Lipid-directed fluorescent probe for cancer cells, preparation method and application thereof

Abstract

The application discloses a kind of lipid-oriented cancer cell fluorescent probes, wherein X1 is selected from C (CH3) 2, O, S or Se;A1 is selected from H, phenyl or substituted phenyl;R1, R2 can be same or different, R1 and R2 are each independently selected from C1-C8 alkyl, C1-C8 hydroxyl, C1-C8 carboxyl, C 1‑8 NR 5 R 6 , C1-C8 ester group, benzyl and substituted benzyl;The substituted benzyl is optionally substituted by C1-C8 alkyl, C1-C8 alkoxy, CN, COOH, NH2, NO2, OH, SH, halogen or C1-C8 haloalkyl;Y ‑ Is selected from halogen negative ion, CIO4 ‑ , PF6 ‑ , CH3COO ‑ Or OTs ‑ . The fluorescent probe utilizes carbon-carbon double bond to connect benzothiazole and quinoline part to form Donor-Accepter structure, has the advantage of nucleic acid response, and the probe molecule can be based on the content of cell membrane lipid Different normal cells / tissues and cancer cells / tissues are distinguished, selectively enter cancer cells and combine with nucleic acid to produce fluorescence. The fluorescent probe can be used for tumor cell or tissue specific fluorescence imaging based on nucleic acid detection.

Description

Technical Field

[0001] This invention relates to the field of fine chemicals, and in particular to a class of lipid-directed fluorescent probes for cancer cells, their preparation methods and applications. Background Technology

[0002] In recent years, the incidence and mortality rates of cancer have been increasing year by year, posing a huge challenge to human life. According to statistics from the International Agency for Research on Cancer (IAPC), more than 18 million new cancer cases were reported globally in 2018 alone, with a mortality rate as high as 52.85%. Rapid diagnosis and early treatment of cancer are effective means to improve the survival rate of cancer patients. Traditional diagnostic methods such as magnetic resonance imaging (MRI), ultrasound (US), and X-ray computed tomography (CT) have limitations such as being time-consuming, having low resolution, and being unable to accurately distinguish lesions smaller than 2 mm. During surgical resection, the inability to accurately distinguish the boundary between normal tissue and cancer cells leads to the possibility of postoperative recurrence. Therefore, the development of high-precision rapid diagnostic methods has received widespread attention from researchers. Among them, fluorescence imaging technology based on small molecule fluorescent probes has been widely used in the early diagnosis of tumors, intraoperative imaging, and prognostic evaluation due to its high sensitivity, high spatiotemporal resolution, and low toxicity.

[0003] The cell membrane, as the protective boundary of a cell, is closely related to many physiological and pathological processes. Tumor cells and normal cells differ in their cell membrane composition. Cancer cells exhibit a strong affinity for lipids and cholesterol. Since lipids and cholesterol in cancer cells are mainly enriched on the cell membrane, distinguishing normal cells from cancer cells based on differences in cell membrane lipid content is of significant research importance in tumor cell or tissue-specific fluorescence imaging. Summary of the Invention

[0004] This invention provides a type of lipid-directed fluorescent probe for cancer cells. The fluorescent probe utilizes carbon-carbon double bonds to link benzothiazole and quinoline portions, forming a donor-acceptor structure. This structure offers the advantage of nucleic acid responsiveness, and the probe molecule can distinguish between normal cells / tissues and cancer cells / tissues based on differences in cell membrane lipid content. It selectively enters cancer cells and binds to nucleic acids, generating fluorescence. This fluorescent probe can be used for tumor cell or tissue-specific fluorescence imaging applications based on nucleic acid detection.

[0005] To achieve the above objectives, the present invention provides the following technical solution: a type of lipid-guided fluorescent probe for cancer cells, having the following general structural formula I.

[0006]

[0007] in,

[0008] X1 is selected from C(CH3)2, O, S or Se;

[0009] A1 is selected from H, phenyl, or substituted phenyl;

[0010] R1 and R2 can be the same or different, and R1 and R2 are each independently selected from C1-C8 alkyl, C1-C8 hydroxy, C1-C8 carboxyl, C 1- 8NR 5 R 6 C1-C8 ester groups, benzyl groups, and substituted benzyl groups; the substituted benzyl groups are optionally substituted by the following groups: C1-C8 alkyl, C1-C8 alkoxy, CN, COOH, NH2, NO2, OH, SH, halogen, or C1-C8 haloalkyl groups;

[0011] Y - Selected from halide anions, ClO4 - PF6 - CH3COO - or OTs - One of them.

[0012] Furthermore, A1 is selected from H;

[0013] R1 and R2 are different; R1 is selected from benzyl or C1-C8 alkyl.

[0014] R2 is selected from C1-C8 alkyl or C1-C8 carboxyl groups;

[0015] X1 is S;

[0016] Furthermore, A1 is selected as free H; R1 is preferably methyl; R2 is selected as 5-carboxypentyl; and X1 is preferably S.

[0017] Furthermore, the fluorescent probe for cancer cells is:

[0018]

[0019] A method for preparing a type of lipid-directed fluorescent probe for cancer cells includes the following steps:

[0020] S1: Prepare a first intermediate and a second intermediate respectively. The first intermediate has the structure of general formula II, and the second intermediate has the structure of general formula III.

[0021]

[0022] In formula II, X1 is selected from C(CH3)2, O, S or Se;

[0023] In general formula III, X2 is selected from F, Cl, Br, or I, and R2 is selected from C1-C8 alkyl, C1-C8 hydroxyl, C1-C8 carboxyl, C1- 8NR 5 R 6 C1-C8 ester groups, benzyl groups, and substituted benzyl groups; the substituted benzyl groups are optionally substituted by the following groups: C1-C8 alkyl, C1-C8 alkoxy, CN, COOH, NH2, NO2, OH, SH, halogen, or C1-C8 haloalkyl groups;

[0024] S2: Mix the first intermediate and the first chemical reagent evenly at a molar ratio of 1:2-5, then add the second intermediate and the catalyst, and react at 80-100℃ for 4-6 hours under an inert atmosphere to obtain the target fluorescent probe.

[0025] Furthermore, in step S2, the reaction temperature is 80°C.

[0026] Further, in step S1, the first intermediate is prepared using the following method:

[0027] The compound having general structural formula IV and a first halogenated reagent were reacted in a second organic solvent under an inert atmosphere to prepare the product.

[0028]

[0029] The reaction time is 6-24 h, the reaction temperature is 80-110 °C, and the molar ratio of compound IV to the first halogenated reagent is 1:2-5. Furthermore, the reaction time is 8-20 h.

[0030] Further, in step S1, the second intermediate is prepared using the following method:

[0031] The compound having the general structural formula V and the second halogenated reagent are reacted in a third organic solvent under an inert atmosphere to prepare the product.

[0032]

[0033] The reaction time is 6-24 h, the reaction temperature is 80-110 °C, and the molar ratio of compound V to the second halogenated reagent is 1:2-5. Further, the reaction time is 8-20 h.

[0034] Further, in step S2, the molar ratio of the first intermediate to the second intermediate is 1:1.2-1.5; the catalyst is selected from inorganic or organic bases; the first chemical reagent is selected from anhydrous ethanol, anhydrous methanol, or anhydrous acetonitrile. Even further, the catalyst is selected from NaHCO3.

[0035] Further, the first halogenated reagent is selected from C1-C8 alkyl, C1-C8 hydroxyl, C1-C8 carboxyl, or C1-C8 substituted with Cl, Br, or I. 1-8 NR5 R 6 C1-C8 ester groups, benzyl groups, and substituted benzyl groups; the substituted benzyl groups are optionally substituted by the following groups: C1-C8 alkyl, C1-C8 alkoxy, CN, COOH, NH2, NO2, OH, SH, halogen, or C1-C8 haloalkyl.

[0036] The second organic solvent is selected from any one of ethanol, methanol, acetonitrile, DMF, or ethylene glycol monomethyl ether.

[0037] Furthermore, the second halogenated reagent is selected from C1-C8 alkyl, C1-C8 hydroxyl, C1-C8 carboxyl, or C1-C8 substituted with Cl, Br, or I. 1-8 NR 5 R 6 C1-C8 ester groups, benzyl groups, and substituted benzyl groups; the substituted benzyl groups are optionally substituted by the following groups: C1-C8 alkyl, C1-C8 alkoxy, CN, COOH, NH2, NO2, OH, SH, halogen, or C1-C8 haloalkyl.

[0038] The third organic solvent is selected from any one of ethanol, methanol, acetonitrile, DMF, or ethylene glycol monomethyl ether.

[0039] Application of a type of lipid-guided fluorescent probe for cancer cells, wherein the fluorescent probe is used in tumor cell or tissue-specific fluorescence imaging based on nucleic acid detection.

[0040] Furthermore, a type of lipid-guided fluorescent probe for cancer cells is being used to detect tumor cells.

[0041] Furthermore, the cancers mentioned include cervical cancer, lung cancer, or breast cancer.

[0042] Furthermore, the method of using lipid-guided fluorescent probes to identify cancer cells is as follows:

[0043] S1. Collect surgical specimens from the patient and prepare slides; or take normal cancer cells / tissue.

[0044] S2. Incubate the tissue section or live cells with the fluorescent probe and perform fluorescence confocal imaging on the tissue section or live cells. When the tissue section or live cells show obvious bright fluorescence signals, it proves that they are cancer cells or cancerous tissue.

[0045] In summary, the present invention has the following beneficial effects:

[0046] 1. Thiazole orange compounds, due to their donor-acceptor structure, can induce intramolecular charge transfer (ICT). The probe molecules exhibit extremely weak autofluorescence, and their positive charge provides excellent affinity for negatively charged nucleic acid molecules, enhancing their binding affinity. After binding to the nucleic acid groove, molecular rotation is restricted, resulting in fluorescence. The fluorescence is enhanced by 46 times (DNA) and 30.75 times (RNA) before and after binding, demonstrating the excellent responsiveness of this invention to nucleic acids.

[0047] 2. Thiazole orange compounds have multiple functional group modification sites, so by introducing hydrophobic alkyl chains and hydrophilic carboxylic acid groups at quinoline, lipid-type fluorescent probes can be constructed and selectively enter cancer cells through cell membrane lipid guidance. Therefore, this invention has a good distinguishing effect between normal cells and tumor cells.

[0048] 3. Since the present invention can selectively enter cancer cells and generate fluorescence without time dependence, it can distinguish between normal cells and cancer cells in a short time (0.5h). Therefore, the present invention can be adapted to flow cytometer and applied to distinguish between normal cells and tumor cells at the single-cell level. Attached Figure Description

[0049] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0050] Figure 1 The images shown in Examples 1-6 of this invention are laser confocal images of the fluorescent probes against normal cells (COS-7 cells) and tumor cells (MCF-7 cells).

[0051] Figure 2 This is the absorption and emission spectrum of the fluorescent probe TO-Ac at different concentrations disclosed in Example 6 of the present invention;

[0052] Figure 3 This is the nucleic acid titration spectrum of the fluorescent probe TO-Ac disclosed in Example 6 of the present invention;

[0053] Figure 4 This is a laser confocal imaging image of normal tissue cells 3T3 cells and COS-7 cells stained by the fluorescent probe TO-Ac disclosed in Example 6 of the present invention;

[0054] Figure 5This is a laser confocal imaging image of HepG2 and MCF-7 tumor tissue cells stained by the fluorescent probe TO-Ac as disclosed in Example 6 of the present invention;

[0055] Figure 6 This is a laser confocal imaging image of four types of fixed cells (HepG2 cells, MCF-7 cells, 3T3 cells and COS-7 cells) stained by the fluorescent probe TO-Ac as disclosed in Example 6 of the present invention;

[0056] Figure 7 This is a laser confocal imaging image of HepG2 cells and COS-7 cells stained with the fluorescent probe TO-Ac as disclosed in Example 6 of the present invention;

[0057] Figure 8 This is a classification test diagram of normal cells and tumor cells using the fluorescent probe TO-Ac in a flow cytometer, as disclosed in Embodiment 6 of the present invention. Detailed Implementation

[0058] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the following will be described in conjunction with the appendices in the embodiments of the present invention. Figure 1-8 The technical solutions in the embodiments of the present invention are clearly and completely described herein. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0059] In this application, unless otherwise expressly stated, percentages and contents are all expressed by mass. Unless otherwise specified, the experimental methods used are conventional methods, and all materials and reagents used are commercially available.

[0060] The fluorescent probe shown in Formula I will be described in detail below with reference to the embodiments.

[0061]

[0062] In formula I,

[0063] X1 is selected from C(CH3)2, O, S or Se;

[0064] A1 is selected from H, phenyl, or substituted phenyl;

[0065] R1 and R2 can be the same or different, and R1 and R2 are each independently selected from C1-C8 alkyl, C1-C8 hydroxy, C1-C8 carboxyl, C 1- 8NR 5 R 6, benzyl and substituted benzyl; the substituted benzyl is optionally substituted by the following groups: C1-C8 alkyl, C1-C8 alkoxy, CN, COOH, NH2, NO2, OH, SH, halogen or C1-C8 haloalkyl;

[0066] Y - Selected from halide anions, ClO4 - PF6 - CH3COO - or OTs - One of them.

[0067] The following are specific examples of fluorescent probes represented by general formula I, but the present invention is not limited to these specific examples.

[0068]

[0069] The reaction mechanism of the compound represented by general formula I in this invention is as follows:

[0070]

[0071] The present invention can be synthesized from compounds represented by general formula I by the methods described below.

[0072] Preparation examples of raw materials and intermediates

[0073] Preparation Example 1

[0074] Synthesis of intermediate 1:

[0075] Dimethylbenzothiazole (4.00 mmol) was added to a two-necked round-bottom flask containing 5 mL of anhydrous acetonitrile, followed by benzyl bromide (20.00 mmol). The entire reaction system was protected under nitrogen atmosphere, magnetically stirred at 200-300 rpm, and the reaction temperature was controlled at approximately 120 °C for 20 h. After the reaction was complete, the reaction solvent was evaporated under vacuum, and then excess ethyl acetate was added to crystallize the mixture. Filtration yielded white intermediate 1, with a yield of 66.70%.

[0076] Preparation Example 2

[0077] Synthesis of intermediate 3:

[0078] The only difference from Preparation Example 1 was that iodomethane (20.00 mmol) was used instead of benzyl bromide (20.00 mmol), and the mixture was filtered to obtain a pale white intermediate 3 with a yield of 82.10%.

[0079] Preparation Example 3

[0080] Synthesis of intermediate 7:

[0081] The only difference from Preparation Example 1 was that 20.00 mmol of benzyl bromide was replaced with 20.00 mmol of iodoethane. The mixture was filtered to obtain a pale white intermediate 7 with a yield of 88.00%.

[0082] Example

[0083] Example 1

[0084] The synthesis uses R1 as benzyl, R2 as methyl, A1 as hydrogen atom, X1 as sulfur atom, and Y as... - For I - The fluorescent probe TO-ph has the following structural formula:

[0085]

[0086] The synthesis of the fluorescent probe TO-ph includes the following steps:

[0087] S1: 2.00 mmol of 4-iodoquinoline was added to a two-necked round-bottom flask containing 5 mL of anhydrous acetonitrile. Then, 10.00 mmol of iodomethane was added to the round-bottom flask. The entire reaction system was protected under nitrogen atmosphere, magnetically stirred, and the reaction temperature was controlled at approximately 120 °C for 20 h. After the reaction was completed, the reaction solvent was evaporated under vacuum, and then excess ethyl acetate was added to crystallize the mixture. The crystals were filtered to obtain a pale yellow intermediate 2 with a yield of 20.55%.

[0088] S2: Intermediate 1 (1.25 mmol) was added to a two-necked round-bottom flask containing 5 mL of anhydrous methanol. Then, intermediate 2 (1.5 mmol) obtained in step S1 was added to the round-bottom flask. Finally, sodium bicarbonate (0.09 mmol) was added. The entire reaction system was protected by nitrogen, magnetically stirred, and the reaction temperature was controlled at about 80 °C. The reaction time was 4-6 h. After the reaction was completed, the reaction solvent was vacuum rotary evaporated. A mixed solution of dichloromethane and methanol (100:5) was selected as the eluent. The pink-orange solid fluorescent dye TO-ph was obtained by column chromatography with a yield of 15.00%.

[0089] 1H NMR (400MHz, DMSO-d6) δ8.67(d,J=7.1Hz,1H),8.54(d,J=8.5Hz,1H),8.07(dd,J=8.2,4.5Hz,2H),8.00(dd,J=9.3,6.3Hz,1H), 7.77(t,J=7.8Hz,2H),7.58(t,J=7.8Hz,1H),7.43–7.35(m,5H),7.30(t,J=6.8Hz,1H),6.98(s,1H),5.95(s,2H),4.19(s,3H).

[0090] Example 2

[0091] The synthesis uses R1 as methyl, R2 as 5-ethoxycarbonylpentyl, A1 as hydrogen atom, X1 as sulfur atom, and Y as... - For I - The fluorescent probe TO-AcOEt has the following structural formula:

[0092]

[0093] The synthesis method of the fluorescent probe TO-AcOEt is as follows:

[0094] S1: 2.00 mmol of 4-iodoquinoline was added to a two-necked round-bottom flask containing 5 mL of anhydrous acetonitrile. Then, 10.00 mmol of ethyl 5-bromopentanoate was added to the round-bottom flask. The entire reaction system was protected under nitrogen atmosphere, magnetically stirred, and the reaction temperature was controlled at approximately 120 °C for 20 h. After the reaction was completed, the reaction solvent was evaporated under vacuum, and then excess ethyl acetate was added to crystallize the mixture. The crystals were filtered to obtain a pale yellow-white intermediate 4, with a yield of 65.20%.

[0095] S2: Intermediate 3 (1.25 mmol) was added to a two-necked round-bottom flask containing 5 mL of anhydrous methanol. Then, intermediate 4 (1.5 mmol) obtained in step S1 was added to the round-bottom flask. Finally, sodium bicarbonate (0.09 mmol) was added. The entire reaction system was protected by nitrogen, magnetically stirred, and the reaction temperature was controlled at about 80 °C. The reaction time was 4-6 h. After the reaction was completed, the reaction solvent was vacuum rotary evaporated. A mixed solution of dichloromethane and methanol (100:5) was selected as the eluent. The pink-orange solid fluorescent dye TO-AcOEt was obtained by column chromatography with a yield of 23.00%.

[0096] 1H NMR (400MHz, DMSO-d6) δ8.81(d,J=8.5Hz,1H),8.66(d,J=7.2Hz,1H),8.14(d,J=8.8Hz,1H),8.06 (d,J=7.9Hz,1H),8.00(t,J=7.8Hz,1H),7.82–7.73(m,2H),7.62(t,J=7.8Hz,1H),7.43(t,J=7.6 Hz,1H),7.36(d,J=7.1Hz,1H),6.93(s,1H),4.60(t,J=7.4Hz,2H),3.11(q,J=7.3Hz,6H),2.88(s ,2H),2.30(t,J=7.3Hz,2H),1.86(p,J=7.1Hz,2H),1.58(p,J=7.4Hz,2H),1.38(p,J=7.8Hz,2H).

[0097] Example 3

[0098] The synthesis uses R1 as methyl, R2 as 6-carboxyhexyl, A1 as hydrogen atom, X1 as sulfur atom, and Y as... - For I - The fluorescent probe TO-6C-Ac has the following structural formula:

[0099]

[0100] The synthesis method of the fluorescent probe TO-6C-Ac is as follows:

[0101] S1: 2.00 mmol of 4-iodoquinoline was added to a two-necked round-bottom flask containing 5 mL of anhydrous acetonitrile. Then, 10.00 mmol of 7-bromoheptanoic acid was added to the round-bottom flask. The entire reaction system was protected under nitrogen atmosphere, magnetically stirred, and the reaction temperature was controlled at approximately 120 °C for 20 h. After the reaction was completed, the reaction solvent was evaporated under vacuum, and then excess ethyl acetate was added to crystallize the mixture. The crystals were filtered to obtain a yellowish-white intermediate 5 with a yield of 20.55%.

[0102] S2: Intermediate 3 (1.25 mmol) was added to a two-necked round-bottom flask containing 5 mL of anhydrous methanol. Then, intermediate 5 (1.5 mmol) obtained in step S1 was added to the round-bottom flask. Finally, sodium bicarbonate (0.09 mmol) was added. The entire reaction system was protected by nitrogen, magnetically stirred, and the reaction temperature was controlled at about 80 °C. The reaction time was 4-6 h. After the reaction was completed, the reaction solvent was vacuum rotary evaporated. A mixed solution of dichloromethane and methanol (100:5) was selected as the eluent. The pink-orange solid fluorescent dye TO-6C-Ac was obtained by column chromatography with a yield of 14.70%.

[0103] 1H NMR (400MHz, DMSO-d6) δ8.80(d,J=8.6Hz,1H),8.64(d,J=7.2Hz,1H),8.15(d,J=8.8Hz,1H) ,8.06(d,J=7.9Hz,1H),8.00(t,J=7.8Hz,1H),7.82–7.74(m,2H),7.62(t,J=7.8Hz,1H),7.4 3(t,J=7.6Hz,1H),7.38(d,J=7.1Hz,1H),6.94(s,1H),4.59(t,J=7.4Hz,2H),4.03(s,3H), 2.19(t,J=7.3Hz,2H), 1.85(t,J=7.3Hz,2H), 1.50(p,J=7.2Hz,2H), 1.34(d,J=10.5Hz,4H).

[0104] Example 4

[0105] The synthesis uses R1 as methyl, R2 as 4-carboxybutyl, A1 as hydrogen atom, X1 as sulfur atom, and Y as... - For I - The fluorescent probe TO-4C-Ac has the following structural formula:

[0106]

[0107] The synthesis method of the fluorescent probe TO-4C-Ac is as follows:

[0108] S1: 2.00 mmol of 4-iodoquinoline was added to a two-necked round-bottom flask containing 5 mL of anhydrous acetonitrile. Then, 10.00 mmol of 5-bromopentanoic acid was added to the round-bottom flask. The entire reaction system was protected under nitrogen atmosphere, magnetically stirred, and the reaction temperature was controlled at approximately 120 °C for 20 h. After the reaction was completed, the reaction solvent was evaporated under vacuum, and then excess ethyl acetate was added to crystallize the mixture. The crystals were filtered to obtain a grayish-white intermediate 6 with a yield of 23.10%.

[0109] S2: Intermediate 3 (1.25 mmol) was added to a two-necked round-bottom flask containing 5 mL of anhydrous methanol. Then, intermediate 6 (1.5 mmol) obtained in step S1 was added to the round-bottom flask. Finally, sodium bicarbonate (0.09 mmol) was added. The entire reaction system was protected by nitrogen, magnetically stirred, and the reaction temperature was controlled at about 80 °C. The reaction time was 4-6 h. After the reaction was completed, the reaction solvent was vacuum rotary evaporated. A mixed solution of dichloromethane and methanol (100:5) was selected as the eluent. The orange solid fluorescent dye TO-4C-Ac was obtained by column chromatography with a yield of 14.70%.

[0110] 1H NMR (400MHz, DMSO-d6) δ8.67(d,J=7.1Hz,1H),8.54(d,J=8.5Hz,1H),8.07(dd,J=8.2,4.5Hz,2H),8.00(dd,J=9.3,6.3Hz,1H), 7.77(t,J=7.8Hz,2H),7.58(t,J=7.8Hz,1H),7.43–7.35(m,5H),7.30(t,J=6.8Hz,1H),6.98(s,1H),5.95(s,2H),4.19(s,3H).

[0111] Example 5

[0112] The synthesis uses R1 as ethyl, R2 as 5-carboxypentyl, A1 as a hydrogen atom, X1 as a sulfur atom, and Y as the base group. - For I - The fluorescent probe EtTO-Ac has the following structural formula:

[0113]

[0114] The synthesis method of the fluorescent probe EtTO-Ac is as follows:

[0115] S1: 2.05 mmol of 4-iodoquinoline was added to a two-necked round-bottom flask containing 5 mL of anhydrous acetonitrile. Then, 10.25 mmol of 6-bromohexanoic acid was added to the round-bottom flask. The entire reaction system was protected under nitrogen atmosphere, magnetically stirred, and the reaction temperature was controlled at approximately 80 °C for 20 h. After the reaction was completed, the reaction solvent was evaporated under vacuum, and then excess ethyl acetate was added to crystallize the mixture. The crystals were filtered to obtain a yellowish-white intermediate 8 with a yield of 20.55%.

[0116] S2: Intermediate 7 (1.25 mmol) was added to a two-necked round-bottom flask containing 5 mL of anhydrous methanol, then intermediate 8 (1.5 mmol) was added to the round-bottom flask, and finally sodium bicarbonate (0.09 mmol) was added. The entire reaction system was protected by nitrogen, magnetically stirred, and the reaction temperature was controlled at about 80 °C for 4-6 h. After the reaction was completed, the reaction solvent was vacuum rotary evaporated, and a mixed solution of dichloromethane and methanol (100:5) was selected as the eluent. The orange solid fluorescent dye EtTO-Ac was obtained by column chromatography with a yield of 15.73%.

[0117] 1H NMR (400MHz, DMSO-d6) δ8.80(d,J=8.5Hz,1H),8.65(d,J=7.2Hz,1H),8.15(d,J=8.8Hz,1H),8 .06(dd,J=8.0,3.5Hz,1H),8.00(t,J=7.9Hz,1H),7.83–7.74(m,2H),7.63(t,J=7.7Hz,1H),7. 47–7.36(m,2H),6.95(d,J=6.6Hz,1H),4.68(t,J=7.2Hz,1H),4.61(t,J=7.5Hz,2H),4.03(s,1 H), 2.22 (t, J = 7.2Hz, 2H), 1.87 (p, J = 7.7Hz, 2H), 1.56 (p, J = 7.4Hz, 2H), 1.39 (q, J = 6.9Hz, 4H).

[0118] Example 6

[0119] The synthesis uses R1 as methyl, R2 as 5-carboxypentyl, A1 as hydrogen atom, X1 as sulfur atom, and Y as... - For I - The fluorescent probe TO-Ac has the following structural formula:

[0120]

[0121] The synthesis method of the fluorescent probe TO-Ac is as follows:

[0122] S1: Same as Example 5;

[0123] S2: The only difference from Example 5 is that intermediate 7 (1.25 mmol) was replaced with intermediate 3 (1.25 mmol), and the orange solid fluorescent dye TO-Ac was obtained by column chromatography with a yield of 21.96%.

[0124] 1H NMR (400MHz, DMSO-d6) δ8.74(dd,J=7.4,2.8Hz,2H),8.08(d,J=8.7Hz,1H),7.98(dd,J=8.0, 1.2Hz, 1H), 7.93 (ddd, J=8.5, 6.9, 1.3Hz, 1H), 7.70 (td, J=8.0, 5.7Hz, 2H), 7.56 (ddd, J=8.4, 7.2,1.3Hz,1H),7.40–7.34(m,1H),7.27(d,J=7.1Hz,1H),6.84(s,1H),4.57(t,J=7.2Hz,2H) ,3.97(s,3H),1.82(q,J=7.8,7.3Hz,4H),1.49(p,J=7.0Hz,2H),1.32(qd,J=8.4,6.1Hz,2H).

[0125] Performance testing

[0126] The fluorescent probes prepared in Examples 1-6 above were used to stain normal cells and tumor cells, respectively, and the results were observed using a confocal laser scanning microscope. (See attached figures.) Figure 1 .

[0127] The concentration of fluorescent probes was 10 μM. Representative areas were selected for imaging using an OLYMPUS FV1000 laser confocal microscope with an excitation wavelength of 488 nm and a receiving wavelength of 520-550 nm.

[0128] Figure 1 The fluorescent probes prepared for Examples 1-6 were used to stain normal cells (COS-7 cells) and tumor cells (MCF-7 cells) (top: COS-7 cells, bottom: MCF-7 cells).

[0129] Depend on Figure 1As can be seen, because the fluorescent probe has a positive charge, it can specifically bind to intracellular nucleic acids to produce fluorescence. Moreover, its molecular weight is small, and it can enter the cell through passive diffusion. Examples 1-6 all have a certain staining ability on normal cells and tumor cells. Among them, the cell uptake experiment and confocal imaging results show that Examples 5 and 6 exhibit good cell differentiation ability and can selectively stain cancer cells without staining normal cells.

[0130] To comprehensively evaluate the performance of the above molecules, the fluorescent probe TO-Ac prepared in Example 6 is used as an example to illustrate the performance of the above molecules in distinguishing between normal cells and tumor cells.

[0131] 1. Fluorescence physical properties experiment

[0132] The fluorescent probe TO-Ac prepared in Example 6 was gradually added to PBS buffer (pH = 7.4) to achieve concentrations of 2, 4, 6, 8, 10, 12, and 14 μM, and the absorption and fluorescence emission spectra were recorded. The test results are as follows: Figure 2 As shown. The instruments used were an FP 6500 UV spectrophotometer and an Agilent Technologies Cary 60 fluorescence spectrophotometer.

[0133] Figure 2 (a) shows the absorption spectra of TO-Ac at different concentrations;

[0134] Figure 2 (b) shows the emission spectra of TO-Ac at different concentrations, with an excitation wavelength of 502 nm.

[0135] from Figure 2 As can be seen, due to the ICT effect of the molecule itself, the maximum absorption wavelength of TO-Ac is 502nm, and there is almost no fluorescence.

[0136] 2. Nucleic acid titration experiment

[0137] The fluorescent probe TO-Ac prepared in Example 6 was prepared to a concentration of 4 μM, and 0-140 μg / ml DNA and RNA solutions were gradually added, respectively. The test system was PBS buffer solution (pH = 7.4). The test results are shown in... Figure 3 .

[0138] Figure 3 (a) is the fluorescence titration spectrum of TO-Ac on DNA;

[0139] Figure 3 (b) is the fluorescence titration spectrum of RNA with TO-Ac;

[0140] Figure 3(c) Comparison of the fluorescence titration spectra of TO-Ac on DNA and RNA;

[0141] from Figure 3 As can be seen, the fluorescent probe TO-Ac can specifically bind to nucleic acids. After the probe molecule binds to the nucleic acid groove, it restricts double bond rotation and produces fluorescence. Nucleic acid titration experiments show that the fluorescent probe TO-Ac has good response to both DNA and RNA, with better response to DNA. The fluorescence is enhanced by 46 times (DNA) and 30.75 times (RNA) before and after binding to nucleic acids. At the same time, the fluorescence difference of the fluorescent probe TO-Ac before and after binding to nucleic acids is large, which can effectively and specifically recognize nucleic acids.

[0142] 3. Normal cell uptake experiment

[0143] The fluorescent probe TO-Ac prepared in Example 6 was prepared to a concentration of 10 μM and added to six culture dishes containing pre-incubated 3T3 and COS-7 cells (cell culture density 10⁵ cells / ml, 70-80% cell bottom coverage). Staining was performed at 37°C and 5% CO₂ for 6 h. Representative areas were imaged using an OLYMPUS FV1000 laser confocal microscope at time points of 0.5 h, 1 h, 2 h, 3 h, 4 h, 5 h, and 6 h. The excitation wavelength of TO-Ac was 502 nm, and the receiving wavelength was 520-550 nm. The experiment was repeated three times. The test results are shown in... Figure 4 .

[0144] Figure 4 (a) is a staining image of 3T3 cells by the fluorescent probe compound TO-Ac;

[0145] Figure 4 (b) is a staining diagram of COS-7 cells by the fluorescent probe compound TO-Ac.

[0146] from Figure 4 As can be seen, the fluorescent probe TO-Ac has difficulty entering 3T3 and COS-7 cells, remaining only on the cell membrane. Since the fluorescent probe does not enter normal cells, it cannot bind to intracellular nucleic acids to generate a fluorescent signal. Therefore, single-photon confocal imaging results show that no fluorescent signal was generated in normal cells within 6 hours of imaging.

[0147] 4. Tumor cell uptake experiment

[0148] The fluorescent probe TO-Ac prepared in Example 6 was prepared to a concentration of 10 μM and added to six culture dishes containing pre-incubated HepG2 and MCF-7 cells (cell culture density 10⁵ cells / ml, 70-80% cell bottom coverage). Staining was performed at 37°C and 5% CO₂ for 6 h. Representative areas were imaged using an OLYMPUS FV1000 laser confocal microscope at time points of 0.5 h, 1 h, 2 h, 3 h, 4 h, 5 h, and 6 h. The excitation wavelength of TO-Ac was 502 nm, and the receiving wavelength was 520-550 nm. The experiment was repeated three times. The test results are shown in... Figure 5 .

[0149] Figure 5 (a) is a staining diagram of HepG2 cells by the fluorescent probe compound TO-Ac;

[0150] Figure 5 (b) is a staining diagram of MCF-7 cells by the fluorescent probe compound TO-Ac.

[0151] As can be seen from Figure 5, the fluorescent probe TO-Ac can rapidly enter tumor cells and specifically bind to intracellular nucleic acids to generate fluorescent signals. Single-photon confocal imaging shows that fluorescent signals are generated inside tumor cells at 0.5 h, and the fluorescent signals remain stable during 6 h interval imaging.

[0152] 5. Fixed cell staining experiment

[0153] The fluorescent probe TO-Ac prepared in Example 6 was prepared to a concentration of 10 μM.

[0154] Four types of fixed cell lines (HepG2, MCF-7, 3T3, and COS-7) were divided into four groups, with three culture dishes in each group. The cells were fixed with ice-cold ethanol, and after 0.5 h, TO-Ac (10 μM) was added and incubated for 2 h. Representative areas were selected for imaging using an OLYMPUS FV1000 laser confocal microscope. The excitation wavelength of TO-Ac was 502 nm, and the receiving band was 520-550 nm. The experiment was repeated three times. The test results are shown in... Figure 6 .

[0155] Figure 6 (a) is a staining diagram of fixed HepG2 cells;

[0156] Figure 6 (b) is a staining image of fixed MCF-7 cells;

[0157] Figure 6 (c) is a staining image of fixed 3T3 cells;

[0158] Figure 6 (d) is a staining image of fixed COS-7 cells.

[0159] from Figure 6 As can be seen, due to the fixation of cells, the cell membrane permeability is enhanced, and the fluorescent probe TO-Ac can enter the fixed cells and generate fluorescence after binding with intracellular nucleic acids. After cell fixation, both normal cells and tumor cells show fluorescent staining, further proving that the fluorescent probe TO-Ac can distinguish between normal cells and cancer cells.

[0160] 6. Mixed cell staining experiment

[0161] The fluorescent probe TO-Ac prepared in Example 6 was prepared to a concentration of 10 μM and added to three culture dishes containing HepG2 cells and COS-7 cells (cell culture density 10⁵ cells / ml, 70-80% coverage of the dish bottom). Staining was performed at 37°C and 5% CO₂ for 0.5 h. Representative areas were selected and imaged using an OLYMPUS FV1000 laser confocal microscope. The excitation wavelength of TO-Ac was 502 nm, and the receiving wavelength was 520-550 nm. The experiment was repeated three times. The test results are shown in... Figure 7 .

[0162] from Figure 7 As can be seen, after co-culturing normal cells and tumor cells, the fluorescent probe TO-Ac can selectively perform fluorescence imaging on HepG2 cells with good selectivity (the red circle represents normal COS-7 cells).

[0163] 7. Flow cytometry staining experiment

[0164] The fluorescent probe TO-Ac prepared in Example 6 was prepared to a concentration of 10 μM and added to a six-well plate containing HepG2 and COS-7 cells (cell culture density 10⁵ cells / ml, 70-80% coverage of the plate bottom). The plate was stained at 37°C and 5% CO₂ for 0.5 h. Then, the cells were digested with 1.5 ml of trypsin at 37°C and 5% CO₂ for 2 min. After scraping off the cells, the plate was centrifuged, and the cell pellet was collected. 0.5 ml of deionized water was added, and the mixture was thoroughly mixed. Cell counting was performed using a Thermo Fisher flow cytometer. The excitation wavelength of TO-Ac was 488 nm, and the receiving wavelength was 520-530 nm. The test results showed... Figure 8 .

[0165] from Figure 8 As can be seen from the results, the fluorescent probe TO-Ac has good distinguishability between cancer cells (tumor cells) and normal cells. The cell flow cytometry results show that the fluorescence intensity of cancer cells is stronger.

[0166] 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 fluorescent probe, characterized in that, It has the following general structural formula I Wherein, R1 is benzyl, R2 is methyl, A1 is a hydrogen atom, X1 is a sulfur atom, and Y... - For I - .

2. The method for preparing a fluorescent probe according to claim 1, characterized in that, Includes the following steps: S1: Prepare a first intermediate and a second intermediate respectively. The first intermediate has the structure of general formula II, and the second intermediate has the structure of general formula III. In formula II, R1 is benzyl, A1 is a hydrogen atom, and X1 is a sulfur atom; In general formula III, X2 is selected from Cl, Br or I, and R2 is a methyl group; S2: Mix the first intermediate and the first chemical reagent evenly at a molar ratio of 1:2-5, then add the second intermediate and the catalyst, and react at 80-100℃ for 4-6 hours under an inert atmosphere to obtain the target fluorescent probe.

3. The method for preparing a fluorescent probe according to claim 2, characterized in that, In step S1, the first intermediate is prepared using the following method: The compound having general structural formula IV and a first halogenated reagent were reacted in a second organic solvent under an inert atmosphere to prepare the product. The reaction time is 6-24 h, the reaction temperature is 80-110 °C, and the molar ratio of compound IV to the first halogenated reagent is 1:2-5.

4. The method for preparing a fluorescent probe according to claim 2, characterized in that, In step S1, the second intermediate is prepared using the following method: The compound having the general structural formula V and the second halogenated reagent are reacted in a third organic solvent under an inert atmosphere to prepare the product. The reaction time is 6-24 h, the reaction temperature is 80-110 °C, and the molar ratio of compound V to the second halogenated reagent is 1:2-5.

5. The method for preparing a fluorescent probe according to claim 2, characterized in that, In step S2, the molar ratio of the first intermediate to the second intermediate is 1:1.2-1.5; The catalyst is selected from inorganic or organic bases; the first chemical reagent is selected from anhydrous ethanol, anhydrous methanol, or anhydrous acetonitrile.

6. The method for preparing a fluorescent probe according to claim 3, characterized in that, The first halogenated reagent is selected from a benzyl group substituted with Cl, Br, or I; The second organic solvent is selected from any one of ethanol, methanol, acetonitrile, DMF, or ethylene glycol monomethyl ether.

7. The method for preparing a fluorescent probe according to claim 4, characterized in that, The second halogenated reagent is selected from methyl groups substituted with Cl, Br, or I; The third organic solvent is selected from any one of ethanol, methanol, acetonitrile, DMF, or ethylene glycol monomethyl ether.

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