A heptamethinylcyanine iodide conjugated 2-pyridone compound and its preparation method and application

Abstract

This invention discloses a heptamethine iodide conjugated with 2-pyridone, its preparation method, and its applications. The 2-pyridone group in this heptamethine iodide conjugated with 2-pyridone can capture singlet oxygen to generate an intra-pyridone peroxide. This intra-pyridone peroxide undergoes a reversible thermal conversion, releasing singlet oxygen and regenerating 2-pyridone in tumor tissue, thereby prolonging the half-life of singlet oxygen and achieving a sustained photodynamic therapy effect. This heptamethine iodide conjugated with 2-pyridone can be used to prepare a fluorescent dye drug delivery system. This delivery system involves liposomes loading the heptamethine iodide conjugated with 2-pyridone and targeting it to the tumor site for use in cell and in vivo imaging, photodynamic therapy of tumors, and combined therapy.

Description

Technical Field

[0001] The invention belongs to the field of biomedical technology, and specifically relates to a heptamethinylcyanine iodide-conjugated 2-pyridone compound and a preparation method and application thereof. Background Art

[0002] Gastric cancer (GC) is one of the most common malignant tumors, and its morbidity and mortality rates remain high. Although some advances have been made in treatment, including surgery, radiotherapy, chemotherapy, targeted therapy, and immunotherapy, most patients are in the late stage when they seek medical treatment and have a poor prognosis. Therefore, early diagnosis and development of new anticancer therapies are urgently needed.

[0003] In recent years, phototherapy has attracted widespread attention as a non-invasive, real-time tumor treatment method. Compared with traditional radiotherapy and chemotherapy, photodynamic therapy (PDT) has been widely studied due to its low invasiveness, low toxicity and side effects, and excellent spatial specificity. The key elements of PDT include light, oxygen, and photosensitizers (PS). Under illumination, photosensitizers absorb energy from lasers of specific wavelengths and transfer energy to surrounding oxygen molecules, generating chemically active reactive oxygen species (ROS), especially singlet oxygen ( 1 O2), causing oxidative stress in cells, destroying proteins and other biomacromolecules, and thus killing tumor cells. However, the effect of photodynamic therapy depends largely on the exposure time and the intensity of the excitation light source. Once the light source is removed, photodynamic therapy can no longer produce singlet oxygen. Therefore, it is of great significance to develop a photosensitizer with a sustained photodynamic therapy effect.

[0004] Fluorescence imaging is an emerging imaging technology with the advantages of simple operation, long duration, high sensitivity and little damage to organisms. It has great advantages in biomedical research and has been widely used in the fields of in vivo imaging, real-time tumor monitoring and treatment. Near-infrared (700-900nm) fluorescence imaging can effectively avoid the self-absorption and autofluorescence interference of biological tissues, becoming the "optical window" for in vivo imaging.

[0005] Liposomes are the first nano drug delivery platform that has successfully transformed from concept to clinical application. They are spherical vesicles formed by phospholipids and other substances. Their bilayer structure is similar to that of biological membranes and has amphiphilicity. This allows liposomes to encapsulate water-soluble and fat-soluble substances, forming a self-enclosed structure that helps prevent drug degradation. The increased vascular permeability of tumor tissues and other reasons make it possible for drug delivery systems with a certain spatial size to be passively targeted to the tumor site. Compared with other drug delivery systems, liposomes have the advantages of good biocompatibility and in vivo degradability, which can achieve targeted release of drugs in tumor tissues, effectively improve the bioavailability of drugs, and reduce toxic side effects. Summary of the invention

[0006] Purpose of the invention: The purpose of the present invention is to provide a heptamethinylcyanine iodide-conjugated 2-pyridone compound and its preparation method and application.

[0007] The 2-pyridone group in the heptamethine cyanine iodide conjugated 2-pyridone compound of the present invention can capture singlet oxygen to generate 2-pyridone endoperoxy compounds, and then the 2-pyridone endoperoxy compounds undergo a reversible reaction through thermal conversion, releasing singlet oxygen and regenerating 2-pyridone in tumor tissue. Therefore, the 2-pyridone endoperoxy compound can continue to release singlet oxygen after the light source is removed during photodynamic therapy, and oxygen is not consumed in the process of releasing singlet oxygen, thereby extending the half-life of singlet oxygen and having the effect of continuous photodynamic therapy.

[0008] Technical solution: The purpose of the present invention is achieved through the following technical solution:

[0009] The present invention provides a heptamethine cyanine iodide conjugated 2-pyridone compound having the following structural formula:

[0010]

[0011] The present invention also provides a method for preparing the compound, comprising the following steps:

[0012] (1) reacting 1,4-di(bromomethyl)benzene with 2-hydroxypyridine to prepare 1-{[4-(bromomethyl)phenyl]methyl}-1,2-dihydro-2-pyridone PhBr-Py;

[0013]

[0014] (2) reacting PhBr-Py obtained in step (1) with 5-iodo-2,3,3-trimethyl-3H-indole, and reacting the obtained intermediate with 2-chloro-3-[(E)-hydroxymethylene]cyclohex-1-ene-1-carbaldehyde to prepare the iodinated heptamethine cyanine conjugated 2-pyridone compound Icy-Py.

[0015]

[0016] Preferably, in step (1), the 1-{[4-(bromomethyl)phenyl]methyl}-1,2-dihydro-2-pyridone is prepared by the following method:

[0017] 2-Hydroxypyridine and 1,4-di(bromomethyl)benzene were dissolved in dioxane, sodium hydride was slowly added in portions, heated and stirred, and after the reaction was completed, water was added to quench the reaction, and the resulting mixture was concentrated under vacuum and purified to obtain 1-{[4-(bromomethyl)phenyl]methyl}-l,2-dihydro-2-pyridone.

[0018] Preferably, in step (2), the intermediate is prepared by the following method:

[0019] 1-{[4-(Bromomethyl)phenyl]methyl}-l,2-dihydro-2-pyridone and 5-iodo-2,3,3-trimethyl-3H-indole were dissolved in acetonitrile and heated to react. After the reaction was completed, the mixture was cooled to room temperature and the precipitate was collected by filtration. After washing and drying, a purple solid was obtained.

[0020] Preferably, in step (2), the heptamethine cyanine iodide-conjugated 2-pyridone compound is prepared by the following method:

[0021] The obtained intermediate and 2-chloro-3-[(E)-hydroxymethylene]cyclohex-1-ene-1-carbaldehyde are dissolved in acetic anhydride and heated to react. After the reaction is completed, the crude product is distilled and purified to obtain the iodinated heptamethine cyanine conjugated 2-pyridone compound Icy-Py.

[0022] A further preferred embodiment of the present invention is that the method for preparing the heptamethine cyanine iodide conjugated 2-pyridone compound comprises the following steps:

[0023] (1) 1,4-di(bromomethyl)benzene and 2-hydroxypyridine are dissolved in dioxane, sodium hydride is slowly added in portions, and the mixture is heated and stirred in an oil bath at a constant rate. After the reaction is completed, water is added to quench the reaction, and the resulting mixture is concentrated under vacuum, purified and eluted to obtain 1-{[4-(bromomethyl)phenyl]methyl}-1,2-dihydro-2-pyridone PhBr-Py;

[0024] (2) PhBr-Py and 5-iodo-2,3,3-trimethyl-3H-indole prepared in step (1) are dissolved in acetonitrile, heated to react, and after the reaction is completed, the precipitate is filtered and collected after cooling to room temperature, washed with ether and then dried in vacuo, and the obtained purple solid and 2-chloro-3-[(E)-hydroxymethylene]cyclohex-1-ene-1-carboxaldehyde are dissolved in acetic anhydride, heated and stirred to react, and after the reaction is completed, the crude product is distilled and purified by silica gel column chromatography to obtain the iodine heptamethine cyanine conjugated 2-pyridone compound Icy-Py.

[0025] As a traditional near-infrared photosensitizer, cyanine dyes have good near-infrared absorption balance, high molar extinction coefficient and excellent stability. The present invention conjugates cyanine dyes with 2-pyridone compounds to generate heptamethine cyanine iodide-conjugated 2-pyridone compounds Icy-Py, which is a new type of fluorescent dye. When illuminated, Icy-Py produces singlet oxygen and endoperoxy compounds Icy-Py-EPOs; when the light source is removed, the 2-pyridone endoperoxy compounds in Icy-Py-EPOs release singlet oxygen through thermal conversion, thereby extending the half-life of singlet oxygen and achieving the effect of continuous photodynamic therapy.

[0026] The present invention also provides the use of the heptamethincyanine iodide conjugated 2-pyridone compound in the preparation of a fluorescent dye drug delivery system.

[0027] The present invention also provides a fluorescent dye drug delivery system, in which the heptamethine cyanine iodide-conjugated 2-pyridone compound is loaded.

[0028] The present invention also provides a method for preparing the fluorescent dye drug delivery system, comprising the following steps: dissolving a mixture of lecithin, phosphatidylethanolamine, cholesterol and DSPE-PEG2000 and a 2-pyridone compound Icy-Py conjugated with heptamethine cyanine iodide in dichloromethane, evaporating the solvent to form a liposome film, adding water to dissolve and then ultrasonicating to form liposome Icy-Py NPs with uniform particle size, i.e., the fluorescent dye drug delivery system.

[0029] Preferably, the molar ratio of the lecithin, phosphatidylethanolamine, cholesterol and DSPE-PEG2000 is 50:25:25:5; the mass ratio of the mixture of the lecithin, phosphatidylethanolamine, cholesterol and DSPE-PEG2000 and the heptamethine cyanine iodide-conjugated 2-pyridone compound is 40:1.

[0030] Preferably, the step of adding water to dissolve uses ultrapure water to dissolve.

[0031] The present invention also provides the use of the fluorescent dye drug delivery system in preparing therapeutic drugs targeting tumors.

[0032] Beneficial effects:

[0033] (1) The hypoxic microenvironment in solid tumors and the further hypoxia formed after photodynamic therapy will inevitably reduce the efficacy of photodynamic therapy. The heptamethine cyanine iodide conjugated 2-pyridone compound of the present invention generates singlet oxygen when irradiated with light. 1O2, and at the same time generate the endoperoxide compound Icy-Py-EPOs. When the light source is removed, the 2-pyridone endoperoxide in Icy-Py-EPOs releases singlet oxygen again through thermal conversion, and the process of releasing singlet oxygen does not depend on oxygen, thereby extending the half-life of singlet oxygen and achieving a sustained photodynamic therapy effect.

[0034] (2) The heptamethine cyanine iodide conjugated with 2-pyridone compound Icy-Py fluorescent dye prepared by the present invention has a stronger tumor cell killing effect than the heptamethine cyanine iodide Icy with a propyl side chain and no 2-pyridone conjugation.

[0035] (3) The iodine heptamethine cyanine conjugated 2-pyridone compound Icy-Py fluorescent dye prepared by the present invention can release singlet oxygen under both light and dark conditions. The research results show that the drug delivery system (Icy-Py NPs) loaded with iodine heptamethine cyanine conjugated 2-pyridone compound can actively target tumor tissues, and exhibits preferential accumulation characteristics in gastric cancer tissues after tail vein injection. Compared with iodine heptamethine cyanine Icy with a propyl side chain and no 2-pyridone conjugation, the Icy-Py fluorescent dye has a stronger tumor inhibition effect. In addition, the fluorescent dye of the present invention has good safety, can be used for photodynamic therapy of gastric cancer, and has a good market prospect. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 This is the mass spectrum of the heptamethincyanine iodide-conjugated 2-pyridone compound Icy-Py before irradiation;

[0037] Figure 2 The mass spectrum of Icy-Py-EPOs is generated after Icy-Py is irradiated with light;

[0038] Figure 3 The mass spectrum of Icy-Py after being irradiated with light and left to stand for half an hour, part of Icy-Py-EPOs releases singlet oxygen and returns to Icy-Py, while part of Icy-Py-EPOs releases a part of singlet oxygen to generate the intermediate product of Icy-Py-EPO;

[0039] Figure 4 It is a characterization test of Icy-Py fluorescent dye drug delivery system; wherein, Figure 4 a is the particle size and transmission electron microscopy results of the fluorescent dye drug delivery system. Figure 4 b is the Zeta potential result diagram of the fluorescent dye drug delivery system. Figure 4 c is a graph showing the stability evaluation results of the fluorescent dye drug delivery system;

[0040] Figure 5Icy-Py fluorescent dye has better tumor cell killing effect than Icy, which has a propyl side chain and is not conjugated with 2-pyridone; Figure 5 a is the uptake result of Icy-Py fluorescent dye drug delivery system in gastric cancer cells at 1h and 2h. Figure 5 b is the live-dead staining result of gastric cancer cell MFC after treatment with corresponding fluorescent dyes. Figure 5 c is the flow cytometry apoptosis result of gastric cancer cell MFC after treatment with corresponding fluorescent dyes. Figure 5 d is the statistical result of apoptosis of gastric cancer cell MFC after treatment with corresponding fluorescent dyes;

[0041] Figure 6 It is multimodal imaging of Icy-Py fluorescent dye; among them, Figure 6 a is the fluorescence imaging result of the distribution of fluorescent dye drug delivery system in tumor-bearing mice. Figure 6 b is the spectrum of photoacoustic imaging of fluorescent dye drug delivery system in tumor-bearing mice. Figure 6 c is the photoacoustic imaging of the fluorescent dye drug delivery system in tumor-bearing mice;

[0042] Figure 7 is the anti-tumor effect of Icy-Py fluorescent dye in vivo; Figure 7 a is a general picture of the tumor in tumor-bearing mice after intervention and treatment with the corresponding fluorescent dye drug delivery system. Figure 7 b is the growth curve of the tumor after intervention with the fluorescent dye drug delivery system. Figure 7 c is the H&E, Ki67 and Tunel staining results of tumor tissue after intervention treatment with fluorescent dye drug delivery system;

[0043] Figure 8 It is the in vivo biosafety assessment of Icy-Py; among them, Figure 8 a is H&E staining of important organs of mice after intervention with fluorescent dye drug delivery system. Figure 8 b is the AST concentration in mouse serum after intervention with the fluorescent dye drug delivery system. Figure 8 c is the ALT concentration in mouse serum after intervention with the fluorescent dye drug delivery system. Figure 8 d is the concentration of Cr in mouse serum after intervention with the fluorescent dye drug delivery system. Figure 8 e is the BUN concentration in mouse serum after intervention with the fluorescent dye drug delivery system. DETAILED DESCRIPTION

[0044] The technical solution of the present invention is described in detail below through specific embodiments, but the protection scope of the present invention is not limited to the embodiments.

[0045] The embodiment of the present invention provides a new fluorescent dye for treating gastric cancer. The fluorescent dye can release singlet oxygen under both light and dark conditions, thereby prolonging the half-life of singlet oxygen. The fluorescent dye drug delivery system is a liposome loaded with iodinated heptamethine cyanine conjugated 2-pyridone compound (Icy-Py NPs) targeted to the tumor site.

[0046] Example 1 Preparation of Heptamethinecyanine Iodide Conjugated 2-Pyridone Compound (Icy-Py)

[0047] (1) Preparation of 1-{[4-(bromomethyl)phenyl]methyl}-1,2-dihydro-2-pyridone

[0048]

[0049] 8.27 g of 1,4-di(bromomethyl)benzene (Compound 1) and 500 mg of 2-hydroxypyridine (Compound 2) were added to a 2000 ml four-necked round-bottom flask under nitrogen protection, and 830 mg of dioxane was added to dissolve. Subsequently, 253 mg of sodium hydride was slowly added in portions at room temperature. The resulting solution was heated and stirred in an oil bath at 70 ° C for 1 hour, and then 3 ml of water was added to quench the reaction. The resulting mixture was concentrated under vacuum, and the crude product was purified by silica gel column chromatography (ethyl acetate: petroleum ether 1:1, v / v) to finally obtain 1.05 g of white solid 1-{[4-(bromomethyl)phenyl]methyl}-l,2-dihydro-2-pyridone PhBr-Py, with a yield of 72%.

[0050] 1 H NMR(400MHz, DMSO-d6)δ(ppm)7.78(dd,J=6.8,1.7Hz,1H),7.44(dd,J=6.8,2.3Hz,1H),7.42-7.39(m,1 H), 7.26 (d, J = 8.1Hz, 1H), 6.41 (d, J = 9.1Hz, 1H), 6.24 (td, J = 6.7, 1.3Hz, 1H), 5.08 (s, 1H), 4.68 (s, 1H).

[0051] 13 C NMR (100MHz, DMSO-d6): δ161.84, 140.61, 139.62, 138.07, 137.73, 129.94, 128.38, 120.36, 106.04, 51.30, 34.63ppm.

[0052] (2) Preparation of 2-[(1E)-2-{2-chloro-3-[(1E)-2-[(2E)-5-iodo-3,3-dimethyl-1-({4-[(2-oxypyridin-1-yl)methyl]phenyl}methyl)-2,3-dihydro-1H-indol-2-ylidene]ethylidene]cyclohex-1-enyl}vinyl]-5-iodo-3,3-dimethyl-1-({4-[(2-oxypyridin-1-yl)methyl]phenyl}methyl)-3H-indole (Icy-Py)

[0053]

[0054] 1.05g 1-{[4-(bromomethyl)phenyl]methyl}-l,2-dihydro-2-pyridone (compound 3) and 285mg 5-iodo-2,3,3-trimethyl-3H-indole (compound 4) were dissolved in 60ml acetonitrile and heated under reflux for 24 hours. After cooling to room temperature, the precipitate was collected by filtration, washed with ether and dried in vacuo. The purple solid obtained was dissolved in 30ml acetic anhydride with 86mg 2-chloro-3-[(E)-hydroxymethylene]cyclohex-1-ene-1-carboxaldehyde under nitrogen protection, and heated and stirred at 50°C for 3 hours. After heating, the crude product was obtained by rotary distillation, and purified by silica gel column chromatography (dichloromethane: methanol 98:2, v / v) to obtain 677mg green solid, i.e., iodine heptamethine cyanine conjugated 2-pyridone compound (Icy-Py), with a yield of 75%.

[0055] 1 H NMR (400MHz, MeOD) δ (ppm) 8.38 (d, J = 14.1 Hz, 2H), 7.94 (d, J = 1.6 Hz, 2H), 7.73 (ddd, J = 9.3, 7.6 ,1.6Hz,4H),7.53(dd,J=8.9,6.7,2.0Hz,2H),7.35(d,J=8.2Hz,4H),7.27(d,J=8.2Hz,4H),7.1 2(d,J=8.4Hz,2H),6.57(d,J=9.1Hz,2H),6.40(td,J=6.7,1.3Hz,2H),6.26(d,J=14.1Hz,2H), 5.40(s,4H),5.20(s,4H),2.50(t,J=5.9Hz,4H),1.80(d,J=5.0Hz,2H),1.77(d,J=4.9Hz,12H).

[0056] 13C NMR (100MHz, MeOD): δ184.03,174.68,162.25,156.03,154.77,153.88,152.40,149.92,149.11,148.31,145.48,143 .01,139.80,139.59,138.13,131.11,124.34,118.88,113.53,99.89,63.23,60.72,38.28,37.18,31.87,10.00ppm.

[0057] HR-MS (ESI, positive charge mode, m / z): [M-Br] + , calculated value 1101.18627, measured value 1101.184093. Comparative Example 1 Preparation of Heptamethine Cyanine Iodide Compound (Icy)

[0058]

[0059] 5-iodo-2,3,3-trimethyl-3H-indole (compound 1, 285 mg) was dissolved in 60 ml of acetonitrile, and then 1-iodopropane (1360 mg) was added and heated to reflux for 24 hours. After cooling to room temperature, the precipitate was collected by filtration, washed with ether and dried in vacuo to obtain 5-iodo-2,3,3-trimethyl-1-propyl-3H-indole (compound 2). Under the protection of argon, compound 2 and 2-chloro-3-[(E)-hydroxymethylene]cyclohex-1-ene-1-carboxaldehyde (compound 3, 86 mg) were dissolved in 30 ml of acetic anhydride solvent and heated and stirred at 50 ° C for 3 hours. After the reaction was complete, the crude product was distilled and purified by silica gel column chromatography (dichloromethane: methanol 98:2, v / v) to obtain 368 mg of blue-green powder with a yield of 80%.

[0060] 1 H NMR (400MHz, CDCl3) δ (ppm) 8.30 (d, J = 14.0Hz, 2H), 7.68 (d, J = 8.1Hz, 2H), 7.63 (s, 2H), 6.99 (d, J = 8.3Hz, 2H), 6.23 (d, J = 14.0H z, 2H), 4.17 (t, J = 6.4Hz, 4H), 2.73 (s, 4H), 1.96 (s, 2H), 1.88 (dd, J = 13.8, 6.9Hz, 4H), 1.69 (s, 12H), 1.04 (t, J = 7.3Hz, 6H) ppm.

[0061] 13C NMR (151MHz, CDCl3) δ171.57,150.88,144.44,143.14,142.25,137.67,131.32,12 8.38,113.02,101.90,88.66,49.22,46.63,28.16,26.80,20.89,20.65,11.67ppm.

[0062] HR-MS (ESI, positive charge mode, m / z): [MI] + , calculated value 791.11204, measured value 791.11041.

[0063] Example 2 Icy-Py fluorescent dye can generate singlet oxygen under both light and dark conditions

[0064] In order to verify that the Icy-Py prepared by the present invention reacts with singlet oxygen under light conditions to generate the endoperoxy compound Icy-Py-EPOs, and that the endoperoxy compound can release singlet oxygen under dark conditions, we placed Icy-Py, Icy-Py after light exposure, and Icy-Py after light exposure in a 37°C oven for half an hour to perform mass spectrometry detection. The experimental results are shown in Figure 1-Figure 3 .

[0065] The results showed that after Icy-Py was irradiated, a peak of Icy-Py-EPOs was generated ( Figure 1 , Figure 2 ) and placed in a 37°C oven away from light for half an hour, some Icy-Py-EPOs released singlet oxygen to return to Icy-Py, and some Icy-Py-EPOs released a portion of singlet oxygen to generate the intermediate product of Icy-Py-EPO ( Figure 3 ). This proves that Icy-Py fluorescent dye can release singlet oxygen under both light and dark conditions.

[0066] Example 3 Preparation of liposome-loaded heptamethine cyanine iodide-conjugated 2-pyridone compound drug delivery system (Icy-PyNPs)

[0067] Phosphatidylcholine, phosphatidylethanolamine, cholesterol, DSPE-PEG2000 (distearoylphosphatidylethanolamine, CAS: 147867-65-0) were weighed in a total amount of 40 mg and 1 mg Icy-Py according to a molar ratio of 50:25:25:5 and dissolved in 5 ml of dichloromethane, and then the solvent was removed by rotary evaporation (42°C, 100 rpm, 20 min) to form a liposome film. Subsequently, 5 ml of ultrapure water was added for dissolution and then ultrasonicated (130 W, 20 kHz, 10 min) to form liposome Icy-Py NPs with uniform particle size, and free unencapsulated Icy-Py was removed by ultrafiltration (2000 g, 5 min) with a 30 kDa filter to obtain a fluorescent dye drug delivery system.

[0068] For cell and animal experiments, the fluorescent dye drug delivery system was sterilized through a 0.45 μm filter and stored at 4° C. The above fluorescent dye drug delivery system was diluted 1:5 with ultrapure water, and its particle size was measured by Nanobrook Omni dynamic light scattering (DLS), and the morphological characteristics of Icy-Py NPs were visualized by transmission electron microscopy.

[0069] in, Figure 4 a is the particle size and transmission electron microscopy results of the fluorescent dye drug delivery system. Electron microscopy analysis shows that the fluorescent dye nanosystem presents a spherical uniform structure. Particle size analysis detects that its particle size is 100-200nm. Figure 4 b is the Zeta potential result of the fluorescent dye drug delivery system. Figure 4 c is the stability evaluation result of the fluorescent dye drug delivery system. It can be seen that the Icy-Py fluorescent dye drug delivery system has good stability.

[0070] Example 4 In vitro tumor cell killing experiment

[0071] The fluorescent dye drug delivery system was used in an in vitro tumor cell killing experiment. The experimental steps are as follows:

[0072] The liposome drug delivery system (Icy-Py NPs) loaded with iodinated heptamethine cyanine conjugated with 2-pyridone compound was added to the gastric cancer cell MFC culture system at 0.8 μM. The MFC cell line was purchased from Wuhan Pronocell Life Science Co., Ltd. The culture conditions of MFC cells were RPMI 1640 complete medium containing 10% FBS and 1% double antibody. The uptake of fluorescent dyes by MFC cells was observed by laser confocal microscopy at 1 and 2 hours. The results are shown in Figure 2. Figure 5 As shown in a, compared with the uptake at 1 h, MFC cells took up more fluorescent dyes at 2 h, indicating that the drug delivery system loaded with heptamethine cyanine iodide conjugated with 2-pyridone compounds can be taken up by gastric cancer cells.

[0073] To further investigate the killing effect of Icy-Py NPs on MFC cells under light conditions, 0.8 μM Icy-Py NPs drug delivery system was co-incubated with MFC cells in a 37°C, 5% CO2 cell culture incubator for 4 h and then irradiated with 808 nm laser (0.5 W / cm 2 , 1min), and then cultured for 24h before flow cytometry analysis and live-dead staining (live-dead staining kit, product number: 40747ES76, Shanghai Yisheng Biology). The experiment was divided into 4 groups, namely, negative control PBS illumination group (+), heptamethine cyanine iodide conjugated 2-pyridone compound drug delivery system non-illumination group Icy-Py NPs (-), heptamethine cyanine iodide drug delivery system illumination group Icy NPs (+), heptamethine cyanine iodide conjugated 2-pyridone compound drug delivery system illumination group Icy-PyNPs (+).

[0074] The results are as follows Figure 5 As shown in bd, Icy-Py significantly promoted the apoptosis of gastric cancer cells MFC under light conditions compared with Icy, which has a propyl side chain and is not conjugated with 2-pyridone.

[0075] Example 5 Icy-Py fluorescent dye multimodal imaging

[0076] 5-week-old, male, 20g BALB / c mice were purchased from Nanjing Jicui Pharmaceutical. MFC gastric cancer cells with good growth status were injected subcutaneously in the right hypochondrium of the mice. The number of gastric cancer cells injected per mouse was 5×10 5 / mouse, and the volume of subcutaneous tumor and the weight of mice were observed every 2 days. The calculation formula of mouse tumor volume was: tumor length × width × width × 0.5.

[0077] When the volume of mouse subcutaneous tumor reaches 600mm 3 , Icy-Py NPs solution was injected into the tail vein at a dose of 3 mg / kg, and the distribution of Icy-Py NPs in tumor-bearing mice was observed.

[0078] The in vivo distribution of Icy-Py NPs drug delivery system was detected by in vivo imaging (CRI maestrosystem (λ ex / λ em =740 / 810nm, CRI, USA), by Figure 6 a It can be seen that 3 hours after administration, Icy-PyNPs injected into the tail vein began to accumulate in the gastric cancer tissue of mice, and the accumulated fluorescence intensity reached the maximum after 24 hours. The tumor and important organs of the mice were removed after 48 hours, and in vitro fluorescence imaging showed that Icy-Py NPs mainly accumulated in the tumor tissue.

[0079] In vivo photoacoustic imaging of the Icy-Py NPs drug delivery system was performed using an animal photoacoustic imager (PA / USVevo LAZR-X Fujifilm Visual Sonics). Figure 6 As shown in bc, the photoacoustic imaging signal reached its peak at 24 hours. The above results indicate that Icy-Py NPs have good targeting ability to gastric cancer tissue and can be used for real-time monitoring and photodynamic therapy of tumors.

[0080] Example 6 In vivo tumor inhibition effect of Icy-Py fluorescent dye

[0081] 5-week-old, male, 20g BALB / c mice were purchased from Nanjing Jicui Pharmaceutical. MFC gastric cancer cells with good growth status were injected subcutaneously in the right hypochondrium of the mice. The number of gastric cancer cells injected per mouse was 5×10 5 / mouse, and the volume of subcutaneous tumor and the weight of mice were observed every 2 days. The calculation formula of mouse tumor volume was: tumor length × width × width × 0.5.

[0082] The volume of mouse ectopic subcutaneous tumors grew to 50 mm 3 The mice were randomly divided into 4 groups, namely, the negative control PBS illumination group (+), the heptamethine cyanine iodide conjugated 2-pyridone compound drug delivery system non-illumination group Icy-Py NPs (-), the heptamethine cyanine iodide drug delivery system illumination group Icy NPs (+), and the heptamethine cyanine iodide conjugated 2-pyridone compound drug delivery system illumination group Icy-Py NPs (+).

[0083] The Icy-Py NPs(+) group and the Icy NPs(+) group were intravenously injected with the corresponding fluorescent dye drug delivery system (3 mg / kg). 24 hours later, the 808 nm laser was irradiated (0.5 W / cm 2 , 5min), twice a week, and the treatment cycle is three weeks.

[0084] The Icy-Py NPs(-) group was injected intravenously with the fluorescent dye drug delivery system (3 mg / kg) without light exposure, while the PBS(+) group was injected intravenously with PBS (200 μl) and then light treatment (0.5 W / cm 2 , 5min).

[0085] During the tumor intervention treatment, the maximum length and width of the tumor were recorded every 2 days with a vernier caliper. The tumor volume was calculated using the formula: length × width × width × 0.5. The tumor growth curve was drawn based on the calculated volume. Figure 7 It can be concluded from ab that Icy-Py has a better anti-tumor effect than Icy, which has a propyl side chain and is not conjugated with 2-pyridone.

[0086] After the treatment course, the tumor tissues of each group were fixed with formalin and embedded in paraffin, and 5 μm sections were cut. H&E was stained with hematoxylin and eosin, and the sections were sealed with neutral resin after staining. After antigen repair and peroxidase blocking, the sections were incubated with Ki67 primary antibody (1:1000) and secondary antibody (1:5000), and DAB was used for color development and microscopic examination. After staining, the sections were sealed. After the sections were permeabilized and fixed, they were treated with a reaction mixture containing rTDT and fluorescein-labeled dUTP, incubated at 37°C for 2 hours, and sealed for subsequent observation. Figure 7 c It can be seen that Icy-Py reduces tumor tissue cell proliferation and increases apoptosis compared with Icy, which has a propyl side chain and is not conjugated with 2-pyridone, and has a better in vivo tumor inhibition effect.

[0087] Example 7 Icy-Py in vivo biosafety assessment

[0088] The method of Example 6 was used to perform in vivo tumor inhibition treatment. After the treatment course, peripheral blood and important organs of the mice were collected, and blood biochemistry and H&E were performed to evaluate the in vivo safety of the fluorescent dye drug delivery system.

[0089] The experimental results are shown in Figure 8 .

[0090] in, Figure 8 a is H&E staining of important organs of mice after intervention with fluorescent dye drug delivery system. Figure 8 b is the AST concentration in mouse serum after intervention with the fluorescent dye drug delivery system. Figure 8 c is the ALT concentration in mouse serum after intervention with the fluorescent dye drug delivery system. Figure 8 d is the concentration of Cr in mouse serum after intervention with the fluorescent dye drug delivery system. Figure 8 e is the BUN concentration in the serum of mice after intervention with the fluorescent dye drug delivery system. The results showed that there was no significant change in the structure of important organs and no significant change in liver and kidney function after intervention with the fluorescent dye drug delivery system.

[0091] It can be seen that Icy-Py NPs have no obvious toxic side effects on important organs such as the heart, liver, spleen, lung, kidney and bone marrow of mice, proving that the fluorescent dye drug delivery system for targeted treatment of gastric cancer constructed by the present invention has good safety.

[0092] The above results show that the present invention provides a novel fluorescent dye for targeted treatment of gastric cancer for continuous photodynamic therapy of tumors, and uses liposomes for tail vein injection to achieve specific enrichment in tumor tissues, thereby significantly inhibiting the growth of gastric cancer.

[0093] In the present invention, unless otherwise specified, the scientific and technical terms used herein are those generally understood by those skilled in the art. In addition, the reagents, materials and operating procedures used herein are reagents, materials and conventional procedures widely used in the corresponding fields.

[0094] Finally, it should be noted that the photosensitizer involved in the present invention is a new type of fluorescent dye, which has not been reported in other studies. The fluorescent dye has the ability to continuously release singlet oxygen, prolongs the half-life of singlet oxygen, and significantly inhibits the growth of gastric cancer.

[0095] As described above, although the present invention has been shown and described with reference to specific preferred embodiments, it should not be construed as limiting the present invention itself. Various changes in form and details may be made without departing from the spirit and scope of the present invention as defined in the appended claims.

Claims

1. A heptamethincyanine iodide-conjugated 2-pyridone compound, characterized in that: It has the following structural formula:

2. A method for preparing the heptamethincyanine iodide-conjugated 2-pyridone compound according to claim 1, characterized in that: The following steps are involved: (1) reacting 1,4-di(bromomethyl)benzene with 2-hydroxypyridine to prepare 1-{[4-(bromomethyl)phenyl]methyl}-1,2-dihydro-2-pyridone PhBr-Py; (2) reacting the PhBr-Py obtained in step (1) with 5-iodo-2,3,3-trimethyl-3H-indole, and reacting the obtained intermediate with 2-chloro-3-[(E)-hydroxymethylene]cyclohex-1-ene-1-carboxaldehyde to prepare the iodinated heptamethine cyanine conjugated 2-pyridone compound Icy-Py 。 3. The preparation method according to claim 2, characterized in that: In step (1), the 1-{[4-(bromomethyl)phenyl]methyl}-1,2-dihydro-2-pyridone PhBr-Py is prepared by the following method: 1,4-di(bromomethyl)benzene and 2-hydroxypyridine were dissolved in dioxane, sodium hydride was slowly added in portions, heated and stirred, and after the reaction was completed, water was added to quench the reaction, and the resulting mixture was concentrated under vacuum and purified to obtain 1-{[4-(bromomethyl)phenyl]methyl}-l,2-dihydro-2-pyridone.

4. The preparation method according to claim 2, characterized in that: In step (2), the intermediate is prepared by the following method: The PhBr-Py and 5-iodo-2,3,3-trimethyl-3H-indole prepared in step (1) are dissolved in acetonitrile and heated for reaction. After the reaction is completed, the mixture is cooled to room temperature and the precipitate is collected by filtration, washed and dried to obtain a purple solid.

5. The preparation method according to claim 2, characterized in that: In step (2), the heptamethine cyanine iodide conjugated 2-pyridone compound Icy-Py is prepared by the following method: The obtained intermediate and 2-chloro-3-[(E)-hydroxymethylene]cyclohex-1-ene-1-carbaldehyde are dissolved in acetic anhydride and heated to react. After the reaction is completed, the crude product is distilled and purified to obtain the iodinated heptamethine cyanine conjugated 2-pyridone compound Icy-Py.

6. Use of the heptamethincyanine iodide-conjugated 2-pyridone compound according to claim 1 in the preparation of a fluorescent dye drug delivery system.

7. A fluorescent dye drug delivery system, characterized in that: The delivery system is loaded with the heptamethine cyanine iodide-conjugated 2-pyridone compound of claim 1.

8. A method for preparing the fluorescent dye drug delivery system according to claim 7, characterized in that: The following steps are involved: A mixture of lecithin, phosphatidylethanolamine, cholesterol and DSPE-PEG2000 and a 2-pyridone compound Icy-Py conjugated with heptamethine cyanine iodide are dissolved in dichloromethane, and the solvent is evaporated to form a liposome film, which is dissolved in water and then sonicated to form liposome Icy-Py NPs with uniform particle size, i.e., the fluorescent dye drug delivery system.

9. The preparation method according to claim 8, characterized in that: The molar ratio of the lecithin, phosphatidylethanolamine, cholesterol and DSPE-PEG2000 is 50:25:25:5; the mass ratio of the mixture of the lecithin, phosphatidylethanolamine, cholesterol and DSPE-PEG2000 and the iodine heptamethine cyanine conjugated 2-pyridone compound is 40:

1.

10. Use of the fluorescent dye drug delivery system according to claim 7 in the preparation of therapeutic drugs targeting gastric cancer.

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