Nile red derivative fluorescence imaging probe molecule with high lipid droplet specificity and light stability and application of Nile red derivative fluorescence imaging probe molecule in cell lipid droplet fluorescence imaging

Abstract

The invention discloses a nile red derivative fluorescence imaging probe molecule with high lipid droplet specificity and light stability and application of the nile red derivative fluorescence imaging probe molecule in cell lipid droplet fluorescence imaging, and belongs to the technical field of biological imaging. The cell lipid droplet fluorescence imaging probe molecule with high lipid droplet specificity and light stability based on the Nile red has fifteen structures which are respectively called NR-1 to NR-15 for short, and the cell lipid droplet fluorescence imaging probe molecule with the fifteen structures based on the Nile red is of a donor and receptor structure. The invention also discloses an application of the fluorescent probe in cell lipid droplet fluorescence imaging, and the cell disclosed by the invention is a HeLa cell. Experiments prove that the fluorescent probe, especially the NR-11 fluorescent probe, has ultrahigh lipid droplet labeling selectivity due to the environment-sensitive TICT emission characteristic, is a lipid droplet fluorescent probe with the advantages of ultrahigh lipid droplet labeling selectivity, high light stability, excellent biocompatibility and the like, and has a huge application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of biological imaging, and in particular relates to a Nile red derivative fluorescent imaging probe molecule with high lipid droplet specificity and photostability and its application in cell lipid droplet fluorescent imaging. Background Art

[0002] Lipid droplets (LDs) are ubiquitous organelles in almost all eukaryotic cells, consisting of a neutral lipid core and a surrounding phospholipid monolayer membrane. LDs have long been considered to be inert fat particles that retain excess lipids in cells. However, recent studies have continuously found that LDs play key roles in various cellular processes, such as energy balance, lipid metabolism, membrane protein production, membrane trafficking, etc. In addition, many metabolic disorders, such as diabetes, coronary artery disease, cardiomyopathy, and fatty liver, have recently been shown to be closely related to abnormal increases in the size and number of LDs. Therefore, in recent years, the study of LDs has become one of the most attractive subjects in the field of cell biology.

[0003] To visualize LDs and clarify their multifunctionality, fluorescence imaging techniques have been widely adopted due to their high spatial resolution, real-time, and non-invasive properties. A variety of lipophilic fluorescent probes for LDs fluorescence imaging have been reported, such as Nile Red, BODIPY493 / 503, solvatochromic probe AP-C12, HClO-responsive probe LDM, AIE-type 1,8-naphthalimide derivatives and TPAPhCN, photoconversion probe SC-P, fluorescent probe LD-FG, ESIPT-type probe PPC, etc. Among these LDs probes, Nile Red is undoubtedly the most attractive. Since the first report of Nile Red-labeled LDs fluorescence imaging published in 1985, this classic probe has been widely used for 40 years, and it is still the most commonly used one for LDs imaging to date. Despite its continuous success over the years, Nile Red has two disadvantages: 1) It is not very specific to LDs, and Nile Red will stain LDs as well as other organelles / regions in cells; 2) Its photostability is relatively low, which can lead to severe photobleaching during time-lapse imaging. With the rapid development of fluorescence imaging technology, these two shortages of Nile Red have become more serious. Undoubtedly, overcoming these two shortages is urgent. However, directly limited by the synthetic route and further functionalization strategy, the synthesis of various Nile Red derivatives is very complicated and time-consuming. Therefore, the shortcomings of LDs specificity and photostability of Nile Red have received little attention and have become a bottleneck for a long time. Summary of the invention

[0004] In view of the shortcomings of the existing lipid droplet imaging fluorescent imaging probe molecule technology, the problem to be solved by the present invention is to provide a Nile red derivative fluorescent imaging probe molecule with high lipid droplet specificity and photostability and its application in cell lipid droplet fluorescent imaging.

[0005] The Nile red derivative fluorescent imaging probe molecule with high lipid droplet specificity and photostability described in the present invention comprises a total of fifteen structures, whose chemical names are 9-(dipropylamino)-5H-benzo[a]benzoxazine-5-one, 9-(dibutylamino)-5H-benzo[a]benzoxazine-5-one, 9-(dipentylamino)-5H-benzo[a]phenoxazine-5-one, 9-(dihexylamino)-5H-benzo[a]benzoxazine-5-one, 5-one, 9-(1-methyl-1H-pyrrol-2-yl)-5H-benzo[a]phenoxazine-5-one, 9-(9H-carbazol-9-yl)-5H-benzo[a]phenoxazine-5-one, 9-(diphenylamino)-5H-benzo[a]phenoxazine-5-one, 9-(diphenylamino)-6-phenyl-5H-benzo[a]benzoxazine-5-one, 9-(diphenylamino)-6-(4-methoxyphenyl)-5H-benzo[a]phenoxazine-5-one [a] benzoxazin-5-one, 6-(4-(dimethylamino)phenyl)-9-(diphenylamino)-5H-benzo[a]benzoxazin-5-one, 4-(9-(diphenylamino)-5-oxo-5H-benzo[a]phenoxazin-6-yl)benzonitrile, 5-(9-(diphenylamino)-5-oxo-5H-benzo[a]phenoxazin-6-yl)pyridinenitrile, 9-(diphenylamino)-6-(pyrimidin-5-yl)-5H- Benzo[a]benzoxazin-5-one, 9-(diphenylamino)-6-(pyridin-4-yl)-5H-benzo[a]benzoxazin-5-one and 9-(diphenylamino)-6-(4-(methylsulfonyl)phenyl)-5H-benzo[a]benzoxazin-5-one, respectively referred to as NR-1 to NR-15, the fifteen structures of Nile red-based cell lipid droplet fluorescence imaging probe molecules are all donor-acceptor structures, and their chemical structures are shown in formula (I):

[0006]

[0007]

[0008] The present invention also provides a method for preparing the Nile red derivative fluorescent imaging probe molecule with high lipid droplet specificity and photostability. The first step is to condense 2-amino-5-bromophenol and 2-chloro-1,4-naphthoquinone under alkaline conditions. The screening process of the reaction catalyst is shown in Table 1:

[0009]

[0010] Table 1: Screening results of reaction catalysts

[0011] Serial number Alkali Dosage Reaction time Solvents Yield 1 <![CDATA[CH3COOK]]> 2eq. 24h <![CDATA[CH2Cl2]]> 8％ 2 <![CDATA[K2CO3]]> 2eq. 24h <![CDATA[CH2Cl2]]> 27％ 3 KOH 2eq. 24h <![CDATA[CH2Cl2]]> 28％ 4 t-BuOK 2eq. 24h <![CDATA[CH2Cl2]]> 16％ 5 DBU 2eq. 12h <![CDATA[CH2Cl2]]> 92％

[0012] Subsequently, the generated bromide intermediate undergoes Buchwald-Hartwig / Suzuki-Miyaura coupling with various amines / borates to generate NR-1 to NR-7; next, NR-7 is successfully monobrominated by NBS, and the brominated product then reacts with various organic boronic acids via Suzuki-Miyaura coupling to generate NR-8 to NR-15; the present invention prepares the fluorescent imaging probe molecule for the first time and applies it to a variety of fluorescent imaging modes. The preparation reaction formula of the fluorescent imaging probe molecule is as follows:

[0013]

[0014]

[0015] In order to significantly improve the specificity of LDs, the present invention adopts two strategies. One strategy is to adjust the hydrophobicity of the molecule, so NR-1 to NR-4 with different alkyl chains were synthesized; considering the hydrophobicity of LDs, NR-1 to NR-4 did show higher LDs specificity than Nile Red. However, the effectiveness of this strategy is limited, and the improvement in the LDs specificity of NR-1 to NR-4 in terms of signal-to-noise ratio is only about 2 times that of Nile Red in fluorescence imaging.

[0016] Another strategy to improve LDs specificity is to tune the emission properties of the molecules. Synthesized molecules NR-5 to NR-7 with twisted intramolecular charge transfer (TICT) properties show strong fluorescence in LDs (low polarity environment) but weak emission in the cytoplasm (high polarity). In contrast, Nile Red with intramolecular charge transfer (ICT) properties exhibits strong emission in both LDs and the cytoplasm. Therefore, the TICT-type molecules NR-5 to NR-7 show much lower background in fluorescence imaging and thus have significantly higher LDs specificity than the ICT-type molecule Nile Red. In fact, the LDs specificity of NR-7 is 8 times that of Nile Red in fluorescence imaging, highlighting the effectiveness of the TICT strategy for LDs specificity.

[0017] Since TICT-type NR-7 showed the highest LDs specificity among these derivatives, we thereafter focused on this fluorophore to further improve the photostability. NR-8 to NR-15 with various substitution groups were synthesized to protect this reactive site, and in fact, these derivatives showed much higher photostability than NR-7. Among these derivatives, NR-11 showed the highest performance in terms of LDs specificity and photostability.

[0018] The cells described in the present invention are HeLa cells.

[0019] The series of cell lipid droplet fluorescent imaging probe molecules NR-11 prepared by the present invention are multifunctional fluorescent imaging probe molecules that can be used for confocal imaging and have TICT emission characteristics, high labeling selectivity, high photostability, environmental sensitivity and excellent biocompatibility (Examples 3 to 7).

[0020] The experimental results confirm that the series of fluorescent imaging probe molecules NR-7 described in the present invention have environmentally sensitive TICT emission characteristics, and its TICT luminescence characteristics are confirmed by solvent-dependent fluorescence spectroscopy. The obtained NR-1 to NR-4, NR-7 to NR-8, NR-11 to NR-15 were compared with the commercial lipid droplet dye Nile Red, and it was found that NR-7 to NR-8 and NR-11 to NR-13 can efficiently and selectively label cell lipid droplets with ultra-high imaging signal-to-noise ratio. Further, NR-11 to NR-15 were compared with the commercial lipid droplet dye Nile Red, and it was found that NR-11 showed the highest stability. The MTT test confirmed that the probe molecule has excellent biocompatibility. Therefore, NR-11 can be used as a lipid droplet fluorescent imaging probe molecule to study the complex physiological functions of cell lipid droplets and promote the development of lipid droplet cell biology. The design strategy of this molecule has important guiding significance for the development of fluorescent imaging probe molecules targeting different organelles.

[0021] In summary, the NR-11 described in the present invention is a new lipid droplet fluorescence imaging probe molecule. Compared with other lipid droplet fluorescence imaging probe molecules, this probe molecule has ultra-high lipid droplet labeling selectivity due to its environmentally sensitive TICT emission characteristics. In view of the above characteristics, its application in cell lipid droplet fluorescence imaging has broad prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] FIG1 : NMR hydrogen spectra of a series of fluorescent imaging probe molecules NR-1 to NR-15 prepared in Example 1 of the present invention, corresponding to Figure 1(a) to Figure 1(o) ; The nuclear magnetic resonance hydrogen spectra of the key intermediates NR-Br-1 and NR-Br-2 of the series of fluorescent imaging probe molecules prepared in Example 1 of the present invention correspond to Figure 1(p) to Figure 1(q) ;

[0023] FIG2 shows the absorption and emission spectra of the series of fluorescent imaging probe molecules NR-1 to NR-15 prepared in Example 1 of the present invention in toluene, chloroform, dichloromethane and acetonitrile solutions (corresponding to Example 2); wherein, Figure 2(a) to Figure 2(o) They correspond to NR-1 to NR-15, respectively, the dotted lines are the absorption spectra, and the solid lines are the emission spectra;

[0024] Figure 3: Quantification of the signal-to-noise ratio of fluorescence imaging probe molecules NR-1 to NR-4, NR-7 to NR-8, NR-11 to NR-15 and the commercial lipid droplet dye Nile Red in HeLa cells (corresponding to Example 7);

[0025] Among them, Figure 3(a) is a confocal photograph of HeLa cells stained with Nile Red and NR-1 to NR-4, NR-7 to NR-8, and NR-11 to NR-15 imaging probe molecules (scale ruler: 10 μm); Figure 3(b) is a statistical bar graph of the signal-to-noise ratio corresponding to the area marked by the circle frame in Figure 3(a) (scale ruler: 1 μm).

[0026] Figure 4: Comparison of the photostability of fluorescent imaging probe molecules NR-11 to NR-15 and the commercial lipid droplet probe Nile Red in HeLa cells (corresponding to Example 6);

[0027] Among them, Figure 4(a) is a confocal photograph of HeLa cells stained with Nile Red and NR-11 to NR-15 imaging probe molecules at the 1st frame (upper figure) and the 50th frame (lower figure) (scale: 10μm); Figure 4(b) is a brightness statistical function diagram of Nile Red and NR-11 to NR-15 in each frame sampled in 50 consecutive frames.

[0028] Figure 5 : A bar graph showing the cell survival rate after HeLa cells were stained with different concentrations of the fluorescent imaging probe molecule NR-11 for 24 hours (corresponding to Example 5). DETAILED DESCRIPTION

[0029] Embodiment 1:

[0030] Synthesis of 9-bromo-5H-benzo[a]phenoxazine-5-one (NR-Br-1)

[0031] To a CH2Cl2 (100 mL) solution of 2-amino-5-bromophenol (5.00 g, 26.6 mmol) and 2-chloro-1,4-naphthoquinone (10.24 g, 53.2 mmol) was added a reaction catalyst DBU (5.96 mL, 39.9 mmol; 1,8-diazabicyclo[5.4.0]undec-7-ene), and the resulting mixture was refluxed for 12 hours. The precipitated solid was washed three times with methanol and dried to obtain 8.09 g (24.8 mmol, 93%) of NR-Br-1 as a pale yellow powder.

[0032] 1H NMR (400MHz, CDCl3): δ8.69(d,J=6.8Hz,1H),8.29(d,J=6.5Hz,1H),7.84–7.74(m,2H),7.68(d,J=8.1Hz,1H),7.48(d,J=7.7Hz,2H),6.44(s,1H).

[0033] Synthesis of 9-(Dipropylamino)-5H-benzo[a]benzoxazin-5-one (NR-1)

[0034] To a mixture of NR-Br-1 (100 mg, 0.306 mmol), dipropylamine (0.155 g, 1.53 mmol), Pd2(dba)3 (10 mg, 0.0366 mmol) and Cs2CO3 (0.198 g, 0.614 mmol) was added degassed toluene (20 mL) and P(t-Bu)3 (0.2 mL, 0.1 mmol, 10 w / v% toluene). The reaction mixture was then refluxed for 24 h. After cooling to room temperature, the mixture was filtered to remove inorganic salts. The filtrate was poured into water and extracted 3 times with CH2Cl2. The combined organic layers were washed with brine, then dried over anhydrous MgSO4 and filtered. After the filtrate was concentrated under reduced pressure, the resulting mixture was purified by silica gel column chromatography to obtain 75.5 mg (0.219 mmol, 71%).

[0035] 1 H NMR (400MHz, CDCl3): δ8.64(d,J=7.8Hz,1H),8.30(d,J=7.6Hz,1H),7.71(t,J=7.4Hz,1H),7.64(t,J=7.3Hz ,1H),7.59(d,J=9.1Hz,1H),6.64(d,J=9.0Hz,1H),6.43(s,1H),6.39(s,1H),3.34(t,J=7.4Hz,4H),1.72–1 .67(m,4H),0.99(t,J=7.2Hz,6H).13CNMR(101MHz,CDCl3): δ183.74,152.17,151.20,146.72,139.95,132. 10,131.75,131.33,131.03,129.92,125.72,124.99,123.79,109.92,105.74,96.48,53.23,20.56,11.40.

[0036] Synthesis of 9-(Dibutylamino)-5H-benzo[a]benzoxazin-5-one (NR-2)

[0037] To a mixture of NR-Br-1 (100 mg, 0.306 mmol), dibutylamine (0.198 g, 1.53 mmol), Pd2(dba)3 (10 mg, 0.0366 mmol) and Cs2CO3 (0.198 g, 0.614 mmol) was added degassed toluene (20 mL) and P(t-Bu)3 (0.2 mL, 0.1 mmol, 10 w / v% toluene). The reaction mixture was then refluxed for 24 h. After cooling to room temperature, the mixture was filtered to remove inorganic salts. The filtrate was poured into water and extracted 3 times with CH2Cl2. The combined organic layers were washed with brine, then dried over anhydrous MgSO4 and filtered. After the filtrate was concentrated under reduced pressure, the resulting mixture was purified by silica gel column chromatography to obtain 81.0 mg (0.219 mmol, 70%).

[0038] 1 H NMR (400MHz, CDCl3): δ8.66(d,J=8.0Hz,1H),8.31(d,J=7.8Hz,1H),7.72(t,J=7.5Hz,1H),7.66–7.62(m,2H),6.66(d,J=8. 9Hz,1H),6.46(s,1H),6.42(s,1H),3.39(t,J=7.5Hz,4H),1.71–1.58(m,4H),1.48–1.35(m,4H),1.00(t,J=7.2Hz,6H).13C NMR (101MHz, CDCl3): δ183.74,152.17,151.16,146.73,139.86,132.11,131.75,131.31,13 1.05,129.89,125.71,124.96,123.79,109.88,105.72,96.41,51.27,29.44,20.32,14.00.

[0039] Synthesis of 9-(Dipentylamino)-5H-benzo[a]phenoxazine-5-one (NR-3)

[0040] To a mixture of NR-Br-1 (100 mg, 0.306 mmol), dipentylamine (0.241 g, 1.53 mmol), Pd2(dba)3 (10 mg, 0.0366 mmol) and Cs2CO3 (0.198 g, 0.614 mmol) was added degassed toluene (20 mL) and P(t-Bu)3 (0.2 mL, 0.1 mmol, 10 w / v% toluene). The reaction mixture was then refluxed for 24 h. After cooling to room temperature, the mixture was filtered to remove inorganic salts. The filtrate was poured into water and extracted 3 times with CH2Cl2. The combined organic layers were washed with brine, then dried over anhydrous MgSO4 and filtered. After the filtrate was concentrated under reduced pressure, the resulting mixture was purified by silica gel column chromatography to obtain 70.5 mg (0.175 mmol, 56%).

[0041] 1 H NMR (400MHz, CDCl3): δ8.65(d,J=7.8Hz,1H),8.31(d,J=7.7Hz,1H),7.72(t,J=7.4Hz,1H),7.69–7.57(m,2H),6.65(d, J=9.0Hz,1H),6.43(d,J=8.1Hz,2H),3.37(t,J=7.5Hz,4H),1.73–1.59(m,4H),1.38(s,8H),0.96(d,J=6.3Hz,6H).13C NMR (101MHz, CDCl3): δ183.64,152.17,151.15,146.77,139.85,132.09,131.69,131.34,131.0 9,129.92,125.73,125.11,123.78,110.02,105.69,96.48,51.56,29.24,27.04,22.59,14.11.

[0042] Synthesis of 9-(Dihexylamino)-5H-benzo[a]benzoxazin-5-one (NR-4)

[0043] To a mixture of NR-Br-1 (100 mg, 0.306 mmol), dihexylamine (0.284 g, 1.53 mmol), Pd2(dba)3 (10 mg, 0.0366 mmol) and Cs2CO3 (0.198 g, 0.614 mmol) was added degassed toluene (20 mL) and P(t-Bu)3 (0.2 mL, 0.1 mmol, 10 w / v% toluene). The reaction mixture was then refluxed for 24 h. After cooling to room temperature, the mixture was filtered to remove inorganic salts. The filtrate was poured into water and extracted 3 times with CH2Cl2. The combined organic layers were washed with brine, then dried over anhydrous MgSO4 and filtered. After the filtrate was concentrated under reduced pressure, the resulting mixture was purified by silica gel column chromatography to obtain 83.1 mg (0.193 mmol, 63%).

[0044] 1 H NMR (400MHz, CDCl3): δ8.65(d,J=7.8Hz,1H),8.31(d,J=7.7Hz,1H),7.72(t,J=7.4Hz,1H),7.68–7.55(m,2H),6 .64(d,J=9.0Hz,1H),6.43(d,J=6.6Hz,2H),3.36(t,J=7.4Hz,4H),1.65(s,4H),1.36(s,12H),0.92(s,6H).13C NMR (101MHz, CDCl3): δ183.65,152.17,151.14,146.77,139.85,132.10,131.70,131.34,131.09,1 29.91,125.72,125.10,123.78,110.01,105.69,96.48,51.60,31.67,27.31,26.78,22.70,14.07.

[0045] Synthesis of 9-(1-methyl-1H-pyrrol-2-yl)-5H-benzo[a]phenoxazine-5-one (NR-5)

[0046] To a mixture of NR-Br-1 (0.20 g, 0.61 mmol), N-methylpyrrole-2-boronic acid pinacol ester (0.25 g, 1.2 mmol), PdCl2(PPh3)2 (39 mg, 0.061 mmol) and K2CO3 (0.25 g, 1.8 mmol) was added a degassed solvent mixture of THF and water (V / V=1:1, 20 mL), and the reaction mixture was refluxed for 12 h. After cooling to room temperature, the mixture was filtered to remove inorganic salts. The filtrate was poured into water and extracted 3 times with CH2Cl2. The combined organic layers were washed with brine, then dried over anhydrous MgSO4 and filtered. After concentrating the filtrate under reduced pressure, the resulting mixture was purified by silica gel column chromatography to obtain 0.16 g (0.50 mmol, 81%) of NR-5 as an orange-red solid.

[0047] 1 H NMR (500MHz, CDCl3): δ8.74(d,J=7.6Hz,1H),8.32(d,J=7.5Hz,1H),7.83(d,J=8.3Hz,1H),7.81–7.71(m,2H),7.43(dd,J=8.3Hz,J=1. 7Hz,1H),7.35(d,J=1.7Hz,1H),6.83–6.79(m,1H),6.47(s,1H),6.44(dd,J=3.6Hz,J=1.6Hz,1H),6.28–6.23(m,1H),3.80(s,3H).13C NMR (101MHz, CDCl3): δ183.89,151.42,146.46,144.20,136.71,132.82,132.10,131.98,131.67, 131.48,131.39,129.82,126.19,125.95,124.90,124.67,114.20,111.14,108.76,107.33,35.78.

[0048] Synthesis of 9-(9H-carbazol-9-yl)-5H-benzo[a]phenoxazine-5-one (NR-6)

[0049] To a mixture of NR-Br-1 (100 mg, 0.306 mmol), carbazole (0.256 g, 1.53 mmol), Pd2(dba)3 (10 mg, 0.0366 mmol) and Cs2CO3 (0.198 g, 0.614 mmol) was added degassed toluene (20 mL) and P(t-Bu)3 (0.2 mL, 0.1 mmol, 10 w / v% toluene). The reaction mixture was then refluxed for 24 h. After cooling to room temperature, the mixture was filtered to remove inorganic salts. The filtrate was poured into water and extracted 3 times with CH2Cl2. The combined organic layers were washed with brine, then dried over anhydrous MgSO4 and filtered. After concentrating the filtrate under reduced pressure, the resulting mixture was purified by silica gel column chromatography to obtain 85.2 mg (0.207 mmol, 67%).

[0050] 1 H NMR (400MHz, CDCl3): δ8.83–8.77(m,1H),8.34(dd,J=7.6Hz,J=1.4Hz,1H),8.17(d,J=7.7Hz,2H),8.06(d,J=8.4Hz,1H),7.88–7.7 7(m,2H),7.65(dd,J=8.4Hz,J=2.2Hz,1H),7.60(dd,J=6.3Hz,J=5.4Hz,3H),7.51–7.43(m,2H),7.36(t,J=7.5Hz,2H),6.51(s,1H).

[0051] Synthesis of 9-(Diphenylamino)-5H-benzo[a]phenoxazine-5-one (NR-7)

[0052] To a mixture of NR-Br-1 (100 mg, 0.306 mmol), diphenylamine (0.256 g, 1.53 mmol), Pd2(dba)3 (10 mg, 0.0366 mmol) and Cs2CO3 (0.198 g, 0.614 mmol) was added degassed toluene (20 mL) and P(t-Bu)3 (0.2 mL, 0.1 mmol, 10 w / v% toluene). The reaction mixture was then refluxed for 24 h. After cooling to room temperature, the mixture was filtered to remove inorganic salts. The filtrate was poured into water and extracted 3 times with CH2Cl2. The combined organic layers were washed with brine, then dried over anhydrous MgSO4 and filtered. After the filtrate was concentrated under reduced pressure, the resulting mixture was purified by silica gel column chromatography to obtain 90.2 mg (0.218 mmol, 71%).

[0053] 1H NMR (400MHz, DMSO-d6): δ8.59(d,J=7.8Hz,1H),8.13(dd,J=7.7Hz,J=1.0Hz,1H),7.89–7.83(m,1H),7.83–7.75(m,1H),7.71(d, J=8.9Hz,1H),7.50–7.42(m,4H),7.29(t,J=7.4Hz,6H),6.83(dd,J=8.9Hz,J=2.5Hz,1H),6.60(d,J=2.5Hz,1H),6.33(s,1H).13C NMR (101MHz, CDCl3) δ183.78,151.64,151.26,145.94,145.54,143.09,131.93,131.73,131.6 7,130.81,130.41,129.89,127.79,126.38,125.83,125.49,124.20,117.23,106.49,105.41.

[0054] Synthesis of 6-bromo-9-(diphenylamino)-5H-benzo[a]benzoxazin-5-one (NR-Br-2)

[0055] To a solution of NR-7 (3.20 g, 7.72 mmol) in CH2Cl2 (100 mL) was slowly added NBS (1.44 g, 8.11 mmol), and the resulting mixture was stirred at room temperature for 1 hour. After the solution was concentrated under reduced pressure, the resulting mixture was purified by silica gel column chromatography to obtain 3.72 g (7.54 mmol, 98%) of NR-Br-2 as a purple solid.

[0056] 1 HNMR (400MHz, CDCl3): δ8.67(dd,J=7.9Hz,J=0.8Hz,1H),8.37(dd,J=7.8Hz,J=1.1Hz,1H),7.79–7.73(m,1H),7.72–7.66(m, 1H),7.62(d,J=8.7Hz,1H),7.40(t,J=7.9Hz,4H),7.25–7.21(m,6H),6.98(dd,J=8.7Hz,J=2.5Hz,1H),6.95(d,J=2.4Hz,1H).

[0057] Synthesis of 9-(Diphenylamino)-6-phenyl-5H-benzo[a]benzoxazin-5-one (NR-8)

[0058] To a mixture of NR-Br-2 (0.15 g, 0.30 mmol), phenylboronic acid (0.110 g, 0.91 mmol), Pd2(dba)3 (34.5 mg, 0.030 mmol), S-Phos (50 mg, 0.12 mmol) and K2CO3 (0.13 g, 0.91 mmol) was added a degassed solvent mixture of toluene, ethanol and water (V / V / V=8:1:1, 25 mL). The reaction mixture was then refluxed for 12 h. After cooling to room temperature, the mixture was filtered to remove inorganic salts. The filtrate was poured into water and extracted 3 times with CH2Cl2. The combined organic layers were washed with brine, then dried over anhydrous MgSO4 and filtered. After concentrating the filtrate under reduced pressure, the resulting mixture was purified by silica gel column chromatography to obtain 0.124 g (0.25 mmol, 83%) of NR-8 as a purple solid.

[0059] 1 H NMR (500MHz, CDCl3): δ8.70(d,J=7.7Hz,1H),8.31(d,J=7.6Hz,1H),7.76(t,J=6.9Hz,1H),7.70(t,J=6.9Hz,1 H),7.63(d,J=8.9Hz,1H),7.39(t,J=7.9Hz,4H),7.24–7.20(m,6H),6.97(dd,J=8.9Hz,J=2.5Hz,1H),6.82(d, J=2.5Hz,1H),6.47(s,1H).13CNMR(101MHz,CDCl3): δ183.77,151.65,151.28,145.92,145.55,143.06,131.9 0,131.72,131.69,130.81,130.42,129.90,127.82,126.38,125.83,125.50,124.20,117.25,106.46,105.39.

[0060] Synthesis of 9-(Diphenylamino)-6-(4-methoxyphenyl)-5H-benzo[a]benzoxazin-5-one (NR-9)

[0061] To a mixture of NR-Br-2 (0.15 g, 0.30 mmol), 4-methoxyphenylboronic acid (0.157 g, 0.91 mmol), Pd2(dba)3 (34.5 mg, 0.030 mmol), S-Phos (50 mg, 0.12 mmol) and K2CO3 (0.13 g, 0.91 mmol) was added a degassed solvent mixture of toluene, ethanol and water (V / V / V=8:1:1, 25 mL). The reaction mixture was then refluxed for 12 h. After cooling to room temperature, the mixture was filtered to remove inorganic salts. The filtrate was poured into water and extracted 3 times with CH2Cl2. The combined organic layers were washed with brine, then dried over anhydrous MgSO4 and filtered. After concentrating the filtrate under reduced pressure, the resulting mixture was purified by silica gel column chromatography to obtain 0.102 g (0.208 mmol, 68%) of NR-9 as a purple solid.

[0062] 1 H NMR (500MHz, CDCl3): δ8.74(d,J=7.9Hz,1H),8.36(d,J=7.6Hz,1H),7.76(t,J=7.5Hz,1H),7.71(t,J=7.6Hz,1H),7.62(d,J=8.9Hz,1 H),7.44(d,J=8.7Hz,2H),7.34(t,J=7.9Hz,4H),7.16(t,J=7.7Hz,6H),6.98(d,J=8.7Hz,2H),6.91(dd,J=8.8Hz,J=2.4Hz,1H),6.72 (d,J=2.4Hz,1H),3.85(s,3H).13CNMR(101MHz,CDCl3): δ182.40,159.10,150.96,146.98,146.17,145.57,143.35,132.01,132.71, 132.67,131.42,130.86,130.07,129.83,127.96,126.39,126.09,125.16,124.03,123.41,118.21,117.93,113.53,106.21,55.30.

[0063] Synthesis of 6-(4-(dimethylamino)phenyl)-9-(diphenylamino)-5H-benzo[a]benzoxazin-5-one (NR-10)

[0064] To a mixture of NR-Br-2 (0.15 g, 0.30 mmol), 4-dimethylaminophenylboronic acid (0.150 g, 0.91 mmol), Pd2(dba)3 (34.5 mg, 0.030 mmol), S-Phos (50 mg, 0.12 mmol) and K2CO3 (0.13 g, 0.91 mmol) was added a degassed solvent mixture of toluene, ethanol and water (V / V / V=8:1:1, 25 mL). The reaction mixture was then refluxed for 12 h. After cooling to room temperature, the mixture was filtered to remove inorganic salts. The filtrate was poured into water and extracted 3 times with CH2Cl2. The combined organic layers were washed with brine, then dried over anhydrous MgSO4 and filtered. After concentrating the filtrate under reduced pressure, the resulting mixture was purified by silica gel column chromatography to obtain 0.110 g (0.207 mmol, 68%) of NR-10 as a purple solid.

[0065] 1 H NMR (500MHz, CDCl3): δ8.71(d,J=7.8Hz,1H),8.36(d,J=7.7Hz,1H),7.75(t,J=7.6Hz,1H),7.69(t,J=7.3Hz,1H),7.58( d,J=8.7Hz,1H),7.44(d,J=8.2Hz,2H),7.33(t,J=7.8Hz,4H),7.15(d,J=8.2Hz,6H),6.93–6.70(m,4H),3.00(s,6H).13C NMR (101MHz, CDCl3): δ182.66,150.76,149.93,146.55,146.31,145.69,143.75,131.85,131.66,131.53,131. 46,130.75,129.92,129.80,128.13,126.37,126.02,124.98,123.98,118.72,117.94,111.82,106.59,40.52.

[0066] Synthesis of 4-(9-(diphenylamino)-5-oxo-5H-benzo[a]phenoxazin-6-yl)benzonitrile (NR-11) To a mixture of NR-Br-2 (0.15 g, 0.30 mmol), 4-cyanophenylboronic acid (0.134 g, 0.91 mmol), Pd2(dba)3 (34.5 mg, 0.030 mmol), S-Phos (50 mg, 0.12 mmol) and K2CO3 (0.13 g, 0.91 mmol) was added a degassed solvent mixture of toluene, ethanol and water (V / V / V=8:1:1, 25 mL). The reaction mixture was then refluxed for 12 h. After cooling to room temperature, the mixture was filtered to remove inorganic salts. The filtrate was poured into water and extracted 3 times with CH2Cl2. The combined organic layers were washed with brine, then dried over anhydrous MgSO4 and filtered. After the filtrate was concentrated under reduced pressure, the resulting mixture was purified by silica gel column chromatography to obtain 0.123 g (0.239 mmol, 79%) of NR-11 as a purple solid.

[0067] 1 H NMR (500MHz, CDCl3): δ8.74(d,J=7.8Hz,1H),8.35(d,J=7.7Hz,1H),7.79(t,J=7.6Hz,1H),7.75–7.69(m,3H),7.64 –7.60(m,3H),7.36(t,J=7.8Hz,4H),7.23–7.14(m,6H),6.95(dd,J=8.9Hz,J=2.3Hz,1H),6.66(d,J=2.4Hz,1H).13C NMR (101MHz, CDCl3): δ181.42,151.44,147.49,145.85,145.26,142.27,136.63,132.05,131.75,131.63,131. 38,131.05,130.44,129.94,127.88,126.42,126.28,125.60,124.14,119.15,118.00,116.49,111.27,105.34.

[0068] Synthesis of 5-(9-(diphenylamino)-5-oxo-5H-benzo[a]phenoxazin-6-yl)pyridinecarbonitrile (NR-12)

[0069] To a mixture of NR-Br-2 (0.15 g, 0.30 mmol), 2-cyano-5-pyridineboronic acid (0.135 g, 0.91 mmol), Pd2(dba)3 (34.5 mg, 0.030 mmol), S-Phos (50 mg, 0.12 mmol) and K2CO3 (0.13 g, 0.91 mmol) was added a degassed solvent mixture of toluene, ethanol and water (V / V / V=8:1:1, 25 mL). The reaction mixture was then refluxed for 12 h. After cooling to room temperature, the mixture was filtered to remove inorganic salts. The filtrate was poured into water and extracted 3 times with CH2Cl2. The combined organic layers were washed with brine, then dried over anhydrous MgSO4 and filtered. After concentrating the filtrate under reduced pressure, the resulting mixture was purified by silica gel column chromatography to obtain 0.120 g (0.232 mmol, 76%) of NR-12 as a purple solid.

[0070] 1 H NMR (500MHz, CDCl3): δ8.86(s,1H),8.74(d,J=7.9Hz,1H),8.35(d,J=7.8Hz,1H),8.03(d,J=8.0Hz,1H),7.83–7.71(m,3H), 7.65(d,J=8.9Hz,1H),7.38(t,J=7.8Hz,4H),7.25–7.15(m,6H),6.97(dd,J=8.9Hz,J=2.4Hz,1H),6.68(d,J=2.4Hz,1H).13C NMR (101MHz, CDCl3): δ180.92,152.65,151.79,147.99,145.62,145.08,141.46,139.41,132.23,131.95,131.71,1 31.36,131.10,131.04,130.62,130.05,127.89,127.78,126.41,125.91,124.18,117.96,117.56,112.82,104.76.

[0071] Synthesis of 9-(diphenylamino)-6-(pyrimidin-5-yl)-5H-benzo[a]benzoxazin-5-one (NR-13) To a mixture of NR-Br-2 (0.15 g, 0.30 mmol), 5-pyrimidineboronic acid (0.113 g, 0.91 mmol), Pd2(dba)3 (34.5 mg, 0.030 mmol), S-Phos (50 mg, 0.12 mmol) and K2CO3 (0.13 g, 0.91 mmol) was added a degassed solvent mixture of toluene, ethanol and water (V / V / V=8:1:1, 25 mL). The reaction mixture was then refluxed for 12 h. After cooling to room temperature, the mixture was filtered to remove inorganic salts. The filtrate was poured into water and extracted 3 times with CH2Cl2. The combined organic layers were washed with brine, then dried over anhydrous MgSO4 and filtered. After the filtrate was concentrated under reduced pressure, the resulting mixture was purified by silica gel column chromatography to obtain 0.105 g (0.213 mmol, 70%) of NR-13 as a purple solid.

[0072] 1 H NMR (500MHz, CDCl3): δ9.19(s,1H),8.99(s,2H),8.75(d,J=8.3Hz,1H),8.39–8.36(m,1H),7.81(t,J=7.6Hz,1H),7.77–7.73(m, 1H),7.66(d,J=8.9Hz,1H),7.37(t,J=7.9Hz,4H),7.23–7.15(m,6H),6.98(dd,J=8.9Hz,J=2.4Hz,1H),6.70(d,J=2.4Hz,1H).13C NMR (101MHz, CDCl3): δ181.15,158.29,157.13,151.73,148.17,145.68,145.12,141.76,132.24,131. 44,131.18,131.08,130.55,130.00,127.90,126.42,126.35,125.80,124.19,117.94,111.23,104.93.

[0073] Synthesis of 9-(diphenylamino)-6-(pyridin-4-yl)-5H-benzo[a]benzoxazin-5-one (NR-14) To a mixture of NR-Br-2 (0.15 g, 0.30 mmol), pyridine-4-boronic acid (0.112 g, 0.91 mmol), Pd2(dba)3 (34.5 mg, 0.030 mmol), S-Phos (50 mg, 0.12 mmol) and K2CO3 (0.13 g, 0.91 mmol) was added a degassed solvent mixture of toluene, ethanol and water (V / V / V=8:1:1, 25 mL). The reaction mixture was then refluxed for 12 h. After cooling to room temperature, the mixture was filtered to remove inorganic salts. The filtrate was poured into water and extracted 3 times with CH2Cl2. The combined organic layers were washed with brine, then dried over anhydrous MgSO4 and filtered. After the filtrate was concentrated under reduced pressure, the resulting mixture was purified by silica gel column chromatography to obtain 0.115 g (0.234 mmol, 77%) of NR-14 as a purple solid.

[0074] 1 H NMR (500MHz, CDCl3): δ8.76(d,J=7.9Hz,1H),8.70(d,J=6.0Hz,2H),8.39(d,J=7.7Hz,1H),7.81(t,J=7.1Hz,1H),7.75(t,J=7.5Hz,1H),7.65 (d,J=8.9Hz,1H),7.47(d,J=6.0Hz,2H),7.38(t,J=7.8Hz,4H),7.24–7.20(m,6H),6.98(dd,J=8.9Hz,J=2.5Hz,1H),6.69(d,J=2.4Hz,1H).13C NMR (101MHz, CDCl3): δ181.18,151.49,149.29,147.67,145.79,145.26,142.17,140.17,132.04,131.41,1 31.37,131.04,130.42,129.94,127.85,126.43,126.27,125.70,125.65,124.11,117.93,115.46,105.27.

[0075] Synthesis of 9-(Diphenylamino)-6-(4-(methylsulfonyl)phenyl)-5H-benzo[a]benzoxazin-5-one (NR-15)

[0076] To a mixture of NR-Br-2 (0.15 g, 0.30 mmol), 2-(methylsulfonyl)pyridine-5-boronic acid (0.183 g, 0.91 mmol), Pd2(dba)3 (34.5 mg, 0.030 mmol), S-Phos (50 mg, 0.12 mmol) and K2CO3 (0.13 g, 0.91 mmol) was added a degassed solvent mixture of toluene, ethanol and water (V / V / V=8:1:1, 25 mL). The reaction mixture was then refluxed for 12 h. After cooling to room temperature, the mixture was filtered to remove inorganic salts. The filtrate was poured into water and extracted 3 times with CH2Cl2. The combined organic layers were washed with brine, then dried over anhydrous MgSO4 and filtered. After concentrating the filtrate under reduced pressure, the resulting mixture was purified by silica gel column chromatography to obtain 0.145 g (0.255 mmol, 84%) of NR-15 as a purple solid.

[0077] 1 H NMR (400MHz, CDCl3): δ8.75 (d, J=7.9Hz, 1H), 8.36 (dd, J=7.7Hz, J=0.9Hz, 1H), 8.00 (d, J=8.4Hz, 2H), 7.80 (td, J=7.7Hz, J=1.3Hz, 1H), 7.75 (dd,J=7.6Hz,J=0.9Hz,1H),7.70(d,J=8.4Hz,2H),7.64(d,J=8.9Hz,1H),7.36(t,J=7.8Hz,4H),7.23–7.14(m,6H),6.96(dd,J=8.9Hz,J=2. 4Hz,1H),6.68(d,J=2.4Hz,1H),3.10(s,3H).13CNMR(101MHz,CDCl3): δ181.59,151.54,147.67,145.83,145.33,142.19,139.25,137.65,1 32.07,131.84,131.43,131.39,131.04,130.44,129.96,127.92,127 .04,126.41,126.21,125.70,124.15,118.10,116.39,105.47,44.64.

[0078] Figure 1(a) to Figure 1(o) Figure 1(p) and Figure 1(q) are the H NMR spectra of the fluorescent imaging probe molecules NR-1 to NR-15 synthesized in Example 1, indicating that the target products NR-1 to NR-15 were prepared. Figure 1(p) and Figure 1(q) are the H NMR spectra of the fluorescent imaging probe molecules NR-Br-1 and NR-Br-2 synthesized in Example 1, indicating that the key intermediates NR-Br-1 and NR-Br-2 were prepared.

[0079] Example 2: Determination of the spectra of the fluorescent imaging probe molecules NR-1 to NR-15 prepared in Example 1 in toluene, chloroform, dichloromethane and acetone solutions

[0080] Absorption-emission spectra in organic solvents: The fluorescent imaging probe molecules NR-1 to NR-15 synthesized in Example 1 were prepared into 10 μM solutions using toluene, chloroform, dichloromethane and acetone solvents. The absorption spectra were collected by ultraviolet absorption spectrometer in the wavelength range of 300 to 800 nm, and the emission spectra were collected by Ocean Optics fiber optic fluorescence spectrometer under the excitation of 470 nm excitation light. Figure 2(a) to Figure 2(o) As shown, the dotted line is the absorption spectrum, and the solid line is the emission spectrum, which illustrates the absorption-emission peak position and its large Stokes shift of the series of fluorescent imaging probe molecules NR-1 to NR-15.

[0081] Example 3: Cell culture

[0082] All percentages in this embodiment are volume percentages.

[0083] HeLa cells were cultured in an incubator at 37°C and 5% CO2 concentration in a high-glucose DMEM medium containing 10% fetal bovine serum and 1% double antibody (penicillin-streptomycin mixture). Among them, fetal bovine serum, double antibody and high-glucose DMEM were purchased directly from a biological reagent company.

[0084] When the cells grow to the logarithmic phase, the cells are passaged: after the original 5mL culture medium in the HeLa cell culture bottle is sucked away, the cell surface is washed with 2mL high-glucose DMEM culture medium (containing 1% double antibody) without fetal bovine serum, and the culture medium is sucked away and then the cells are digested with 1mL trypsin for 2 minutes. After most of the cells are detached from the wall, 2mL high-glucose DMEM culture medium containing 10% fetal bovine serum and 1% double antibody is added and blown evenly. The treated cell suspension is transferred to a 15mL centrifuge tube, centrifuged at 1500r / min for 5 minutes, and the supernatant is removed after centrifugation. 1mL high-glucose DMEM culture medium containing 10% fetal bovine serum and 1% double antibody is added and blown evenly again. The cell suspension is taken out at a ratio of 1:3 and 1:10 (volume ratio) and transferred to a new cell culture bottle and culture dish respectively, and placed in a CO2 cell culture incubator for culture. The cells in the culture dish can be used for subsequent passage or confocal imaging experiments when the concentration is appropriate.

[0085] Example 4: Confocal imaging

[0086] The confocal experiment was performed on a cell culture dish, and the sample preparation method was as described in Example 3. HeLa cells were cultured in a 20 mm diameter glass-bottomed culture dish and propagated in a CO2 incubator for 1 day. After taking it out of the incubator, the original DMEM culture medium in the culture dish was removed. For standard confocal imaging, 1 mL of DMEM culture medium containing a target dye molecule (NR-1 to NR-15 and Nile Red) was added; wherein the molecular concentration was 2 μM, and the cells were placed in a cell culture incubator and continued to be cultured for 2 hours. After taking it out, it was washed 3 times with HBSS solution, and then confocal fluorescence imaging was performed in HBSS solution. The confocal imaging system is a Leica DMI8 imaging system, and the specific operation is as follows: the treated cell culture dish is placed on the stage, and an appropriate amount of lens oil is applied to the bottom of the dish, and then synchronous observation and focusing operation are performed under a wide-field fluorescence microscope, and the focus adjustment is completed when the cells in the field of view are clearly focused, and then the focus correction is performed under the confocal field of view, and the excitation intensity and detector width, and the detector gain are set to 530nm; 550nm-795nm; 800%, respectively. After the settings are completed, you can start shooting and obtain confocal fluorescence images.

[0087] Example 5: Test of cytotoxicity of the fluorescent imaging probe molecule NR-11 prepared in Example 1

[0088] We used 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to test the cytotoxicity of the fluorescent imaging probe NR-11 (HeLa cells). HeLa cells were seeded on a 96-well plate (the number of cells in each well was about 1*10 4 ), and cultured in a CO2 cell culture incubator for 24 hours. Afterwards, the culture medium in the culture wells of the 6×10 array area in the central area was replaced with a culture medium containing different concentrations (0, 0.5, 1.0, 2.0, 5.0 and 10.0 μM) of fluorescent imaging probe molecule NR-11 and 1% (volume fraction) DMSO (10 groups of parallel experiments were set for each concentration). After another 24 hours of culture, MTT reagent (10 μL per well) was added to these wells, and they were returned to the cell culture incubator for continued culture for 4 hours. After removing the original culture medium in these wells, DMSO (100 μL per well) was added to dissolve the generated formazan crystals. After being placed on a shaker at room temperature for 30 minutes, the absorbance of each well was measured at 490 nm using an enzyme marker. Since only living cells can react with the MTT reagent to generate formazan crystals, the average absorbance value of the wells with different concentrations in each group can be compared with the average absorbance value of the control group (10 wells with a probe molecule concentration of 0) to calculate the cell survival rate. The results are shown as follows. Figure 5 As shown, it indicates that the fluorescent imaging probe molecule NR-11 has basically no cytotoxicity, and the probe molecule at a concentration of 10.0 μM will not affect the normal growth of HeLa cells within 24 hours.

[0089] Example 6: Comparison of the photostability of the fluorescent imaging probe molecules NR-11 to NR-15 prepared in Example 1 and the commercial lipid droplet dye Nile Red in HeLa cells

[0090] After we took out the two culture dishes filled with HeLa cells in Example 3 from the cell culture incubator, removed the original DMEM culture medium, added DMEM culture medium (containing 1% DMSO) containing 2μM NR-11 to NR-15 and 2μM Nile Red, respectively, put them back into the incubator and continued to culture for 2 hours, then took out the two culture dishes, washed them three times with HBSS solution, and then performed fluorescence imaging. The confocal images in the 500-700nm channel were recorded using a Nikon A1RMP microscope, and the excitation laser power was the same and 50 times higher than the conventional excitation intensity (488nm), as shown in Figure 2. Figure 4(a) to Figure 4(b) The total signal intensity of each image was measured by ImageJ and normalized with the value of the first image, and then plotted as a function of the number of confocal images recorded, as shown in Figure 4(b). The results show that NR-11 has higher photostability than Nile Red.

[0091] Example 7: Comparison of the imaging signal-to-noise ratio of the fluorescent imaging probe molecules NR-1 to NR-4, NR-7 to NR-8, NR-11 to NR-15 prepared in Example 1 and the commercial lipid droplet dye Nile Red in HeLa cells

[0092] After taking out the two culture dishes filled with HeLa cells in Example 3 from the cell culture incubator, the original DMEM culture medium was removed, and DMEM culture medium (containing 1% DMSO) containing 2 μM NR-1 to NR-4, NR-7 to NR-8, NR-11 to NR-15 and 2 μM Nile Red was added respectively, and the dishes were returned to the incubator for further culture for 2 hours. The two culture dishes were taken out, washed three times with HBSS solution respectively, and then fluorescence imaging was performed, as shown in FIG. Figure 3(a) to Figure 3(b) We selected areas in two culture dishes and enlarged them, and compared the signal-to-noise ratio of the selected area in the enlarged area, as shown in Figure 3(b). We found that the fluorescent imaging probe molecule NR-11 showed an imaging signal-to-noise ratio much higher than that of Nile Red, which shows its ultra-high lipid droplet labeling selectivity and confirms the effectiveness of the environmentally sensitive TICT emission characteristics in improving the labeling selectivity of fluorescent imaging probes.

Claims

1. A Nile red derivative fluorescent imaging probe molecule with high lipid droplet specificity and photostability, the structural formula of which is shown in one of the following:

2. Use of a Nile red derivative fluorescent imaging probe molecule with high lipid droplet specificity and photostability as claimed in claim 1 in cellular lipid droplet fluorescent imaging.

3. Use of a Nile red derivative fluorescent imaging probe molecule with high lipid droplet specificity and photostability in cell lipid droplet fluorescence imaging as claimed in claim 2, characterized in that: The cells are HeLa cells.

Images