A naphthyridine-based compound fluorescent dye and its application in tumor cell imaging

Abstract

The present invention belongs to the field of biomedical technologies, and specifically provides a naphthyridene-based compound fluorescent dye and its application in tumor cell imaging. The fluorescent dye includes a naphthyridyl group as an electron acceptor and an amino-substituted phenyl group as an electron donor, and the two are connected by an alkenyl group to form a conjugated donor-acceptor conjugate system (D-A system) to achieve luminescence. Among them, the naphthyridyl group of the electron acceptor is composed of two pyridine rings joined together. Compared with the existing pyridine, pyrimidine, pyrazine, quinoline groups, etc., it can optimize the luminescence performance of the dye more effectively, contribute to obtaining higher fluorescence brightness and prominent photostability, and is suitable for use in tumor cell fluorescence imaging. Similarly, at least one hydroxyalkyl group is included in the amino-substituted phenyl group of the electron donor, and the dipole moment of the molecule will be further increased, making the molecule more sensitive to the tumor cell environment. It is particularly suitable for in vitro detection and in vivo imaging of tumors, showing imaging advantages of high resolution and high signal-to-noise ratio.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a naphthyridine-based compound fluorescent dye and its application in tumor cell imaging. Background Art

[0002] Biological imaging is an important research method for understanding the organizational structure of organisms and clarifying various physiological functions of organisms. Due to its advantages such as high sensitivity, high resolution, intuitive imaging, fast imaging speed, and non-destructive detection, biological imaging has been widely used in the fields of scientific research and biomedical diagnosis. Biological imaging also has important practical application values in exploring the pathogenesis, clinical manifestations, and genetic lesions of diseases, understanding the corresponding physiological and pathological information, diagnosing diseases, and developing new medical methods. Currently, in biological imaging, fluorescence imaging technology has become a hot spot in the field of tumor diagnosis research due to its advantages of timely and accurate detection of tumors, continuous and non-invasive observation of tumor changes, fast detection time, and no need to inject substrates.

[0003] In recent years, in order to achieve non-invasive monitoring of tumors in situ, in real-time, and in a targeted manner, as well as higher-precision fluorescence imaging, higher requirements have been put forward for fluorescent dyes. Traditional fluorescent dyes can be classified into the following major categories: acridine orange, ethidium bromide, and propidium iodide, DAPI, Hoechst dyes, EthD III, 7-AAD, RedDot1, RedDot2, etc. Acridine orange has membrane permeability and can penetrate the cell membrane, staining nuclear DNA and RNA green and red respectively, thus making the cell nucleus show green or yellow-green fluorescence. Ethidium bromide is a highly sensitive fluorescent stain that emits an orange-red signal when excited at a standard 302 nm. DAPI is a blue fluorescent dye that can penetrate the cell membrane. When it binds to DNA, it can produce fluorescence more than 20 times stronger than that of DAPI itself, while there is no fluorescence enhancement when binding to single-stranded DNA. The staining sensitivity of DAPI to double-stranded DNA is higher than that of EB and propidium iodide (PI), and its fluorescence intensity is lower than that of Hoechst, but its photostability is higher than that of Hoechst. Hoechst dyes are a class of fluorescent dyes that label DNA in microscopic observations. The two most common ones are Hoechst33342 and Hoechst33258. Both of these dyes are excited at ultraviolet 350 nm and emit cyan / blue fluorescence near the maximum emission wavelength at 461 nm. RedDot 1 dye has extremely strong nuclear selectivity, and RedDot1 dye can be excited by several common lasers and can emit fluorescence in the far-infrared region. The red near-infrared fluorescence of RedDot1 can effectively distinguish it from other commonly used fluorescent probes. PI, propidium iodide, cannot pass through the live cell membrane but can penetrate the dead cell membrane and stain the nucleus. PI is the first choice as a red fluorescent counterstain and is often used in combination with fluorescent probes such as Calcein-AM or FDA to distinguish dead / live cells. EthD III, 7-AAD, and RedDot 2 also cannot penetrate the live cell membrane but can distinguish necrotic cells and are more suitable for the detection of apoptosis and necrosis experiments. The optimal excitation wavelengths of traditional dyes targeting the cell nucleus, such as the DAPI series and the Hoechst series, are only about 360 nm, which cannot be effectively excited by existing commercial lasers. Moreover, the light energy at this wavelength is relatively high, not only causing greater phototoxicity to cells but also easily resulting in the phenomenon of photobleaching of the dye.

[0004] As can be seen from the above, excellent fluorescent dyes should have good photostability, biocompatibility, high absorbance, high brightness, and lower cytotoxicity, etc. However, among the fluorescent dyes on the domestic and foreign markets at present, there are not many compound systems that meet the above requirements, and some staining procedures are complex, requiring washing and being inconvenient to operate. Summary of the Invention

[0005] Based on the above technical problems, the present invention provides a naphthyridine-based compound fluorescent dye and its application in tumor cell imaging. The fluorescent dye has high staining intensity, stable fluorescence emission, fast imaging speed, and certain stability and anti-photobleaching properties, showing imaging advantages of high resolution and high signal-to-noise ratio.

[0006] A naphthyridine-based compound fluorescent dye proposed by the present invention has the following general structural formula:

[0007]

[0008] Wherein, R1 or R2 is independently hydrogen or an organic electron-donating group, R3 is an alkyl group or a hydroxyalkyl group, and n is 1-5.

[0009] In the present invention, the fluorescent dye includes a naphthyridinyl group as an electron acceptor and an amino-substituted phenyl group as an electron donor. The two are connected by an alkenyl group to form a conjugated donor-acceptor conjugate system (D-A system) to achieve luminescence. Among them, the naphthyridinyl group of the electron acceptor is composed of two pyridine rings joined together. Compared with existing pyridine, pyrimidine, pyrazine, quinoline groups, etc., it can optimize the luminescence performance of the dye more, help obtain higher fluorescence brightness and outstanding photostability, and is suitable for use in tumor cell fluorescence imaging. Similarly, at least one hydroxyalkyl group is included in the amino-substituted phenyl group of the electron donor, and the dipole moment of the molecule will further increase, making the molecule more sensitive to the tumor cell environment, and is particularly suitable for in vitro detection and in vivo imaging of tumors.

[0010] In the present invention, when R1 or R2 is an organic electron-donating group, after the "organic electron-withdrawing group" is used as a substituent to replace the hydrogen on the naphthyridinyl group, the electron cloud density at the N atom on the naphthyridinyl group will increase accordingly, and its basicity will also increase. In this way, it is easier to accept protons, so it is more sensitive to the tumor cell environment and ultimately more suitable for in vitro detection and in vivo imaging of tumors. In actual use, R1 or R2 is independently a hydroxy group, an amino group, a mercapto group, a C1-C6 alkyl group, or a C1-C6 alkoxy group.

[0011] Preferably, R1 or R2 is hydrogen, R3 is methyl, and n is 2;

[0012] The structural formula of the fluorescent dye is as follows:

[0013] 。

[0014] Preferably, R1 or R2 is hydrogen, R3 is 2-hydroxyethyl, and n is 2;

[0015] The structural formula of the fluorescent dye is as follows:

[0016] 。

[0017] Preferably, the fluorescent dye is obtained by performing an aldol condensation reaction on the compound shown in Structural Formula I and the compound shown in Structural Formula II;

[0018]

[0019] Preferably, the molar ratio of the compound shown in Structural Formula I to the compound shown in Structural Formula II is 1:0.9 - 1.1.

[0020] The present invention also proposes an application of the above fluorescent dye in tumor cell imaging.

[0021] Preferably, the application is to use the fluorescent dye configured into a dye solution for tumor cell imaging.

[0022] The present invention simultaneously proposes an application of the above fluorescent dye in the preparation of a tumor diagnostic imaging agent.

[0023] Preferably, the tumor includes breast cancer or lung cancer.

[0024] Compared with existing fluorescent dyes, the fluorescent dye of the present invention has the properties of low toxicity and strong fluorescence emission, has a very strong imaging ability for tumor cells, can quickly image in cells, and has a certain stability and anti-photobleaching property. Therefore, it can be well applied to cell imaging in vivo and in vitro, especially for in vitro detection and in vivo imaging of tumors, showing imaging advantages of high resolution and high signal-to-noise ratio. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 It is a fluorescence imaging photo of the fluorescent dye described in Example 1 of the present invention for 4T1 orthotopic breast cancer tumor mice;

[0026] Figure 2 It is a fluorescence imaging photo of the fluorescent dye described in Example 1 of the present invention for MDA MB231 tumor mice;

[0027] Figure 3 It is a fluorescence imaging photo of the fluorescent dye described in Example 1 of the present invention for MDA MB435 tumor mice;

[0028] Figure 4 It is a fluorescence imaging photo of the fluorescent dye described in Example 1 of the present invention for A549R subcutaneous tumor mice;

[0029] Figure 5 It is a fluorescence imaging photo of the fluorescent dye described in Example 1 of the present invention for 4T1 lung metastasis tumor mice. DETAILED DESCRIPTION OF THE INVENTION

[0030] ​Next, the present invention will specifically illustrate the technical solution through specific embodiments. However, it should be clearly stated that these embodiments are for illustrative purposes only and are not to be construed as limiting the scope of the present invention.

[0031] Example 1

[0032] This example presents a naphthyridine-based compound fluorescent dye, and its structural formula is shown as follows:

[0033]

[0034] The specific synthesis method is as follows:

[0035] After completely dissolving the compound shown in structural formula a1 (1.44 g, 1 eq) and p-toluenesulfonamide (1.71 g, 1 eq) in toluene (15 mL), then adding the compound shown in structural formula b1 (1.78 g, 1 eq), stirring and refluxing for 96 h, cooling to room temperature, spinning off the solvent, adding the obtained solid to ethanol for recrystallization, filtering by suction, and washing with alcohol, the fluorescent dye is obtained (yield 93.2%, purity 99.7%);

[0036]

[0037] The NMR characterization of the product is as follows: 1 H NMR (400 MHz, DMSO) δ 8.96( d, J = 8.1 Hz, 1H ), 8.51( dd, J = 12.6, 7.5 Hz, 2H ), 7.84( d, J = 8.7 Hz, 2H ), 7.76 - 7.61( m, 2H ), 7.58 - 7.46( m, 2H ), 6.78( d, J = 8.9 Hz, 2H ), 4.75( t, J = 5.9 Hz, 1H ), 3.61( q, J = 7.0 Hz, 2H ), 3.48( t, J = 7.5 Hz, 2H ), 3.06( s, 3H ).

[0038] Example 2

[0039] This example presents a naphthyridine-based compound fluorescent dye, and its structural formula is shown as follows:

[0040]

[0041] The specific synthesis method is as shown in Example 1, except that the compound shown in structural formula b1 is replaced with an equivalent amount of the compound shown in structural formula b2;

[0042]

[0043] The NMR characterization of the product is as follows: 11H NMR (400 MHz, DMSO) δ 9.02 (d, J = 7.6 Hz, 1H), 8.46 (dd, J = 13.8, 8.2 Hz, 2H), 7.85 - 7.75 (m, 2H), 7.67 - 7.55 (m, 2H), 7.50 (d, J = 9.3 Hz, 1H), 7.32 (d, J = 13.5 Hz, 1H), 6.84 (d, J = 7.9 Hz, 2H), 4.79 (s, 2H), 3.61 (t, J = 6.1 Hz, 4H), 3.47 (t, J = 6.1 Hz, 4H).

[0044] Comparative Example 1

[0045] This comparative example presents a naphthyridine-based compound fluorescent dye, whose structural formula is as follows:

[0046]

[0047] The specific synthesis method is as shown in Example 1, except that the compound shown in structural formula b1 is replaced with an equivalent amount of the compound shown in structural formula b3;

[0048]

[0049] The NMR characterization of the product is as follows: 1 1H NMR (400 MHz, DMSO) δ 9.07 (d, J = 8.3 Hz, 1H), 8.39 (d, J = 10.5 Hz, 2H), 7.91 - 7.82 (m, 2H), 7.63 (dd, J = 8.2 Hz, 2H), 7.52 (d, J = 8.9 Hz, 1H), 7.25 - 7.35 (m, 1H), 6.82 (d, J = 8.4 Hz, 2H), 3.09 (s, 6H).

[0050] Comparative Example 2

[0051] This comparative example presents a pyridine-based compound fluorescent dye, whose structural formula is as follows:

[0052]

[0053] The specific synthesis method is as shown in Example 1, except that the compound shown in structural formula a1 is replaced with an equivalent amount of the compound shown in structural formula a2;

[0054]

[0055] The NMR characterization of the product is as follows: 11H NMR (400 MHz, DMSO) δ 8.56 (d, J = 8.4 Hz, 1H), 7.73 (d, J = 8.7 Hz, 2H), 7.54 (dd, J = 11.2, 7.5 Hz, 1H), 7.59 - 7.41 (m, 2H), 7.32 (d, J = 8.0 Hz, 2H), 6.69 (d, J = 8.9 Hz, 2H), 4.73 (t, J = 5.6 Hz, 1H), 3.57 (q, J = 6.5 Hz, 2H), 3.45 (t, J = 6.8 Hz, 2H), 3.03 (s, 3H).

[0056] Comparative Example 3

[0057] This comparative example presents a pyrimidine-ene compound fluorescent dye, whose structural formula is shown as follows:

[0058]

[0059] The specific synthesis method is as shown in Example 1, except that the compound shown in structural formula a1 is replaced with an equivalent amount of the compound shown in structural formula a3;

[0060]

[0061] The NMR characterization of the product is as follows: 1 1H NMR (400 MHz, DMSO) δ 8.76 (d, J = 8.3 Hz, 2H), 7.71 (d, J = 8.0 Hz, 2H), 7.57 (d, J = 8.5 Hz, 1H), 7.02 (d, J = 8.9 Hz, 2H), 6.87 (d, J = 8.0 Hz, 2H), 4.73 (t, J = 6.5 Hz, 1H), 3.60 (q, J = 6.6 Hz, 2H), 3.49 (t, J = 6.8 Hz, 2H), 3.03 (s, 3H).

[0062] Comparative Example 4

[0063] This comparative example presents a pyrazine-ene compound fluorescent dye, whose structural formula is shown as follows:

[0064]

[0065] The specific synthesis method is as shown in Example 1, except that the compound shown in structural formula a1 is replaced with an equivalent amount of the compound shown in structural formula a4;

[0066]

[0067] The NMR characterization of the product is as follows: 11H NMR (400 MHz, DMSO) δ 8.84 (s, 1H), 7.81 (d, J = 8.3 Hz, 2H), 7.73 (d, J = 8.5 Hz, 2H), 7.32 (d, J = 8.3 Hz, 2H), 6.57 (d, J = 9.0 Hz, 2H), 4.76 (t, J = 4.7 Hz, 1H), 3.59 (q, J = 5.8 Hz, 2H), 3.47 (t, J = 5.9 Hz, 2H), 3.02 (s, 3H).

[0068] Comparative Example 5

[0069] This comparative example presents a quinoline-ene compound fluorescent dye, whose structural formula is shown as follows:

[0070]

[0071] The specific synthesis method is as shown in Example 1, except that the compound shown in structural formula a1 is replaced with an equivalent amount of the compound shown in structural formula a5;

[0072]

[0073] The NMR characterization of the product is as follows: 1 1H NMR (400 MHz, DMSO) δ 8.27 (d, J = 8.6 Hz, 1H), 7.91 (dd, J = 12.6, 8.0 Hz, 2H), 7.79 (d, J = 8.7 Hz, 1H), 7.76 - 7.68 (m, 2H), 7.58 - 7.46 (m, 3H), 7.18 (d, J = 16.3 Hz, 1H), 6.74 (d, J = 8.9 Hz, 2H), 4.73 (t, J = 5.4 Hz, 1H), 3.57 (q, J = 6.0 Hz, 2H), 3.45 (t, J = 6.1 Hz, 2H), 3.00 (s, 3H).

[0074] Comparative Example 6

[0075] This comparative example presents a quinoline-ene compound fluorescent dye, whose structural formula is shown as follows:

[0076]

[0077] The specific synthesis method is as shown in Example 2, except that the compound shown in structural formula a1 is replaced with an equivalent amount of the compound shown in structural formula a5;

[0078]

[0079] The NMR characterization of the product is as follows: 11H NMR (400 MHz, DMSO) δ 8.26 (d, J = 8.6 Hz, 1H), 7.93 (d, J = 8.3 Hz, 1H), 7.89 (d, J = 8.0 Hz, 1H), 7.79 (d, J = 8.7 Hz, 1H), 7.75 - 7.66 (m, 2H), 7.51 (dd, J = 15.8, 8.0 Hz, 3H), 7.17 (d, J = 16.2 Hz, 1H), 6.75 (d, J = 8.8 Hz, 2H), 4.82 (s, 2H), 3.58 (t, J = 6.1 Hz, 4H), 3.48 (t, J = 6.1 Hz, 4H).

[0080] Comparative Example 7

[0081] This comparative example presents a quinoline-ene compound fluorescent dye, whose structural formula is as follows:

[0082]

[0083] The specific synthesis method is as shown in Example 2, except that the compound shown in structural formula a1 is replaced with an equivalent amount of the compound shown in structural formula a6;

[0084]

[0085] The NMR characterization of the product is as follows: 1 1H NMR (400 MHz, DMSO) δ 8.65 (d, J = 8.5 Hz, 1H), 8.46 (d, J = 5.5 Hz, 1H), 8.00 - 7.85 (m, 3H), 7.76 (t, J = 7.5 Hz, 1H), 7.71 - 7.60 (m, 4H), 6.75 (d, J = 8.4 Hz, 2H), 4.81 (t, J = 5.3 Hz, 2H), 3.67 - 3.53 (m, 4H), 3.49 (d, J = 5.9 Hz, 4H).

[0086] Comparative Example 8

[0087] This comparative example presents a quinoxaline-ene compound fluorescent dye, whose structural formula is as follows:

[0088]

[0089] The specific synthesis method is as shown in Example 1, except that the compound shown in structural formula a1 is replaced with an equivalent amount of the compound shown in structural formula a7;

[0090]

[0091] The NMR characterization of the product is as follows:1 1H NMR (400 MHz, DMSO) δ 8.78 (s, 1H), 8.02 - 7.98 (m, 2H), 7.79 (d, J = 15.7 Hz, 1H), 7.76 - 7.69 (m, 1H), 7.65 - 7.58 (m, 1H), 7.53 (d, J = 8 Hz, 2H), 7.23 (d, J = 15.0 Hz, 1H), 6.71 (d, J = 8.5 Hz, 2H), 4.73 (t, J = 5.4 Hz, 1H), 3.57 (q, J = 6.0 Hz, 2H), 3.45 (t, J = 6.1 Hz, 2H), 3.00 (s, 3H).

[0092] The following cell culture medium, Matrigel, buffer, fetal bovine serum, etc. were purchased from Sigma Reagent Company; culture dishes, syringes, etc. were purchased from Thermo Fisher Scientific; the mouse breast cancer 4T1 cell line was obtained from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences; the human lung cancer cisplatin-resistant cell line A549R was from Shanghai Fuming Gene Biotechnology Co., Ltd.; all cells were placed in cell culture medium containing DMEM (a medium containing various amino acids and glucose) and 10% fetal bovine serum, and cultured under the conditions of 5% CO2 and 37 °C.

[0093] All animals were used for the care and use of laboratory animals in accordance with the guidelines, and the procedures were approved by the Animal Protection and Utilization Committee of the University of Science and Technology of China.

[0094] 4T1 orthotopic breast cancer tumor mice:

[0095] The cultured 4T1 breast tumor cells were centrifuged and collected, and a mixture of cell culture medium and Matrigel (mass ratio 3:1) was added. After mixing, the cell density was 500,000 per 100 μL; 100 μL of the mixture was injected near the mammary gland of 4 - 8-week-old NOD / SCID mice; the mice injected with tumors were cultured for 1 - 2 weeks to obtain 4T1 orthotopic breast cancer tumor mice.

[0096] MDA MB231 tumor mice:

[0097] The method was the same as that for 4T1 orthotopic breast cancer tumor mice, except that MDA MB231 tumor cells (human breast cancer cell line) were used instead of 4T1 breast tumor cells, and the mice used were 4 - 8-week-old BALB / cA-nu mice, and 3 million cells were implanted into each mouse, and the tumors grew for 2 weeks.

[0098] MDA MB435 tumor mice:

[0099] The method was the same as that for the 4T1 orthotopic breast cancer tumor mice, except that MDA MB435 tumor cells (human breast cancer cell line) were used instead of 4T1 breast tumor cells, and the mice used were 4-8-week-old BALB / cA-nu mice. 3 million cells were implanted into each mouse, and the tumors grew for 2 weeks.

[0100] A549R subcutaneous tumor mice:

[0101] The method was the same as that for the 4T1 orthotopic breast cancer tumor mice, except that A549R tumor cells were used instead of 4T1 breast tumor cells, and 100 μL of the mixture was injected subcutaneously into the abdomen of the mice. At the same time, the mice used were 4-8-week-old NOD / scid mice. 3 million cells were implanted into each mouse, and the tumors grew for 2 weeks.

[0102] 4T1 lung metastasis tumor mice:

[0103] The method was the same as that for the 4T1 orthotopic breast cancer tumor mice, except that the mice used were 4-8-week-old NOD / scid mice. 3 million cells were implanted into each mouse, and the tumors grew for 2 weeks.

[0104] Application Example 1

[0105] The fluorescent dyes, rhodamine B, and safranin T described in the above examples or comparative examples were used for imaging of 4T1 orthotopic breast cancer tumor mice:

[0106] 5 mg of the fluorescent dye was suspended in 1.0 mL of 0.5% sodium carboxymethylcellulose solution to prepare a uniform suspension, which was the contrast agent solution. Eight 4T1 orthotopic breast cancer tumor mice were divided into two groups (4 mice in each group), namely the experimental group and the control group.

[0107] The mice were fasted for more than 4 h. The experimental group was intragastrically administered 0.2 mL of the contrast agent solution, and the control group was administered 0.2 mL of sodium carboxymethylcellulose solution. After the mice continued to fast for 2 h, they were fed. 2-4 h after intragastric administration, the mice were anesthetized, and sodium pentobarbital with a mass ratio of 80 mg / kg was injected into the abdominal cavity of the mice to anesthetize the mice for 10-20 min. The chest cavity of the mice was cut open with a scalpel to expose the tumor tissue and part of the normal tissue. The fluorescence signal of the live mice was observed using an MVX10 stereomicroscope of Olympus Corporation. The imaging field of view was 2 cm × 1.5 cm.

[0108] Imaging of the tumor and surrounding muscle tissue was performed in the fluorescence mode with an excitation light wavelength of 472 ± 15 nm and an accepted emission wavelength of 520 ± 17.5 nm. The results are as Figure 1 shown. Figure 1This is a fluorescence imaging photo of the fluorescent dye described in Example 1 of the present invention used for 4T1 orthotopic breast cancer tumor mice. The bright part is the tumor, and the dark part is the normal tissue area.

[0109] Table 1 shows the signal-to-noise ratio data of the fluorescent dyes described in the examples and comparative examples of the present invention for the corresponding fluorescence imaging of 4T1 orthotopic breast cancer tumor mice.

[0110]

[0111] Note: B-G: The excitation light wavelength is 472 ± 15 nm, and the received emission wavelength is 520 ± 17.5 nm; B-R: The excitation light wavelength is 475 ± 25 nm, and the received emission wavelength is 590 ± 52 nm; G-R: The excitation light wavelength is 562 ± 20 nm, and the received emission wavelength is 641 ± 35.5 nm;

[0112] As can be seen from the results in Table 1 above, the naphthyridineene-based compound fluorescent dye of the present invention has excellent fluorescence and has a good discrimination effect on 4T1 orthotopic breast cancer.

[0113] Application Example 2

[0114] The above-mentioned fluorescent dyes described in the examples or comparative examples, as well as rhodamine B and safranin T, were used for imaging of MDA MB231 tumor mice:

[0115] Specifically referring to Application Example 1, except that MDA MB231 tumor mice were used instead of 4T1 orthotopic breast cancer tumor mice, and the skin of the mice was incised with a scalpel.

[0116] Figure 2 This is a fluorescence imaging photo of the fluorescent dye described in Example 1 of the present invention used for MDA MB231 tumor mice. Similarly, the bright part is the tumor, and the dark part is the normal tissue area.

[0117] Table 2 shows the signal-to-noise ratio data of the fluorescent dyes described in the examples and comparative examples of the present invention for the corresponding fluorescence imaging of MDA MB231 tumor mice.

[0118]

[0119] Note: B-G: The excitation light wavelength is 472 ± 15 nm, and the received emission wavelength is 520 ± 17.5 nm; B-R: The excitation light wavelength is 475 ± 25 nm, and the received emission wavelength is 590 ± 52 nm; G-R: The excitation light wavelength is 562 ± 20 nm, and the received emission wavelength is 641 ± 35.5 nm;

[0120] As can be seen from the results in Table 2 above, the naphthyridineene-based compound fluorescent dye of the present invention has excellent fluorescence and has a good discrimination effect on MDA MB231 tumors.

[0121] Application Example 3

[0122] The fluorescent dyes described in the above embodiments or comparative examples, as well as rhodamine B and safranin T, were used for imaging MDA MB435 tumor-bearing mice:

[0123] Specifically referring to Application Example 1, except that MDA MB435 tumor-bearing mice were used instead of 4T1 orthotopic breast cancer tumor-bearing mice, and the skin of the mice was incised with a scalpel.

[0124] Figure 3 The following is a fluorescence imaging photograph of the fluorescent dye described in Example 1 of the present invention for MDA MB435 tumor-bearing mice. The equally bright parts are tumors, and the dark parts are normal tissue areas.

[0125] Table 3 shows the signal-to-noise ratio data of the fluorescent dyes described in the embodiments and comparative examples of the present invention for the corresponding fluorescence imaging of MDA MB435 tumor-bearing mice.

[0126]

[0127] Note: B-G: The excitation light wavelength is 472 ± 15 nm, and the received emission wavelength is 520 ± 17.5 nm; B-R: The excitation light wavelength is 475 ± 25 nm, and the received emission wavelength is 590 ± 52 nm; G-R: The excitation light wavelength is 562 ± 20 nm, and the received emission wavelength is 641 ± 35.5 nm;

[0128] As can be seen from the results in Table 3 above, the naphthyridine-based compound fluorescent dye of the present invention has excellent fluorescence and a good discrimination effect on MDA MB435 tumors.

[0129] Application Example 4

[0130] The fluorescent dyes described in the above embodiments or comparative examples were used for imaging A549R subcutaneous tumor-bearing mice:

[0131] Specifically referring to Application Example 1, except that A549R subcutaneous tumor-bearing mice were used instead of 4T1 orthotopic breast cancer tumor-bearing mice, and the skin of the mice was incised with a scalpel.

[0132] Figure 4 The following is a fluorescence imaging photograph of the fluorescent dye described in Example 1 of the present invention for A549R subcutaneous tumor-bearing mice. The equally bright parts are tumors, and the dark parts are normal tissue areas.

[0133] Table 4 shows the signal-to-noise ratio data of the fluorescent dyes described in the embodiments and comparative examples of the present invention for the corresponding fluorescence imaging of A549R subcutaneous tumor-bearing mice.

[0134]

[0135] Note: B-G: The excitation light wavelength is 472 ± 15 nm, and the received emission wavelength is 520 ± 17.5 nm; G-R: The excitation light wavelength is 562 ± 20 nm, and the received emission wavelength is 641 ± 35.5 nm;

[0136] As can be seen from the results in Table 4 above, the naphthyridene-based compound fluorescent dye described in the present invention has excellent fluorescence and has a good discrimination effect on A549R subcutaneous tumors.

[0137] Application Example 5

[0138] The fluorescent dye described in the above embodiments or comparative examples was used for imaging of 4T1 lung metastasis tumor mice:

[0139] Specifically referring to Application Example 1, except that 4T1 lung metastasis tumor mice were used instead of 4T1 orthotopic breast cancer tumor mice.

[0140] Figure 5 This is a fluorescence imaging photo of the fluorescent dye described in Example 1 of the present invention for 4T1 lung metastasis tumor mice. The equally bright parts are tumors, and the dark parts are normal tissue areas.

[0141] Table 5 shows the signal-to-noise ratio data of the fluorescent dye described in the examples and comparative examples of the present invention for the corresponding fluorescence imaging of 4T1 lung metastasis tumor mice.

[0142]

[0143] Note: B-G: The excitation light wavelength is 472 ± 15 nm, and the received emission wavelength is 520 ± 17.5 nm; B-R: The excitation light wavelength is 475 ± 25 nm, and the received emission wavelength is 590 ± 52 nm;

[0144] As can be seen from the results in Table 5 above, the naphthyridene-based compound fluorescent dye described in the present invention has excellent fluorescence and has a good discrimination effect on 4T1 lung metastasis tumors.

[0145] Application Example 6

[0146] The acute toxicity of the fluorescent dye described in the above embodiments was measured.

[0147] The acute toxicity test method is the "up-and-down method". For the specific experimental process, please refer to the literature ("An Up-and-Down Procedure for Acute Toxicity Testing", Robert D. Bruce, Fundamental and Applied Toxicology. 1985(5): 151-7), which is a method recommended by the Organization for Economic Cooperation and Development (OECD) and the United States Environmental Protection Agency (EPA) to test the maximum tolerance of living animals to dyes.

[0148] Six healthy female SD rats (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.) were selected and fasted for 12 h, and then acute toxicity tests were conducted on them: first, the rats were weighed. One of them (blank control) was administered 1.5 mL of 0.5% sodium carboxymethylcellulose solution, and the remaining five were gavaged with the amount of the fluorescent dye described in Example 1 or 2 at 5 g per kilogram of the rat body weight. All the rats were weighed within 15 days, and their weights all increased relatively. According to the acute toxicity classification of foreign compounds, it can be seen that the toxicity of the fluorescent dye described in Example 1 or 2 is extremely low, and its median lethal dose LD50 > 5000 mg / kg.

[0149] As described above, the above are only the preferred specific embodiments of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention, according to the technical solution and inventive concept of the present invention, makes equivalent substitutions or changes, and all should be covered within the protection scope of the present invention.

Claims

1. A naphthyridine-based compound fluorescent dye, characterized in that, The general structural formula of the fluorescent dye is as follows: Wherein, R1 or R2 is independently hydrogen, R3 is methyl or hydroxyethyl, and n is 2; The structural formula of the fluorescent dye is as follows: or 。 2. A method for preparing the naphthyridine-based compound fluorescent dye according to claim 1, characterized in that, Including: The naphthyridine-based compound fluorescent dye is obtained by carrying out an aldol condensation reaction on the compound shown in Structural Formula I and the compound shown in Structural Formula II; 。 3. The preparation method of the naphthyridine-based compound fluorescent dye according to claim 2, characterized in that, The molar ratio of the compound shown in Structural Formula I to the compound shown in Structural Formula II is 1:0.9 - 1.

1.

4. Use of the fluorescent dye according to claim 1 in the preparation of a tumor diagnostic imaging agent, characterized in that, The tumor is breast cancer or lung cancer.

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