WO2020228627A1 - 一种新型双模态小分子造影剂及其制备方法与应用 - Google Patents

一种新型双模态小分子造影剂及其制备方法与应用 Download PDF

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WO2020228627A1
WO2020228627A1 PCT/CN2020/089319 CN2020089319W WO2020228627A1 WO 2020228627 A1 WO2020228627 A1 WO 2020228627A1 CN 2020089319 W CN2020089319 W CN 2020089319W WO 2020228627 A1 WO2020228627 A1 WO 2020228627A1
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contrast agent
compound
small molecule
linker
nir
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PCT/CN2020/089319
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English (en)
French (fr)
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徐庆祥
周益舟
帅天白
王乐天
王景霖
任陇飞
王锦涛
赵丽
李玉艳
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南京鼓楼医院
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/0002—General or multifunctional contrast agents, e.g. chelated agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
    • A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
    • A61K49/0032—Methine dyes, e.g. cyanine dyes
    • A61K49/0034—Indocyanine green, i.e. ICG, cardiogreen
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
    • A61K49/0052—Small organic molecules
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/04—X-ray contrast preparations
    • A61K49/0433—X-ray contrast preparations containing an organic halogenated X-ray contrast-enhancing agent
    • A61K49/0438—Organic X-ray contrast-enhancing agent comprising an iodinated group or an iodine atom, e.g. iopamidol
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D209/00—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D209/02—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom condensed with one carbocyclic ring
    • C07D209/04—Indoles; Hydrogenated indoles
    • C07D209/08—Indoles; Hydrogenated indoles with only hydrogen atoms or radicals containing only hydrogen and carbon atoms, directly attached to carbon atoms of the hetero ring

Definitions

  • the invention belongs to the field of contrast agents, and specifically relates to a novel bimodal small molecule contrast agent and a preparation method and application thereof.
  • Liver cancer is one of the malignant tumors with the highest mortality rate. About 383,000 people die of liver cancer in my country each year, accounting for 51% of the global deaths from liver cancer. The severe situation has brought a heavy burden to society and medical care. Early onset of liver cancer is quite insidious, with no obvious symptoms and signs. When patients are diagnosed with liver cancer, they are often staged late, with a poor prognosis, and the 5-year survival rate is not high. Therefore, the development of an effective and reliable diagnostic method can enable early detection of liver cancer and early treatment to improve the survival rate of patients, which has high clinical application value.
  • Computed Tomography has the advantages of high resolution, low cost, and high imaging efficiency, and has become one of the most commonly used imaging methods in clinical practice.
  • CT takes advantage of the difference in absorption and transmittance of X-rays by different tissues of the human body to image the examination site, so as to find subtle lesions in the body.
  • CT contrast agents on the market contain iodine compounds. Iodine has a large atomic number, and most of the iodine-containing compounds have strong tissue penetrating power. Enrichment in relevant areas can increase imaging brightness and improve diagnosis accuracy. Sodium diatrizoate, iohexol, ioverol, iodixanol, etc.
  • contrast agents are widely used clinically, and reflect the changes of local tissue blood perfusion through perfusion imaging, so as to obtain rich diagnostic information.
  • the existing clinical CT contrast agents do not have the ability to target liver cancer. Therefore, the development of contrast agents that can be used for early diagnosis of liver cancer is an urgent problem to be solved.
  • Near-infrared fluorescence imaging has become the current research focus due to its high sensitivity, no ionizing radiation, easy operation, low cost and real-time imaging.
  • the near-infrared fluorescence imaging detection signal is in the near-infrared region (700-900nm), in which the fluorescence background interference of organisms is small and the penetration ability is relatively strong.
  • Indocyanine Green (ICG) is the only near-infrared imaging reagent approved by the FDA, which is excreted by the hepatobiliary system after intravenous injection. By detecting the content in the blood, it can detect whether the liver function is normal. Indocyanine green can target liver cancer tissues, but due to its weak penetration and the imaging depth is only 5-10mm, it can only detect tumor tissues on the liver surface and cannot identify tumors inside the liver, which limits its use.
  • the technical problem to be solved by the present invention is to overcome the weak penetrating power of existing near-infrared imaging reagents such as indocyanine green, and the imaging depth is only 5-10mm, which can only detect tumor tissue on the liver surface, but cannot identify the tumor inside the liver. And other shortcomings, but provides a new bimodal small molecule contrast agent and its preparation method.
  • a new type of bimodal small molecule contrast agent is a compound represented by formula (1),
  • CT stands for iodinated organic molecules
  • Linker represents the connection chain, the structure is Indicates the connection position, M is selected from -COO-, -CO-, -NH- or -O-; N is selected from -COO-, -CO-, -NH- or -O-; X is -O-; m is 0, 1, 2, 3, 4, 5 or 6; m'is 2, 3, 4, 5 or 6;
  • NIR stands for cyanine dye derivatives, the structure is R 1 , R 2 , R 3 , R 4 , R 5 and R 6 are the same or different and are independently selected from carboxyl group, amino group, hydroxyl group, sulfonic acid group or hydrogen atom; Y is selected from carboxylic acid anion, sulfonic acid anion or Phosphate anion; R x is H or halogen; R y and R z are H, or R y and R z form a 3-6 membered carbocyclic ring with the carbon to which they are connected; n is 1, 2, 3 or 4;
  • W represents alkali metal
  • the iodinated organic molecule is an iodinated organic molecule containing a carboxyl group, wherein the carbonyl group after the carboxyl group is removed from the hydroxyl group is connected to the Linker.
  • the iodinated organic molecule is an aromatic iodine-containing compound, multiple iodine atoms may exist on the aromatic ring, and the structure is wherein, at least one of R c1 , R c2 , R c3 , R c4 and R c5 is I, and the rest are selected from I, -COOH, -NH 2 , -CONH 2 or -CONHCH 3 ; more preferably, R c1 , R At least three of c2 , R c3 , R c4, and R c5 are I.
  • the Where M and N are -NH- or -O-; m is 0, 2 or 3; m'is 2, 3 or 4; more preferably, when m is 0, m'is 2, 3 or 4; m is When 2 or 3, m'is 2.
  • R 1 , R 3 , R 4 and R 6 are the same and are selected from hydrogen atoms;
  • R 2 and R 5 are the same and are selected from carboxyl groups; when R y and R z are H, R x is H; when R y and When R z and the carbon to which it is connected together form a 3-6 membered carbocyclic ring, R x is a halogen selected from F, Cl, Br or I; n is 2 or 3.
  • R y and R z form a 3-6 membered carbocyclic ring with the carbon to which they are connected, the 3-6 membered carbocyclic structure is
  • R 1 , R 2 or R 3 of the NIR cyanine dye derivative is connected to the left Linker, and R 4 , R 5 or R 6 is connected to the right Linker.
  • W is selected from Na or K.
  • the structure of the CT is
  • the structure of the Linker is
  • the structure of the NIR is NIR
  • the contrast agent is selected from any one of compounds having the following structures:
  • the present invention also provides a method for preparing a novel bimodal small molecule contrast agent, which includes the following steps: under the action of a base and a condensing agent, the organic iodinated molecule (CT-Linker) connecting Linker and NIR are at room temperature It is obtained by condensation reaction, and the reaction is as follows:
  • the molar ratio of the NIR to the organic iodinated molecules connected to the Linker is 1:(2.3-2.5); the molar ratio of NIR to the base is 1:(3-3.5); the molar ratio of NIR to the condensing agent is 1:(3 -3.5);
  • CT stands for iodinated organic molecules
  • Linker represents the connection chain, the structure is M is selected from -COO-, -CO-, -NH- or -O-; N is selected from -COO-, -CO-, -NH- or -O-; X is -O-; m is 0, 1, 2, 3, 4, 5 or 6; m'is 2, 3, 4, 5 or 6;
  • NIR stands for cyanine dye derivatives, the structure is R 1 , R 2 , R 3 , R 4 , R 5 and R 6 are the same or different and are independently selected from carboxyl group, amino group, hydroxyl group, sulfonic acid group or hydrogen atom; Y is selected from carboxylic acid anion, sulfonic acid anion or Phosphate anion; R x is H or halogen; R y and R z are H, or R y and R z form a 3-6 membered carbocyclic ring with the carbon to which they are connected; n is 1, 2, 3 or 4;
  • W represents alkali metal
  • the solvent used in the reaction is one or more of amide solvents, ketone solvents, nitrile solvents and sulfoxide solvents. More preferably, the solvent is one or more of N,N-dimethylformamide, acetone, acetonitrile and dimethyl sulfoxide.
  • the base is an inorganic base or an organic base; the inorganic base is selected from one or more of sodium bicarbonate, potassium bicarbonate, sodium carbonate and potassium carbonate; the organic base is selected from triethyl One or more of amine, diisopropylethylamine and pyridine.
  • the base is only an organic base, it is necessary to perform a salt-forming reaction in the presence of an alkali metal hydroxide after the reaction is completed.
  • the condensing agent is cyclohexylcarbodiimide, 1-hydroxybenzotriazole, 1-hydroxy-7-azobenzotriazole, 1-(3-dimethylaminopropyl)-3 -Ethylcarbodiimide hydrochloride, 2-(7-benzotriazole oxide)-N,N,N',N'-tetramethylurea hexafluorophosphate and O-benzotriazole -One or more of tetramethylurea hexafluorophosphate.
  • the reaction time may not be specifically limited.
  • the end of the reaction is when the NIR disappears or the reaction no longer proceeds.
  • Compound 2 is obtained by Fisher indole synthesis reaction between p-hydrazinobenzoic acid and 3-methyl-2-butanone.
  • Compound 2 reacts with 1,3-propane sultone or 1,4-butane sultone to obtain 3 series of compounds.
  • the 3 series compounds and 4b undergo nucleophilic addition and elimination reactions to obtain 6 series compounds.
  • Linker series compounds react with di-tert-butyl dicarbonate to form 8-series compounds with unilateral Boc protection.
  • Compound 7 reacts with 8 series compounds to obtain 9 series compounds.
  • Compound 9 was de-Boc with trifluoroacetic acid to obtain 10 series compounds.
  • the final product is obtained by condensation of the 10 series compound and the 5 series compound or the 6 series compound.
  • the method for synthesizing the aforementioned 9 series compound includes the following steps: the 7 series compound reacts with the Linker series compound in the presence of a base.
  • the base is preferably an inorganic base and/or an organic base.
  • the inorganic base is preferably one or more of sodium bicarbonate, potassium bicarbonate, sodium carbonate and potassium carbonate.
  • the organic base is preferably one or more of triethylamine, diisopropylethylamine and pyridine.
  • the molar ratio of the 7 series compound to the Linker series compound is preferably 1: (1.3-1.5).
  • the reaction temperature is preferably room temperature.
  • the synthesis method of the final product includes the following steps: the 5 series compound reacts with the 10 series compound in the presence of a base and a condensing agent.
  • the solvent is preferably one or more of amide solvents, ketone solvents, nitrile solvents and sulfoxide solvents, more preferably N,N-dimethylformamide, acetone, acetonitrile and One or more of dimethyl sulfoxide.
  • the base is preferably an inorganic base and/or an organic base.
  • the inorganic base is preferably one or more of sodium bicarbonate, potassium bicarbonate, sodium carbonate and potassium carbonate.
  • the organic base is preferably one or more of triethylamine, diisopropylethylamine and pyridine.
  • the base is only an organic base, after the reaction is completed, it is necessary to perform a salt-forming reaction in the presence of an alkali metal hydroxide to obtain the target compound.
  • the condensing agent is preferably cyclohexylcarbodiimide, 1-hydroxybenzotriazole, 1-hydroxy-7-azobenzotriazole, 1-(3-dimethylaminopropyl)-3-ethyl Carbodiimide hydrochloride, 2-(7-benzotriazole oxide)-N,N,N',N'-tetramethylurea hexafluorophosphate and O-benzotriazole-tetrazole One or more of methylurea hexafluorophosphate.
  • the molar ratio of the 5 series compound to the 10 series compound is preferably 1:(2.3-2.5).
  • the molar ratio of the 5 series compound to the base is preferably 1:(3-3.5).
  • the molar ratio of the 5 series compound to the condensing agent is preferably 1:(3-3.5).
  • the reaction temperature of the 5 series compound and the 10 series compound is preferably room temperature.
  • the synthesis method of the final product includes the following steps: the 6 series compound reacts with the 10 series compound in the presence of a base and a condensing agent.
  • the solvent is preferably one or more of amide solvents, ketone solvents, nitrile solvents and sulfoxide solvents, more preferably N,N-dimethylformamide, acetone, acetonitrile and One or more of dimethyl sulfoxide.
  • the base is preferably an inorganic base and/or an organic base.
  • the inorganic base is preferably one or more of sodium bicarbonate, potassium bicarbonate, sodium carbonate and potassium carbonate.
  • the organic base is preferably one or more of triethylamine, diisopropylethylamine and pyridine.
  • the base is only an organic base, after the reaction is completed, it is necessary to perform a salt-forming reaction in the presence of an alkali metal hydroxide to obtain the target compound.
  • the condensing agent is preferably cyclohexylcarbodiimide, 1-hydroxybenzotriazole, 1-hydroxy-7-azobenzotriazole, 1-(3-dimethylaminopropyl)-3-ethyl Carbodiimide hydrochloride, 2-(7-benzotriazole oxide)-N,N,N',N'-tetramethylurea hexafluorophosphate and O-benzotriazole-tetrazole One or more of methylurea hexafluorophosphate.
  • the molar ratio of the 6 series compound to the 10 series compound is preferably 1:(2.3-2.5).
  • the molar ratio of the 6 series compound to the base is preferably 1:(3-3.5).
  • the molar ratio of the 6 series compound to the condensing agent is preferably 1:(3-3.5).
  • the reaction temperature of the 6 series compound and the 10 series compound is preferably room temperature.
  • the synthesis method of the final product preferably includes the following steps: mixing the 5 series compound or the 6 series compound and the condensing agent in an ice bath (activation), and then adding a solution of the 10 series compound, wherein the solvent is preferably the same as the reaction solvent, and proceeding The reaction.
  • post-treatment operations include salt formation and recrystallization.
  • the molar ratio of sodium hydroxide to 5 series compound or 6 series compound used in the salt formation process is preferably 1: (1.05-1.15).
  • the solvent for recrystallization is a mixed solution of water and isopropanol with a molar ratio of 1:(15-25), a mixed solution of methanol and dichloromethane, a mixed solution of methanol and chloroform, or a mixed solution of methanol and ethyl acetate.
  • the aforementioned diatrizoic acid can also be replaced with 2,3,5-triiodobenzoic acid or 2,4,6-triiodobenzoic acid.
  • the present invention also provides the application of the prepared compound represented by formula (1) as a contrast agent in near-infrared/CT imaging.
  • the compound represented by formula (1) of the present invention when used as a contrast agent, it can be formulated for intravenous injection.
  • commonly used excipients are sterile water for injection and poloxamer 188, the mass fraction of poloxamer is 2-4%, and the mass fraction of the compound of formula (1) is 9-11% , The rest is sterile water.
  • room temperature refers to 10-30°C.
  • CT refers to computer tomography technology, which uses X-ray tomography, which is received by the electrophoton detector, and converts the signal into a digital input to an electronic computer, and then the computer is converted into an image, thereby distinguishing parts of different density.
  • the present invention utilizes the characteristic that ICG can be selectively enriched in the liver cancer area and forms a strong contrast with the surrounding tissues, and its key structure is selected as a carrier, and it is compared with CT imaging with iodine-rich groups such as diatrienic acid and iohexol.
  • the functional groups of the agent are connected to increase the penetration ability while obtaining the targeting of liver cancer.
  • the small molecule near-infrared/CT dual-modal contrast agent is designed and synthesized. In vivo and in vitro experiments have confirmed that the small molecule contrast agent of the present invention has good water solubility and low toxicity.
  • the two contrast modes confirm each other, enrich diagnostic information, improve diagnostic accuracy, and provide a feasible new contrast agent for clinical early diagnosis of liver cancer.
  • the compound prepared by the present invention can be specifically absorbed by liver cancer tissue, so that the contrast agent has significant liver cancer targeting characteristics.
  • the compound prepared by the present invention has good water solubility, the solubility in water can reach 100 mg/mL, and can be administered by intravenous injection.
  • Figure 1 is a comparison diagram of in vitro CT values of the compound prepared in Example 1 of the present invention and sodium diatrizoate.
  • Figure 2 is a linear graph of the in vitro CT value of diatrizoate sodium.
  • Figure 3 is a linear graph of the in vitro CT value of the compound prepared in Example 1 of the present invention.
  • Figure 4 is a graph showing the MTT cell viability test of the compound prepared in Example 1 of the present invention and ICG.
  • Figure 5 is a graph showing the cellular uptake evaluation of the compound prepared in Example 1 of the present invention in liver cancer cells and normal liver cells.
  • Fig. 6 is a near-infrared imaging image of the compound prepared in Example 1 of the present invention and ICG in living small animals.
  • Fig. 7 is a CT image of a small animal in vivo of the compound prepared in Example 1 of the present invention.
  • DCM dichloromethane
  • Step 2 Put 4g compound 2, 11.93mL 1,4-butane sultone, 50mL o-dichlorobenzene, nitrogen protection, reflux at 180°C for 9h into a 250mL three-necked flask in turn; after the reaction, a large amount of solids precipitate out and filter with suction. Washed with acetone three times to obtain a pink solid compound 3 with a yield of 93%.
  • Step 3 Add 2g of compound 3, 784mg of pentadienal diphenylamine hydrochloride, 30mL of acetic anhydride, 18mL of glacial acetic acid to a 250mL three-necked flask in sequence, and finally add 808.8mg of sodium acetate under nitrogen protection and reflux at 120°C for 45min; After the reaction, 50mL of anhydrous ether was added, and the precipitated solid was suction filtered to obtain a crude product, and then recrystallized. The solvent was a mixed solution of isopropanol and water with a molar ratio of 4:1 to obtain green compound 5a with a yield of 76% .
  • Step 4 In a 250 mL three-necked flask, dissolve 5.8 mL of ethylenediamine in 15 mL of dry DCM. Ice bath, nitrogen protection, anhydrous reaction, start stirring. 3.2 mL of di-tert-butyl dicarbonate was dissolved in 65 mL of dry DCM and slowly added dropwise to the reaction system. After the addition, the ice bath was removed and replaced with an oil bath at 25°C for 18 hours. After the reaction, the by-products were removed by filtration, and saturated sodium bicarbonate solution was added to the residue. It was extracted with DCM, and the organic phases were combined and concentrated to obtain a pale yellow oil 8-1 with a yield of 71%.
  • Step 5 Put 6g of diatrizoic acid, 60mL of thionyl chloride, anhydrous reaction in a 250mL three-necked flask, and reflux at 75°C for 3h. After the completion of the reaction, the thionyl chloride was removed under reduced pressure, and the residue was washed with n-hexane to obtain a pale yellow solid compound 7 with a yield of 75%.
  • Step 6 Add 2 g of compound 7, 1.32 mL of triethylamine, and 30 mL of dry N,N-dimethylformamide (DMF) into a 250 mL three-necked flask, and react without water under nitrogen protection. 708 mg of compound 8-1 dissolved in DMF was added dropwise at 25°C. After the addition is complete, continue to react at 25°C for 12 hours. After the completion of the reaction, the solvent was removed under reduced pressure, a large amount of solid was precipitated after adding DCM, washed with water, and filtered with suction to obtain a crude product. Adding DCM to make slurry, suction filtration to obtain a pale yellow solid compound 9-1 with a yield of 84%.
  • DMF dry N,N-dimethylformamide
  • Step 7 Add 3.09 g of compound 9-1, 3 mL of DCM, and 3 mL of trifluoroacetic acid into a 25 mL three-necked flask, under nitrogen protection, and react at 25° C. for 3 hours. After the completion of the reaction, the trifluoroacetic acid was removed under reduced pressure, and the reaction mixture was washed with acetone and DCM to obtain a yellowish solid compound 10-1 with a yield of 82%.
  • Step 8 Under ice bath, mix 1g compound 5a, 784mg 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride, (EDCI), 0.55mg 1-hydroxybenzotriazole (HOBt ), 0.57mL triethylamine, 60mL of anhydrous DMF into a 250mL three-necked flask, anhydrous reaction, nitrogen protection, stirring for 1.5h. Drop 1.97 g of the DMF solution of compound 10-1, after the addition, remove the ice bath, and react at room temperature for 12 hours.
  • EDCI 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride
  • HOBt 1-hydroxybenzotriazole
  • the solvent is a mixture of isopropanol and water with a molar ratio of 20:1 to obtain Dark green solid CNCI-1, yield 42%.
  • the preparation method is the same as in Example 1, except that 1,3-propanediamine is used in step 4 instead of ethylenediamine for reaction, and the rest of the synthesis steps remain unchanged to obtain the final product compound CNCI-2 with a yield of 39%.
  • the preparation method is the same as in Example 1, except that 1,4-butanediamine is used to replace ethylenediamine for reaction in step 4, and the rest of the synthesis steps remain unchanged to obtain the final product compound CNCI-3 with a yield of 30%.
  • the preparation method is the same as in Example 1, except that in step 4, 1,8-diamino-3,6-dioxa-octane is used instead of ethylenediamine for the reaction, and the rest of the synthesis steps remain unchanged to obtain the final product compound CNCI-4.
  • the rate is 30.2%.
  • the preparation method is the same as in Example 1, except that 2(2,2'–(2,2'-oxybis(ethane-2,1-diyl)bis(oxy))bis(ethylamine)) is used in step 4 instead Ethylenediamine was reacted with the rest of the synthesis steps unchanged to obtain the final product compound CNCI-5 with a yield of 24%.
  • Step 2 In a 25 mL single-neck flask, add 173 mg of compound 3, 87 mg of compound 4, 68 mg of sodium acetate, 1 mL of acetic acid and 2 mL of acetic anhydride in sequence, under nitrogen protection, reflux and stir at 120°C for 45 min.
  • Example 1 After the green solid 5b is obtained, the method in Example 1 is continued, except that compound 5b is substituted for compound 5a for the reaction, and the rest of the synthesis steps remain unchanged to obtain the final product compound CNCII-1 with a yield of 15.3%.
  • the preparation method is the same as that in Example 6, except that 1,3-propanediamine is used instead of ethylenediamine for the reaction, and the rest of the synthesis steps remain unchanged to obtain the final product compound CNCII-2 with a yield of 14.2%.
  • the preparation method is the same as that in Example 6, except that 1,4-butanediamine is used instead of ethylenediamine for the reaction, and the rest of the synthesis steps remain unchanged to obtain the final product compound CNCII-3 with a yield of 21%.
  • the preparation method is the same as in Example 6, except that 1,8-diamino-3,6-dioxa-octane is used instead of ethylenediamine for the reaction, and the rest of the synthesis steps remain unchanged to obtain the final product compound CNCII-4 with a yield of 17.1% .
  • the preparation method is the same as in Example 6, except that 2(2,2'–(2,2'-oxybis(ethane-2,1-diyl)bis(oxy))bis(ethylamine)) is used instead of ethylenediamine
  • the reaction is carried out, and the rest of the synthesis steps remain unchanged to obtain the final product compound CNCII-5 with a yield of 15.6%.
  • the preparation method is the same as in Example 1, except that in step 2, 1,3-propane sultone is used instead of 1,4-butane sultone to react with compound 2, and the rest of the synthesis steps remain unchanged to obtain the final product compound CNCIII-1 , The yield is 28.8%.
  • the preparation method is the same as that in Example 11, except that 1,3-propanediamine is used instead of ethylenediamine for the reaction, and the rest of the synthesis steps remain unchanged to obtain the final product compound CNCIII-2 with a yield of 28.2%.
  • the preparation method is the same as in Example 11, except that 1,4-butanediamine is used instead of ethylenediamine for the reaction, and the rest of the synthesis steps remain unchanged to obtain the final product compound CNCIII-3 with a yield of 19.1%.
  • the preparation method is the same as in Example 11, except that 1,8-diamino-3,6-dioxaoctane is used instead of ethylenediamine for the reaction, and the rest of the synthesis steps remain unchanged to obtain the final product compound CNCIII-4 with a yield of 46.8% .
  • the preparation method is the same as in Example 11, except that 2(2,2'–(2,2'-oxybis(ethane-2,1-diyl)bis(oxy))bis(ethylamine)) is used instead of ethylenediamine
  • the reaction is carried out, and the rest of the synthesis steps remain unchanged to obtain the final product compound CNCIII-5 with a yield of 29.8%.
  • the preparation method is the same as in Example 6, except that 1,3-propane sultone is used instead of 1,4-butane sultone to react with compound 2, and the rest of the synthesis steps remain unchanged to obtain the final product compound CNCIV-1 with a yield 20.7%.
  • the preparation method is the same as that in Example 16, except that 1,3-propanediamine is used instead of ethylenediamine for the reaction, and the rest of the synthesis steps remain unchanged to obtain the final product compound CNCIV-2 with a yield of 14.2%.
  • the preparation method is the same as that in Example 16, except that 1,4-butanediamine is used instead of ethylenediamine for the reaction, and the rest of the synthesis steps remain unchanged to obtain the final product compound CNCIV-3 with a yield of 14%.
  • the preparation method is the same as in Example 16, except that 1,8-diamino-3,6-dioxa-octane is used instead of ethylenediamine for the reaction, and the rest of the synthesis steps remain unchanged to obtain the final product compound CNCIV-4 with a yield of 19.2% .
  • the preparation method is the same as in Example 16, except that 2(2,2'–(2,2'-oxybis(ethane-2,1-diyl)bis(oxy))bis(ethylamine)) is used instead of ethylenediamine
  • the reaction is carried out, and the rest of the synthesis steps remain unchanged to obtain the final product compound CNCIV-5 with a yield of 20%.
  • the preparation method is the same as in Example 2, except that 2,3,5-triiodobenzoic acid is used instead of diatrizoic acid for the reaction, and the rest of the synthesis steps remain unchanged to obtain the final product compound CNCV-2 with a yield of 46%.
  • the preparation method is the same as in Example 21, except that 1,3-ethylene glycol is used instead of 1,3-ethylenediamine for the reaction, and the rest of the synthesis steps remain unchanged to obtain the final product compound CNCV-3 with a yield of 26%.
  • the preparation method was the same as that in Example 3, except that 2,4,6-triiodobenzoic acid was used instead of diatrizoic acid for the reaction, and the rest of the synthesis steps remained unchanged to obtain the final product compound CNCVI-2 with a yield of 43%.
  • the preparation method is the same as that in Example 23, except that 1,3-ethylene glycol is used instead of 1,3-ethylenediamine for the reaction, and the rest of the synthesis steps remain unchanged to obtain the final product compound CNCVI-3 with a yield of 29%.
  • the cell experiment of the present invention selects HepG2 (human liver cancer cells) and L02 (human normal liver cells) cells for in vitro culture.
  • the HepG2 cells are from Shanghai Institute of Cells, Chinese Academy of Sciences
  • the L02 cells are from Shanghai Fusheng Biotechnology Co., Ltd.
  • the HepG2 cells were cultured in HyClone DMEM medium containing 10% FBS, 100 IU/mL penicillin and 100 mg/mL streptomycin.
  • the L02 cells were cultured in RPMI 1640 medium containing 10% FBS, 100 IU/mL penicillin and 100 mg/mL streptomycin.
  • the model mice in the animal experiment of the present invention are ordinary nude mice inoculated with HepG2 cells under the armpit, and the nude mice are fed for 1 week to obtain a tumor model.
  • the model rabbit in the animal experiment of the present invention is a common big-eared rabbit liver that is inoculated with VX2 (rabbit hepatocarcinoma cells) in situ, and the rabbits are raised for 2-3 weeks to obtain a tumor model.
  • VX2 rabbit hepatocarcinoma cells
  • FIG. 1 for the comparison of the in vitro CT intensity of the compound CNCI-1 and sodium diatrizoate prepared in Example 1.
  • the compound CNCI-1 and sodium diatrizoate were respectively prepared in a 1.5 mL EP tube with an equal volume of concentration gradient solution ( 40mg I/mL-0) was placed under Siemens Inveon PET/CT for imaging, and the imaging results were analyzed. Within the concentration gradient range shown in the figure, the two contrast agents have similar CT signal intensities.
  • FIG. 4 for the MTT cell viability test diagram of the compound CNCI-1 and ICG prepared in Example 1.
  • the cells were seeded on a 96-well plate (6 ⁇ 10 3 cells/well) at 37° C. and incubated for 24 hours under 5% CO 2 conditions. Subsequently, CNCI-1 or ICG (0-100 ⁇ M) was added to the incubation system in the fresh medium. After 48 hours, standard MTT assay was performed to detect cell viability.
  • the abscissa refers to the compound and ICG dosage, and the ordinate refers to the inhibitory rate of the compound and ICG on cells.
  • the figure shows that when the concentration of compound CNCI-1 and ICG is less than 100 ⁇ M, the cell survival rate of HepG2 is above 80%, indicating that the contrast agent has good cell compatibility and low toxicity.
  • FIG. 5B is stained, and the MERGE image in FIG. 5C is a superimposed image of FIGS. 5A and 5B.
  • the experimental results showed that the compound CNCI-1 was swallowed into liver cancer cells, but this phenomenon did not occur in the normal liver cells, indicating that the uptake efficiency of the compound CNCI-1 liver cancer cells was much higher than that of the normal liver cells.
  • Fig. 7 for a CT image of a small animal in vivo of the most preferred compound of the contrast agent of the present invention.
  • big-eared rabbits were used to inoculate rabbit-derived liver cancer VX2 cells in situ to construct liver cancer models.
  • the experimental rabbits were given a rapid intravenous bolus injection (compound concentration 100mg/mL, administration 7mL).
  • the rabbit liver was perfused imaging with Siemens Inveon PET/CT, and the CT image of the rabbit liver was obtained.
  • A is the cross-sectional radiography of the rabbit liver.
  • Fig. B is an angiogram of the rabbit liver in a lateral section
  • C is an angiogram of the rabbit’s liver in a vertical section.
  • the position of the ruler indicates the heterogeneous blood vessel near the liver cancer
  • D is the stereoscopic image of the liver part fitted by the instrument.
  • the arrow indicates Fit the generated stereoscopic image of heterogeneous blood vessels.

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Abstract

一种新型双模态小分子造影剂及其制备方法与应用，利用ICG可选择性富集于肝癌区域，与周围组织形成较强对比的特点，选取其关键结构作为载体，与泛影酸、碘海醇等富含碘基团的CT造影剂官能团相连接，在获取肝癌靶向性的同时，增加穿透能力，设计合成了小分子近红外/CT双模态造影剂。该小分子造影剂水溶性好，毒性低，两种造影模态相互印证，丰富诊断信息，提高诊断精度，为临床上肝癌的早期诊断提供可行的造影剂。

Description

一种新型双模态小分子造影剂及其制备方法与应用 技术领域
本发明属于造影剂领域，具体涉及一种新型双模态小分子造影剂及其制备方法与应用。
背景技术
肝癌是死亡率最高的恶性肿瘤之一，我国每年约有38.3万人死于肝癌，占全球肝癌死亡病例数的51％，严峻的形势给社会和医疗带来了沉重的负担。肝癌早期发病颇隐匿，无明显的症状和体征。患者被确诊肝癌时，往往分期较晚，预后差，5年生存率不高。因此发展一种有效可靠的诊断手段可以使得肝癌能够被早期发现，早期治疗以提高病患的存活率，在临床上具有较高的应用价值。
计算机断层扫描(Computed Tomography，CT)具有分辨率高、成本低、造影效率高等优点，已成为临床上使用最普遍的造影手段之一。CT利用人体不同组织对X线的吸收与透过率的不同，对检查部位进行成像，从而能发现体内细微病变。目前，已上市CT造影剂多含碘化合物。碘的原子序数较大，含碘化合物多具有较强的组织穿透力，富集在相关区域可增加成像亮度，提高诊断精度。临床上广泛使用的有泛影酸钠、碘海醇、碘佛醇、碘克沙醇等，通过灌注成像等方式反映局部组织血流灌注量的改变，从而获得丰富的诊断信息。临床现有的CT造影剂不具备对肝癌的靶向能力，因此，开发能够用于肝癌早期诊断的造影剂是亟待解决的难题。
近红外荧光成像以其高灵敏度、无电离辐射、操作简便、费用低以及可实时成像成为目前的研究热点。近红外荧光成像检测信号处于近红外区域(700～900nm)，在这个区域内生物体的荧光背景干扰小、穿透能力相对较强。吲哚菁绿(Indocyanine Green，ICG)是FDA唯一批准的近红外成像试剂，经静脉注射后由肝胆系统排泄。通过检测血液中含量，可检测肝功能是否正常。吲哚菁绿能够靶向肝癌组织，但由于穿透力弱，成像深度仅有5-10mm，只能检测肝脏表面的肿瘤组织，无法识别出肝脏内部的肿瘤，使其使用受到限制。
发明内容
本发明所要解决的技术问题是为了克服现有近红外成像试剂例如吲哚菁绿穿透力弱，成像深度仅有5-10mm，只能检测肝脏表面的肿瘤组织，无法识别出肝脏内部的肿瘤等的缺陷，而提供了一种新型双模态小分子造影剂及其制备方法。
本发明是通过以下技术方案实现的：
一种新型双模态小分子造影剂，为如式(1)所示的化合物，
CT-Linker-NIR-Linker-CT·W +
                    (I)，
其中，CT表示碘化有机分子；
Linker表示连接链，结构为
Figure PCTCN2020089319-appb-000001
表示连接位置，M选自-COO-、-CO-、-NH-或-O-；N选自-COO-、-CO-、-NH-或-O-；X为-O-；m为0、1、2、3、4、5或6；m’为2、3、4、5或6；
NIR表示菁类染料衍生物，结构为
Figure PCTCN2020089319-appb-000002
R 1、R 2、R 3、R 4、R 5和R 6相同或不同，彼此独立地选自羧基、氨基、羟基、磺酸基或氢原子；Y选自羧酸阴离子、磺酸阴离子或磷酸阴离子；R x为H或卤素；R y和R z为H，或R y和R z与其相连的碳一起形成3-6元碳环；n为1、2、3或4；
W表示碱金属。
优选地，所述碘化有机分子为含有羧基的碘化有机分子，其中，羧基脱去羟基后的羰基与Linker相连。
优选地，所述碘化有机分子为芳香族含碘化合物，芳环上可存在多个碘原子，结构为
Figure PCTCN2020089319-appb-000003
其中，R c1、R c2、R c3、R c4和R c5中至少一个为I，其余选自I、-COOH、-NH 2、-CONH 2或-CONHCH 3；更 优选地，R c1、R c2、R c3、R c4和R c5中至少三个为I。
优选地，所述
Figure PCTCN2020089319-appb-000004
中，M和N为-NH-或-O-；m为0、2或3；m’为2、3或4；更优选地，m为0时m’为2、3或4；m为2或3时m’为2。
优选地，所述
Figure PCTCN2020089319-appb-000005
中，R 1、R 3、R 4和R 6相同，选自氢原子；R 2和R 5相同，选自羧基；当R y和R z为H时，R x为H；当R y和R z与其相连的碳一起形成3-6元碳环时，R x为卤素，选自F、Cl、Br或I；n为2或3。
优选地，当R y和R z与其相连的碳一起形成3-6元碳环时，所述的3-6元碳环结构为
Figure PCTCN2020089319-appb-000006
Figure PCTCN2020089319-appb-000007
优选地，NIR菁类染料衍生物的R 1、R 2或R 3与左侧Linker相连，R 4、R 5或R 6与右侧Linker相连。
优选地，W选自Na或K。
优选地，所述CT的结构为
Figure PCTCN2020089319-appb-000008
Figure PCTCN2020089319-appb-000009
优选地，所述Linker的结构为
Figure PCTCN2020089319-appb-000010
Figure PCTCN2020089319-appb-000011
优选地，所述NIR的结构为
Figure PCTCN2020089319-appb-000012
Figure PCTCN2020089319-appb-000013
优选地，所述造影剂选自如下结构的化合物中的任一种：
Figure PCTCN2020089319-appb-000014
Figure PCTCN2020089319-appb-000015
Figure PCTCN2020089319-appb-000016
Figure PCTCN2020089319-appb-000017
Figure PCTCN2020089319-appb-000018
Figure PCTCN2020089319-appb-000019
Figure PCTCN2020089319-appb-000020
Figure PCTCN2020089319-appb-000021
本发明还提供一种新型双模态小分子造影剂的制备方法，其包括以下步骤：在碱和缩合剂的作用下，将连接Linker的有机碘化分子(CT-Linker)与NIR在室温下进行缩合反应得到，反应如下所示：
CT-Linker+NIR→CT-Linker-NIR-Linker-CT·W +
                                    (I)，
所述NIR与连接Linker的有机碘化分子的摩尔比为1:(2.3-2.5)；NIR与碱的摩尔比为1:(3-3.5)；NIR与缩合剂的摩尔比为1:(3-3.5)；
其中，CT表示碘化有机分子；
Linker表示连接链，结构为
Figure PCTCN2020089319-appb-000022
M选自-COO-、-CO-、-NH-或-O-；N选自-COO-、-CO-、-NH-或-O-；X为-O-；m为0、1、2、3、4、5或6；m’为2、3、4、5或6；
NIR表示菁类染料衍生物，结构为
Figure PCTCN2020089319-appb-000023
R 1、R 2、R 3、R 4、R 5和R 6相同或不同，彼此独立地选自羧基、氨基、羟基、磺酸基或氢原子；Y选自羧酸阴离子、磺酸阴离子或磷酸阴离子；R x为H或卤素；R y和R z为H，或R y和R z与其相连的碳一起形成3-6元碳环；n为1、2、3或4；
W表示碱金属。
优选地，所述反应所用的溶剂为酰胺类溶剂、酮类溶剂、腈类溶剂和亚砜类溶剂中的一种或多种。更优选地，所述溶剂为N,N-二甲基甲酰胺、丙酮、乙腈和二甲基亚砜中的一种或多种。
优选地，所述碱为无机碱或有机碱；所述的无机碱选自碳酸氢钠、碳酸氢钾、碳酸钠和碳酸钾中的一种或多种；所述的有机碱选自三乙胺、二异丙基乙胺和吡啶中的一种或多种。当所述碱仅为有机碱时，反应结束后，需要在碱金属氢氧化物的存在下，进行成盐反应。
优选地，所述缩合剂为环己基碳二亚胺、1-羟基苯并三唑、1-羟基-7-偶氮苯并三氮唑、1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐、2-(7-氧化苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯和O-苯并三氮唑-四甲基脲六氟磷酸盐中的一种或多种。
优选地，所述的制备方法中，反应的时间可不作具体限定，优选以NIR消失或不再进行反应时作为反应的终点。
具体制备过程如下：
将对肼基苯甲酸与3-甲基-2-丁酮经Fisher吲哚合成反应得到化合物2。
Figure PCTCN2020089319-appb-000024
化合物2与1,3-丙磺酸内酯或1,4-丁磺酸内酯反应得到3系列化合物。
Figure PCTCN2020089319-appb-000025
3系列化合物与戊二烯醛缩二苯胺盐酸盐发生亲核加成消除反应得到5系列化合物。
Figure PCTCN2020089319-appb-000026
三氯氧磷，N,N-二甲基甲酰胺，环己酮，苯胺反应得到4b。
Figure PCTCN2020089319-appb-000027
3系列化合物与4b发生亲核加成消除反应得到6系列化合物。
Figure PCTCN2020089319-appb-000028
Linker系列化合物与二碳酸二叔丁酯反应生成单边Boc保护的8系列化合物。
泛影酸与氯化亚砜反应得到化合物7。
Figure PCTCN2020089319-appb-000029
化合物7与8系列化合物反应得到9系列化合物。
Figure PCTCN2020089319-appb-000030
化合物9通过三氟醋酸脱Boc得到10系列化合物。
Figure PCTCN2020089319-appb-000031
10系列化合物与5系列化合物或6系列化合物经缩合得到终产物。
Figure PCTCN2020089319-appb-000032
优选地，上述9系列化合物的合成方法包括下列步骤：7系列化合物在碱的存在下与Linker系列化合物反应。所述碱优选为无机碱和/或有机碱。所述的无机碱优选碳酸氢钠、碳酸氢钾、碳酸钠和碳酸钾中的一种或多种。所述的有机碱优选三乙胺、二异丙基乙胺和吡啶中的一种或多种。当所述碱仅为有机碱时，反应结束后，需要在碱金属氢氧化物的存在下，进行成盐反应，得到目标化合物。7系列化合物与Linker系列化合物的摩尔比优选为1:(1.3-1.5)。反应温度优选为室温。
优选地，终产物的合成方法包括下列步骤：5系列化合物在碱和缩合剂的存在下与10系列化合物反应。终产物的合成方法中，溶剂优选为酰胺类溶剂、酮类溶剂、腈类溶剂和亚砜类溶剂中的一种或多种，更优选N,N-二甲基甲酰胺、丙酮、乙腈和二甲基亚砜中的一种或多种。所述碱优选为无机碱和/或有机碱。所述的无机碱优选碳酸氢钠、碳酸氢钾、碳酸钠和碳酸钾中的一种或多种。所述的有机碱优选三乙胺、二异丙基乙胺和吡啶中的一种或多种。当所述碱仅为有机碱时，反应结束后，需要在碱金属氢氧化物的存在下，进行成盐反应，得到目标化合物。所述缩合剂优选为环己基碳二亚胺、1-羟基苯并三唑、1-羟基-7-偶氮苯并三氮唑、1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐、2-(7-氧化苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷 酸酯和O-苯并三氮唑-四甲基脲六氟磷酸盐中的一种或多种。5系列化合物与10系列化合物的摩尔比优选为1:(2.3-2.5)。5系列化合物与碱的摩尔比优选为1:(3-3.5)。5系列化合物与缩合剂的摩尔比优选为1:(3-3.5)。5系列化合物与10系列化合物反应的温度优选为室温。
优选地，终产物的合成方法包括下列步骤：6系列化合物在碱和缩合剂的存在下与10系列化合物反应。终产物的合成方法中，溶剂优选为酰胺类溶剂、酮类溶剂、腈类溶剂和亚砜类溶剂中的一种或多种，更优选N,N-二甲基甲酰胺、丙酮、乙腈和二甲基亚砜中的一种或多种。所述碱优选为无机碱和/或有机碱。所述的无机碱优选碳酸氢钠、碳酸氢钾、碳酸钠和碳酸钾中的一种或多种。所述的有机碱优选三乙胺、二异丙基乙胺和吡啶中的一种或多种。当所述碱仅为有机碱时，反应结束后，需要在碱金属氢氧化物的存在下，进行成盐反应，得到目标化合物。所述缩合剂优选为环己基碳二亚胺、1-羟基苯并三唑、1-羟基-7-偶氮苯并三氮唑、1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐、2-(7-氧化苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯和O-苯并三氮唑-四甲基脲六氟磷酸盐中的一种或多种。6系列化合物与10系列化合物的摩尔比优选为1:(2.3-2.5)。6系列化合物与碱的摩尔比优选为1:(3-3.5)。6系列化合物与缩合剂的摩尔比优选为1:(3-3.5)。6系列化合物与10系列化合物反应的温度优选为室温。
优选地，终产物的合成方法优选包括下列步骤：将5系列化合物或6系列化合物与缩合剂在冰浴下混合(活化)，然后加入10系列化合物的溶液，其中溶剂优选与反应溶剂相同，进行所述反应。
优选地，终产物的合成方法结束后，若碱选用的是有机碱，还包括后处理的操作。所述后处理的操作包括成盐与重结晶。所述成盐过程使用氢氧化钠与5系列化合物或6系列化合物的摩尔比优选为1:(1.05-1.15)。所述重结晶的溶剂为摩尔比为1:(15-25)的水与异丙醇混合溶液，甲醇与二氯甲烷混合溶液，甲醇与氯仿混合溶液或甲醇与乙酸乙酯混合溶液。
优选地，上述的泛影酸还可以替换成2,3,5-三碘基苯甲酸或2,4,6-三碘基苯甲酸。
本发明还提供所制备的述如式(1)所示的化合物作为造影剂在近红外/CT成像中的应用。
本发明所述如式(1)所示的化合物作为造影剂使用时，可配制静脉注射剂使用。在配制静脉注射剂时，常用辅料为注射用无菌用水与泊洛沙姆188，泊洛沙姆质量分数为2-4％，所述如式(1)的化合物的质量分数为9-11％，其余为无菌水。
本发明中，室温是指10-30℃。
本发明中，CT是指计算机断层扫描技术，利用X线断层扫描，电光子探测器接收，并把信号转化为数字输入电子计算机，再由计算机转化为图像，从而区分不同密度部位的一种手段。
本发明的有益效果为：
(1)本发明利用ICG可选择性富集于肝癌区域，与周围组织形成较强对比的特点，选取其关键结构作为载体，与泛影酸、碘海醇等富含碘基团的CT造影剂官能团相连接，在获取肝癌靶向性的同时，增加穿透能力，设计合成了小分子近红外/CT双模态造影剂。体内外实验证实，本发明的小分子造影剂水溶性好，毒性低，两种造影模态相互印证，丰富诊断信息，提高诊断精度，为临床上肝癌的早期诊断提供可行的新型造影剂。
(2)本发明所制备的化合物可被肝癌组织特异性吸收，使得该造影剂具有显著的肝癌靶向特征。
(3)本发明所制备的化合物在近红外/CT成像中的成像效果均与单模态阳性药效果类似，从而可结合两类造影手段的优势。
(4)本发明所制备的化合物具有较好的水溶性，在水中溶解度可达到100mg/mL，可通过静脉注射给药。
附图说明
图1为本发明实施例1制备的化合物与泛影酸钠的体外CT值对比图。
图2为泛影酸钠的体外CT值线性图。
图3为本发明实施例1制备的化合物的体外CT值线性图。
图4为本发明实施例1制备的化合物与ICG的MTT细胞活力测试图。
图5为本发明实施例1制备的化合物在肝癌细胞与正常肝细胞中的细胞摄取评价图。
图6为本发明实施例1制备的化合物与ICG的活体小动物近红外成像图。
图7为本发明实施例1制备的化合物的活体小动物CT成像图。
具体实施方式
下面结合附图和实施例对本发明的发明内容作进一步地说明。
实施例1
制备化合物CNCI-1，结构式如下：
Figure PCTCN2020089319-appb-000033
步骤1：将4g 4-肼基苯甲酸、3.96mL 3-甲基-2-丁酮、4.32g醋酸钠、60mL醋酸投入250mL三颈瓶中，氮气保护；25℃搅拌3h，随后120℃反应7h；反应结束后，用水将混合物转移二氯甲烷(DCM)萃取，合并浓缩有机相；采用柱层析分离(DCM:甲醇＝50:1)，浓缩得到黄色固体化合物2，收率61％。
1H NMR(300MHz,CDCl 3)δ(ppm):8.17(d,J＝8.19Hz,1H),8.08(s,1H),7.67(d,J＝8.19Hz,1H),2.41(s,3H,),1.40(s,6H)。
步骤2：向250mL三颈瓶中依次投入4g化合物2，11.93mL1,4-丁磺酸内酯，50mL邻二氯苯，氮气保护，180℃回流9h；反应结束后大量固体析出，抽滤，丙酮洗涤三次，得到粉红色固体化合物3，收率93％。
1H NMR(300MHz,DMSO)δ(ppm)：8.40(s,1H),8.17(dd,2H),4.52(t,2H),2.90(s,3H),2.51(t,2H),1.97(m,2H),1.77(m,2H),1.58(s,6H)。
步骤3：依次向250mL三颈瓶中加入2g化合物3，784mg戊二烯醛缩二苯胺盐酸盐，30mL醋酸酐，18mL冰醋酸，最后加入808.8mg醋酸钠，氮气保护，120℃回流45min；反应结束后，加入50mL无水乙醚，将析出的固体抽滤得到粗品，随后进行重结晶，溶剂选用摩尔比为4:1的异丙醇与水混合溶液，得到绿色化合物5a，收率76％。
1H NMR(300MHz,DMSO)δ(ppm):8.08(d,2H,J＝1.2Hz),7.98(dd,2H,J＝1.1,8.2Hz),7.95(m,5H),7.51(d,2H,J＝8.7Hz),6.65(t,2H,J＝12.4Hz),6.54(d,2H,J＝13.6Hz),4.11(m,4H),3.09(m,4H),1.75(m,8H),1.67(m,12H)。
步骤4：在250mL三颈瓶中，将5.8mL乙二胺溶于15mL干燥的DCM中。冰浴，氮气保护，无水反应，开始搅拌。将3.2mL二碳酸二叔丁酯溶于65mL干燥的DCM中，缓慢滴加至反应体系。滴加完毕，撤除冰浴，换成25℃油浴反应18h。反应结束后，过滤除去副产物，向残留物中加入饱和碳酸氢钠溶液。用DCM萃取，合并浓缩有机相，得淡黄色油状物8-1，收率71％。
1H NMR(300MHz,CDCl3)δ(ppm):3.15(t,J＝6.5Hz,2H),2.77(t,J＝6.5Hz,2H),1.47(s,9H)。
步骤5：向250mL三颈瓶中投入6g泛影酸，60mL氯化亚砜，氮气保护，无水反应，75℃回流3h。待反应结束后，减压除去氯化亚砜，正己烷洗涤残留物，得到淡黄色固体化合物7，收率75％。
步骤6：将2g化合物7，1.32mL三乙胺，30mL干燥的N,N-二甲基甲酰胺(DMF)加入250mL三颈瓶中，无水反应，氮气保护。25℃下滴加DMF溶解的708mg化合物8-1。滴加完毕后，继续25℃反应12h。反应结束后，减压除去溶剂，加入DCM有大量固体析出，水洗，抽滤得到粗品。加入DCM打浆，抽滤得淡黄色固体化合物9-1，收率84％。
1H NMR(300MHz,DMSO)δ(ppm):9.99(m,2H),3.17(m,4H),2.02(s,6H),1.39(s,9H)。
步骤7：将3.09g化合物9-1，3mLDCM，3mL三氟乙酸加入25mL三颈瓶中，氮气保护，25℃反应3h。反应结束后，减压除去三氟醋酸，分别用丙酮与DCM各洗涤反应混合物，得微黄色固体化合物10-1，收率82％。
1H NMR(300MHz,DMSO)δ(ppm):10.02(m,2H),3.44(m,2H),2.98(t,2H),2.02(s,6H)。
步骤8：冰浴下，将1g化合物5a，784mg 1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐、(EDCI)，0.55mg1-羟基苯并三唑(HOBt)，0.57mL三乙胺，无水DMF 60mL投入250mL三颈瓶，无水反应，氮气保护，搅拌1.5h。滴加1.97g化合物10-1的DMF溶液，滴加完毕，撤除冰浴，室温反应12h。减压旋干溶剂，柱层析分离(DCM:甲醇＝2:1-4:1)，合并浓缩收集的组分，旋干溶剂得到深绿色固体。将固体溶于甲醇，加入氢氧化钠(54mg,1.36mmol)，回流2min，将溶剂旋至固体将要析出，进行重结晶，溶剂选用摩尔比为20:1的异丙醇与水混合溶液，得到深绿色固体CNCI-1，收率42％。
1H NMR(300MHz,DMSO)δ(ppm):9.96(m,4H),8.81-8.40(m,4H),8.01(s,2H),7.90-7.88(m,5H),7.48(d,J＝8.13Hz,2H),6.62-6.48(m,4H),4.11(br,4H),3.52(m,8H),2.51(m,4H),2.02(s,12H),1.75(m,8H),1.66(s,12H)。
制备流程如下所示。
Figure PCTCN2020089319-appb-000034
Figure PCTCN2020089319-appb-000035
实施例2
制备化合物CNCI-2，结构式如下：
Figure PCTCN2020089319-appb-000036
制备方法同实施例1，除了步骤4中用1,3-丙二胺代替乙二胺进行反应，其余合成步骤不变，得到终产品化合物CNCI-2，收率39％。
1H NMR(300MHz,DMSO)δ(ppm):9.95(m,4H),8.63-8.46(m,4H),8.03(s,2H),8.03-7.88(m,5H),7.48(d,J＝8.13Hz,2H),6.66-6.48(m,4H),4.11(br,4H),3.40(m,8H),2.53(m,4H),2.01(s,12H),1.85(m,4H),1.74(m,8H),1.66(s,12H)。
实施例3
制备化合物CNCI-3，结构式如下：
Figure PCTCN2020089319-appb-000037
制备方法同实施例1，除了步骤4中用1,4-丁二胺代替乙二胺进行反应，其余合成步骤不变，得到终产品化合物CNCI-3，收率30％。
1H NMR(300MHz,DMSO)δ(ppm):9.95(m,4H),8.63-8.46(m,4H),8.02-7.87(m,7H),7.47(d,J＝7.65Hz,2H),6.62-6.49(m,4H),4.11(br,4H),3.42(m,8H),2.54(m,4H),2.01(s,12H),1.92-1.73(m,16H),1.66(s,12H)。
实施例4
制备化合物CNCI-4，结构式如下：
Figure PCTCN2020089319-appb-000038
制备方法同实施例1，除了步骤4中用1,8-二氨基-3,6-二氧杂辛烷代替乙二胺进行反应，其余合成步骤不变，得到终产品化合物CNCI-4，收率30.2％。
1H NMR(300MHz,DMSO)δ(ppm):9.95(m,4H),8.63-8.46(m,4H),8.02-7.87(m,7H),7.47(d,J＝7.65Hz,2H),6.62-6.49(m,4H),4.11(br,4H),3.42(m,16H),3.38(m,8H),3.24(m,4H),2.01(s,12H),1.92-1.73(m,12H),1.66(s,12H)。
实施例5
制备化合物CNCI-5，结构式如下：
Figure PCTCN2020089319-appb-000039
制备方法同实施例1，除了步骤4中用2(2,2'–(2,2'-氧双(乙烷-2,1-二基)双(氧))二(乙胺))代替乙二胺进行反应，其余合成步骤不变，得到终产品化合物CNCI-5，收率24％。
1H NMR(300MHz,DMSO)δ(ppm):10.02-9.91(m,4H),8.7-8.59(m,4H),8.03(s,2H),7.95-7.81(m,5H),7.47(d,J＝8.52Hz,2H),6.61(t,2H),6.50(d,2H),4.10(m,4H),3.54(m,24H),3.44(m,8H),2.50(m,4H),2.01(s,12H),1.76-1.65(m,8H),1.56(s,12H)。
实施例6
制备化合物CNCII-1，结构式如下：
Figure PCTCN2020089319-appb-000040
制备的前两个步骤同实施例1，得到化合物3。
然后再制备NIR，具体步骤如下：
步骤一：在250mL单颈瓶中，加入26mL DMF，磁力搅拌，冰浴下滴加22mL POCl 3。滴加完毕后，继续 冰浴搅拌30min，撤除冰浴，加入11mL环己酮，氮气保护，加热回流1h。降至室温，改用机械搅拌，滴加36mL摩尔比1:1的苯胺与乙醇混合溶液。滴加完毕后，继续搅拌1h。加入220mL摩尔比10:1的为水和HCl的混合溶液，冰浴搅拌2h。抽滤，冰水、丙酮、乙醚洗涤滤饼。将固体打浆洗涤(PE:EA＝2:1)，得紫色固体4，收率32％。
1H NMR(300MHz,DMSO)δ(ppm):8.40(s,2H),7.50-7.38(m,8H),7.21-7.16(m,2H),2.71(t,4H),1.80(m,2H)。
步骤二：在25mL单颈瓶中，依次加入173mg化合物3，87mg化合物4，68mg醋酸钠，1mL醋酸和2mL醋酸酐，氮气保护，120℃回流搅拌45min。溶液变为绿色，采用柱层析分离(PE:EA＝2:1)监测至反应完全，停止加热，降至室温，将反应液倒入10mL乙醚中，绿色固体析出。抽滤，乙醚洗涤固体，柱层析分离(DCM:甲醇＝3:1)，得绿色固体5b，收率46％。
1H NMR(300MHz,DMSO)δ(ppm):8.27(d,J＝14.2,2H),8.07(d,J＝1.5,2H),7.97(d,J＝1.6,2H),7.51(d,J＝8.4,2H),6.60(d,J＝13.8,2H),4.44–4.34(m,4H),2.58(d,J＝6.7,4H),2.04(dt,4H),1.70(s,12H)。
得到绿色固体5b后，继续按照实施例1中的方法，除了将化合物5b代替化合物5a进行反应，其余合成步骤不变，得到终产品化合物CNCII-1，收率15.3％。
1H NMR(300MHz,DMSO)δ(ppm):10.02-9.91(m,4H),8.75-8.20(m,4H),7.91(d,J＝12.24Hz,2H),7.79(s,2H),7.72(d,J＝8.28Hz,2H),6.96(d,J＝8.07Hz,2H),5.56(d,J＝11.58Hz,2H),3.83(m,4H),3.67(m,8H),2.56(m,4H),2.50(m,4H),1.99(s,12H),1.66-1.57(m,22H)。
制备流程如下所示。
Figure PCTCN2020089319-appb-000041
Figure PCTCN2020089319-appb-000042
实施例7
制备化合物CNCII-2，结构式如下：
Figure PCTCN2020089319-appb-000043
制备方法同实施例6，除了用1,3-丙二胺代替乙二胺进行反应，其余合成步骤不变，得到终产品化合物CNCII-2，收率14.2％。
1H NMR(300MHz,DMSO)δ(ppm):10.00-9.90(m,4H),8.59-8.27(m,4H),7.87-7.73(m,6H),6.96(d,J＝7.77Hz,2H),5.56(d,J＝13.56Hz,2H),3.77(m,4H),3.32-3.24(m,8H),2.56(m,4H),2.50(m,4H),1.99(s,12H),1.88-1.66(m,26H)。
实施例8
制备化合物CNCII-3，结构式如下：
Figure PCTCN2020089319-appb-000044
制备方法同实施例6，除了用1,4-丁二胺代替乙二胺进行反应，其余合成步骤不变，得到终产品化合物CNCII-3，收率21％。
1H NMR(300MHz,DMSO)δ(ppm):9.98-9.87(m,4H),8.59-8.24(m,4H),7.92-7.73(m,6H),6.95(d,J＝7.83Hz,2H),5.56(d,J＝13.2Hz,2H),3.84(m,4H),3.31-3.21(m,8H),2.55(m,4H),2.50(m,4H),1.99(s,12H),1.87-1.57(m,30H)。
实施例9
制备化合物CNCII-4，结构式如下：
Figure PCTCN2020089319-appb-000045
制备方法同实施例6，除了用1,8-二氨基-3,6-二氧杂辛烷代替乙二胺进行反应，其余合成步骤不变，得到终产品化合物CNCII-4，收率17.1％。
1H NMR(300MHz,DMSO)δ(ppm):9.98-9.87(m,4H),8.74-8.55(m,4H),7.91-7.74(m,6H),6.95(d,J＝8.13Hz,2H),5.56(d,J＝13.4Hz,2H),3.77(m,4H),3.56(m,16H),3.39(m,8H),2.56(m,4H),2.50(m,4H),1.99(s,12H),1.68-1.57(m,22H)。
实施例10
制备化合物CNCII-5，结构式如下：
Figure PCTCN2020089319-appb-000046
制备方法同实施例6，除了用2(2,2'–(2,2'-氧双(乙烷-2,1-二基)双(氧))二(乙胺))代替乙二胺进行反应，其余合成步骤不变，得到终产品化合物CNCII-5，收率15.6％。
1H NMR(300MHz,DMSO)δ(ppm):10.00-9.89(m,4H),8.68-8.32(m,4H),7.92-7.74(m,6H),6.95(d,J＝8.32Hz,2H),5.56(d,J＝12.8Hz,2H),3.76(m,4H),3.51(m,24H),3.39(m,8H),2.56(m,4H),2.49(m,4H),1.99(s,12H),1.67-1.57(m,22H)。
实施例11
制备化合物CNCIII-1，结构式如下：
Figure PCTCN2020089319-appb-000047
制备方法同实施例1，除了步骤2中用1,3-丙磺酸内酯代替1,4-丁磺酸内酯与化合物2进行反应，其余合成步骤不变，得到终产品化合物CNCIII-1，收率28.8％。
1H NMR(300MHz,DMSO)δ(ppm):9.97(m,4H),8.78-8.39(m,4H),8.00(s,2H),7.96-7.86(m,5H),7.52(d,J＝7.95Hz,2H),6.56(m,4H),4.23(br,4H),3.49-3.44(m,8H),2.56(m,4H), 2.00(s,12H),1.96(m,4H),1.64(s,12H)。
实施例12
制备化合物CNCIII-2，结构式如下：
Figure PCTCN2020089319-appb-000048
制备方法同实施例11，除了用1,3-丙二胺代替乙二胺进行反应，其余合成步骤不变，得到终产品化合物CNCIII-2，收率28.2％。
1H NMR(300MHz,DMSO)δ(ppm):9.98(m,4H),8.76-8.48(m,4H),8.04(s,2H),7.93-7.90(m,5H),7.55(d,J＝7.71Hz,2H),6.59(m,4H),4.28(m,4H),3.40(m,8H),2.56(m,4H),2.03(m,16H),1.86(m,4H),1.68(s,12H,4CH3)。
实施例13
制备化合物CNCIII-3，结构式如下：
Figure PCTCN2020089319-appb-000049
制备方法同实施例11，除了用1,4-丁二胺代替乙二胺进行反应，其余合成步骤不变，得到终产品化合物CNCIII-3，收率19.1％。
1H NMR(300MHz,DMSO)δ(ppm):9.97(m,4H),8.61-8.43(m,4H),8.00-7.86(m,7H),7.50(d,J＝7.80Hz,2H),6.60-6.52(m,4H),4.23(br,4H),3.21-3.19(m,8H),2.54(m,4H),1.99(m,16H),1.66(m,20H)。
实施例14
制备化合物CNCIII-4，结构式如下：
Figure PCTCN2020089319-appb-000050
制备方法同实施例11，除了用1,8-二氨基-3,6-二氧杂辛烷代替乙二胺进行反应，其余合成步骤不变，得到终产品化合物CNCIII-4，收率46.8％。
1H NMR(300MHz,DMSO)δ(ppm):9.95(m,4H),8.72-8.52(m,4H),8.02-7.87(m,7H),7.50(d,J＝7.83Hz,2H),6.55-6.52(m,4H),4.23(br,4H),3.51(m,16H),3.35(m,8H),2.56(m,4H),1.99(s,16H),1.63(s,12H)。
实施例15
制备化合物CNCIII-5，结构式如下：
Figure PCTCN2020089319-appb-000051
制备方法同实施例11，除了用2(2,2'–(2,2'-氧双(乙烷-2,1-二基)双(氧))二(乙胺))代替乙二胺进行反应，其余合成步骤不变，得到终产品化合物CNCIII-5，收率29.8％。
1H NMR(300MHz,DMSO)δ(ppm):9.94(m,4H),8.77-8.58(m,4H),8.01(s,2H),7.89-7.84(m,5H),7.53(d,J＝8.52Hz,2H),6.55(m,4H),4.24(br,4H),3.52(m,24H),3.42(m,8H),2.55 (m,4H),1.99(m,16H),1.64(s,12H)。
实施例16
制备化合物CNCIV-1，结构式如下：
Figure PCTCN2020089319-appb-000052
制备方法同实施例6，除了用1,3-丙磺酸内酯代替1,4-丁磺酸内酯与化合物2进行反应，其余合成步骤不变，得到终产品化合物CNCIV-1，收率20.7％。
1H NMR(300MHz,DMSO)δ(ppm):10.03-9.97(m,4H),8.88-8.2(m,4H),7.90(d,2H,J＝12.24Hz),7.80(s,2H),7.72(d,2H,J＝8.46Hz),7.02(d,J＝7.71Hz,2H),5.66(d,2H),3.91(br,4H),3.60(m,8H),2.58(m,4H),2.00(s,12H),1.89(m,4H),1.72(m,2H),1.59(s,12H)。
实施例17
制备化合物CNCIV-2，结构式如下：
Figure PCTCN2020089319-appb-000053
制备方法同实施例16，除了用1,3-丙二胺代替乙二胺进行反应，其余合成步骤不变，得到终产品化合物CNCIV-2，收率14.2％。
1H NMR(300MHz,DMSO)δ(ppm):10.05-9.95(m,4H),8.63-8.32(m,4H),7.94(d,2H,J＝12.93Hz),7.84(s,2H),7.76(d,2H,J＝8.13Hz),6.99(d,J＝8.25Hz,2H),5.58(d,2H,J＝12.66Hz),3.80(br,4H),3.27(m,8H),2.59(m,4H),2.00(s,12H),1.82(m,4H),1.74(m,6H),1.62(s,12H)。
实施例18
制备化合物CNCIV-3，结构式如下：
Figure PCTCN2020089319-appb-000054
制备方法同实施例16，除了用1,4-丁二胺代替乙二胺进行反应，其余合成步骤不变，得到终产品化合物CNCIV-3，收率14％。
1H NMR(300MHz,DMSO)δ(ppm):10.02-9.91(m,4H),8.76-8.27(m,4H),7.94(d,2H,J＝12.69Hz),7.83(s,2H),7.76(d,2H,J＝8.28Hz),6.98(d,J＝7.92Hz,2H),5.58(d,2H,J＝12.84Hz),3.81(br,4H),3.29(m,8H),2.59(m,4H),2.51(m,4H),2.00(s,12H),1.80(m,14H),1.62(s,12H)。
实施例19
制备化合物CNCIV-4，结构式如下：
Figure PCTCN2020089319-appb-000055
制备方法同实施例16，除了用1,8-二氨基-3,6-二氧杂辛烷代替乙二胺进行反应，其余合成步骤不变，得到终产品化合物CNCIV-4，收率19.2％。
1H NMR(300MHz,DMSO)δ(ppm):10.03-9.92(m,4H),8.78-8.39(m,4H),8.00-7.86(m,7H),7.52(d,J＝8.13Hz,2H),5.56(d,J＝13.4Hz,2H),3.77(m,4H),3.56(m,16H),3.39(m,8H),2.56(m,4H),2.50(m,4H),1.99(s,12H),1.68-1.57(m,22H)。
实施例20
制备化合物CNCIV-5，结构式如下：
Figure PCTCN2020089319-appb-000056
制备方法同实施例16，除了用2(2,2'–(2,2'-氧双(乙烷-2,1-二基)双(氧))二(乙胺))代替乙二胺进行反应，其余合成步骤不变，得到终产品化合物CNCIV-5，收率20％。
1H NMR(300MHz,DMSO)δ(ppm):10.01-9.90(m,4H),8.68-8.31(m,4H),7.90(d,2H,J＝12.30Hz),7.82(s,2H),7.73(d,2H,J＝7.41Hz),7.02(d,J＝8.52Hz,2H),5.66(d,2H,J＝12.84Hz),3.91(br,4H),3.52(m,24H),3.40(m,8H),2.58(m,4H),1.99(s,12H),1.89(m,4H),1.71(m,2H),1.57(s,12H)。
实施例21
制备化合物CNCV-2，结构式如下：
Figure PCTCN2020089319-appb-000057
制备方法同实施例2，除了用2,3,5-三碘基苯甲酸代替泛影酸进行反应，其余合成步骤不变，得到终产品化合物CNCV-2，收率46％。
1H NMR(300MHz,DMSO)δ(ppm):9.95(m,4H),8.65-8.48(m,4H),8.03(s,2H),8.01-7.90(m,5H),7.76(s,2H),7.62(s,2H),7.45(d,J＝8.13Hz,2H),6.65-6.49(m,4H),4.12(br,4H),3.42(m,8H),2.53(m,4H),1.85(m,4H),1.76(m,8H),1.68(s,12H)。
实施例22
制备化合物CNCV-3，结构式如下：
Figure PCTCN2020089319-appb-000058
制备方法同实施例21，除了用1,3-乙二醇代替1,3-乙二胺进行反应，其余合成步骤不变，得到终产品化合物CNCV-3，收率26％。
1H NMR(300MHz,DMSO)δ(ppm):9.98(m,4H),8.60-8.46(m,4H),8.31(s,2H),7.99-7.86(m,5H),7.75(s,2H),7.62(s,2H),7.49(d,J＝8.13Hz,2H),6.62-6.48(m,4H),4.09(br,4H),3.40(m,8H),2.54(m,4H),1.81(m,4H),1.72(m,8H),1.61(s,12H)。
实施例23
制备化合物CNCVI-2，结构式如下：
Figure PCTCN2020089319-appb-000059
制备方法同实施例3，除了用2,4,6-三碘基苯甲酸代替泛影酸进行反应，其余合成步骤不变，得到终产品化合物CNCVI-2，收率43％。
1H NMR(300MHz,DMSO)δ(ppm):10.03(m,4H),8.78-8.57(m,4H),8.03(s,2H),8.03-7.88(m,5H),7.82(s,4H),7.48(d,J＝8.13Hz,2H),7.21(s,4H),6.62-6.45(m,4H),4.23(br,4H),3.39(m,8H),2.57(m,4H),1.95(m,4H),1.83(m,8H),1.58(s,12H)。
实施例24
制备化合物CNCVI-3，结构式如下：
Figure PCTCN2020089319-appb-000060
制备方法同实施例23，除了用1,3-乙二醇代替1,3-乙二胺进行反应，其余合成步骤不变，得到终产品化合物CNCVI-3，收率29％。
1H NMR(300MHz,DMSO)δ(ppm):10.04(m,4H),8.59-8.38(m,4H),8.06(s,2H),8.11-7.91(m, 5H),7.77(s,4H),7.51(d,J＝8.13Hz,2H),6.88-6.47(m,4H),4.21(br,4H),3.36(m,8H),2.58(m,4H),1.87(m,4H),1.74(m,8H),1.66(s,12H)。
应用实验
本发明的细胞实验选用HepG2(人肝癌细胞)与L02(人正常肝细胞)细胞进行体外培养。其中，所述的HepG2细胞来自中国科学院上海细胞所，所述的L02细胞来自于上海复生生物科技有限公司。所述HepG2细胞在含有10％FBS，100IU/mL青霉素和100mg/mL链霉素的HyClone DMEM培养基中培养。所述L02细胞在含有10％FBS，100IU/mL青霉素和100mg/mL链霉素的RPMI 1640培养基中培养。
本发明的动物实验中的模型鼠为普通裸鼠经腋下接种HepG2细胞，饲养裸鼠1周，得到肿瘤模型。
本发明的动物实验中的模型兔为普通大耳兔肝脏原位接种VX2(兔源肝癌细胞)，饲养兔2-3周，得到肿瘤模型。
参见图1是实施例1制备的化合物CNCI-1与泛影酸钠体外CT强度对照图，将化合物CNCI-1与泛影酸钠在1.5mL的EP管中分别配制等体积的浓度梯度溶液(40mg I/mL-0)置于Siemens Inveon PET/CT下进行成像，分析成像结果，在图示浓度梯度范围内，这两种造影剂具有近似的CT信号强度。
参见图2和图3分别是泛影酸钠与实施例1制备的化合物CNCI-1的体外CT强度线性图，在上述浓度梯度范围内，将CT值绘制成回归曲线，横坐标为两种造影剂的含碘量(mg I/mL)，纵坐标为CT值，两条曲线的R 2均大于0.99，说明这两种造影剂在图示浓度梯度具有良好的线性关系。
参见图4是实施例1制备的化合物CNCI-1与ICG的MTT细胞活力测试图。测试化合物CNCI-1和ICG对HepG2细胞的细胞毒性，于37℃将细胞接种到96孔板(6×10 3细胞/孔)上，5％CO 2条件下共孵育24小时。随后，在新鲜培养基中加入CNCI-1或ICG(0-100μM)加入至孵育体系。48小时后，进行标准MTT测定检测细胞活力。其中横坐标指化合物以及ICG用量，纵坐标指化合物及ICG对细胞的抑制率。图中表明，化合物CNCI-1与ICG的浓度小于100μM时，HepG2的细胞均存活率在80％以上，说明该造影剂具有良好的细胞相容性且毒性较低。
参见图5是实施例1制备的化合物CNCI-1在肝癌细胞HepG2与正常肝细胞L02中的体外细胞摄取评价图。首先，将HepG2在5％CO 2，37℃下在12孔板(2.0×10 5细胞/孔)上孵育24小时，随后用含有化合物CNCI-1(25μM)的培养基替换原培养基，孵育1小时。经PBS洗涤两次并用4％多聚甲醛溶液固定，通过共聚焦显微镜(Olympus，Japan)分析CLSM图像。以相同方法处理L02细胞作为对照。图5A中DAPI为活细胞细胞核染色，图5B中化合物CNCI-1染色，图5C中MERGE图为图5A和图5B叠加在一起的图。实验结果显示化合物CNCI-1被吞噬进入肝癌细胞内，而在这正常肝细胞中没有此现象发生，说明化合物CNCI-1肝癌细胞中的摄取效率远高于正常肝细胞。
参见图6是实施例1制备的化合物CNCI-1与ICG的活体小动物近红外成像图。IVIS成像光谱系统用于进行NIRF荧光成像实验，并通过IVIS 4.5 Living Imaging软件(PerkinElmer，U.S.)在此参数(λex＝780nm，λem＝831nm，曝光时间＝5.8秒)下进行分析。实验选取两组HepG2腋下接种裸鼠，分别经尾静脉注射浓度为10mg/kg的化合物CNCI-1与ICG的2％泊洛沙姆溶液0.2mL，在不同时间段对实验鼠进行近红外成像，记录不同时间段的活体模型鼠近红外成像结果。图中圆圈部位指示肿瘤位置，肿瘤区域成像亮度远高于周围组织。因此，在近红外成像下均能显示化合物CNCI-1与ICG在肿瘤区域的富集，且化合物CNCI-1具有更好的对比度。
参见图7为本发明造影剂最优选化合物的活体小动物CT成像图。实验采用大耳兔原位接种兔源肝癌VX2细胞，建造肝癌模型。实验兔经静脉快速推注给药(化合物浓度100mg/mL,给药7mL)，利用Siemens Inveon PET/CT对兔子进行肝脏灌注成像，得到兔子肝部的CT图像，A为兔子肝部横切面造影图，B为兔子肝部侧切面造影图，C为兔子肝部竖切面造影图，标尺位置指示肝癌附近的异生血管，D为仪器拟合出其肝脏部位的立体图像，箭头所指示即为拟合生成的异生血管立体图像。体内CT实验结果显示，肝癌处异生的血管显像明显，呈现高亮，通过处理且能拟合出异生血管的3D影像，可用于指示肝癌位置。
以上所述的仅是本发明的优选实施方式，应当指出，对于本领域的普通技术人员来说，在不脱离本发明创造构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。

Claims (10)

  1. 一种新型双模态小分子造影剂，其特征在于：为如式(1)所示的化合物，
    CT-Linker-NIR-Linker-CT·W +
    (I)，
    其中，CT表示碘化有机分子；
    Linker表示连接链，结构为
    Figure PCTCN2020089319-appb-100001
    M选自-COO-、-CO-、-NH-或-O-；N选自-COO-、-CO-、-NH-或-O-；X为-O-；m为0、1、2、3、4、5或6；m’为2、3、4、5或6；
    NIR表示菁类染料衍生物，结构为
    Figure PCTCN2020089319-appb-100002
    R 1、R 2、R 3、R 4、R 5和R 6相同或不同，彼此独立地选自羧基、氨基、羟基、磺酸基或氢原子；Y选自羧酸阴离子、磺酸阴离子或磷酸阴离子；R x为H或卤素；R y和R z为H，或R y和R z与其相连的碳一起形成3-6元碳环；n为1、2、3或4；
    W表示碱金属。
  2. 根据权利要求1所述的一种新型双模态小分子造影剂，其特征在于：
    所述碘化有机分子为芳香族含碘化合物，结构为
    Figure PCTCN2020089319-appb-100003
    其中，R c1、R c2、R c3、R c4和R c5中至少一个为I，其余选自I、-COOH、-NH 2、-CONH 2或-CONHCH 3；
    所述
    Figure PCTCN2020089319-appb-100004
    中，M和N为-NH-或-O-；m为0、2或3；m’为2、3或4；
    所述
    Figure PCTCN2020089319-appb-100005
    中，R 1、R 3、R 4和R 6相同，选自氢原子；R 2和R 5相同，选自羧基；当R y和R z为H时，R x为H；当R y和R z与其相连的碳一起形成3-6元碳环时，R x为卤素，选自F、Cl、Br或I；n为2或3；
    W选自Na或K。
  3. 根据权利要求2所述的一种新型双模态小分子造影剂，其特征在于：
    所述CT的结构为
    Figure PCTCN2020089319-appb-100006
    Figure PCTCN2020089319-appb-100007
    所述Linker的结构为
    Figure PCTCN2020089319-appb-100008
    Figure PCTCN2020089319-appb-100009
    所述NIR的结构为
    Figure PCTCN2020089319-appb-100010
    Figure PCTCN2020089319-appb-100011
    Figure PCTCN2020089319-appb-100012
  4. 根据权利要求1-3任一项所述的一种新型双模态小分子造影剂，其特征在于：所述造影剂选自如下结构的化合物中的任一种：
    Figure PCTCN2020089319-appb-100013
    Figure PCTCN2020089319-appb-100014
    Figure PCTCN2020089319-appb-100015
    Figure PCTCN2020089319-appb-100016
    Figure PCTCN2020089319-appb-100017
    Figure PCTCN2020089319-appb-100018
    Figure PCTCN2020089319-appb-100019
    Figure PCTCN2020089319-appb-100020
  5. 一种权利要求1所述的新型双模态小分子造影剂的制备方法，其特征在于：所述造影剂是在碱和缩合剂的作用下，将连接Linker的有机碘化分子与NIR在室温下进行缩合反应得到，反应如下所示：
    CT-Linker+NIR→CT-Linker-NIR-Linker -CT·W +
    (I)，
    所述NIR与连接Linker的有机碘化分子的摩尔比为1:(2.3-2.5)；NIR与碱的摩尔比为1:(3-3.5)；NIR与缩合剂的摩尔比为1:(3-3.5)；
    其中，CT表示碘化有机分子；
    Linker表示连接链，结构为
    Figure PCTCN2020089319-appb-100021
    M选自-COO-、-CO-、-NH-或-O-；N选自-COO-、-CO-、-NH-或-O-；X为-O-；m为0、1、2、3、4、5或6；m’为2、3、4、5或6；
    NIR表示菁类染料衍生物，结构为
    Figure PCTCN2020089319-appb-100022
    R 1、R 2、R 3、R 4、R 5和R 6相同或不同，彼此独立地选自羧基、氨基、羟基、磺酸基或氢原子；Y选自羧酸阴离子、磺酸阴离子或磷酸阴离子；R x为H或卤素；R y和R z为H，或R y和R z与其相连的碳一起形成3-6元碳环；n为1、2、3或4；
    W表示碱金属。
  6. 根据权利要求5所述的新型双模态小分子造影剂的制备方法，其特征在于：所述反应所用的溶剂为酰胺类溶剂、酮类溶剂、腈类溶剂和亚砜类溶剂中的一种或多种。
  7. 根据权利要求6所述的新型双模态小分子造影剂的制备方法，其特征在于：所述溶剂为N,N-二甲基甲酰胺、丙酮、乙腈和二甲基亚砜中的一种或多种。
  8. 根据权利要求5所述的新型双模态小分子造影剂的制备方法，其特征在于：所述碱为无机碱或有机碱；所述的无机碱选自碳酸氢钠、碳酸氢钾、碳酸钠和碳酸钾中的一种或多种；所述的有机碱选自三乙胺、二异丙基乙胺和吡啶中的一种或多种。
  9. 根据权利要求5所述的新型双模态小分子造影剂的制备方法，其特征在于：所述缩合剂为环己基碳二亚胺、1-羟基苯并三唑、1-羟基-7-偶氮苯并三氮唑、1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐、2-(7-氧化苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯和O-苯并三氮唑-四甲基脲六氟磷酸盐中的一种或多种。
  10. 一种如权利要求1-3任一项所述的造影剂在近红外/CT成像中的应用。
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