Near-infrared small-molecule fluorescent dye with fifteen-methine conjugated chain and preparation method and application thereof

Abstract

The present application relates to a kind of near-infrared small molecule fluorescent dyes with fifteen methine conjugated chain and its preparation method and application, the near-infrared small molecule dye has the structure shown in general formula A, is based on two novel dye matrix of benzindole structure connected by fifteen methine chain, the functional dye molecule with absorption emission peak wavelength in near-infrared region obtained by substituent group on matrix structure regulation, different substituents can be introduced on fifteen methine chain and indole N atom.Its absorption and emission wavelength is in the near-infrared region of 1100-1300nm, with high brightness, excellent light stability, small cytotoxicity, simple synthesis and structure easy to modify and the like characteristics.After water-soluble modification, it can be widely used in tumor targeted imaging, in vivo blood vessel imaging and other biomedical fields.General formula A

Description

Technical Field

[0001] This invention relates to the field of biomaterials technology, particularly to the field of biomedical imaging detection, specifically to a near-infrared small molecule fluorescent dye with a decadecylamine conjugated chain, its preparation method, and its application. Background Technology

[0002] Fluorescence imaging, with its rapid response, high spatiotemporal resolution, and real-time imaging capabilities, has excellent applications in pathology and physiology, such as early diagnosis and treatment of diseases and the study of their mechanisms. Moreover, compared to other clinical imaging methods, such as positron emission tomography (PET), computed tomography (CT), and single-photon emission computed tomography (SPECT), fluorescence imaging has the advantage of not emitting harmful radiation. Organic small-molecule fluorescent probes, due to their low toxicity and rapid metabolism, exhibit good safety and biocompatibility among many fluorescent probes, such as carbon nanotubes, rare-earth nanoparticles, and quantum dots, and have applications in angiography, lymph node imaging, and targeted tumor imaging.

[0003] However, the main bottleneck of fluorescence imaging lies in its shallow tissue penetration depth and the low signal-to-noise ratio caused by the high absorption, scattering, and autofluorescence of biological tissues. In recent years, near-infrared II imaging (NIR-II, 1000-1700 nm), also known as short-wave infrared (SWIR), has shown great promise due to its ability to avoid the autofluorescence region of biological tissues and reduce light scattering. Compared to traditional visible light and near-infrared I imaging, it offers higher resolution and tissue penetration depth. Furthermore, the near-infrared II region is further divided into near-infrared IIa (1300-1400 nm) and near-infrared IIb (1500-1700 nm). Although the tissue absorption coefficient in the near-infrared II region is slightly higher than that in the near-infrared I region, the significant reduction in scattered light and autofluorescence results in higher resolution and penetration depth. While near-infrared II fluorescent probes offer advantages such as long wavelength and high contrast, the limited variety of conjugated chains in fluorescent probe molecules leads to lower molar absorptivity and fluorescence quantum yield, and limited modifiability on the conjugated chains.

[0004] In terms of fluorescent dye performance, the absorption and fluorescence wavelengths of cyanine dyes are significantly improved with the increase of polymethyl cyanide chains, thereby enhancing important performance parameters such as penetration depth and signal-to-background ratio in bioimaging. However, current methods for synthesizing long polymethyl cyanide chains are lacking.

[0005] However, in terms of synthesis, as the polymethyl cyanide chain lengthens, the synthesis of cyanine dyes becomes more difficult, and the stability of the dyes is also poorer.

[0006] Therefore, designing and synthesizing near-infrared II organic small molecule fluorescent probes with high molar absorptivity, high brightness, excellent photostability, low cytotoxicity, simple synthesis, and novel and easily modifiable structure is an important goal in the field of fluorescent probe synthesis. Summary of the Invention

[0007] The purpose of this invention is to address the aforementioned problems by providing a near-infrared small-molecule fluorescent dye with a 15-membered methine conjugated chain, its preparation method, and its applications, with a structure that is easily modified. The near-infrared small-molecule dye provided by this invention is based on a novel dye parent structure of benzoindole with two 15-membered methine chains. The absorption and emission peak wavelengths of the obtained functional dye molecule are controlled in the near-infrared region by substituents on the parent structure. Different substituents can be introduced onto the 15-membered methine chain and the N atom of the indole. This application improves important performance parameters of the fluorescent dye, such as penetration depth and signal-to-background ratio, by lengthening the chain length of the methine chain. Furthermore, this application simplifies the synthesis of the dye while ensuring its stability during the process of lengthening the methine chain.

[0008] The objective of this invention is achieved through the following technical solution:

[0009] The first objective of this invention is to provide a near-infrared small molecule fluorescent dye having a decadecylamine conjugated chain, said small molecule fluorescent dye comprising one or more of the structure shown in general formula A and a solvate of the structure shown in general formula A, wherein general formula A is as follows:

[0010]

[0011] In the formula:

[0012] R1 and R2 are each independently selected from C1-C6 alkyl groups, aromatic rings, and substituted aromatic rings, and chlorine atoms;

[0013] R3 is independently selected from C1-C6 alkyl, C1-C6 terminal alkyne, and C1-C6 sulfonic acid substituted alkanes;

[0014] n is independently selected from 1 or 2;

[0015] X is independently selected from bromine, tetraphenylboronic acid, and tetra(pentafluorophenyl)boronic acid.

[0016] Furthermore, the near-infrared small molecule dye has a maximum absorption wavelength between 1100-1300 nm and a maximum emission wavelength between 1150-1300 nm; its molecular weight is between 700-1300 Da.

[0017] Furthermore, the brightness of the near-infrared small molecule dye reaches 4.8-115.1 M. -1 cm -1 The molar extinction coefficient reaches 4.8 × 10⁻⁶. 4 -2.6×10 5 M -1 cm -1 .

[0018] Furthermore, the structure of the small molecule fluorescent dye is that of general formula A, or an isomer of the structure of general formula A, or a solvate of the structure of general formula A.

[0019] More preferably, the structure of the small molecule fluorescent dye is that shown in general formula A.

[0020] Furthermore, the solvate of the structure represented by general formula A is a solvate formed by general formula A and solvents such as dichloromethane, methanol, and dimethyl sulfoxide.

[0021] More preferably, the structure of the small molecule fluorescent dye is any one of the following formulas B1-B14:

[0022]

[0023]

[0024] A second objective of this invention is to provide a method for preparing the above-mentioned near-infrared small molecule dye, comprising the following steps:

[0025] Step 1: The compound shown in formula (1) undergoes a carbon-nitrogen coupling reaction with a halogenated compound or a sulfonate lactone compound to obtain the compound shown in formula (2).

[0026] Step 2: The compound shown in formula (2) undergoes a dehydration condensation reaction with the aldehyde compound to obtain the compound shown in formula (3);

[0027] Step 3: The compound shown in formula (3) undergoes a Suzuki coupling reaction to obtain the compound shown in formula (4);

[0028] Step 4: The compound shown in formula (3) or formula (4) undergoes a Knoevenagel condensation reaction with cyclopentanone to obtain the compound shown in formula (5);

[0029] Step 5: The compound shown in formula (5) undergoes a nucleophilic addition reaction under the action of lithium reagent or Grignard reagent to obtain the compound shown in general formula A, or the compound is obtained by reacting with the corresponding anionic salt after the nucleophilic addition reaction.

[0030] Wherein, equation (1) is Equation (2) is Equation (3) is Equation (4) is Equation (5) is

[0031] Furthermore, the preparation method includes the following steps:

[0032]

[0033] Further, in step one, the compound shown in formula (1) undergoes a carbon-nitrogen coupling reaction with a halogenated compound or a sulfonate lactone compound to obtain the compound shown in formula (2), including:

[0034] In a first organic solvent environment, the compound shown in formula (1) is heated and stirred with a halogenated compound or with a sulfonyl lactone compound to obtain the compound shown in formula (2);

[0035] The halogenated product is an alkyl or terminal alkyne halogenated product;

[0036] The halogenated compound or sulfonyl lactone compound has the structure shown in any one of the following formulas C1, C2, and C3:

[0037]

[0038] In equations C1, C2, and C3:

[0039] R3 is independently selected from C1-C6 alkyl groups and C1-C6 terminal alkynes;

[0040] Where C1 and C2 are halogenated compounds, and C3 is a sulfonate lactone compound;

[0041] The first organic solvent is selected from one or more of benzene, toluene, xylene, chlorobenzene, dichlorobenzene, dichloromethane, chloroform, acetonitrile, N,N-dimethylformamide, dimethyl sulfoxide, tetrahydrofuran, diethyl ether, 1,4-dioxane, and glacial acetic acid.

[0042] Furthermore, in step one, the heating and stirring conditions are as follows: heating temperature is 100-120℃, stirring speed is 500-1000rpm, and heating and stirring time is 24-72h.

[0043] Furthermore, the molar ratio of the compound shown in formula (1) to the halogenated compound or the compound shown in formula (1) to the sulfonyl lactone compound is 1:1.2-1:10.

[0044] Further, in step two, the compound shown in formula (2) undergoes a dehydration condensation reaction with an aldehyde compound to obtain the compound shown in formula (3), which includes:

[0045] In a second organic solvent environment, the compound shown in formula (2) was stirred with an aldehyde compound to obtain the compound shown in formula (3);

[0046] The aldehyde compound has the structure shown in either formula D1 or D2:

[0047]

[0048] The second organic solvent is selected from one or more of benzene, toluene, xylene, chlorobenzene, dichlorobenzene, dichloromethane, chloroform, acetonitrile, N,N-dimethylformamide, dimethyl sulfoxide, acetic acid, tetrahydrofuran, diethyl ether, 1,4-dioxane, ethanol, and methanol.

[0049] Furthermore, in step two, the stirring conditions are as follows: stirring speed of 500-1000 rpm and stirring time of 9-24 h.

[0050] Furthermore, the molar ratio of the compound shown in formula (2) to the aldehyde compound is 1:1 to 1:3.

[0051] Further, in step three, the compound shown in formula (3) undergoes a Suzuki coupling reaction to obtain the compound shown in formula (4), which includes:

[0052] Under the third organic solvent environment and inert gas protection, the compound shown in formula (3) and the boric acid compound were heated and stirred with palladium catalyst and base as catalyst to obtain the compound shown in formula (4) through Suzuki coupling reaction.

[0053] The boric acid compound has the structure shown in formula E below:

[0054] R1-B(OH)2

[0055] E

[0056] In formula E:

[0057] R1 is independently selected from C1-C6 alkyl groups, aromatic rings, and substituted aromatic rings;

[0058] The palladium catalyst is selected from one or more of tetrakis(triphenylphosphine)palladium, tris(dibenzylacetone)dipalladium, bis(dibenzylacetone)palladium, and palladium acetate.

[0059] The alkali is selected from one or more of potassium tert-butoxide, sodium tert-butoxide, potassium carbonate, cesium carbonate, and potassium phosphate.

[0060] The third organic solvent is selected from one or more of benzene, toluene, xylene, N,N-dimethylformamide, dimethyl sulfoxide, tetrahydrofuran, and diethyl ether.

[0061] Furthermore, in step three, under inert gas protection, the heating and stirring conditions are as follows: heating temperature is 80-120℃, stirring speed is 500-1000rpm, and heating and stirring time is 12-48h.

[0062] Furthermore, the molar ratio of the compound shown in formula (3) to the boric acid compound is 1:1 to 1:3.

[0063] Further, in step four, the compound shown in formula (3) or formula (4) undergoes a Knoevenagel condensation reaction with cyclopentanone to obtain the compound shown in formula (5), which includes:

[0064] In a fourth organic solvent environment, the compound shown in formula (3) or formula (4) is heated and stirred with cyclopentanone and base, wherein the base is used as a catalyst, and the compound shown in formula (5) is obtained by Knoevenagel condensation reaction.

[0065] The alkali is selected from one or more of sodium hydroxide, sodium tert-butoxide, sodium ethoxide, and potassium hydroxide;

[0066] The fourth organic solvent is selected from one or more of tetrahydrofuran, diethyl ether, 1,4-dioxane, ethanol, and methanol.

[0067] Furthermore, in step four, the heating and stirring conditions are as follows: heating temperature is 80-120℃, stirring speed is 500-1000rpm, and heating and stirring time is 12-72h.

[0068] Furthermore, the molar ratio of the compound shown in formula (3) to cyclopentanone is 1:0.5-1:1.

[0069] Further, in step five, the compound shown in formula (5) undergoes a nucleophilic addition reaction under the action of a lithium reagent or a Grignard reagent to obtain the compound shown in general formula A, or a portion of the compound obtained after the nucleophilic addition reaction reacts with the corresponding anionic salt to obtain the compound shown in general formula A, including:

[0070] In the fifth organic solvent environment, under the protection of an inert gas, the compound shown in formula (5) is reacted with a lithium reagent or the compound shown in formula (5) is reacted with a Grignard reagent at low temperature, and after a nucleophilic addition reaction, the compound shown in general formula A is obtained, or a portion of the compound obtained after the nucleophilic addition reaction is reacted with the corresponding anionic salt to exchange anions and react to obtain the compound shown in general formula A.

[0071] The lithium reagent or Grignard reagent has the structure shown in either formula F1 or F2:

[0072]

[0073] In formulas F1 and F2:

[0074] R2 is independently selected from C1-C6 alkyl groups, aromatic rings, and substituted aromatic rings;

[0075] The anionic salt is selected from one or more of sodium tetraphenylborate and potassium tetra(pentafluorophenyl)borate.

[0076] The fifth organic solvent is selected from one or more of ultra-dry tetrahydrofuran, diethyl ether, and 1,4-dioxane.

[0077] Furthermore, in step five, under the protection of an inert gas, the reaction conditions at low temperature are as follows: the low temperature is -87℃, the stirring rate is 500-1000 rpm, and the stirring time is 0.5-3h.

[0078] Furthermore, the molar ratio of the compound shown in formula (5) to the lithium reagent or the compound shown in formula (5) to the Grignard reagent is 1:1 to 1:20.

[0079] Furthermore, the molar ratio of the compound shown in formula (5) to the anionic salt is 1:1 to 1:20.

[0080] The near-infrared small molecule dyes provided by this invention are novel dye parent structures based on two benzoindole structures linked by a 15-membered mesylate chain. Functional dye molecules with absorption and emission peak wavelengths in the near-infrared region are obtained by modulating the absorption and emission peak wavelengths through substituents on the parent structure. Different substituents can be introduced onto the 15-membered mesylate chain and the N atom of the indole. Their maximum absorption wavelength is between 1100-1300 nm, and their maximum emission wavelength is between 1150-1300 nm; their molecular weight is between 700-1300 Da, and they exhibit high brightness of 4.8-115.1 M. -1 cm -1 It has a high molar extinction coefficient of 4.8 × 10⁻⁶. 4 -2.6×10 5 M -1 cm -1 It features excellent photostability, low cytotoxicity, simple synthesis, and easy structural modification.

[0081] The third objective of this invention is to provide an application of a near-infrared small molecule fluorescent dye with a decylmethylhexane conjugated chain. The near-infrared small molecule dye provided by this invention can be used for tumor-targeted imaging, in vivo vascular imaging, fluorescence imaging, etc.

[0082] Compared with the prior art, the beneficial effects of the present invention are reflected in the following aspects:

[0083] This invention provides a near-infrared small-molecule fluorescent dye with a pentadecanylene conjugated chain and its preparation method. It exhibits longer absorption and emission wavelengths, enabling NIR-IIb imaging. Compared to traditional visible and NIR-I imaging, it demonstrates better signal-to-background ratio (SNR) and imaging performance. This overcomes the limitations of NIR-II imaging, which suffers from fewer molecular structures and shorter wavelengths. Its maximum absorption wavelength is between 1100-1300 nm, and its maximum emission wavelength is between 1150-1300 nm. It features high brightness, high molar extinction coefficient, excellent photostability, low cytotoxicity, simple synthesis, and easy structural modification, making it suitable for tumor-targeted imaging, in vivo angiography, and NIR-II fluorescence imaging. Attached Figure Description

[0084] Figure 1 The normalized absorption spectrum and photoluminescence (PL) spectrum of the small molecule fluorescent dyes B1-B10 in dichloromethane are shown.

[0085] Figure 2 The small molecule fluorescent dyes B3 and B7 nanoparticles and ICG of this invention were prepared into PBS solutions with a concentration of 0.15 mM. Fluorescence imaging test images of different long-pass filters LP1300 and LP1500 were obtained using a chemiluminescence fluorescence imaging analysis system, where (a) LP1300 and (b) LP1500.

[0086] Figure 3 The small molecule fluorescent dye B3 of this invention was prepared into PBS solutions with concentrations of 0-150 μM nanoparticles. 4T1 cells were incubated with B3 material at a density of 8000 cells / well for 12 hours. The diagram shows the survival rate of the 4T1 cells.

[0087] Figure 4 The diagram shows a NIR-IIb imaging of the hind limb blood vessels of Balb / c-nude mice, prepared by using the small molecule fluorescent dye B3 nanoparticles of this invention in a PBS solution with a concentration of 0.15 mM.

[0088] Figure 5 This is a schematic diagram of mouse NIR-II vascular imaging using the small molecule fluorescent dye B3 of this invention.

[0089] Figure 6 This is a schematic diagram of mouse NIR-II tumor imaging using the small molecule fluorescent dye B3 of this invention. Detailed Implementation

[0090] The present invention will now be described in detail with reference to specific embodiments, but this is by no means a limitation thereof. Any preparation methods, materials, structures, or compositional ratios not explicitly described in this technical solution are considered common technical features disclosed in the prior art.

[0091] The following embodiments will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention. These all fall within the scope of protection of the present invention.

[0092] In this embodiment, the proton nuclear magnetic resonance (¹H NMR) spectrum of the compound was determined by a Bruker AVANCE III HD 400 or a Bruker AVANCE III HD 500; all reagents used were commercially available.

[0093] Example 1

[0094] This embodiment provides a method for preparing fluorescent dye B1. The compound structure and specific synthetic route are as follows:

[0095]

[0096] (1) Synthesis of compound B1-(2)

[0097] 2,3,3-trimethyl-4,5-benzo-3H-indole (1.0 g, 4.8 mmol) was added to a 100 mL round-bottom flask, followed by acetonitrile (36 mL) and 1-iodopropane (702 μL, 7.2 mmol). The reaction was heated for 2 days at 110 °C with a stirring rate of 800 rpm. After cooling to room temperature, the solution was concentrated by rotary evaporation. The remaining oily solution was washed with diethyl ether to give product B1-(2), a dark brown solid, 1.34 g, with a yield of 97%. H-NMR (400MHz, CDCl3, 25℃) δ = 8.08 (2H, t, J = 8Hz, ar.CH) and 8.04(1H,d,J=8Hz,ar.CH)part.overlapped,7.92(1H,d,J=8Hz,ar.CH),7.83(1H,d,J=8Hz,ar.CH),7.66(1H,t,J=8Hz,ar.CH),4.8 0(2H,t,J=8Hz,-NCH2-),3.20(3H,s,-CH3),2.11-2.02(2H,m,-NCH2CH2CH2),1.87(6H,s,-CH3),1.11(3H,t,J=8Hz,-NCH2CH2CH3); 13C-NMR (100MHz, CDCl3, 25℃) δ = 195.3, 189.7, 150.1, 138.5, 138.0, 136.9, 13 3.5,132.1,131.2,130.0,127.3,55.8,55.2,22.6,21.6,16.8,11.2; ESI-MS m / z calculated for C 18 H 22 N + 252.17; obs.:252.2

[0098] (2) Synthesis of compound B1-(3)

[0099] A mixture of compound B1-(2) (5 mmol, 1.9 g) and 2-chloro-1-formyl-3-(hydroxymethylene)cyclohexene (6 mmol, 1.0 g) was weighed and dissolved in a mixture of toluene (37 mL) and acetic acid (13 mL). The mixture was stirred at room temperature for 12 h at a stirring rate of 800 rpm and concentrated by rotary evaporation. The resulting compound was purified by silica gel column chromatography (PE:EA = 9:1) to give compound B1-(3), a red solid of 1.0 mmol (427 mg), with a separation yield of 21.1%.

[0100] 1 H NMR(400MHz,Chloroform-d)δ10.30(s,1H),8.07(d,J=8.6Hz,1H),8.00(d,J=12.6Hz, 1H),7.85(d,J=8.2Hz,1H),7.80(d,J=8.7Hz,1H),7.56–7.46(m,1H),7.36–7.29(m,1H ),7.13(d,J=8.7Hz,1H),5.57(d,J=12.8Hz,1H),3.79(t,J=6.0Hz,2H),2.61(t,J=5.9 Hz,2H),2.52(t,J=6.0Hz,2H),2.01(s,6H),1.89–1.78(m,4H),1.06(t,J=7.2Hz,3H). 13 C NMR(101MHz,Chloroform-d)δ190.8,164.3,148.8,141.6,131.3,130.1,129.9,129.6,129.5,128.9,128.3 ,127.0,122.8,122.7,121.7,109.4,92.5,48.6,44.2,27.5,26.7,24.6,21.0,20.1,11.7.HRMS:calculated for C 26 H28 ClNO[M+H] + 406.1932, found 406.1942.

[0101] (3) Synthesis of compound B1-(4)

[0102] A mixture of B1-(3) (1 mmol, 410 mg), phenylboronic acid (2 mmol, 250 mg), K2CO3 (2.2 mmol, 300 mg), and Pd(PPh3)4 (tetraphenylphosphine palladium) (0.06 mmol, 60 mg) was added to a Shrek flask under argon protection. Dry DMF (0.1 mmol, 8.5 mL) and deionized water (0.08 mmol, 1.5 mL) were added to the flask, and the mixture was stirred at 90 °C for 24 h at 800 rpm. After cooling to room temperature, the reaction mixture was poured into brine and extracted with ethyl acetate. The organic phase was dried over Na2SO4, filtered, and concentrated by rotary evaporation. Purification by silica gel column chromatography (PE:EA = 9:1) yielded compound B1-(4) as a red solid (0.87 mmol, 389 mg), with a separation yield of 87.0%.

[0103] 1 H NMR(400MHz,Chloroform-d)δ9.26(s,1H),7.85(d,J=8.6Hz,1H),7.83(d,J=8.1Hz,1H),7 .76(d,J=8.7Hz,1H),7.47–7.37(m,4H),7.25–7.19(m,3H),7.01(d,J=8.7Hz,1H),6.57(d, J=12.9Hz,1H),5.55(d,J=12.9Hz,1H),3.69(t,J=7.2Hz,2H),2.59(t,J=5.8Hz,2H),2.52( t,J=6.1Hz,2H),1.92–1.83(m,2H),1.82–1.70(m,2H),1.43(s,6H),1.00(t,J=7.4Hz,3H). 13 C NMR(101MHz,Chloroform-d)δ193.1,162.7,158.5,141.7,137.6,133.9,131.1,130.3,129.9,129.8,129.4,129.1,128.8 ,128.6,127.9,127.5,126.7,122.4,121.6,109.3,92.5,47.8,44.1,27.0,25.4,22.5,21.5,20.0,11.7.HRMS:calculated for C 32 H33 NO[M+H] + 448.2635, found 448.2638.

[0104] (4) Synthesis of compound B1-(5)

[0105] A mixture of B1-(4) (0.5 mmol, 225 mg), cyclopentanone (0.25 mmol, 23 μL), and NaOH (2 mmol, 80 mg) was added to a round-bottom flask. 0.2 mmol of anhydrous ethanol (EtOH) was added to the flask, and the mixture was stirred at 80 °C for 48 h at a stirring rate of 800 rpm. After cooling to room temperature, the mixture was filtered and washed with ethanol to obtain compound B1-(5), a blue solid of 0.19 mmol (183 mg), with a separation yield of 77%. 1 H NMR(400MHz,Chloroform-d)δ7.85(d,J=8.4Hz,2H),7.75(d,J=8.0Hz,2H),7.68(d,J=8.7Hz,2H ),7.43–7.35(m,8H),7.22–7.18(m,2H),7.14–7.08(m,4H),7.02–6.96(m,4H),6.23(d,J=12.8Hz ,2H),5.48(d,J=12.8Hz,2H),3.65(t,J=7.0Hz,4H),3.05–2.97(m,4H),2.78(t,J=5.9Hz,4H),2 .58(t,J=5.6Hz,4H),1.98–1.90(m,4H),1.80–1.72(m,4H),1.43(s,12H),1.00(t,J=7.4Hz,6H). 13 C NMR(101MHz,Chloroform-d)δ159.5,150.0,142.1,140.1,135.0,134.4,131.1,130.5,130.1,129.7,129.5,129.2,129.1,1 29.0,128.7,128.0,127.0,126.5,122.0,121.7,109.1,92.8,47.4,44.0,28.7,26.9,16.7,25.0,22.5,20.0,11.7.MALDI-FT ICR MS:calculated forC 69 H 70 N2O[M] + 942.5488, found 942.5444.

[0106] (5) Synthesis of compound B1

[0107] Add B1-(5) (0.02 mmol, 20 mg) to a Shrek flask under argon protection. Add 2 mL of ultra-dry THF (ultra-dry tetrahydrofuran) to the flask and cool to 0 °C in an ice-water bath. Then, add 1 M phenyl magnesium bromide (0.04 mmol, 40 μL) dropwise to the solution. Stir in a cryogenic reactor for 0.5 h at a stirring rate of 800 rpm, and quench the reaction with 2 mL of methanol. Purify the remaining liquid by alumina (300-400 mesh) chromatography (DCM:MeOH = 25:1) to obtain compound B1, 0.004 mmol, 4.2 mg, black solid, with a separation yield of 19.3%.

[0108] 1 H NMR(400MHz,Chloroform-d)δ8.00–7.79(m,6H),7.52–7.49(m,2H),7.41–7.28(m,11H),7.05–6.87(m,11H),6.61(d,J=7.1Hz,2H),6.17(d,J =14.2Hz,1H),4.42(t,J=6.5Hz,3H),3.00–2.65(m,9H),2.08–1.98(m,8H),1.91–1.82(m,4H),1.41(s,12H),1.03(t,J=7.3Hz,6H).MALDI-FT ICR MS:calculated for C 75 H 75 BrN2[M-Br] + 1003.5925,found1003.5925,[M-2H-Br] + 1001.5768, found 1001.5726.

[0109] Example 2

[0110] This embodiment provides a method for preparing fluorescent dye B3. The compound structure and specific synthetic route are as follows:

[0111]

[0112] (1) Compound B1-(2) is the same as in the synthesis of B1.

[0113] (2) Synthesis of compound B3-(3)

[0114] A mixture of B1-(2) (10 mmol, 3.8 g) and 2-chloro-1-formyl-3-(hydroxymethylene)cyclopentene (15 mmol, 2.4 g) was dissolved in a mixture of toluene (347 mmol, 37 mL) and acetic acid (20 g, 13 mL). The mixture was stirred at room temperature for 12 h at a stirring rate of 800 rpm and concentrated by rotary evaporation. The residual organic matter was purified by silica gel column chromatography (PE:EA = 9:1) to give compound B1-(3), a red solid of 1.6 mmol (636.2 mg), with a separation yield of 16.3%.

[0115] 1 H NMR(400MHz,Chloroform-d)δ10.01(s,1H),8.02(d,J=8.5Hz,1H),7.81(d,J=8.2Hz,1H),7.76(d,J=8.7Hz,1H),7.50–7.39(m,2H),7.31–7.26(m,1 H),7.08(d,J=8.7Hz,1H),5.31(d,J=12.9Hz,1H),3.75(t,J=6.2Hz,2H), 2.80–2.73(m,4H),1.94(s,6H),1.84–1.77(m,2H),1.02(t,J=7.4Hz,3H). 13 C NMR(101MHz,Chloroform-d)δ186.7,162.5,149.4,141.8,135.6,133.9,130.0,129.8,129.6,129.2, 129.0,126.9,125.0,122.7,121.7,109.4,93.4,48.4,27.6,26.2,24.9,20.0,11.7.HRMS:calculated forC 25 H 26 ClNO[M+H] + 392.1776, found 392.1776.

[0116] (3) Synthesis of compound B3-(4)

[0117] A mixture of B1-(3) (0.5 mmol, 200 mg), 4-methoxyphenylboronic acid (1 mmol, 152 mg), K2CO3 (1 mmol, 138 mg), and Pd(PPh3)4 (0.2 mmol, 231 mg) was added to a Shrek flask under argon protection. Dry DMF (0.1 mmol, 8.5 mL) and deionized water (0.08 mmol, 1.5 mL) were added to the flask, and the mixture was stirred at 90 °C for 24 h at 800 rpm. After cooling to room temperature, the reaction mixture was poured into brine and extracted with ethyl acetate. The composite organic layer was dried on Na2SO4, filtered, and concentrated under vacuum. Compound B3-(4) was purified by silica gel flash chromatography (PE:EA = 6:1) to obtain 0.39 mmol (179 mg) red solid, with a separation yield of 77.4%.

[0118] 1 H NMR(400MHz,Chloroform-d)δ9.57(s,1H),7.92(d,J=8.6Hz,1H),7.77(d,J=8.2Hz,1H) ,7.71(d,J=8.7Hz,1H),7.44–7.37(m,1H),7.31(d,J=8.6Hz,2H),7.25–7.20(m,1H),7. 06–7.00(m,3H),6.94(d,J=12.8Hz,1H),5.38(d,J=12.8Hz,1H),3.92(s,3H),3.70(t,J =7.2Hz,2H),2.89–2.80(m,4H),1.83–1.74(m,2H),1.71(s,6H),1.01(t,J=7.4Hz,3H). 13 C NMR(101MHz,Chloroform-d)δ189.6,162.4,160.9,160.0,142.0,140.5,140.0,131.0,129.8,129.7,129.4,129.0,128,7,126 .7,126.2,125.4,122.3,121.6,116.1,114.8,113.8,109.3,94.2,55.4,47.8,44.0,27.7,27.2,26.5,20.0,11.7.MALDI-FTICR MS: calculated for C 32 H 33 NO2[M] + 463.2511, found 463.2495.

[0119] (4) Synthesis of compound B3-(5)

[0120] A mixture of B3-(4) (0.1 mmol, 46 mg), cyclopentanone (0.05 mmol, 4.5 μL), and NaOH (0.2 mmol, 8 mg) was added to a round-bottom flask. Ethanol (1 mL) was added to the flask, and the mixture was stirred at 80 °C for 48 h at a stirring rate of 800 rpm. After cooling to room temperature, the reaction mixture was filtered and washed with ethanol to give compound B3-(5), a blue solid of 0.04 mmol (41 mg), with a separation yield of 85%.

[0121] 1 H NMR(400MHz,Chloroform-d)δ7.90(d,J=8.5Hz,2H),7.74(d,J=8.1Hz,2H),7.67(d ,J=8.7Hz,2H),7.41–7.35(m,2H),7.22–7.15(m,6H),7.05–6.87(m,8H),6.68(d,J= 12.6Hz,2H),5.36(d,J=11.8Hz,2H),3.94–3.85(m,6H),3.70–3.62(m,4H),3.17–2 .99(m,4H),2.90–2.78(m,4H),1.82–1.73(m,16H),1.00(t,J=7.4Hz,6H).MALDI-FT ICR MS: calculated for C 69 H 70 N2O3[M] + 974.5386, found 974.5343.

[0122] (5) Synthesis of compound B3

[0123] B3-(5) (0.02 mmol, 20 mg) was added to a Schlenk flask under argon protection. Ultra-dry THF (2 mL) was added to the flask, and the mixture was cooled to 0°C in an ice-water bath. Then, 1 M 4-methoxyphenyl magnesium bromide (0.2 mmol, 0.2 mL) was added dropwise to the solution. The mixture was stirred in a cryogenic reactor for 0.5 h at 800 rpm, and the reaction was quenched by adding 2 mL of methanol. The solution was purified by alkaline alumina column chromatography (DCM:MeOH = 25:1) to give compound 4c as a black solid (0.009 mmol, 9.7 mg), with a separation yield of 41.2%.

[0124] 1H NMR(400MHz,Chloroform-d)δ7.98–7.77(m,6H),7.49–7.30(m,6H),7.15–7.08(m,4H),6.95–6.88(m,8H),6.82–6.75(m,4H),6.68–6.64(m,1H),6 .07(d,J=13.8Hz,1H),4.69–4.12(m,4H),3.75–3.60(m,9H),3.25–3.00( m,8H),2.05–1.78(m,8H),1.67(s,12H),0.97(t,J=7.2Hz,6H).MALDI-FT ICR MS:calculated for C 66 H 67 BrN2O3[M-Br] + 1065.5929,found 1065.5929,[M-2H-Br] + 1063.5772, found 1063.5616.

[0125] Example 3

[0126] This embodiment provides a method for preparing the fluorescent dye B13. The compound structure and specific synthetic route are as follows:

[0127]

[0128] (1) Compound B1-(2) is the same as in the synthesis of B1.

[0129] (2) Compound B3-(3) is the same as B3 in synthesis.

[0130] (3) Synthesis of compound B13-(4)

[0131] A mixture of B3-(3) (0.5 mmol, 197 mg), phenylboronic acid (1 mmol, 122 mg), K2CO3 (1 mmol, 138 mg), and Pd(PPh3)4 (0.05 mmol, 50 mg) was added to a Shrek flask under argon protection. Dry DMF (0.05 mmol, 4 mL) and deionized water (0.04 mmol, 0.8 mL) were added to the flask, and the mixture was stirred at 90 °C for 24 h at 800 rpm. After cooling to room temperature, the reaction mixture was poured into brine and extracted with ethyl acetate. The organic phase was dried on Na2SO4, filtered, and concentrated by rotary evaporation. The obtained organic compound was purified by column chromatography with silica gel (PE:EA = 6:1) to give compound B13-(4), a red solid of 0.45 mmol (197 mg), with a separation yield of 90.9%.

[0132] 1 H NMR(400MHz,Chloroform-d)δ9.55(s,1H),7.90(d,J=8.4Hz,1H),7.77(d,J=8.4Hz, 1H),7.71(d,J=8.8Hz,1H),7.53–7.44(m,3H),7.42–7.33(m,3H),7.25–7.20(m,1H) ,7.03(d,J=8.7Hz,1H),6.88(d,J=12.1Hz,1H),5.37(d,J=13.1Hz,1H),3.69(t,J=6 .7Hz,2H),2.92–2.81(m,4H),1.83–1.72(m,2H),1.68(s,6H),1.01(t,J=6.8Hz,3H). 13 CNMR(101MHz,Chloroform-d)δ189.6,163.0,161.2,141.9,140.5,140.2,133.3,129.8,129.8,129.7,129.6,129.5,128.9, 128.8,128.7,128.3,126.7,126.6,122.4,121.6,115.4,109.3,94.2,47.9,44.0,27.8,27.2,26.5,11.7.HRMS:calculated for C 31 H 31 NO[M+H] + 434.2478, found 434.2494.

[0133] (4) Synthesis of compound B13-(5)

[0134] A mixture of 0.2 mmol (87 mg) of B13-(4), 0.1 mmol (8.9 μL) of cyclopentanone, and 4 mmol (32 mg) of NaOH was added to a round-bottom flask. 2 mL of ethanol was added to the flask, and the mixture was stirred at 80 °C for 48 h at a stirring rate of 800 rpm. After cooling to room temperature, the reaction mixture was filtered and washed with ethanol to obtain compound B13-(5), a blue solid of 0.08 mmol (76 mg), with a separation yield of 83%.

[0135] 1H NMR(400MHz,Chloroform-d)δ7.89(d,J=9.0Hz,2H),7.74(d,J=8.1Hz,2H),7.67(d ,J=8.7Hz,2H),7.46–7.33(m,8H),7.25–7.14(m,8H),6.99(d,J=8.8Hz,2H),6.62( d,J=12.6Hz,2H),5.35(d,J=11.4Hz,2H),3.75–3.58(m,4H),3.21–2.97(m,8H),2. 94–2.77(m,4H),1.80–1.72(m,4H),1.68(s,12H),1.00(t,J=7.4Hz,6H).MALDI-FT ICR MS: calculated for C 67 H 66 N2O[M] + 914.5175, found 914.5050.

[0136] (5) Synthesis of compound B13

[0137] B13-(5), 0.01 mmol, 9 mg was added to a Schlenk flask under argon protection. Ultra-dry THF (200 mmol, 2 mL) was added to the flask, and the mixture was cooled to 0°C in an ice-water bath. Then, 1 M phenyl magnesium bromide (0.2 mmol, 0.2 mL) was added dropwise to the solution. The mixture was stirred in an ice-water bath for 0.5 h at a stirring rate of 800 rpm, and the reaction was quenched by adding 2 mL of methanol. The obtained organic compound was purified by alkaline alumina (300-400 mesh) column chromatography (DCM:MeOH = 25:1) to give 4 mg of the compound as a black solid (0.004 mmol, 4.0 mg), with a yield of 41.0%. The obtained solid was dissolved in acetonitrile, and sodium tetraphenylborate (0.2 mmol, 68 mg) was added. The mixture was stirred at room temperature for 5 hours at a stirring rate of 800 rpm. The resulting organic compound was purified by alkaline alumina (300-400 mesh) column chromatography (DCM:MeOH 99:1) to give compound B13, 2.4 mg, as a black solid (0.002 mmol, 2.4 mg), with a separation yield of 46.3%.

[0138] 1H NMR(400MHz,Chloroform-d)δ7.96–7.92(m,2H),7.90–7.81(m,4H),7.49–7.39(m,13H),7.08–6.99(m,10H),6.89–6.82(m,3H),5.84(d,J= 14.0Hz,1H),3.79(t,J=7.2Hz,2H),3.67(t,J=6.8Hz,2H),3.49–2.88(m,8H),1.79–1.62(m,20H),0.91(t,J=7.2Hz,6H).HRMS:calculated for C 73 H 71 BrN2[M-Br] + 975.5612, found 975.5620.

[0139] Example 4

[0140] This embodiment provides a method for preparing fluorescent dye B7. The compound structure and specific synthetic route are as follows:

[0141]

[0142] (1) Compound B1-(2) is the same as in the synthesis of B1.

[0143] (2) Compound B3-(3) is the same as B3 in synthesis.

[0144] (3) Compound B13-(4) is the same as in the synthesis of B13.

[0145] (4) Compound B13-(5) is the same as in the synthesis of B13.

[0146] (5) Synthesis of compound B7

[0147] B13-(5) (0.01 mmol, 9 mg) was added to a Schlenk flask under argon protection. Ultra-dry THF (200 mmol, 2 mL) was added to the flask, and the mixture was cooled to 0°C in an ice-water bath. Then, 1 M phenyl magnesium bromide (0.2 mmol, 0.2 mL) was added dropwise to the solution. The mixture was stirred in an ice-water bath for 0.5 h at 800 rpm, and the reaction was quenched by adding 2 mL of methanol. The obtained organic compound was purified by alkaline alumina (300-400 mesh) column chromatography (DCM:MeOH = 25:1) to give compound B7, 4 mg, as a black solid (0.004 mmol, 4.0 mg), with a yield of 41.0%.

[0148] 1H NMR(400MHz,Chloroform-d)δ7.96–7.92(m,2H),7.90–7.81(m,4H),7.49–7.39(m,13H),7.08–6.99(m,10H),6.89–6.82(m,3H),5.84(d,J= 14.0Hz,1H),3.79(t,J=7.2Hz,2H),3.67(t,J=6.8Hz,2H),3.49–2.88(m,8H),1.79–1.62(m,20H),0.91(t,J=7.2Hz,6H).HRMS:calculated for C 73 H 71 BrN2[M-Br] + 975.5612, found 975.5620.

[0149] The preparation methods of B2 and B4-B12 in general formula A dye B1-B12 are basically the same as those in Example 1, except that the corresponding reactants used are different.

[0150] The preparation method of general formula dyes B13-B14 is basically the same as the reaction conditions in Example 3, the difference being the different reactants used.

[0151] Test case

[0152] The general formula dyes B1-B10 prepared in the above examples were respectively formulated to a concentration of 1×10⁻⁶. -5 The DCM solution of M was scanned and plotted, along with its UV-Vis-NIR absorption and fluorescence emission spectra, as shown in the attached figure. Figure 1 As shown. The relevant photophysical properties are shown in Table 1. The maximum absorption wavelength (λ) of the visible general formula A series dyes is... abs The maximum emission wavelength (λ) is between 1100-1300 nm. em The molar extinction coefficient (ε) is 4.8 × 10⁻⁶ between 1150 and 1300 nm. 4 -2.6×10 5 M -1 cm -1 The brightness ranges from 4.8 to 115.1 MHz. -1 cm -1 Between these, the fluorescence quantum yield (Φ) is relatively high.

[0153] Table 1 shows the photophysical data of dye A.

[0154]

[0155] The general formula dyes B3 and B7 nanoparticles and ICG (indocyanine green) prepared in the above examples were respectively formulated into 0.15 mM PBS solutions. Fluorescence imaging test images were obtained using a chemiluminescence fluorescence imaging analysis system with different long-pass filters LP1300 and LP1500, as shown in the attached figures. Figure 2 As shown, by Figure 2 It can be seen that, at LP1300, the brightness of B3 and B7 is 5.1 and 4.1 times that of ICG at the same concentration, respectively. At LP1500, the brightness of B3 and B7 is 9.6 and 5.1 times that of ICG at the same concentration, respectively.

[0156] The general formula A dye B3 prepared in the above examples was formulated into PBS solutions of nanoparticles with concentrations ranging from 0 to 150 μM. 4T1 cells (8000 cells / well) were co-incubated with the B3 material for 12 hours. The 4T1 cell viability is shown in the attached figure. Figure 3 As shown, by Figure 3 It can be seen that B3 nanoparticles at a concentration of 150 μM do not exhibit significant cytotoxicity.

[0157] The general formula A dye B3 nanoparticles prepared in the above examples were formulated into a 0.15 mM PBS solution, and NIR-IIb imaging of the hind limb vessels of Balb / c-nude mice was performed as shown in the attached figure. Figure 4 As shown, by Figure 4 It is known that B3 nanoparticles can clearly distinguish the femoral artery, vein, and multiple calf vessels when used for hind limb vascular imaging in mice, with a signal-to-background ratio as high as 6.8.

[0158] Mouse NIR-II vascular imaging using the general formula A dye B3 prepared in the above examples is as follows: Figure 5 As shown, by Figure 5 It can be seen that B3 can clearly distinguish the blood vessels in the chest, abdomen and hind limbs of mice.

[0159] Mouse NIR-II tumor imaging using the general formula A dye B3 prepared in the above examples is as follows: Figure 6 As shown, by Figure 6 It can be seen that B3 is enriched in mouse orthotopic mammary tumors, and the ratio of tumor background signal to normal tissue can reach 4.8.

[0160] Specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to these embodiments; the descriptions of the embodiments are provided to enable those skilled in the art to understand and use the invention. Those skilled in the art will readily make various modifications to these embodiments and apply the general principles described herein to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from its scope should be within the protection scope of the present invention.

Claims

1. A near-infrared small molecule fluorescent dye having a pentadecylamine conjugated chain, characterized in that, The structure of the small molecule fluorescent dye is any one of the following formulas B1-B14: 。 2. The near-infrared small molecule dye with a pentadecylamine conjugated chain according to claim 1, characterized in that, The maximum absorption wavelength of the near-infrared small molecule dye is between 1100-1300 nm, and the maximum emission wavelength is between 1150-1300 nm; the molecular weight is between 700-1300 Da. The brightness of the near-infrared small molecule dye is 4.8-115.1 M. -1 cm -1 The molar extinction coefficient is 4.8 × 10⁻⁶. 4 -2.6×10 5 M -1 cm -1 .

3. The application of a near-infrared small molecule dye having a pentadecylamine conjugated chain as described in any one of claims 1-2 in the preparation of fluorescent probes for tumor-targeted imaging, in vivo vascular imaging, and fluorescence imaging.

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