reproducibility. Compared to the previously reported 1,7-position juulonidine-modified Aza-BODIPY dye (NJ1060), it has a higher quantum yield and a more pronounced ACQ effect. Furthermore, compared to earlier NIR-I region ACQ probes (P2), the near-infrared fluorescent compound dyes provided by this invention have higher signal penetration depth, stronger ACQ effect, and weaker in vivo reproducibility. Therefore, when used for in vivo tracking of drug carriers, they offer higher sensitivity and accuracy.

[0316] Furthermore, the experiment revealed that the near-infrared fluorescent compound dye provided by this invention did not exhibit significant photoquenching or photobleaching phenomena under normal operating conditions and in an environment without light protection, indicating that its chemical and optical properties are highly stable.

[0317] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A near-infrared fluorescent dye, characterized in that: The fluorescent dye is a compound having the general formula (I): Among them, general formula (I) is an Aza-BODIPY structure modified by jurodin at positions 3 and 5; Ar1 and Ar2 are independently selected from: benzene ring, thiophene ring, furan ring, pyrrole ring or pyridine ring.

2. A near-infrared fluorescent dye, characterized in that: The near-infrared fluorescent dye is either of the following two structural formulas: R1 and R2 are substituents on the benzene ring, with 1 to 5 substituents on each benzene ring; R3 and R4 are substituents on the thiophene ring, with 1-3 substituents on each thiophene ring; R1, R2, R3, and R4 are independently selected from the following groups: H, C1-C8 alkoxy groups, NR groups. a R b -COR c Halogen, C1-C8 alkyl, C3-C6 cycloalkyl, hydroxyl, amino, nitro, aldehyde, -CF3, -CN, carboxyl; Among them, R a R b R c The group is independently selected from the following groups: H, C1-C10 alkyl, C3-C10 cycloalkyl, C2-C10 alkenyl, C6-C20 aryl, and C5-C14 heteroaryl.

3. The near-infrared fluorescent dye as described in claim 2, characterized in that: R1 and R2 are independently H or C1-C6 alkoxy groups.

4. The near-infrared fluorescent dye as described in claim 3, characterized in that: The C1-C6 alkoxy groups are straight-chain or branched-chain alkoxy groups.

5. The near-infrared fluorescent dye as described in claim 4, characterized in that: The straight-chain alkoxy group is a straight-chain dialkoxy group.

6. The near-infrared fluorescent dye as described in claim 2, characterized in that: In the structural formula (II), R1 and / or R2 are NR a R b hour, R a and R b And onto the benzene ring, or, R a and R b Form a ring; In the structural formula (Ⅲ), R3 and / or R4 are NR a R b hour, R a and R b And onto the thiophene ring, or, R a and R b Forming a ring.

7. The near-infrared fluorescent dye of the above, characterized in that: The near-infrared fluorescent dye has the following structural formula: R5, R6, R7 and R8 are independently selected from C1-C4 alkyl groups.

8. The near-infrared fluorescent dye as described in claim 7, characterized in that: R5 and / or R6 are fused to the benzene ring, and / or R7 and / or R8 are fused to the benzene ring.

9. A near-infrared fluorescent dye, characterized in that, The near-infrared fluorescent dye is specifically one of the following compounds:

10. A fluorescent composite material, characterized in that: The near-infrared fluorescent dye according to any one of claims 1-9 is encapsulated inside a carrier, wherein the carrier has a hydrophobic matrix structure or a hydrophobic core-hydrophilic shell structure, and the near-infrared fluorescent dye is encapsulated in the hydrophobic core.

11. The fluorescent composite material as described in claim 10, characterized in that: The carrier is nanoscale, and the hydrophobic matrix structure is selected from at least one of the following: polymer nanoparticles / microspheres, solid lipid microparticles / nanoparticles, microemulsions, nanoemulsions, and nanocrystals. The hydrophobic core-hydrophilic shell structure is a nanopolymer micelle.

12. The fluorescent composite material as described in claim 10, characterized in that: Used in fluorescence imaging, diagnosis, and in vivo fluorescence tracing of drug carriers.

Images