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CN115340543B - Positive ion type cyclopean and application thereof in preparing pharmaceutical preparation for removing photodynamic therapy residues - Google Patents

Positive ion type cyclopean and application thereof in preparing pharmaceutical preparation for removing photodynamic therapy residues Download PDF

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CN115340543B
CN115340543B CN202210970847.7A CN202210970847A CN115340543B CN 115340543 B CN115340543 B CN 115340543B CN 202210970847 A CN202210970847 A CN 202210970847A CN 115340543 B CN115340543 B CN 115340543B
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npbox
chlorin
photodynamic therapy
hiporfin
npmebipy
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CN115340543A (en
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黎占亭
张丹维
孙健达
王辉
刘亚敏
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Fudan University
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    • C07ORGANIC CHEMISTRY
    • C07DHETEROCYCLIC COMPOUNDS
    • C07D471/00Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
    • C07D471/22Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed systems contains four or more hetero rings
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    • A61K41/00Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A61K41/0057Photodynamic therapy with a photosensitizer, i.e. agent able to produce reactive oxygen species upon exposure to light or radiation, e.g. UV or visible light; photocleavage of nucleic acids with an agent
    • A61K41/0071PDT with porphyrins having exactly 20 ring atoms, i.e. based on the non-expanded tetrapyrrolic ring system, e.g. bacteriochlorin, chlorin-e6, or phthalocyanines
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    • A61K47/50Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
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Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a positive ion type cyclopa and application thereof in preparation of a photodynamic therapy residue removal preparation. The positive ion type cyclopean provided by the invention is a water-soluble positive ion box-shaped macrocyclic molecule containing naphthalene units, is marked as NpBox, and specifically comprises three structural formulas. The high-strength combination of the porphin sodium, the chlorin e6, the hematoporphyrin and other hydrophobic anion type photodynamic therapeutic drugs can be realized, so that the photosensitization effect is reduced. The cyclopa can therefore be used for the preparation of pharmaceutical formulations for the removal of photodynamic therapy residues; animal experiments show that the cyclop can obviously reduce the residue of the photosensitizer on the surface of animal skin, and the effect of photodynamic therapy cannot be affected when the cyclop is used after photodynamic therapy.

Description

Positive ion type cyclopean and application thereof in preparing pharmaceutical preparation for removing photodynamic therapy residues
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to application of water-soluble positive ion type cyclopa in preparation of a medicinal preparation for removing photodynamic therapy residues.
Background
Cancer is one of the most serious diseases endangering human health and safety in the 21 st century, and the treatment of cancer is a urgent problem to be solved in public health. Due to the complexity and variety of tumors, treatments for cancer are also evolving. From 1903 Tappenier and Jesionek found that eosin can treat tumors under the excitation of light with specific wavelength, to the fact that porphin sodium is approved by FDA to be marketed for treating lung cancer, esophageal cancer, bladder cancer and other diseases, photodynamic therapy (PDT) is gradually developed into a reliable cancer treatment means. Compared with the traditional chemotherapy, PDT has the advantages of high efficiency, low toxicity, space-time controllability, minimally invasive and the like. Photodynamic therapy generally refers to a therapeutic approach that uses photosensitizers to convert oxygen molecules into a large amount of Reactive Oxygen Species (ROS) under excitation of light of a specific wavelength to cause necrosis or apoptosis of diseased cells. Currently, the vast majority of photosensitizers in clinical trials are porphyrins and their derivatives, such as porphin sodium (Photofrin), hematoporphyrin (HiPorfin), temoporfin (Temoporfin), palipofen (Padoporfin), and Talaporfin (Talaporfin), among which porphin sodium is the most representative. The porphin sodium has higher intersystem crossing efficiency and lower dark toxicity, and is a photosensitizer with wider application. However, the hydrophobic structure characteristics of these photosensitizers make them extremely prone to enrichment on skin tissue and long-term residue, making the patient's skin photosensitive for a long period of time after photodynamic therapy is completed, even requiring severe photophobia for up to several weeks, and bringing great inconvenience to daily life. Pharmacokinetic studies show that porphin sodium remains in reticuloendothelial cells for a long time, and published test reports show that after treatment with the recommended dose of 2mg/kg of porphin sodium, patients can develop obvious skin photoreaction, such as urticaria-like erythema, if exposed to about 1 time of sunlight or a strong light environment equivalent thereto, within a period of 1-3 months.
Disclosure of Invention
The invention aims to provide a positive ion type cyclopean and application thereof in preparing a medicinal preparation for removing photodynamic therapy residues.
The invention provides a positive ion type cyclopean which is a water-soluble positive ion box-shaped macrocyclic molecule containing naphthalene units, is marked as NpBox, and specifically comprises three structural formulas which are respectively marked as 2,6-NpBox, 1,4-NpBox and 1,5-NpBox according to the substitution positions of pyridine on the naphthalene units, wherein the three structural formulas are respectively as follows:
Figure BDA0003796665160000021
the NpBox provided by the invention is a positive ion type cyclopean and has the advantages of water solubility, high stability, low toxicity and the like.
As the photosensitizer used in photodynamic therapy contains carboxylic acid anions and porphyrin hydrophobic planes, various interactions such as electrostatic interaction, pi-pi accumulation and hydrophobic interaction are utilized. The NpBox provided by the invention can be combined with the photosensitizer efficiently, so that the photosensitizer is eliminated. Therefore, the invention also provides the application of the NpBox in preparing a pharmaceutical preparation of a medicine for removing residues in photodynamic therapy, in particular to the application of combining a photosensitizer through electrostatic action, pi-pi accumulation, hydrophobic action and the like by utilizing a hydrophobic cavity and high electropositivity of water-soluble NpBox, so that the concentration of the photosensitizer in skin tissues is reduced, the generation capacity of singlet oxygen is weakened, and the purpose of reducing and eliminating phototoxicity of the photosensitizer is realized. The use of positive ion type cyclopean does not affect the effect of photodynamic therapy.
In the invention, the pharmaceutical preparation for removing residues of photodynamic therapy further comprises pharmaceutically acceptable additives.
In the invention, the application of the NpBox in preparing a medicine preparation for removing photodynamic therapy residues is specifically performed as follows: dissolving NpBox in 5% glucose aqueous solution to form injection, wherein the concentration of the NpBox aqueous solution is 0.1mg/mL-1.5mg/mL; 200 μl of photosensitizer is tail-injected into mice and the formulated NpBox solution is injected over a period of time (e.g., within one hour) after the photodynamic therapy is completed.
In the invention, the medicine used for photodynamic therapy is a broad-spectrum cancer photodynamic therapy medicine.
In the invention, the model of the photodynamic therapy drug is porphin sodium, chlorin e6 or hematoporphyrin.
The positive ion type cyclopean provided by the invention can obviously reduce the accumulation of the medicine on the surface of animal skin, and does not influence the photodynamic treatment effect.
The invention also provides a preparation method of the positive ion type cyclop, which comprises the following steps of:
(1) Performing Suzuki coupling reaction on dibromonaphthalene and 4-pyridine borate to prepare dipyridine substituted naphthalene, which is marked as NpBipy;
(2) Nucleophilic substitution reaction is carried out on NpBipy and 1, 4-di (bromomethyl) benzene to prepare a corresponding compound NpBnBipy with two ends substituted by bromomethyl benzyl;
(3) Nucleophilic substitution reaction of NpBipy and NpBnBipy is carried out again to obtain corresponding cycloparaffin.
In the invention, an in vitro dynamic light scattering experiment, a nuclear magnetic titration experiment, a fluorescence experiment, a singlet oxygen generation experiment, a nanosecond transient absorption experiment, a cytotoxicity experiment, a hemolysis experiment, a mouse photosensitization inhibition experiment and the like are carried out on an NpBox and photosensitizer system. In vitro combination experiments show that the NpBox provided by the invention has obvious combination effect with different types of photosensitizers and can adjust the photophysical and chemical properties of the photosensitizers. Cytotoxicity experiments and hemolysis experiments show that the NpBox has higher biological safety; related animal experiments show that the NpBox can obviously reduce the accumulation of photosensitizers such as porphin sodium and the like on animal skin and inhibit the photosensitization. Among them, 2,6-NpBox has the most remarkable effect.
The experimental results show that the NpBox has good biocompatibility, can realize the combination of hydrophobic negative ion type porphyrin photodynamic therapy drugs in organisms and inhibit the photosensitivity of the drugs, and has wide application prospects in photodynamic therapy and clinical problems solving.
Drawings
FIG. 1 shows the scheme for the synthesis of cycloparaffin.
FIG. 2 is a scheme for the synthesis of the linearity control compound NpMeBipy.
FIG. 3 shows the crystal structure of NpBox. (a) 2,6-NpBox; (b) 1,4-NpBox; (c) 1,5-NpBox.
FIG. 4 is a graph showing the minimum energy model (DFT) of the complex of cycloparaffin and photosensitizer inclusion. Wherein (a)
Figure BDA0003796665160000031
(b)/>
Figure BDA0003796665160000032
FIG. 5 is a schematic illustration of the related compounds in waterDynamic light scattering particle diameter D in (1) H (nm), wherein the concentration of photosensitizer and NpBox is 0.1mM and the concentration of npmebipy is 0.2mM.
FIG. 6 shows the dynamic light scattering particle size D of a mixture of porphin sodium (Photofrin) and different cationic compounds in water H (nm), wherein the concentration ratio of photosensitizer and NpBox is 1:1 (both 0.1 mM) to NpMeBipy is 1:2 (NpMeBipy is 0.2 mM).
FIG. 7 shows the dynamic light scattering particle size D of Chlorin e6 (chlorine e 6) and a mixture of different cationic compounds in water H (nm), wherein the concentration ratio of photosensitizer and NpBox is 1:1 (both 0.1 mM) to NpMeBipy is 1:2 (NpMeBipy is 0.2 mM).
FIG. 8 shows the dynamic light scattering particle size D of hematoporphyrin (HiPorfin) and a mixture of different cationic compounds in water H (nm), wherein the concentration ratio of photosensitizer and NpBox is 1:1 (both 0.1 mM) to NpMeBipy is 1:2 (NpMeBipy is 0.2 mM).
FIG. 9 is a graph showing the dynamic light scattering particle size D of 2,6-NpBox and its derivatives with various photosensitizers in 20% DMSO aqueous solution H (nm), wherein the concentration ratio of the photosensitizer and the NpBox is 1:1 (both 0.1 mM).
FIG. 10 shows Chlorin e6 (chlorine e 6) and varying proportions of 2,6-NpBox at D 2 In O 1 HNMR spectra (400 MHz,25 ℃, [ Chlorin e 6)]=0.2 mM). Wherein, (a) Chlorin e6, (b) 2,6-NpBox (0.2 mM), (c) [2,6-NpBox]/[Chlorin e6]=0.5,(d)[2,6-NpBox]/[Chlorin e6]=1。
FIG. 11 shows the ratio of Chlorin e6 (chlorine e 6) to different ratios of 2,6-NpBox in CD 3 In OD 1 HNMR spectra (400 MHz,25 ℃, [ Chlorin e 6)]=0.2 mM). Wherein, (a) 2,6-NpBox (0.2 mM), (b) chlorine e6, (c) [2,6-NpBox]/[Chlorin e6]=0.05,(d)[2,6-NpBox]/[Chlorin e6]=0.1,(e)[2,6-NpBox]/[Chlorin e6]=0.2,(f)[2,6-NpBox]/[Chlorin e6]=0.3,(g)[2,6-NpBox]/[Chlorin e6]=0.4,(h)[2,6-NpBox]/[Chlorin e6]=0.6,(i)[2,6-NpBox]/[Chlorin e6]=0.8,(j)[2,6-NpBox]/[Chlorin e6]=1,(k)[2,6-NpBox]/[Chlorin e6]=1.2,(l)[2,6-NpBox]/[Chlorin e6]=1.4。
FIG. 12 shows Chlorin e6 (chlorine e 6) and 1,4-NpBox at D 2 In O 1 HNMR spectra (400 MHz,25 ℃, [ Chlorin e 6)]=0.2 mM). Wherein, (a) Chlorin e6, (b) 1,4-NpBox (0.2 mM), (c) [1,4-NpBox]/[Chlorin e6]=1
FIG. 13 shows Chlorin e6 (chlorine e 6) and 1,5-NpBox at D 2 In O 1 HNMR spectra (400 MHz,25 ℃, [ Chlorin e 6)]=0.2mM)。(a)Chlorin e6,(b)1,5-NpBox(0.2mM),(c)[1,5-NpBox]/[Chlorin e6]=1)。
FIG. 14 shows the ratio of Chlorin e6 (chlorine e 6) to 1,4-NpBox in CD 3 In OD 1 HNMR spectra (400 MHz,25 ℃, [ Chlorin e 6)]=0.2 mM). Wherein, (a) chlorine e6, (b) [1,4-NpBox]/[Chlorin e6]=0.4,(c)[1,4-NpBox]/[Chlorin e6]=0.8,(d)[1,4-NpBox]/[Chlorin e6]=1.2(e)[1,4-NpBox]/[Chlorin e6]=1.6,(f)[1,4-NpBox]/[Chlorin e6]=2,(g)[1,4-NpBox]/[Chlorin e6]=2.4。
FIG. 15 shows the ratio of Chlorin e6 (chlorine e 6) to 1,5-NpBox in CD 3 In OD 1 HNMR spectra (400 MHz,25 ℃, [ Chlorin e 6)]=0.2 mM). Wherein (a) chlorine e6, (b) [1,5-NpBox]/[Chlorin e6]=0.4,(c)[1,5-NpBox]/[Chlorin e6]=0.8,(d)[1,5-NpBox]/[Chlorin e6]=1.2(e)[1,5-NpBox]/[Chlorin e6]=1.6,(f)[1,5-NpBox]/[Chlorin e6]=2,(g)[1,5-NpBox]/[Chlorin e6]=2.4。
FIG. 16 shows the ratio of Chlorin e6 (chlorine e 6) to different ratios of 2,6-NpMeBipy in CD 3 In OD 1 HNMR spectra (400 MHz,25 ℃, [ Chlorin e 6)]=0.2 mM). Wherein (a) Chlorin e6, (b) [2,6-NpMeBipy]/[Chlorin e6]=1,(c)[2,6-NpMeBipy]/[Chlorin e6]=2,(d)[2,6-NpMeBipy]/[Chlorin e6]=4。
FIG. 17 shows the ratio of Chlorin e6 (chlorine e 6) to 1,4-NpMeBipy in CD 3 In OD 1 HNMR spectra (400 MHz,25 ℃, [ Chlorin e 6)]=0.2 mM). Wherein (a) Chlorin e6, (b) [1,4-NpMeBipy]/[Chlorin e6]=1,(c)[1,4-NpMeBipy]/[Chlorin e6]=2,(d)[1,4-NpMeBipy]/[Chlorin e6]=4。
FIG. 18 shows the ratio of Chlorin e6 (chlorine e 6) to 1,5-NpMeBipy in CD 3 In OD 1 HNMR spectra (400 MHz,25 ℃, [ Chlorin e 6)]=0.2 mM). Wherein (a) chlorine e6,(b)[1,5-NpMeBipy]/[Chlorin e6]=1,(c)[1,5-NpMeBipy]/[Chlorin e6]=2,(d)[1,5-NpMeBipy]/[Chlorin e6]=4。
FIG. 19 shows hematoporphyrin (HiPorfin) and different proportions of 2,6-NpBox at D 2 In O 1 HNMR spectra (400 MHz,25 ℃, [ HiPorfin)]=0.2 mM). Wherein, (a) HiPorfin, (b) 2,6-NpBox (0.2 mM),
(c)[2,6-NpBox]/[HiPorfin]=0.5,(d)[2,6-NpBox]/[HiPorfin]=1。
FIG. 20 shows hematoporphyrin (HiPorfin) and 1,4-NpBox at D 2 In O 1 HNMR spectra (400 MHz,25 ℃, [ HiPorfin)]=0.2 mM). Wherein, (a) HiPorfin, (b) 1,4-NpBox (0.2 mM), (c) [1, 4-NpBox)]/[HiPorfin]=1。
FIG. 21 shows hematoporphyrin (HiPorfin) and 1,5-NpBox at D 2 In O 1 HNMR spectra (400 MHz,25 ℃, [ HiPorfin)]=0.2mM)。(a)HiPorfin,(b)1,5-NpBox(0.2mM),(c)[1,5-NpBox]/[HiPorfin]=1。
FIG. 22 shows hematoporphyrin (HiPorfin) and different ratios of 2,6-NpBox at 20% CD 3 OD/D 2 In O solution 1 HNMR spectra (400 MHz,25 ℃, [ HiPorfin)]=0.2 mM). Wherein, (a) 2,6-NpBox (0.2 mM), (b) HiPorfin, (c) [2,6-NpBox]/[HiPorfin]=0.05,(d)[2,6-NpBox]/[HiPorfin]=0.1,(e)[2,6-NpBox]/[HiPorfin]=0.2,(f)[2,6-NpBox]/[HiPorfin]=0.3,(g)[2,6-NpBox]/[HiPorfin]=0.4,(h)[2,6-NpBox]/[HiPorfin]=0.6,(i)[2,6-NpBox]/[HiPorfin]=0.8,(j)[2,6-NpBox]/[HiPorfin]=1,(k)[2,6-NpBox]/[HiPorfin]=1.2,(l)[2,6-NpBox]/[HiPorfin]=1.4。
FIG. 23 shows hematoporphyrin (HiPorfin) and different ratios of 1,4-NpBox at 20% CD 3 OD/D 2 In O solution 1 HNMR spectra (400 MHz,25 ℃, [ HiPorfin)]=0.2 mM). Wherein (a) HiPorfin, (b) 1,4-NpBox]/[HiPorfin]=0.4,(c)[1,4-NpBox]/[HiPorfin]=0.8,(d)[1,4-NpBox]/[HiPorfin]=1.2(e)[1,4-NpBox]/[HiPorfin]=1.6,(f)[1,4-NpBox]/[HiPorfin]=2,(g)[1,4-NpBox]/[HiPorfin]=2.4。
FIG. 24 shows hematoporphyrin (HiPorfin) and different ratios of 1,5-NpBox at 20% CD 3 OD/D 2 In O solution 1 HNMR spectra (400 MHz,25 ℃, [ HiPorfin)]=0.2 mM). Wherein,,(a)HiPorfin,(b)[1,5-NpBox]/[HiPorfin]=0.4,(c)[1,5-NpBox]/[HiPorfin]=0.8,(d)[1,5-NpBox]/[HiPorfin]=1.2(e)[1,5-NpBox]/[HiPorfin]=1.6,(f)[1,5-NpBox]/[HiPorfin]=2,(g)[1,5-NpBox]/[HiPorfin]=2.4。
FIG. 25 shows hematoporphyrin (HiPorfin) and different ratios of 2,6-NpMeBipy at 20% CD 3 OD/D 2 In O solution 1 HNMR spectra (400 MHz,25 ℃, [ HiPorfin)]=0.2mM)。(a)HiPorfin,(b)[2,6-NpMeBipy]/[HiPorfin]=1,(c)[2,6-NpMeBipy]/[HiPorfin]=2,(d)[2,6-NpMeBipy]/[HiPorfin]=4。
FIG. 26 shows hematoporphyrin (HiPorfin) at 20% CD with different ratios of 1,4-NpMeBipy 3 OD/D 2 In O solution 1 HNMR spectra (400 MHz,25 ℃, [ HiPorfin)]=0.2mM)。(a)HiPorfin,(b)[1,4-NpMeBipy]/[HiPorfin]=1,(c)[1,4-NpMeBipy]/[HiPorfin]=2,(d)[1,4-NpMeBipy]/[HiPorfin]=4。
FIG. 27 shows hematoporphyrin (HiPorfin) at 20% CD with different ratios of 1,5-NpMeBipy 3 OD/D 2 In O solution 1 HNMR spectra (400 MHz,25 ℃, [ HiPorfin)]=0.2mM)。(a)HiPorfin,(b)[1,5-NpMeBipy]/[HiPorfin]=1,(c)[1,5-NpMeBipy]/[HiPorfin]=2,(d)[1,5-NpMeBipy]/[HiPorfin]=4。
FIG. 28 shows porphin sodium (Photofrin) with different cationic cyclophanes in 20% DMSO-d 6 /D 2 In O solution 1 HNMR spectra (400 MHz,25 ℃ C., photofrin monomer concentration 0.2 mM). Wherein (a) Photofrin, (b) [2,6-NpBox](0.2mM),(c)[2,6-NpBox]/[Photofrin]=1,(d)[1,4-NpBox](0.2mM),(e)[1,4-NpBox]/[Photofrin]=1,(f)[1,5-NpBox](0.2mM),(g)[1,5-NpBox]/[Photofrin]=1。
FIG. 29 shows porphin sodium (Photofrin) with different cationic linear compounds in 20% DMSO-d 6 /D 2 In O solution 1 HNMR spectra (400 MHz,25 ℃ C., photofrin monomer concentration 0.2 mM). Wherein (a) Photofrin, (b) [2,6-NpMeBipy](0.4mM),(c)[2,6-NpMeBipy]/[Photofrin]=2,(d)[1,4-NpMeBipy](0.4mM),(e)[1,4-NpMeBipy]/[Photofrin]=2,(f)[1,5-NpMeBipy](0.4mM),(g)[1,5-NpMeBipy]/[Photofrin]=2。
FIG. 30 is the fluorescence spectrum (excitation wavelength 410 nm) of porphin sodium (Photofrin, 10. Mu.M) mixed with different proportions of 2,6-NpBox in phosphate buffer (20 mM, pH 7.4). Wherein, (a) the sample has an emission spectrum of 570-750 nm; (b) a fluorescence intensity profile of the sample at 670 nm.
FIG. 31 is a fluorescence spectrum (excitation wavelength 420 nm) of Chlorin e6 (chlorine e6, 5. Mu.M) mixed with varying proportions of 2,6-NpBox in phosphate buffered saline (20 mM, pH 7.4). Wherein, (a) the sample has an emission spectrum of 570-750 nm; (b) a fluorescence intensity profile of the sample at 660 nm.
FIG. 32 shows fluorescence spectra (excitation wavelength 430 nm) of hematoporphyrin (HiPorfin, 5. Mu.M) mixed with different proportions of 2,6-NpBox in phosphate buffer solution (20 mM, pH 7.4). Wherein, (a) the sample has an emission spectrum of 570-750 nm; (b) a fluorescence intensity profile of the sample at 670 nm.
FIG. 33 is the fluorescence spectrum (excitation wavelength 410 nm) of porphin sodium (Photofrin, 10. Mu.M) mixed with different proportions of 1,4-NpBox in phosphate buffer (20 mM, pH 7.4). Wherein, (a) the sample has an emission spectrum of 570-750 nm; (b) a fluorescence intensity profile of the sample at 670 nm.
FIG. 34 is a fluorescence spectrum (excitation wavelength 420 nm) of Chlorin e6 (Chlorin e6, 5. Mu.M) mixed with varying proportions of 1,4-NpBox in phosphate buffered saline (20 mM, pH 7.4). Wherein, (a) the sample has an emission spectrum of 570-750 nm; (b) a fluorescence intensity profile of the sample at 660 nm.
FIG. 35 shows fluorescence spectra (excitation wavelength 430 nm) of hematoporphyrin (HiPorfin, 5. Mu.M) mixed with different proportions of 1,4-NpBox in phosphate buffer solution (20 mM, pH 7.4). Wherein, (a) the sample has an emission spectrum of 570-750 nm; (b) a fluorescence intensity profile of the sample at 670 nm.
FIG. 36 shows fluorescence spectra (excitation wavelength 410 nm) of porphin sodium (Photofrin, 10. Mu.M) mixed with different proportions of 1,5-NpBox in phosphate buffer (20 mM, pH 7.4). Wherein, (a) the sample has an emission spectrum of 570-750 nm; (b) a fluorescence intensity profile of the sample at 670 nm.
FIG. 37 is a fluorescence spectrum (excitation wavelength 420 nm) of Chlorin e6 (chlorine e6, 5. Mu.M) mixed with varying proportions of 1,5-NpBox in phosphate buffered saline (20 mM, pH 7.4). Wherein, (a) the sample has an emission spectrum of 570-750 nm; (b) Fluorescence intensity profile of sample at 660nm
FIG. 38 is a fluorescence spectrum (excitation wavelength 430 nm) of hematoporphyrin (HiPorfin, 5. Mu.M) mixed with 1,5-NpBox in different proportions in phosphate buffer solution (20 mM, pH 7.4). Wherein, (a) the sample has an emission spectrum of 570-750 nm; (b) a fluorescence intensity profile of the sample at 670 nm.
FIG. 39 is the fluorescence spectrum (excitation wavelength 410 nm) of porphin sodium (Photofrin, 10. Mu.M) mixed with varying proportions of 2,6-NpMeBipy in phosphate buffer (20 mM, pH 7.4). Wherein, (a) the sample has an emission spectrum of 570-750 nm; (b) a fluorescence intensity profile of the sample at 670 nm.
FIG. 40 is the fluorescence spectrum (excitation wavelength 420 nm) of Chlorin e6 (chlorine e6, 5. Mu.M) mixed with varying proportions of 2,6-NpMeBipy in phosphate buffered saline (20 mM, pH 7.4). Wherein, (a) the sample has an emission spectrum of 570-750 nm; (b) a fluorescence intensity profile of the sample at 660 nm.
FIG. 41 is a fluorescence spectrum (excitation wavelength 430 nm) of hematoporphyrin (HiPorfin, 5. Mu.M) mixed with different proportions of 2,6-NpMeBipy in phosphate buffer solution (20 mM, pH 7.4). Wherein, (a) the sample has an emission spectrum of 570-750 nm; (b) a fluorescence intensity profile of the sample at 670 nm.
FIG. 42 is the fluorescence spectrum (excitation wavelength 410 nm) of porphin sodium (Photofrin, 10. Mu.M) mixed with different proportions of 1,4-NpMeBipy in phosphate buffer (20 mM, pH 7.4). Wherein, (a) the sample has an emission spectrum of 570-750 nm; (b) a fluorescence intensity profile of the sample at 670 nm.
FIG. 43 is the fluorescence spectrum (excitation wavelength 420 nm) of Chlorin e6 (chlorine e6, 5. Mu.M) mixed with varying proportions of 1,4-NpMeBipy in phosphate buffered saline (20 mM, pH 7.4). Wherein, (a) the sample has an emission spectrum of 570-750 nm; (b) a fluorescence intensity profile of the sample at 660 nm.
FIG. 44 is a fluorescence spectrum (excitation wavelength 430 nm) of hematoporphyrin (HiPorfin, 5. Mu.M) mixed with different proportions of 1,4-NpMeBipy in phosphate buffer solution (20 mM, pH 7.4). Wherein, (a) the sample has an emission spectrum of 570-750 nm; (b) a fluorescence intensity profile of the sample at 670 nm.
FIG. 45 is the fluorescence spectrum (excitation wavelength 410 nm) of porphin sodium (Photofrin, 10. Mu.M) mixed with different proportions of 1,5-NpMeBipy in phosphate buffer (20 mM, pH 7.4). Wherein, (a) the sample has an emission spectrum of 570-750 nm; (b) a fluorescence intensity profile of the sample at 670 nm.
FIG. 46 is the fluorescence spectrum (excitation wavelength 420 nm) of Chlorin e6 (chlorine e6, 5. Mu.M) mixed with varying proportions of 1,5-NpMeBipy in phosphate buffered saline (20 mM, pH 7.4). Wherein, (a) the sample has an emission spectrum of 570-750 nm; (b) a fluorescence intensity profile of the sample at 660 nm.
FIG. 47 is a fluorescence spectrum (excitation wavelength 430 nm) of hematoporphyrin (HiPorfin, 5. Mu.M) mixed with different proportions of 1,5-NpMeBipy in phosphate buffer solution (20 mM, pH 7.4). Wherein, (a) the sample has an emission spectrum of 570-750 nm; (b) a fluorescence intensity profile of the sample at 670 nm.
FIG. 48 shows fluorescence intensity (Photofrin, 10. Mu.M; chlorin e6, 5. Mu.M; hiPorfin, 5. Mu.M) at a given wavelength in phosphate buffer (20 mM, pH 7.4) after mixing the photosensitizer with a ratio of NpBox (1:1) or NpMeBipy (1:2).
FIG. 49 is a graph showing the change in fluorescence intensity difference ΔEmission at 666nm in methanol (ΔEmission is the difference between fluorescence intensity at zero concentration of 2,6-NpBox and corresponding fluorescence intensity) after mixing Chlorin e6 (chlorine e6, 5. Mu.M) with different proportions of 2,6-NpBox, and the complexation constant Ka in 1:1 binding mode obtained by nonlinear fitting.
FIG. 50 shows the variation of the difference ΔEmission between the fluorescence intensities of hematoporphyrin (HiPorfin, 5. Mu.M) and different proportions of 2,6-NpBox in methanol at 670nm (difference between the fluorescence intensity and the corresponding fluorescence intensity when the ΔEmission is 2,6-NpBox concentration is zero) and the complexing constant Ka in the 1:1 binding mode obtained by nonlinear fitting.
Fig. 51 is a singlet oxygen generation curve (DPBF assay, [ DPBF ] =25 μm) of porphin sodium (2 μm) before and after 1:1 mixing with different npbs ox in PBS solution.
Fig. 52 is a singlet oxygen generation curve before and after 1:1 mixing of Chlorin e6 (chlorine e6,2 μm) with different NpBox in PBS solution (DPBF assay, [ DPBF ] =25 μm).
FIG. 53 shows singlet oxygen generation curves before and after 1:1 mixing of hematoporphyrin (HiPorfin, 2. Mu.M) with different NpBox in PBS (DPBF assay, [ DPBF ] = 25. Mu.M).
FIG. 54 shows porphin sodium (Photofrin, 30. Mu.M) at 20mM NaH 2 PO 4 Nanosecond transient absorption Spectroscopy (lambda) in buffer (pH 7.4) ex =420 nm). And for 5X 10 -7 ~2×10 -3 The absorption difference (DeltaAbs) at 430nm in s-range is fitted to a single exponential to obtain the triplet lifetime τ T
FIG. 55 shows Chlorin e6 (Chlorin e6, 7.5. Mu.M) at 20mM NaH 2 PO 4 Nanosecond transient absorption Spectroscopy (lambda) in buffer (pH 7.4) ex =420 nm). And for 5X 10 -7 ~2×10 -3 The absorption difference (DeltaAbs) at 430nm in s-range is fitted to a single exponential to obtain the triplet lifetime τ T
FIG. 56 shows hematoporphyrin (HiPorfin, 30. Mu.M) in 20mM NaH 2 PO 4 Nanosecond transient absorption Spectroscopy (lambda) in buffer (pH 7.4) ex =420 nm). And for 5X 10 -7 ~2×10 -3 The absorption difference (DeltaAbs) at 430nm in s-range is fitted to a single exponential to obtain the triplet lifetime τ T
FIG. 57 shows the mixture of porphin sodium (30. Mu.M) with NpBox in 20mM NaH before and after mixing 2 PO 4 Nanosecond transient absorption Spectroscopy (lambda) in buffer (pH 7.4) ex =420nm,λ obs =430 nm). Wherein, (a) linear abscissa; (b) logarithmic abscissa.
FIG. 58 shows a mixture of Chlorin e6 (Chlorin e6, 7.5. Mu.M) with NpBox at 20mM NaH 2 PO 4 Nanosecond transient absorption Spectroscopy (lambda) in buffer (pH 7.4) ex =420nm,λ obs =430 nm). Wherein, (a) linear abscissa; (b) logarithmic abscissa.
FIG. 59 shows 20mM NaH before and after mixing hematoporphyrin (HiPorfin, 30. Mu.M) with NpBox 2 PO 4 Nanosecond transient absorption Spectroscopy (lambda) in buffer (pH 7.4) ex =420nm,λ obs =430 nm). Which is a kind ofIn, (a) linear abscissa; (b) logarithmic abscissa.
FIG. 60 shows the cellular activities of H9C2 (a) and L02 (b) after treatment with different concentrations of 2,6-NpBox (MTT method, incubation 24H, n=6).
FIG. 61 shows the cellular activities of H9C2 (a) and L02 (b) after treatment with different concentrations of 1,4-NpBox (MTT method, incubation 24H, n=6).
FIG. 62 shows the cellular activities of H9C2 (a) and L02 (b) after treatment with different concentrations of 1,5-NpBox (MTT method, incubation 24H, n=6).
FIG. 63 shows the hemolytic activity (545 nm) of human and rat erythrocytes after treatment with 2,6-NpBox at various concentrations.
FIG. 64 is a graph showing the body weight of Balb/c mice (6 in each group, 3 in each case) over time after tail vein injection of 2,6-NpBox glucose solutions (5%) at various concentrations.
FIG. 65 shows the results of animal model experiments. Wherein, (a) animal experiments simplify the flow; (b) Photographs of the blank (PBS), negative (Photofrin) and experimental (Photofrin+2, 6-NpBox) groups at different time periods; (c) Fluorescence intensity of Photofrin in mouse tissues of negative control and experimental group (heart, liver, spleen, lung, kidney, skin, n=3); (d) three groups of mouse skin tissue H & E sections; (e) three groups of mouse tumor growth curves; (f) the size of the three groups of mouse tumors at the end of the observation; (g) H & E, TUNEL and Ki-67 characterization of three groups of mouse tumors (scale: 200 μm).
Fig. 66 is a photograph (dose modified versus time, n=5) of a group of mice (photofrin+2, 6-NpBox) during the experiment.
Fig. 67 shows the body weight changes (n=5) during the experiment in the blank (PBS), negative control (Photofrin) and experimental mice (photofrin+2, 6-NpBox).
Fig. 68 is a photograph (n=5) of mice of the experimental group (chlorine6+2, 6-NpBox) during the experiment.
Fig. 69 is a photograph (n=5) of mice of the experimental group (hiporfin+2, 6-NpBox) during the experiment.
Detailed Description
The invention is further described by the following examples, which should not be construed as limiting the invention.
Example 1: preparation of water-soluble cationic cyclop ibox and linear compound NpMeBipy.
The preparation route of the water-soluble positive ion type cycloparaffin NpBox (compound 4) provided by the invention is shown in figure 1: dibromonaphthalene 1 at different positions is taken as a raw material, is coupled with 4-pyridine borate to obtain a compound 2, and is subjected to nucleophilic substitution reaction with 1, 4-di (bromomethyl) benzene for 2 times to form a cyclic molecule 4. Taking 2, 6-dibromonaphthalene 1a as a raw material to synthesize the cyclopean 2,6-NpBox (4 a), the specific operation is as follows: 2, 6-Dibromonaphthalene (2.5 g,8.7 mmol), pyridine 4-borate (2.5 g,20.3 mmol) and Na 2 CO 3 (2.5 g,20.6 mmol) in DMF/H 2 To 165mL of a mixed solvent of O (10:1), pd (PPh) was added 3 ) 4 (1 g,0.87 mmol) was reacted under nitrogen at 90℃for 36 hours, cooled to room temperature and insoluble matter was removed by filtration through celite. After removal of the solvent, 100mLCH was added 2 Cl 2 The organic phase was washed three times with 100mL of water, the solvent was removed, and the resulting solid was dissolved in 200mL of hydrochloric acid solution (ph=1), insoluble impurities were removed by filtration, and the pH of the solution was adjusted to 7 to 8, to precipitate a white solid. 2, 6-npbbiy (2 a,2.2g, 89%): 1 H NMR(400MHz,CDCl 3 )δ8.73(d,J=5.1Hz,4H),8.17(s,2H),8.06(d,J=8.4Hz,2H),7.83(d,J=9.9Hz,2H),7.67(d,J=6.3Hz,4H);1,4-NpBipy(2b,1.6g,65%): 1 H NMR(400MHz,DMSO-d 6 )δ8.75(d,J=6.0Hz,4H),7.86(d,J=8.4Hz,2H),7.67–7.59(m,2H),7.56(d,J=6.1Hz,6H);1,5-NpBipy(2c,1.7g,69%): 1 H NMR(400MHz,DMSO-d 6 )δ8.76(d,J=6.0Hz,4H),7.87(dd,J=6.5,3.3Hz,2H),7.68–7.54(m,8H)。
a solution of 2,6-NpBipy (400 mg,1.4 mmol) in acetonitrile (60 mL) was added in portions to refluxing 1, 4-bis (bromomethyl) benzene (4.6 g,17.4 mmol) in CH 2 Cl 2 In solution (30 mL), reflux was continued for 48h, cooled to room temperature, and filtered to give a solid, using CH 2 Cl 2 (10 mL. Times.3) washing. Subsequently, the solid was formulated as a saturated solution of DMF and 10mL of saturated aqueous ammonium hexafluorophosphate solution and 5 volumes of water were added. Filtration, drying, and obtaining a yellow solid. 2,6-NpBnBipy (3 a,1.2g, 90%): 1 H NMR(400MHz,CD 3 CN)δ8.80(d,J=6.8Hz,4H),8.60(d,J=1.9Hz,2H),8.41(d,J=6.9Hz,4H),8.29(d,J=8.7Hz,2H),8.08(dd,J=8.6,1.8Hz,2H),7.68–7.39(m,8H),5.73(s,4H),4.61(s,4H); 13 C NMR(101MHz,DMSO-d 6 )δ154.51,144.94,144.01,134.06,133.07,132.83,130.66,129.12,128.76,127.18,125.69,125.40,62.43,39.60;MS calcd for C 36 H 30 Br 2 N 2 PF 6 [M-PF 6 ] + :795.0395;found:795.0397;1,4-NpBnBipy(3b,1.1g,83%): 1 H NMR(400MHz,DMSO-d 6 )δ9.37(d,J=6.8Hz,,4H),8.41(d,J=6.7Hz,4H),7.98(dd,J=6.5,3.3Hz,2H),7.86(s,2H),7.74(dd,J=6.5,3.3Hz,2H),7.65(d,J=8.2Hz,4H),7.58(d,J=8.3Hz,4H),5.96(s,4H),4.75(s,4H); 13 C NMR(101MHz,DMSO-d 6 )δ155.81,144.90,139.42,136.32,134.28,130.16,129.90,129.45,129.32,128.43,127.75,125.46,62.52,33.67;MS calcd for C 36 H 30 Br 2 N 2 PF 6 [M-PF 6 ] + :795.0400;found:795.0400;1,5-NpBnBipy(3c,1.2g,90%): 1 H NMR(400MHz,DMSO-d 6 )δ9.34(d,J=6.4Hz,4H),8.39(d,J=6.8Hz,4H),8.07(dd,J=7.6,1.9Hz,2H),7.85–7.74(m,4H),7.65(d,J=8.1Hz,4H),7.58(d,J=8.2Hz,4H),5.91(s,4H),4.75(s,4H); 13 CNMR(101MHz,DMSO-d 6 )δ155.45,144.87,139.42,134.70,134.35,130.18,129.84,129.47,129.39,129.19,127.38,127.28,62.46,33.71;MS calcd for C 36 H 30 Br 2 N 2 PF 6 [M-PF 6 ] + :795.0406;found:795.0406。
2,6-NpBnBipy (200 mg,0.21 mmol), 2,6-NpBipy (57.2 mg,0.20 mmol), TBAI (15 mg,0.041 mmol) and pyrene (212 mg,1.05 mmol) were dissolved in 200mL of dry acetonitrile and reacted at 30℃for 7 days. After the reaction, add (t-Bu) 4 The reaction was quenched with 2-3mL of NCl in saturated acetonitrile. The crude product is separated by column chromatography (eluent CH 3 OH:H 2 O:sat.NH 4 Cl=6:3:1) to give a yellow solid. 2,6-NpBox (4 a,51.1mg, 26%): 1 H NMR(400MHz,D 2 O)δ8.96(d,J=6.4Hz,8H),8.37(s,4H),8.27(d,J=9.5Hz,8H),8.06(d,J=9.3Hz,4H),7.87(d,J=7.7Hz,4H),7.71(s,8H),5.83(s,8H); 13 C NMR(101MHz,DMSO-d 6 )δ154.13,144.39,136.49,133.84,132.41,130.36,129.99,129.13,125.44,125.30,61.20;MS calcd for C 56 H 44 N 4 [M] 4+ :193.0886;found:193.0904;1,4-NpBox(4b,53.5mg,27%): 1 H NMR(400MHz,D 2 O)δ9.09(d,J=6.0Hz,8H),8.17(d,J=6.1Hz,8H),7.91–7.82(m,4H),7.75(s,8H),7.62(d,J=5.6Hz,4H),7.41(s,4H),5.93(s,8H); 13 C NMR(101MHz,DMSO-d 6 )δ155.61,144.48,136.31,136.02,130.21,129.96,129.22,129.17,128.88,125.48,61.93;MS calcd for C 56 H 44 N 4 P 2 F 12 [M-2PF 6 ] 2+ :531.1419;found:531.1419;1,5-NpBox(4c,63.2mg,32%): 1 H NMR(400MHz,D 2 O)δ9.08(d,J=6.3Hz,8H),8.12(d,J=4.6Hz,8H),7.83(s,8H),7.80(d,J=8.9Hz,4H),7.48–7.42(m,8H),5.94(s,8H); 13 C NMR(101MHz,DMSO-d 6 )δ156.16,144.25,136.63,134.42,130.27,129.55,129.40,129.21,127.23,126.83,62.21;MS calcd for C 56 H 44 N 4 P 2 F 12 [M-2PF 6 ] 2+ :531.1419;found:531.1419。
as a control compound, the present invention also synthesizes a pyridine terminal methyl-substituted positive ion type linear compound NpMeBipy (5), as shown in FIG. 2. Taking 2,6-NpBipy (2 a) as a raw material to synthesize 2,6-NpMeBipy (5 a), specific operations are as follows: compound 2,6-NpBipy (100 mg,0.35 mmol) and MeI (0.5 g,3.5 mmol) were dissolved in DMF (10 mL) and reacted at 50℃for 12h. After the completion of the reaction, the reaction mixture was cooled to room temperature, and 50mL of Et was added 2 O, the solid was collected by filtration, and the methylated chloride salt was obtained by ion exchange. 2,6-NpMeBipy (5 a,98.0mg, 72%): 1 H NMR(400MHz,DMSO-d 6 )δ9.10(d,J=6.6Hz,4H),8.89(s,2H),8.68(d,J=6.8Hz,4H),8.35(d,J=8.7Hz,2H),8.29(d,J=10.4Hz,2H),4.38(s,6H); 13 C NMR(101MHz,D 2 O)δ155.65,145.05,133.41,131.06,130.24,128.65,125.37,125.18,45.32;MS calcd for C 22 H 20 N 2 [M] 2+ :156.0808;found:156.0806;1,4-NpMeBipy(5b,84.4mg,62%): 1 H NMR(400MHz,D 2 O)δ8.95(d,J=6.6Hz,4H),8.29(d,J=6.6Hz,4H),8.00(dd,J=6.5,3.3Hz,2H),7.82(s,2H),7.74(dd,J=6.5,3.2Hz,2H),4.52(s,6H); 13 C NMR(101MHz,D 2 O)δ156.70,145.03,136.35,130.10,128.93,128.36,127.49,125.16,47.51;MS calcd for C 22 H 20 N 2 [M] 2+ :156.0808;found:156.0808;1,5-NpMeBipy(5c,89.9mg,66%): 1 H NMR(400MHz,D 2 O)δ8.93(d,J=6.5Hz,4H),8.28(d,J=6.3Hz,4H),8.08(dd,J=6.3,3.2Hz,2H),7.81–7.75(m,4H),4.51(s,6H); 13 C NMR(101MHz,D 2 O)δ157.10,144.93,134.56,130.00,128.97,127.13,127.05,47.30;MS calcd for C 22 H 20 N 2 [M] 2+ :156.0808;found:156.0818。
the solid phase structure of the three cyclophanes was confirmed by their crystal structure (fig. 3). All three cyclophanes are square box-shaped, forming a hydrophobic cavity inside, the cavity dimensions 2,6-NpBox >1,5-NpBox ≡1,4-NpBox seen from the distance of two opposite phenyl groups, possibly in combination with size matching compounds. Theoretical calculations have shown that (FIG. 4), chlorin e6, hiPorfin et al can enter the cavity of the cyclopean 2,6-NpBox and form inclusion complexes therewith.
Example 2: dynamic light scattering experiments of positive ion type cyclopean and its linear control compound and photodynamic therapy drug.
Dynamic light scattering experiments provide particle size distribution in solution, and FIGS. 5-9 respectively represent the particle sizes of a photosensitizer aqueous solution with a certain concentration, a cyclop NpBox aqueous solution, a linear compound NpMeBipy aqueous solution and a specific ratio of photosensitizer to cyclop NpBox (1:1) or a linear compound NpMeBipy (1:2) in the aqueous solution. As shown in FIG. 5, D of 2,6-NpBox and Photofrin in aqueous solution at the tested concentrations H The particle diameters of the rest samples are about 1nm and about 10nm and about 30nm respectively. As shown in fig. 6-8, the 1:1 mixture of the three photosensitizers with the cyclopean 1, 4-or 1,5-NpBox, respectively, all formed particles with a particle size greater than 1000nm in aqueous solution, indicating that they stacked to form large particles through various weak interactions; the 1:1 mixture of photosensitizer and cycloparaffin 2,6-NpBox in aqueous solution has a smaller particle size of about 20-30nm. The assembly behavior of the photosensitizer and the linear compound is also different, and the detection is carried outUnder the test conditions, the hydration particle size of the 1:2 mixture of the three photosensitizers and the 2,6-NpMeBipy is also larger than 1000nm, the hydration particle size of the 1:2 mixture of the photosensitizers and the 1, 4-or 1,5-NpMeBipy is smaller than 100nm, and the hydration particle size of the 1:2 mixture of HiPorfin and the 1, 4-or 1,5-NpMeBipy is smaller than 10nm, which indicates that the stacking modes are different. The hydrated particle sizes of 2,6-NpBox and the compound of the 2,6-NpBox and the three photosensitizers in 20% DMSO aqueous solution are all about 1nm (figure 9), and compared with the dynamic light scattering particle sizes in the corresponding compound aqueous solution, the hydrated particle sizes in the aqueous solution are all larger, which indicates that the compounds have certain piling effect in the aqueous solution.
Example 3: nuclear magnetic titration experiments of positive ion type cyclopa and its control compound and photodynamic therapy medicine.
The assembled form of the photosensitizer with the cationic cyclopean or linear compound of the present invention in solution was further determined by nuclear magnetic titration experiments. The nuclear magnetic titration experiments of 2,6-NpBox cycloparaffin and Chlorin e6 in heavy water are exemplified. The specific operation is as follows: heavy aqueous solutions of Chlorin e6 (0.2 mM) and 2,6-NpBox (2 mM) were prepared, respectively, 2,6-NpBox was gradually dropped into the Chlorin e6 solution in proportion, and the nuclear magnetic resonance hydrogen spectrum of the solution system after each drop was measured (FIG. 10). The research of nuclear magnetic titration experiments shows that the positive ion type cyclopean 2,6-NpBox and photosensitizer Chlorin e6 drop in heavy water with sharply reduced resolution, and have wider signal peak and obvious chemical shift change at the ratio of 1:1. In CD 3 The nuclear magnetic titration experiment in OD (FIG. 11) observed a gradual increase in the cyclopean 2,6-NpBox, a widening of the Chlorin e6 signal peak, and a gradual change in the peak and chemical shift of the 2,6-NpBox signal peak. After the combination of Chlorine6 with cyclopean 1,4-NpBox (FIG. 12) or 1,5-NpBox (FIG. 13) in heavy water at a ratio of 1:1, the Chlorine6 aromatic zone had little signal and was not effectively resolved, while the cyclopean signal was not significantly changed, at CD 3 In OD, there was no significant change in the signal of both the photosensitizer and the cycloparaffin with increasing proportions of cycloparaffin 1,4-NpBox (FIG. 14) or 1,5-NpBox (FIG. 15). In CD 3 When nuclear magnetic titration experiments were performed on Chlorin e6 using three linear compounds in OD, no Chlorin e6 signal was observed as the proportion of linear compounds increasedThe peaks were significantly changed, and at the same time, the peak patterns and chemical shifts of the signal peaks of the three linear compounds were not significantly changed (fig. 16 to 18). The above experimental results show that in aqueous solution, the cationic cyclopean of the present invention has strong interaction with photosensitizer, and the binding mode of 1,4-NpBox or 1,5-NpBox and Chlorin e6 is similar, and the binding mode of 2,6-NpBox and Chlorin e6 is different from the binding mode of 1,4-NpBox or 1,5-NpBox and Chlorin e 6. In combination with theoretical calculation experiments (FIG. 4 a), chlorin e6 may enter the cavity of 2,6-NpBox to form inclusion, while stacking with 1,4-NpBox or 1,5-NpBox may occur outside the ring. In heavy water (FIGS. 19-21) or 20% CD 3 OD/D 2 In O (FIGS. 22-24), nuclear magnetic titration experiments of HiPorfin with cyclopean showed similar results to that of Chlorin e 6. At 20% CD 3 OD/D 2 The results of nuclear magnetic titration experiments on HiPorfin with three linear compounds in O were also similar to that of Chlorin e6 (FIGS. 25-27). The results of nuclear magnetic titration experiments demonstrate that HiPorfin and three cyclophanes interact in a similar mode of action to Chlorin e 6. Photofrin showed broad peaks in the nuclear magnetic resonance hydrogen spectrum, especially in the low field region, and when three cyclophan with equimolar amounts of Photofrin monomers were added separately (FIG. 28), similar experimental results to that of Chlorin e6, hiPorfin were shown, indicating strong interactions of Photofrin with cyclophan in the solution phase. When three linear compounds were added in equimolar amounts to the Photofrin monomer respectively (fig. 29), the nuclear magnetic hydrogen spectrum signal still exhibited a broad peak with very low resolution.
Example 4: fluorescence experiments of cationic cyclopean and photodynamic therapy drugs in aqueous solution.
The combination of the photosensitizer with the cationic cycloparaffin or linear compound of the present invention in solution phase was characterized by fluorescence experiments. The specific operation is as follows: preparing a photosensitizer solution with a certain concentration, dropwise adding an NpBox or NpMeBipy solution, and performing fluorescence test. With the addition of the latter, the fluorescence intensity of the photosensitizer decreases until equilibrium, indicating that there is some degree of charge transfer or energy transfer between the photosensitizer and NpBox and NpMeBipy. As shown in FIGS. 30 to 47, the positive ion type cyclopeanox and the linear compound N can be determined by fluorescence testpMeBipy has some interaction with photosensitizers. From FIG. 48, the quenching effect of NpBox and NpMeBipy on photosensitizer fluorescence can be intuitively compared, and the following conclusions can be drawn: (1) 2,6-NpBox can most significantly quench the fluorescence of the photosensitizer; (2) The fluorescence quenching effect of NpBox is more pronounced than that of the corresponding NpMeBipy photosensitizer. Fluorescence experiments showed that the binding constants of 2,6-NpBox to Chlorine6 (fig. 49) and HiPorfin (fig. 50) in methanol solution in 1:1 mode were (1.89.+ -. 0.31). Times.10, respectively 6 And (2.86.+ -. 0.57). Times.10 6
Example 5: effect of cationic cyclop on singlet oxygen yield of photodynamic therapy drugs.
Preparing a photosensitizer solution with a certain concentration, adding a certain proportion of NpBox (1:1), and adding 1, 3-diphenyl isobenzofuran (DPBF) with a certain concentration as a singlet oxygen indicator. The absorbance of the solution in the wavelength range of 300-500 nm is measured before laser irradiation, and the absorbance in the same wavelength range is measured after a certain time of laser irradiation. Singlet oxygen yield was calculated from the decay rate of the DPBF characteristic absorption at 427nm over time. As shown in fig. 51-53, the experimental results demonstrate that the addition of the cationic cyclopean can reduce the singlet oxygen yield of the photosensitizer. Of the three cyclophanes, 2,6-NpBox most significantly reduced the singlet oxygen yield of the photosensitizer, indicating that energy or charge transfer between 2,6-NpBox and the photosensitizer prevented the photosensitizer from generating singlet oxygen. Consistent with fluorescence experiments in inhibition ability.
Example 6: effect of positive ion type cyclopean on triplet lifetime of photodynamic therapy drug.
The generation of singlet oxygen depends on the triplet state of the photosensitizer, and in general, the longer the triplet lifetime of the photosensitizer, the more advantageous it is to sensitize oxygen to singlet oxygen. As can be seen from FIGS. 54 to 56, the triplet lifetime of the three photosensitizers is on the order of 10 -5 ~10 -4 s. Preparing a photosensitizer solution with a certain concentration, adding a certain proportion of NpBox (1:1), measuring the absorption change value of the photosensitizer at 430nm under 420nm excitation wavelength by nanosecond transient absorption spectrum, and experimental results show (figures 57-59) that after adding a certain proportion of NpBox (1:1), the triplet state of the photosensitizer is shownAbsorption is significantly changed, wherein 2,6-NpBox can be measured on the resolution scale of the instrument (10 -9 s) complete quenching of the triplet state of the photosensitizer, indicating that after addition of 2,6-NpBox, the triplet lifetime of all three photosensitizers is less than 10 -9 s. The combination of 1,4-NpBox and 1,5-NpBox with photosensitizer also significantly reduces the concentration of free triplet photosensitizer in the system.
Example 7: cytotoxicity experiments of positive ion type cyclopean.
By using a cell counting kit Cell Counting Kit-8 (CCK-8), we studied the cytotoxicity of three positive ion type cyclopeans, 2,6-NpBox, 1,4-NpBox and 1,5-NpBox, in vitro using normal hepatocytes (L-02) and rat cardiomyocytes (H9C 2) as models. The results showed that when the content of the cyclop sample was 0-100. Mu.g/mL, the viability of both cells was 85% or more (FIGS. 60-62), indicating that the cytotoxicity of the three cyclop-2, 6-NpBox, 1,4-NpBox and 1,5-NpBox was low.
Example 8: acute toxicity test and hemolysis test of 2,6-NpBox animals.
Animal experiments were performed following the ethical principles of experimental animals, taking 2,6-NpBox as an example. Biocompatibility was first verified by hemolysis experiments. As shown in FIG. 63, the experiment shows that 2,6-NpBox does not have obvious hemolysis phenomenon under the concentration of 0-100 mug/mL, and the 2,6-NpBox has good biocompatibility. The specific operation process of the acute toxicity experiment is as follows: ICR mice weighing 18 to 24 grams were selected as biological models for testing the biotoxicity of 2,6-NpBox (three male and three female mice were selected for each set of experiments); weigh and record the initial body weight of the mice; the 2,6-NpBox injection is injected into the mice through tail vein at one time, the dosages are respectively 0mg/kg, 5mg/kg, 10mg/kg and 15mg/kg, the injection volume is controlled at 0.2mL, and the injection time is 60 seconds; the mice were then weighed and recorded every 24 hours for 14 days (fig. 64). This study showed that 2,6-NpBox had low acute toxicity to animals at the concentrations used (4 mg/kg) and no death occurred at doses up to 15mg/kg (3.75 times the effective dose), demonstrating high biosafety.
Example 9: study of the phototoxic effects of 2,6-NpBox on photosensitizer photodynamic therapy and post-treatment.
On an animal level, we studied the effect of 2,6-NpBox on photosensitizer tumor treatment effect and post-treatment photosensitization (figure 65). Taking porphin sodium (Phorofrin) as an example, firstly, injecting a photosensitizer into tail vein, injecting 2,6-NpBox into tail vein after a period of time, and respectively carrying out operations such as illumination, photographing, observation, sampling, slicing and the like on the skin of the back of an animal after different times of darkroom feeding. As shown in fig. 65b, the skin photosensitization experiment result shows that the experimental animal injected with only photofurin showed severe photosensitization, the skin showed obvious redness and red spots, and the skin photosensitization of the animal injected with 2,6-NpBox was significantly inhibited, and the inhibition effect was related to the dose of 2,6-NpBox and the time interval to light after injection (fig. 66). The fluorescence intensity of the photosensitizer was significantly reduced in the skin and major organs of animals injected with 2,6-NpBox compared to the photosensitizer group (fig. 65 c). The staining pattern of the skin sections can observe that the photosensitizer group has capillary rupture and red blood cell exudation and hair follicle reduction; while the 2,6-NpBox group remained essentially identical to the blank group with no obvious abnormalities (fig. 65 d). At the same time, the antitumor effect was not affected (fig. 65e to 65 g). Throughout the experiment, the body weights of the three animals were not significantly changed (fig. 67). Similar results were obtained for animal model experiments with photosensitizers chlorin e6 (FIG. 68) or hematoporphyrin (FIG. 69). The experimental results show that the 2,6-NpBox has obvious inhibition effect on phototoxicity after photodynamic therapy and does not influence the photodynamic therapy effect.
The foregoing description of the preferred embodiments of the invention is not intended to limit the invention to the particular embodiments disclosed, but on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the invention.

Claims (7)

1. A positive ion type cyclopean is a water-soluble positive ion box-shaped macrocyclic molecule containing naphthalene units, is marked as NpBox, and specifically comprises three structural formulas which are respectively marked as 2,6-NpBox, 1,4-NpBox and 1,5-NpBox according to the substitution positions of pyridine on the naphthalene units, wherein the three structural formulas are respectively:
Figure DEST_PATH_IMAGE001
2. use of a cationic cyclopean according to claim 1 for the preparation of a pharmaceutical preparation for the removal of residues in photodynamic therapy.
3. The use according to claim 2, wherein the pharmaceutical formulation further comprises a pharmaceutically acceptable additive.
4. The use according to claim 2, characterized by the following specific operations: dissolving NpBox in 5% glucose aqueous solution to form injection, wherein the concentration of the NpBox aqueous solution is 0.1mg/mL-1.5mg/mL; 200 μl of photosensitizer tail was injected intravenously into mice and the formulated NpBox solution was injected one hour after the end of photodynamic therapy.
5. The use according to claim 2, 3 or 4, wherein the medicament for photodynamic therapy is a broad spectrum cancer photodynamic therapy medicament.
6. The use according to claim 5, wherein the model of the photodynamic therapy drug is porphin sodium, chlorin e6 or hematoporphyrin.
7. A process for the preparation of a cationic cyclop according to claim 1, characterized in that dibromonaphthalene substituted in different positions is subjected to suzuki coupling reaction and then to two nucleophilic substitution reactions to obtain the cationic cyclop, comprising the following specific steps:
(1) Performing Suzuki coupling reaction on dibromonaphthalene and 4-pyridine borate to prepare dipyridine substituted naphthalene, which is marked as NpBipy;
(2) Nucleophilic substitution reaction is carried out on NpBipy and 1, 4-di (bromomethyl) benzene to prepare a corresponding compound NpBnBipy with two ends substituted by bromomethyl benzyl;
(3) Nucleophilic substitution reaction of NpBipy and NpBnBipy is carried out again to obtain corresponding cycloparaffin.
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108864116A (en) * 2017-10-24 2018-11-23 陈志龙 A kind of novel chlorin e 6 derivative and the preparation method and application thereof
CN113616809A (en) * 2021-07-01 2021-11-09 复旦大学 Application of supramolecular organic framework material in removal of residual medicine in photodynamic therapy

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108864116A (en) * 2017-10-24 2018-11-23 陈志龙 A kind of novel chlorin e 6 derivative and the preparation method and application thereof
CN113616809A (en) * 2021-07-01 2021-11-09 复旦大学 Application of supramolecular organic framework material in removal of residual medicine in photodynamic therapy

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