Anionic pentamethine cyanine and preparation method and application thereof

Abstract

The application belongs to the field of chemical medicines, and specifically discloses an anionic pentamethine cyanine and a preparation method and application thereof. The anionic pentamethine cyanine with a specific structure is obtained by taking tricyano furan (2-(3-cyano-4,5,5-trimethyl furan-2(5H)-yl) malonitrile) as an end group precursor. The anionic pentamethine cyanine disclosed by the application can take into account near-infrared fluorescence imaging and optical treatment effects, and can be used as a fluorescent agent and a photosensitizer, and applied to near-infrared fluorescence imaging guided optical treatment, so as to realize tumor diagnosis and treatment integration.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of chemical drugs, and more particularly relates to an anionic pentamethine cyanine and a preparation method and application thereof. BACKGROUND

[0002] Optical therapy is a minimally invasive treatment with advantages of non-invasiveness and spatiotemporal selectivity, and has important application prospects in the biomedical field such as tumor treatment and antibacterial and anti-inflammatory. Photosensitizers used for optical therapy usually have multiple excited state energy relaxation pathways, thus exhibiting properties such as fluorescence emission, reactive oxygen species generation and heat generation. Therefore, optical therapy guided by fluorescence imaging can realize the integration of diagnosis and treatment, avoid missing small lesions, improve treatment efficiency and reduce toxic side effects. Compared with the current clinically used visible light band-based fluorescence imaging technology, near-infrared fluorescence imaging technology has the advantages of high penetration depth and high imaging signal-to-noise ratio, and can diagnose diseases such as tumors located deep in the body and further guide the development of treatment programs such as optical therapy.

[0003] At present, a variety of organic dye molecules have been selected as fluorescence imaging agents or photosensitizers for optical therapy. Among them, cationic heptamethine cyanine, such as indocyanine green (ICG), is widely used due to its narrow half-width, high-intensity near-infrared absorption and fluorescence, good biocompatibility, and extremely low dark cytotoxicity. However, the inherent defects of this type of cyanine limit its further application in near-infrared fluorescence imaging-guided optical therapy. On the one hand, the reactive oxygen species yield of cyanine-based photosensitizers is low. The current improvement methods mainly involve introducing heavy atoms and other chemical structure modifications, but the improvement effect is limited, and the dark cytotoxicity or synthesis difficulty is inevitably increased. On the other hand, the fluorescence quantum yield of cyanine in the near-infrared window is relatively low, and uncontrollable aggregation behavior also leads to further decline in fluorescence and other properties, which seriously restricts the application potential of cyanine in vivo. SUMMARY

[0004] In view of the defects in the prior art, the purpose of the present application is to provide an anionic pentamethine cyanine and a preparation method and application thereof. By improving the chemical structure of the cyanine end group, an anionic pentamethine cyanine with a specific structure is obtained, which has both near-infrared fluorescence imaging effect and reactive oxygen species generation ability.

[0005] To achieve the above-mentioned purpose, the first aspect of the present application provides an anionic pentamethine cyanine with tricyano furan as an end group, which has the structure shown in formula I:

[0006]

[0007] wherein X +represents one of metal cations, quaternary ammonium salt cations, quaternary phosphonium salt cations and imidazolium salt cations; R1, R2 and R3 are each independently selected from one of hydrogen, halogen, unsubstituted or substituted C 1-10 alkyl, unsubstituted or substituted C 1-10 alkoxy, unsubstituted or substituted aryl, preferably hydrogen.

[0008] Further, X + is preferably a sodium ion, a dodecyltriphenylphosphonium ion or a (2-(2-(2-(2-hydroxyethyloxy)hydroxyethyloxy)hydroxyethyloxy)ethyl)triphenylphosphonium ion, and R is preferably hydrogen.

[0009] The second aspect of the present application provides a preparation method of an anionic pentamethine cyanine, characterized in that the method comprises the following steps:

[0010] (1) condensing tricyano furan, sodium acetate and a pentamethine chain precursor in acetic anhydride to obtain an anionic pentamethine cyanine with sodium ion as a cation, the structure of which is shown in formula II:

[0011]

[0012] wherein R1, R2 and R3 are each independently selected from one of hydrogen, halogen, unsubstituted or substituted C 1-10 alkyl, unsubstituted or substituted C 1-10 alkoxy, unsubstituted or substituted aryl;

[0013] Alternatively, the anionic pentamethine cyanine with sodium ion as a cation obtained in step (1) is prepared into an anionic pentamethine cyanine containing different cations through ion exchange.

[0014] Further, the tricyano furan is tricyano furan (2-(3-cyano-4,5,5-trimethylfuran-2(5H)-yl)propanedinitrile), the structure of which is shown in formula III:

[0015]

[0016] The pentamethine chain precursor is a propandial bis(phenylimine) hydrochloride derivative; the structural formula of the propandial bis(phenylimine) hydrochloride derivative is shown in formula IV:

[0017]

[0018] wherein R1, R2 and R3 are each independently selected from one of hydrogen, halogen, unsubstituted or substituted C 1-10 alkyl, unsubstituted or substituted C 1-10 alkoxy, unsubstituted or substituted aryl.

[0019] Further, step (1) specifically comprises: mixing tricyanofuran, sodium acetate and malondialdehyde bis (phenylimine) hydrochloride derivative in acetic anhydride under nitrogen or inert gas atmosphere, stirring until the reaction is completed, extracting with dichloromethane and water, combining the organic phase after extraction, drying with anhydrous sodium sulfate, concentrating under reduced pressure to obtain a crude product, then purifying by silica gel column chromatography and reprecipitating with dichloromethane to obtain an anionic pentamethine cyanine with sodium ions as cations as shown in formula II.

[0020] Further, the molar ratio of the malondialdehyde bis (phenylimine) hydrochloride derivative to tricyanofuran is 1: (1.5-4).

[0021] Further, the molar ratio of the malondialdehyde bis (phenylimine) hydrochloride derivative to sodium acetate is 1: (2-10).

[0022] Further, the reaction temperature of the reaction is 0-40℃; the reaction time of the reaction is 0.5-6h.

[0023] Further, the ion exchange comprises: ion exchange of the anionic pentamethine cyanine with sodium ions as cations and a corresponding cation salt in a solution to prepare an anionic pentamethine cyanine containing different cations.

[0024] Further, the cation salt is a salt formed by one of metal cations, quaternary ammonium salt cations, quaternary phosphonium salt cations and imidazole salt cations.

[0025] Further, the molar ratio of the anionic pentamethine cyanine with sodium ions as cations to the corresponding cation salt is 1: (1-2.5).

[0026] Further, the solvent used in the solution is a mixed solvent composed of one or more of N, N-dimethylformamide, dichloromethane, acetic anhydride, ethanol, tetrahydrofuran, acetone, toluene and water; the reaction time of the ion exchange is 1-36h.

[0027] The third aspect of the present application provides an application of an anionic pentamethine cyanine, which is applied in the preparation of a fluorescent imaging agent for near-infrared fluorescence imaging or a photosensitizer for optical therapy. The anionic pentamethine cyanine can be used as a near-infrared fluorescent agent and a photosensitizer for optical therapy, and can be applied to optical therapy guided by near-infrared fluorescence imaging to realize tumor diagnosis and treatment integration.

[0028] Further, the anionic pentamethine cyanine is coated in nanoparticles or liposomes.

[0029] The method for coating the anionic pentamethine cyanine in nanoparticles comprises: mixing the anionic pentamethine cyanine and F-127 (chemical structural formula is HO (C2H4O) m -(C3H6O) n H, m and n are positive integers) is dissolved in an organic solvent, then distilled into a film under reduced pressure, then injected into a phosphate buffer, ultrasonic treatment, to obtain an anionic pentamethine cyanine nanoparticle dispersion;

[0030] The liposome includes adriamycin liposome TLCD99, amphotericin B liposome, daunorubicin liposome and gentamicin liposome, etc.

[0031] Compared with the prior art, the above technical scheme conceived by the present application can achieve the following

[0032] Beneficial effects:

[0033] (1) The anionic pentamethine cyanine provided by the present application improves the chemical structure of the cyanine end group, uses tricyano furan as the end group, obtains anionic pentamethine cyanine with a specific structure, and can balance the near-infrared fluorescence imaging effect and the active oxygen generation ability, thereby providing a new way for near-infrared fluorescence imaging guided optical therapy.

[0034] (2) The anionic pentamethine cyanine provided by the present application with tricyano furan as the end group has an absorption peak and a fluorescence peak in the near-infrared region, and has bright near-infrared fluorescence in the aqueous solution and nanoparticles, thereby realizing high-resolution in vivo fluorescence imaging. The nanoparticles composed of the anionic pentamethine cyanine show good tumor enrichment ability and can realize in-situ diagnosis of tumors.

[0035] (3) The anionic pentamethine cyanine provided by the present application with tricyano furan as the end group shows excellent active oxygen generation ability in the aqueous solution and nanoparticles, can effectively reduce the tumor volume, and shows excellent optical therapy effect. Therefore, as a chemical drug, the anionic pentamethine cyanine can balance the near-infrared fluorescence imaging and the optical therapy effect, can be applied to near-infrared fluorescence imaging guided optical therapy, and realizes tumor diagnosis and treatment integration.

[0036] (4) The anionic pentamethine cyanine provided by the present application has simple structure and preparation method, is easy to purify and control, has high yield, good repeatability, and is convenient for popularization and application. DETAILED DESCRIPTION

[0037] Figure 1 The structural general formula of the anionic pentamethine cyanine described in the present application.

[0038] Figure 2 The synthesis route diagram of C5T-Na, C5T-Pc and C5T-Pco in Example 1.

[0039] Figure 3 Hydrogen nuclear magnetic spectrum of C5T-Na in Example 1.

[0040] Figure 4 Hydrogen nuclear magnetic spectrum of C5T-Pc in Example 1.

[0041] Figure 5 Hydrogen nuclear magnetic spectrum of C5T-Pco in Example 2.

[0042] Figure 6 Normalized absorption spectra, excitation spectra (emission wavelength at 790 nm) and emission spectra (excitation wavelength at 700 nm) of (a) C5T-Na, (b) C5T-Pc and (c) C5T-Pco in dilute tetrahydrofuran solution (5 μg / mL) in Example 3.

[0043] Figure 7 Normalized absorption spectra, excitation spectra (emission wavelength at 824 nm) and fluorescence spectra (excitation wavelength at 700 nm) of (a) C5T-Na, (b) C5T-Pc, (c) C5T-Pco in concentrated tetrahydrofuran solution and (d) C5T-Pco nanoparticles (chlorin concentration at 0.1 mg / mL) in Example 3.

[0044] Figure 8 Photoluminescence spectra of (a) ICG (indocyanine green) aqueous solution or C5T-Pco nanoparticles (chlorin concentration at 0.1 mg / mL) for absolute fluorescence quantum yield calculation, inset is the near-infrared second zone fluorescence imaging photo of C5T-Pco nanoparticles; (b) capillary filled with ICG aqueous solution or C5T-Pco nanoparticles (chlorin concentration at 0.1 mg / mL) under different thickness of fat emulsion cover (excitation wavelength at 808 nm, long wave cutoff wavelength at 1100 nm) in Example 3.

[0045] Figure 9 Near-infrared second zone fluorescence imaging photos of tumor-bearing mice at different time points after intravenous injection of C5T-Pco nanoparticles (excitation wavelength at 808 nm, long wave cutoff wavelength at 1100 nm) in Example 3.

[0046] Figure 10(a) Relative fluorescence intensity curves at 525 nm of 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) solution containing ICG aqueous solution or C5T-Pco nanoparticles after irradiation by 808 and 760 nm lasers, respectively, in Example 4; (b) Relative fluorescence intensity curves at 525 nm of DCFH-DA solution containing C5T-Pco nanoparticles after irradiation by 808 and 760 nm lasers, respectively; (c) Laser scanning confocal fluorescence imaging of tumor cells co-stained by DCFH-DA and ICG aqueous solution and C5T-Pco nanoparticles after laser irradiation for 5 min.

[0047] Figure 11 (a) Schematic diagram of the process of optical therapy of anionic pentamethine cyanine nanoparticles in Example 5, (b) body weight curves and (c) relative tumor growth curves of mice in different groups, and (d) weights and (e) photos of tumors of mice in different groups after optical therapy.

[0048] Figure 12 Hematoxylin-eosin (H&E) staining results of tumors of mice in different groups after optical therapy in Example 5.

[0049] Figure 13 Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and Ki-67 staining results of tumors of mice in different groups after optical therapy in Example 5.

[0050] Figure 14 H&E staining results of each important organ of mice in different groups after optical therapy in Example 5. DETAILED DESCRIPTION

[0051] In order to make the objects, technical solutions and advantages of the present application clearer, the present application will be further described in detail below with reference to the drawings and examples. It should be understood that the specific examples described herein are only used to explain the present application and do not limit the present application. In addition, the technical features involved in each embodiment of the present application described below can be combined with each other as long as they do not conflict with each other.

[0052] First of all, it needs to be explained that:

[0053] The term "metal cation" used herein refers to a cation formed by losing an electron from a metal element (except for ammonium ion).

[0054] The term "quaternary ammonium salt cation" used herein refers to a cation formed by substituting four hydrogen atoms in an ammonium ion.

[0055] As used herein, the term "quaternary phosphonium salt cation" refers to a cation formed by replacing the nitrogen element in a quaternary ammonium salt cation with a phosphorus element.

[0056] As used herein, the term "imidazolium salt cation" refers to a cation formed by replacing the hydrogen on the nitrogen element of imidazole.

[0057] As used herein, the term "alkyl" refers to a straight chain or branched chain saturated hydrocarbon group.C 1-10 Examples of alkyl groups are methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, or decyl.

[0058] As used herein, the term "alkoxy" refers to a derivative group of an alkyl group single-bonded to an oxygen atom.C 1-10 Examples of alkoxy groups include methoxy, ethoxy, propoxy, butoxy, pentoxy, hexoxy, heptoxy, octoxy, nonoxy, or decoxy.

[0059] As used herein, the term "aromatic group" refers to an organic cyclic group having aromaticity. Examples of aromatic groups include phenyl, naphthyl, anthryl, phenanthryl, indenyl, and indanyl. As used herein, the term "aromatic group" also includes aromatic heterocyclic groups. As used herein, the term "aromatic heterocyclic group" refers to a group formed by replacing the carbon of the cyclic portion of an aromatic group with one or more heteroatoms. The heteroatoms in an aromatic heterocyclic group are typically at least one element selected from oxygen, sulfur, nitrogen, selenium. Examples of aromatic heterocyclic groups include pyridyl, pyrazinyl, pyrimidinyl, furanyl, thienyl, pyrazolidinyl, pyrrolyl, oxazolyl, oxadiazolyl, isoxazolyl, thiazolyl, thiadiazolyl, isothiazolyl, imidazolyl, pyrazolyl, quinolyl, or isoquinolyl.

[0060] As used herein, the term "substituted" refers to an organic group having one or more substituents selected from alkyl, alkoxy, aromatic group (as defined herein), alkenyl, hydroxy, carboxy, ester, acyl, amido, acyloxy, amino, nitro, cyano, halo, sulfonic acid, thiol, sulfonyl, phosphonic acid, phosphate, phosphonic acid ester, or phosphonic acid ester group.

[0061] As used herein, the term "halo" refers to replacement of a hydrogen atom with at least one halogen atom. The halogen atom is selected from one or more of fluorine, chlorine, bromine, or iodine.

[0062] As used herein, the term "aggregated state" refers to a supramolecular state of a material formed by non-covalent interactions between molecules under conditions of high concentration solution, poor solvent, and nanoparticles, etc. The formation of aggregated state can lead to changes in optical, electrical, and chemical properties of the material, and has important influences on the performance and behavior of dyes in practical applications.

[0063] In general, the application provides an anionic pentamethine cyanine with tricyanofuran as end group, which has the structure shown in formula I.

[0064]

[0065] wherein X represents one of metal cation, quaternary ammonium salt cation, quaternary phosphonium salt cation, imidazolium salt cation and the like; R1, R2 and R3 are each independently selected from one of hydrogen, halogen, unsubstituted or substituted C + alkyl, unsubstituted or substituted C 1-10 alkoxy, unsubstituted or substituted aryl. 1-10

[0066] X represents one of metal cation, quaternary ammonium salt cation, quaternary phosphonium salt cation, imidazolium salt cation and the like; R1, R2 and R3 are each independently selected from one of hydrogen, halogen, unsubstituted or substituted C + alkyl, unsubstituted or substituted C 1-10 alkoxy, unsubstituted or substituted aryl.

[0067] The end group precursor is tricyanofuran, which has the structure shown in formula III:

[0068]

[0069] The synthesis route of the anionic pentamethine cyanine in the application can be summarized as follows: first, condensation of tricyanofuran and pentamethine chain precursor to obtain an anionic pentamethine cyanine with sodium ion as cation, and then ion exchange with corresponding cation salt to obtain an anionic pentamethine cyanine containing different cations.

[0070] wherein X represents one of metal cation, quaternary ammonium salt cation, quaternary phosphonium salt cation, imidazolium salt cation and the like; R1, R2 and R3 are each independently selected from one of hydrogen, halogen, unsubstituted or substituted C

[0071]

[0072] wherein X represents one of metal cation, quaternary ammonium salt cation, quaternary phosphonium salt cation, imidazolium salt cation and the like; R1, R2 and R3 are each independently selected from one of hydrogen, halogen, unsubstituted or substituted C 1-10 alkyl, unsubstituted or substituted C 1-10 alkoxy, unsubstituted or substituted aryl.

[0073] The structure of the malondialdehyde bis(phenylimine) hydrochloride derivative is shown in formula IV:

[0074]

[0075] wherein R1, R2 and R3 are each independently selected from one of hydrogen, halogen, unsubstituted or substituted C 1-10 unsubstituted or substituted C 1-10 unsubstituted or substituted C

[0076] In some embodiments, the molar ratio of the malondialdehyde bis(phenylimine) hydrochloride derivative to the sodium acetate is 1:2-10.

[0077] In some embodiments, the molar ratio of the malondialdehyde bis(phenylimine) hydrochloride derivative to the sodium acetate is 1:2-10.

[0078] In some embodiments, the reaction temperature of the reaction is 0-40℃; and the reaction time of the reaction is 0.5-6h.

[0079] In some embodiments, the anionic pentamethine cyanine with sodium ion as cation and the corresponding cationic salt are ion-exchanged in a solution to prepare the anionic pentamethine cyanine containing different cations.

[0080] In some embodiments, the molar ratio of the anionic pentamethine cyanine with sodium ion as cation to the corresponding cationic salt is 1:1-2.5.

[0081] In some embodiments, the solvent is a mixed solvent consisting of one or more of N,N-dimethylformamide, dichloromethane, acetic anhydride, ethanol, tetrahydrofuran, acetone, toluene or water.

[0082] The R is preferably hydrogen, X + When the R is selected from the group consisting of dodecyltriphenylphosphonium cation and (2-(2-(2-(2-hydroxyethyloxy)hydroxyethyloxy)hydroxyethyloxy)ethyl)triphenylphosphonium cation, the prepared anionic pentamethine cyanine has a structure as shown in C5T-Pc and C5T-Pco of Formula V.

[0083]

[0084] The present application improves the chemical structure of the end group of the fluorin, selects tricyano furan (2-(3-cyano-4,5,5-trimethyl furan-2(5H)-yl) malonitrile) as the end group precursor, and obtains anionic pentamethine fluorin with a specific structure. Moreover, the synthesis route of the fluorin is simple and can be flexibly adjusted. Based on the fluorin provided by the present application, the near-infrared fluorescence imaging effect and the active oxygen generation capacity can be considered, and the fluorin can be used as a near-infrared fluorescent agent and an optical treatment photosensitizer at the same time, and is applied to optical treatment guided by near-infrared fluorescence imaging. When used for treating tumor carrying mice, the nanoparticles based on the fluorin can be enriched at the tumor site, emit bright near-infrared fluorescence, generate active oxygen, and reduce the tumor volume, so as to realize the integration of tumor diagnosis and treatment.

[0085] It should be noted that in the pentamethine fluorin provided by the present application, R1, R2 and R3 are each independently selected from one or more of hydrogen, halogen, unsubstituted or substituted C 1-10 alkyl, unsubstituted or substituted C 1-10 alkoxy and unsubstituted or substituted aromatic group. The substituent structures are similar and do not significantly affect the conjugated structure of the anionic pentamethine fluorin. The prepared anionic pentamethine fluorin should have similar optical properties. In addition, experiments prove that in the anionic pentamethine fluorin provided by the present application, the cation itself does not significantly change the optical properties of the anionic pentamethine fluorin in a non-aggregated state. In addition, the anionic pentamethine fluorin described in the present application has similar properties, and each cation in the examples does not have near-infrared luminescence and optical treatment ability. It can be inferred that the near-infrared fluorescence imaging effect and the active oxygen generation capacity of the examples described in the present application are both from the special structure of the anionic pentamethine fluorin. When C5T-Pco is used as a chemical drug, good near-infrared fluorescence imaging and optical treatment effect can be achieved, and it is further applied to optical treatment guided by near-infrared fluorescence imaging, and good tumor diagnosis and treatment effect can be achieved.

[0086] Example 1

[0087] The general structure of C5T-Na, C5T-Pc and C5T-Pco is as shown in Figure 1 The synthesis route is as shown in Figure 2 .

[0088] Synthesis of compound C5T-Na:

[0089] Tricyano furan (2-(3-cyano-4,5,5-trimethylfuran-2(5H)-yl)propanedinitrile, 385 mg, 1.93 mmol), sodium acetate (190 mg, 2.32 mmol) and malondialdehyde bis(phenylimine) hydrochloride (200 mg, 0.77 mmol) were dissolved in 5 mL of acetic anhydride. The reaction was stirred at 40 °C for 3 h, extracted with dichloromethane and deionized water. The combined organic phase was dried over anhydrous sodium sulfate and concentrated by evaporation under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography (dichloromethane / methanol 20 / 1 by volume as eluent) and reprecipitated by dichloromethane to obtain black powder C5T-Na (269 mg, yield 76 %). As shown in Figure 3 1 H NMR (600 MHz, Acetone-d6) δ 7.80 (t, J = 13.2 Hz, 2H, =CH-), 6.44 (t, J = 12.6 Hz, 1H, =CH-), 6.08 (d, J = 13.8 Hz, 2H, =CH-), 1.66 (s, 12H, -CH3), indicating the successful preparation of the product.

[0090] Synthesis of compound C5T-Pc:

[0091] C5T-Na (50 mg, 0.11 mmol) and PcBr (dodecyltriphenylphosphonium bromide, 67 mg, 0.13 mmol) were dissolved in 1 mL of dichloromethane / N,N-dimethylformamide (10 / 1 by volume). The reaction was stirred at room temperature for 1 h, extracted with dichloromethane / deionized water, and the combined organic phase was concentrated by evaporation under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography (dichloromethane / methanol 100 / 1 by volume as eluent) and recrystallized by dichloromethane / acetone to obtain black powder C5T-Pc (74 mg, yield 78 %). As shown in Figure 4 1 H NMR (400 MHz, Chloroform-d) δ 7.90-7.82 (m, 3H, ArH), 7.78-7.71 (m, 6H, ArH), 7.70-7.62 (m, 6H, ArH), 6.15 (t, J = 12.4 Hz, 1H, =CH-), 5.83 (br, s, 2H, =CH-), 3.24-3.11 (m, 2H, -PCH2-), 1.69-1.55 (m, 16H, -CH2-), 1.36-1.15 (m, 16H, -CH2- and -CH3), 0.86 (t, J = 7.2 Hz, 3H, -CH3).

[0092] Example 2

[0093] ​​Synthesis of compound C5T-Pco:

[0094] C5T-Na (152 mg, 0.33 mmol) and PcoBr ((2-(2-(2-(2-hydroxyethyloxy)hydroxyethyloxy)hydroxyethyloxy)ethyl)triphenylphosphonium bromide, 204 mg, 0.40 mmol) were dissolved in 3 mL of dichloromethane / N,N-dimethylformamide (10 / 1 by volume). The reaction system was stirred at room temperature for 1 h, then extracted with dichloromethane and deionized water. The combined organic phase was concentrated by evaporation under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography (dichloromethane / methanol as eluent, 100 / 1 by volume) and recrystallized by dichloromethane / acetone to obtain black powder C5T-Pco (240 mg, yield 83%). As shown in Figure 5 1 H NMR (600 MHz, Chloroform-d) δ 7.87-7.81 (m, 3H, ArH), 7.77-7.68 (m, 12H, ArH), 6.16 (t, J = 12.6 Hz, 1H, =CH-), 5.80 (br, s, 2H, =CH-), 3.85 (dt, J = 21.6, 5.4 Hz, 2H, -PCH2-), 3.74-3.68 (m, 2H, -CH2-), 3.62-3.53 (m, 6H, -CH2-), 3.49-3.44 (m, 2H, -CH2-), 3.41-3.33 (m, 4H, -CH2-), 1.62 (s, 12H, -CH3).

[0095] Example 3

[0096] Optical properties of compounds C5T-Na, C5T-Pc and C5T-Pco and performance of C5T-Pco nanoparticles as near-infrared fluorescence imaging agent:

[0097] Anionic pentamethine cyanines represented by C5T-Na, C5T-Pc and C5T-Pco exhibit highly consistent spectral characteristics in dilute tetrahydrofuran solution (5 pg / mL), as shown in Figure 6 They not only exhibit strong near-infrared absorption peaks centered around 763 nm, as well as fluorescence excitation spectra highly overlapping with the absorption spectra. They also exhibit highly consistent emission spectral characteristics, with the maximum emission peak centered around 796 nm, and have a relatively high fluorescence quantum yield (25%), which is conducive to achieving near-infrared imaging effect. In addition, the above results show that the structural changes of ions have little effect on the optical properties of cyanines in non-aggregated state. However, the excitation characteristic peaks and fluorescence emission characteristic peaks of the three anionic pentamethine cyanines in concentrated tetrahydrofuran solution (0.1 mg / mL) are all obviously red-shifted, as shown in Figure 7 ​As shown in the figure. It is noteworthy that, unlike dilute solutions, the fluorescence excitation and absorption spectra of concentrated solutions of anionic pentamethrin show significant differences, with almost no overlap. This indicates that the fluorescence intensity of aggregates formed by this type of anionic pentamethrin decreases substantially after photoexcitation in the 730–780 nm wavelength range. Simultaneously, the excitation spectrum of these aggregates exhibits a new strong fluorescence excitation peak after 800 nm, suggesting that the fluorescence relaxation pathway of the excited state energy of these aggregates exhibits excitation wavelength dependence, potentially enabling wavelength-selective applications.

[0098] To improve drug biocompatibility, drugs are often encapsulated in nanoparticles or liposomes in practical applications. The nanoparticles in this example were prepared using a modified thin-film hydration method: a tetrahydrofuran solution of C5T-X (0.4 mL, 0.5 mg / mL) and... F-127 (chemical structural formula: HO(C2H4O)) m -(C3H6O) n A dichloromethane solution (1.0 mL, 50 mg / mL) of H was mixed in a sample vial, and the mixture was distilled under reduced pressure at -70 kPa and 50 °C to form a film. 2 mL of phosphate buffer solution was slowly injected into the sample vial, and the vial was ultrasonically treated in a 50 °C water bath for 10 min to obtain a C5T-X nanoparticle dispersion. The concentration of C5T-X in the prepared C5T-X nanoparticle dispersion was 0.1 mg / mL. The concentration of F-127 is 25 mg / mL.

[0099] Taking C5T-Pco as an example, its nanoparticles exhibit optical properties highly consistent with those of concentrated solutions, such as... Figure 7 As shown in d, the fluorescence quantum yield of C5T-Pco nanoparticles after excitation with 808 nm light in the wavelength range of 814–1200 nm was 2.11%, which is 53 times that of ICG aqueous solution (0.1 mg / mL, fluorescence quantum yield 0.04%). Figure 8 a) This also surpasses most near-infrared fluorescent dyes. Furthermore, the fluorescence emission spectrum of C5T-Pco nanoparticles still exhibits fluorescence signals in the 1000–1200 nm wavelength range, with a fluorescence quantum yield of 0.05%, which meets the requirements for near-infrared II fluorescence imaging. Figure 8 The illustration in figure a shows this. Furthermore, fat emulsion injection was used to simulate biological tissue in in vivo imaging to investigate the tissue penetration ability of C5T-Pco nanoparticles in the near-infrared II region. (See illustration a). Figure 8 As shown in b, compared to the 1mm tissue penetration depth of ICG, the fluorescence of C5T-Pco nanoparticles can penetrate 6mm thick biological tissue, significantly improving the resolution and detection depth of fluorescence imaging, which is beneficial for in situ diagnosis of tumors in vivo.

[0100] In view of the above fluorescent test results, the C5T-Pco nanoparticles were injected into the tumor-bearing mice by intravenous injection method to study their tumor enrichment ability in the blood circulation in vivo. Figure 9 The near-infrared two-zone fluorescence imaging results of mice after inoculation of C5T-Pco nanoparticles were shown: the fluorescence signal of C5T-Pco nanoparticles reached a peak at the tumor site within 8-12 h, completed the enrichment process, had good tumor enrichment ability, and completed the metabolism after 36 h. The above experimental results showed that anionic pentamethine cyanine could be used as a near-infrared fluorescent imaging agent for in situ detection of tumors.

[0101] Example 4

[0102] Performance of C5T-Pco nanoparticles as photosensitizers for photodynamic therapy:

[0103] DCFH-DA was used as a reactive oxygen species fluorescent probe to detect the reactive oxygen species generation ability of ICG and C5T-Pco nanoparticles under 808 and 760 nm excitation, respectively. After 0.3 W / cm 2 The reactive oxygen species production of C5T-Pco nanoparticles was significantly better than that of ICG, which was 2.9 times that of the latter after 5 min of laser irradiation Figure 10 a). Notably, the reactive oxygen species production of C5T-Pco nanoparticles excited by 760 nm was significantly higher than that excited by 808 nm, which was 4.2 times that of the latter, as shown in Figure 10 b. It showed that the relaxation pathway of the excited state energy of anionic pentamethine cyanine aggregates showed excitation wavelength selectivity, which was conducive to improving the utilization rate of excitons, thereby realizing efficient tumor diagnosis and treatment integration.

[0104] Further, DCFH-DA was used as a reactive oxygen species probe to carry out 4T1 cell co-staining experiments, and the fluorescence signals were observed by laser scanning confocal microscopy to detect the reactive oxygen species generation ability of ICG and C5T-Pco nanoparticles in cells. As shown in Figure 10 c, after laser irradiation, the fluorescence signal in 4T1 cells co-cultured with ICG was weak, while bright fluorescence could be observed in cells co-cultured with C5T-Pco nanoparticles, proving that C5T-Pco nanoparticles also had excellent reactive oxygen species generation ability in cells.

[0105] Example 5

[0106] Performance of C5T-Pc and C5T-Pco nanoparticles as photosensitizers for photodynamic therapy:

[0107] Figure 11a is a schematic diagram of the optical therapy process of anionic pentamethine cyanine nanoparticle: the tumor-bearing mice were randomly divided into eight groups (5 mice per group), and when the tumor grew to about 70 mm 3 Afterwards, the mice were intravenously injected with phosphate buffered saline (group 1), ICG aqueous solution (group 3 and group 6), C5T-Pc nanoparticle (group 4 and group 7) and C5T-Pco nanoparticle (group 5 and group 8) at a solvent volume of 0.1 mL and a cyanine concentration of 0.1 mg / mL on the first day, the third day and the fifth day, respectively. Eight hours after the injection, the tumor sites of the mice were irradiated with laser (0.3 W cm -2 , 5 min), in which group 2 (without injection of any drug), group 7 and group 8 were irradiated with 760 nm laser, and group 6 was irradiated with 808 nm laser. Moreover, the body weight and tumor size of the mice in each group were measured every two days. There was no significant difference between the body weights of the mice in the eight groups Figure 11 b), indicating that the anionic pentamethine cyanine nanoparticle has good biocompatibility and low dark cytotoxicity. Compared with the tumors of the mice in each control group (group 1 to group 5), the volume and weight of the tumors of the mice in group 6 to group 8 were significantly reduced Figure 11 c-e), indicating that the optical therapy effect of C5T-Pc nanoparticle is better than that of ICG, and C5T-Pco nanoparticle has the best optical therapy effect.

[0108] Further, H&E staining, TUNEL and Ki-67 staining experiments were performed on the tumor tissue sections of each group to evaluate the apoptosis and proliferation of tumor cells after different treatments. As shown in Figure 12 , there was no significant difference in the H&E staining results of the tumors of the mice in group 1 to group 5, while the necrosis area and apoptosis degree of the tumors of the mice in group 6 to group 8 were increased in turn. In addition, according to the TUNEL and Ki-67 staining results Figure 13 ), the proportion of apoptosis (bright spots) of the tumors of the mice in group 6 to group 8 was increased in turn, while the proportion of tumor cell proliferation (bright spots) was decreased in turn. The above results show that under the corresponding laser irradiation, the anionic pentamethine cyanine nanoparticle exhibits a better tumor optical therapy effect than ICG, especially C5T-Pco. It is worth noting that there is no significant difference in the staining results between group 1 to group 5, indicating that the anionic pentamethine cyanine has small side effects. In addition, as shown in Figure 14 , the H&E staining results of the main organs of the mice in each group also showed no morphological changes and tissue lesions. The above results show that the anionic pentamethine cyanine has a significant tumor optical therapy effect under the guidance of near-infrared fluorescence imaging, and has small side effects.

[0109] In summary, the anionic pentamethine cyanine described in the patent can take into account the near-infrared fluorescence imaging and the optical treatment effect, especially can be used as a fluorescent agent and a photosensitizer, applied to the near-infrared fluorescence imaging guided optical treatment, realizes the tumor diagnosis and treatment integration. In the research process, it is found that the anionic pentamethine cyanine prepared based on tricyanofuran has an absorption peak and a fluorescence peak in the near-infrared wave band, and can still realize excellent near-infrared fluorescence imaging and optical treatment effect in aqueous solution and nanoparticles. The nanoparticles based on the anionic pentamethine cyanine not only show good tumor enrichment capacity, realize the in-situ diagnosis of tumors based on the near-infrared fluorescence imaging technology, but also have excellent optical treatment capacity, effectively reduce the tumor volume, so as to realize the tumor diagnosis and treatment integration.

[0110] Those skilled in the art will easily understand that the above description is only the preferred embodiment of the present application, and is not used to limit the present application, and any modification, equivalent replacement and improvement made within the spirit and principle of the present application should be included in the protection scope of the present application.

Claims

1. Use of an anionic pentamethine cyanine for the preparation of a composition for the treatment of skin, characterized in that, The application of the anionic pentamethine cyanine in the preparation of a fluorescent imaging agent for near-infrared fluorescence imaging or a photosensitizer for optical therapy; the structural formula of the anionic pentamethine cyanine is as follows: X + represents a cation; R1, R2and R3are hydrogen.

2. Use of an anionic pentamethine cyanine according to claim 1, characterized in that, The anionic pentamethine cyanine is coated in a nanoparticle or a liposome; The method for coating the anionic pentamethine cyanine in a nanoparticle comprises the following steps: dissolving the anionic pentamethine cyanine and Pluronic F-127 in an organic solvent, then performing film distillation under reduced pressure, injecting into a phosphate buffer, and performing ultrasonic treatment to obtain an anionic pentamethine cyanine nanoparticle dispersion; The liposome comprises one or more of an adriamycin liposome TLCD99, an amphotericin B liposome, a daunorubicin liposome, and a gentamicin liposome.

3. Use of the anionic pentamethine cyanine of claim 1, characterized in that, The preparation method of the anionic pentamethine cyanine comprises the following steps: The anionic pentamethine cyanine with sodium ions as cations is obtained by condensing tricyano furan, sodium acetate, and a precursor of a pentamethine chain in acetic anhydride, and the condensation reaction is performed at a temperature of 0-40 DEG C; the structure is as shown in formula II: Formula II R1, R2, and R3 are hydrogen; The tricyano furan is tricyano furan (2-(3-cyano-4,5,5-trimethyl furan-2(5H)-yl) propanedinitrile), and the structure is as shown in formula III: Formula III The precursor of the pentamethine chain is a malondialdehyde bis(phenylimine) hydrochloride derivative; the structural formula of the malondialdehyde bis(phenylimine) hydrochloride derivative is as shown in formula IV: Formula IV R1, R2, and R3 are hydrogen.

4. Use of an anionic pentamethine cyanine according to claim 3, characterized in that, The preparation method specifically comprises the following steps: mixing tricyano furan, sodium acetate, and the malondialdehyde bis(phenylimine) hydrochloride derivative in acetic anhydride under nitrogen or inert gas atmosphere, stirring until the reaction is completed, extracting and drying, then performing reduced pressure evaporation and concentration to obtain a crude product, and then performing silica gel column chromatography purification and dichloromethane reprecipitation to obtain the anionic pentamethine cyanine with sodium ions as cations as shown in formula II.

5. Use of an anionic pentamethine cyanine according to claim 4, characterized in that, The molar ratio of the malondialdehyde bis(phenylimine) hydrochloride derivative to tricyano furan is 1:(1.5-4); And / or, the molar ratio of the malondialdehyde bis(phenylimine) hydrochloride derivative to sodium acetate is 1:(2-10); and the reaction time is 0.5-6 h.

6. Use of an anionic pentamethine cyanine according to any one of claims 3 to 5, characterized in that, The preparation method further comprises: performing ion exchange on the anionic pentamethine cyanine with sodium ions as cations and other cation salts in a solution to obtain an anionic pentamethine cyanine containing other cations; The other cation salt is a salt formed by one of metal cations, quaternary ammonium salt cations, quaternary phosphonium salt cations, and imidazole salt cations, and does not include sodium ion salt.

7. Use of an anionic pentamethine cyanine according to claim 6, characterized in that, The molar ratio of the anionic pentamethine cyanine with sodium ions as cations to the corresponding cation salt is 1:(1-2.5); And / or, the solvent used in the solution is a mixed solvent composed of one or more of N,N-dimethylformamide, dichloromethane, acetic anhydride, ethanol, tetrahydrofuran, acetone, toluene, and water; and the ion exchange reaction time is 1-36 h.

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