Nano CT contrast agent compound as well as preparation method and application thereof

Abstract

The invention provides a self-assembled nano CT contrast agent compound and a pharmaceutically acceptable salt thereof, a method for preparing the compound, and a CT contrast agent containing the compound. The invention also provides application of the compound and the pharmaceutically acceptable salt thereof or the CT contrast agent in in-vivo tumor CT detection imaging. According to the compound disclosed by the invention, the self-assembled nano CT contrast agent is constructed by utilizing a size aggregation effect strategy, and the compound has an amphiphilic structure, has relatively good tumor targeting capability and can improve in-vivo tumor CT imaging.

Description

Technical Field

[0001] The present invention belongs to the field of chemical biology technology and relates to a self-assembled nano CT contrast agent compound. Specifically, the present invention relates to a method for constructing a self-assembled nano CT contrast agent compound by utilizing a size aggregation effect strategy, and a method for using the nano CT contrast agent compound for in vivo tumor CT imaging. Background Art

[0002] X-ray computed tomography (CT) has become a key non-invasive diagnostic tool in clinical practice due to its special features such as high resolution and deep tissue penetration. However, the inherent limitations of CT imaging, especially in differentiating soft tissues of similar density, require the use of exogenous contrast agents to improve imaging accuracy.

[0003] In the prior art, clinical CT imaging uses traditional small-molecule iodinated contrast agents (ICAs), which exhibit disadvantages such as low molecular weight, widespread distribution in the body, and rapid renal clearance, resulting in a narrow imaging time window. In addition, existing nanoscale ICAs face challenges such as complex preparation protocols, structural variability, limited reproducibility, low iodine content, and uniformity issues. Therefore, there is an urgent need to develop innovative nanoscale ICAs with unique, well-defined structures, simple but reproducible preparation procedures, and high-performance properties for precise CT imaging in the tumor environment. Summary of the invention

[0004] In view of these shortcomings in the prior art, the purpose of the present invention is to provide a self-assembled nano CT contrast agent constructed by utilizing the size aggregation effect strategy for improving in vivo tumor CT imaging, as well as a method for preparing the nano contrast agent and its application in tumor-bearing mice.

[0005] According to one aspect of the present application, the present application provides a compound and a pharmaceutically acceptable salt thereof, wherein the compound has a structure shown in formula (I):

[0006]

[0007] Among them: (1) the value of n can be between 12 and 24;

[0008] (2) R can be a stimuli-responsive group.

[0009] According to certain embodiments of the present application, the stimulus responsive group is selected from a hydrogen peroxide responsive group, a hypochlorous acid responsive group, an ·OH responsive group, a t-BuOO· responsive group, a peroxynitrite responsive group, a cysteine ​​responsive group, a glutathione responsive group, a hydrogen sulfide responsive group, a nitroreductase responsive group, and a phosphatase responsive group.

[0010] According to certain embodiments of the present application, the pharmaceutically acceptable salt includes sodium salt, potassium salt, ammonium salt, and calcium salt.

[0011] According to certain embodiments of the present application, the compound of formula (I) is selected from:

[0012]

[0013]

[0014]

[0015] According to one aspect of the present application, the present application provides a method for preparing the compound, the method comprising:

[0016] 1) Dissolve the precursor compound 1 aminotetraphenylethylene and the precursor compound 2 N-Fmoc-N'-Boc-L-ornithine, the condensation reagent HOBT, the condensation reagent HBTU and N,N-diisopropylethylamine in N,N-dimethylformamide (20 mL), stir at room temperature for 2 hours, and then quickly add 1-(4-aminophenyl)-1,2,2-triphenylethylene. Stir the resulting mixture at room temperature until TLC analysis shows that the reaction is complete. The reaction mixture is evaporated on a rotary evaporator to obtain a solid residue, which is purified by column chromatography (petroleum ether / ethyl acetate = 3 / 1) to obtain a white solid precursor compound 1;

[0017] 2) Precursor compound 3 was dissolved in a mixture of dichloromethane (10 mL) and piperidine (2 mL) to remove Fmoc at 0°C for 30 minutes, and the mixture was washed with water, the organic layer was separated and eluted with anhydrous Na 2 SO 4 After drying, spin dry. 2,3,5-triiodobenzoic acid, HOBT, and HBTU were dissolved in N,N-dimethylformamide (20 mL), reacted in an ice bath for 30 min, and then the spin-dried system was added, and the resulting mixed system was stirred at room temperature until the reaction was completed. The solvent dichloromethane was removed on a rotary evaporator to obtain a solid residue, which was purified by column chromatography (dichloromethane / methanol = 200 / 1) to obtain a white solid precursor compound 5;

[0018] 3) The precursor compound 5 was dissolved in a mixture of dichloromethane (10 mL) and trifluoroacetic acid (2 mL), reacted at room temperature for 10 minutes to remove the Boc protecting group, and then the solvent dichloromethane was removed on a rotary evaporator to obtain a white solid mixture. The precursor compound 6 of compound A, triethylamine and 4-dimethylaminopyridine were then dissolved in dichloromethane (20 mL), and the above mixture was added, and the resulting mixture was stirred at room temperature until the reaction was completed, and then the mixture was washed with water, the organic layer was separated and the reaction mixture was washed with anhydrous Na 2 SO4 Next, p-tetrabutylammonium fluoride was added to the obtained mixture, and stirred at room temperature for 1 hour, and evaporated on a rotary evaporator to obtain a solid residue, which was purified by column chromatography (dichloromethane / ethyl acetate=2 / 1) to obtain a precursor of compound A 7 as a light blue solid;

[0019] 4) Precursor compound 8, precursor compound 7, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine were dissolved in dichloromethane (5 mL). The reaction mixture was stirred at room temperature for 6 hours. After the reaction was complete, the reaction mixture was washed with 1M HCl solution (10 mL), saturated NaHCO 3 (10 ml) and saturated NaCl (10 mmL). The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated to give a crude product, which was purified by column chromatography (dichloromethane / methanol=10 / 1) to give the compound of formula (I) as a light green solid.

[0020] According to certain embodiments of the present application, the method comprises:

[0021]

[0022] According to one aspect of the present application, the present application provides a CT contrast agent, which comprises the compound according to any one of claims 1 to 3 and a pharmaceutically acceptable salt thereof.

[0023] According to certain embodiments of the present application, the iodine content of the CT contrast agent is 12%-40%.

[0024] According to one aspect of the present application, the present application provides a method for preparing the CT contrast agent, wherein the compound BioDHU-CT of formula (I) and a pharmaceutically acceptable salt thereof are dissolved in an organic solvent, and then added to a PBS phosphate buffer solution at pH 7-8.

[0025] According to certain embodiments of the present application, the organic solvent is selected from N,N-dimethylformamide, ethanol, dimethyl sulfoxide, acetonitrile, and tetrahydrofuran.

[0026] According to one aspect of the present application, the present application provides use of the compound and a pharmaceutically acceptable salt thereof or the CT contrast agent in the preparation of a drug for in vivo tumor CT detection imaging.

[0027] Beneficial effects of the present invention:

[0028] 1) This invention proposes for the first time a strategy of utilizing size aggregation effect to construct a self-assembled nano-CT contrast agent for improving in vivo tumor CT imaging.

[0029] 2) The compounds of formula (I), BioDHU-CT, have good tumor targeting capabilities, as well as their rapid aggregation in the tumor area after activation. This unique property improves the retention efficiency of CT contrast agents, thereby extending the imaging time for optimal visualization. It is worth noting that after activation, the iodine content within the nanoparticles increases significantly from 14% to 40%, further improving the overall performance of CT imaging in the tumor environment.

[0030] 3) The CT contrast agent compound of formula (I) BioDHU-CT of the present invention has an amphiphilic structure and can easily form nanoparticles of a certain size in an aqueous solution.

[0031] 4) After the CT contrast agent compound of the present invention is injected into tumor-bearing mice through the tail vein, it will quickly be enriched in the tumor area through the EPR effect.

[0032] 5) The present invention constructs a single-component nano CT contrast agent, compound BioDHU-CT, of formula (I) using the AIE fluorophore tetraphenylethylene. After activation, the nanoparticles of the compound BioDHU-CT of formula (I) exhibit a unique size aggregation effect. Both in vitro and in vivo studies have shown that the nanoparticles of the compound BioDHU-CT of formula (I) exhibit excellent responsiveness and rapidly form larger aggregates within the tumor region after activation. This aggregation phenomenon improves the retention efficiency of the CT contrast agent and prolongs the imaging time window. Description of the drawings:

[0033] Figure 1 The precursor compound 3 described in Example 1 of the present invention 1 H NMR spectrum.

[0034] Figure 2 The precursor compound 3 described in Example 1 of the present invention 13 C NMR spectrum.

[0035] Figure 3 The precursor compound 5 described in Example 2 of the present invention 1 H NMR spectrum.

[0036] Figure 4 The precursor compound 5 described in Example 2 of the present invention 13 C NMR spectrum.

[0037] Figure 5 The compound A precursor compound 7 described in Example 3 of the present invention 1 H NMR spectrum.

[0038] Figure 6The compound A precursor compound 7 described in Example 3 of the present invention 13 C NMR spectrum.

[0039] Figure 7 The compound BioDHU-CT compound A of formula (I) described in Example 4 of the present invention 1 H NMR spectrum.

[0040] Figure 8 The compound BioDHU-CT compound A of formula (I) described in Example 4 of the present invention is 13 C NMR spectrum.

[0041] Fig. 9 The transmission electron microscope image and water and particle size image of the compound BioDHU-CT of formula (I) described in the present invention.

[0042] Fig.10 The response behavior of the BioDHU-CT compound 1 described in the present invention to ROS. From A to J are pure BioDHU-CT, H 2 O 2 , O 2 -, TBHP, ROO·, NO, t-BuOO·, ·OH, ONOO-, HOCl. ·OH, t-BuOO·, ONOO- and HOCl are all active oxygen species, which can react with BioDHU-CT to dissociate the nanoparticles and release the fluorescent methylene blue fluorophore. The concentration of the BioDHU-CT compound 1 is 10 μM, and the concentration of the active oxygen species is 40 μM. The fluorescence intensity at 686 nm is collected, and the excitation wavelength is 620 nm.

[0043] Fig.11 The fluorescence spectrum of the reaction of the BioDHU-CT compound 1 of the present invention with ·OH, t-BuOO·, ONOO- and HOCl. The concentration of the BioDHU-CT compound 1 is 10 μM, the active oxygen concentration is 40 μM, and the excitation wavelength is 620 nm.

[0044] Fig.12The following are verification diagrams of the size increase of the BioDHU-CT compound 1 described in the present invention after the reaction with hypochlorous acid. Figure a is a transmission electron microscope image of the BioDHU-CT compound 1 after the reaction with hypochlorous acid. Figure b is a maldi-tof image of the BioDHU-CT compound 1 after the reaction with hypochlorous acid. Figure c is the fluorescence of tetraphenylethylene in a system with different water contents (ethanol: water) after the BioDHU-CT compound 1 responds to hypochlorous acid, with the water content ranging from 10% to 80%, the concentration of the BioDHU-CT compound 1 being 10 μM, the concentration of hypochlorous acid being 40 μM, and the excitation wavelength being 360 nm. Figure d shows the fluorescence and CT signals of tetraphenylethylene in the filtrate tested after the BioDHU-CT compound 1 responded to different concentrations of hypochlorous acid and was filtered with a filter membrane with a pore size of 220 nm. The concentration of the BioDHU-CT compound 1 was 50 μM, and the concentrations of hypochlorous acid were 0 μM, 5 μM, 10 μM, 15 μM, 20 μM, 30 μM, and 50 μM, respectively, and the excitation wavelength was 360 nm.

[0045] Fig.13 The CT imaging diagram of the intratumoral injection of the BioDHU-CT compound 1 of the present invention in vivo. Subcutaneous tumors of Hela cells were planted in the left and right hind legs of mice, and iohexol, a CT contrast agent commonly used in clinical practice, was selected as a control. The BioDHU-CT was injected into the left tumor of the mouse, and iohexol was injected into the right tumor. Then, CT imaging was performed at 10 minutes, 40 minutes, 1 hour, 1.5 hours, 2 hours, 3 hours, 6 hours, 12 hours, and 24 hours. The concentration of the BioDHU-CT was 8 mg / mL, and the injection volume was 20 μL. The arrow in the figure indicates BioDHU-CT compound A NPs (8 mg I / mL, 20 μL), and the circle indicates the control iohexol (8 mg I / mL, 20 μL). DETAILED DESCRIPTION DETAILED DESCRIPTION OF THE INVENTION

[0047] CT imaging contrast agents

[0048] X-ray computed tomography (CT) has become the most commonly used non-invasive diagnostic tool in clinical practice due to its high resolution, deep tissue penetration, low cost, and wide availability. Small molecule iodinated contrast agents (ICAs) (such as iohexol and iodixanol) are commonly used in clinical practice to more effectively visualize soft tissues and pathological areas. However, due to their low molecular weight, large volume distribution, and rapid renal clearance (excreted by the kidneys only a few minutes after intravenous administration), the time window for CT imaging of specific areas of interest after administration is usually very narrow (usually <60 minutes). Beyond this window, CT images may lack sufficient contrast, may miss important areas, and lead to incorrect assessment of the disease.

[0049] In order to address some of the shortcomings of small molecule ICAs, iodine-containing organic nanoparticles for in vivo CT imaging have recently been designed and explored in the prior art, including nanoemulsions, liposomes, nanogels, and dendritic polymers (multi-components). These nanoscale ICAs significantly prolong the blood pool residence time and reduce renal clearance. However, they still face many problems, such as preparation refolding, structural heterogeneity, poor reproducibility, low iodine content, etc. (multi-component disadvantages). In addition, in tumor imaging, nanoparticles (NP) with a diameter of less than 30 can penetrate deeply once entering the tumor, while larger nanoparticles (about 150nm or more) show high retention efficiency. Therefore, it is challenging to achieve good penetration depth and high retention efficiency of single-sized nano contrast agents.

[0050] Nanoparticle (NP)

[0051] Nanoparticles are tiny particles with nanometer size. Currently, many iodinated organic nanoparticles with a size of about 200nm, such as nanoemulsions, liposomes, dendrimers, etc., have been developed as CT contrast agents for tumor imaging.

[0052] Single-component nanoformulations with size-aggregation effects after activation in the tumor microenvironment (TME)

[0053] Single-component nanoformulations with size aggregation effects after activation in the tumor microenvironment (TME) provide a new approach to address the above problems. First, single-component self-assembly ensures that the structure of nanocontrast agents is single, clear, and reproducible. Second, due to the adjustable size, nanoICAs can maintain a small size before activation, which ensures that they have good penetration depth, and can grow rapidly after activation, thereby extending the imaging window. In addition, due to the increased mass ratio of iodine after the reaction, the required amount of contrast agent can be reduced, thereby reducing potential biological toxicity.

[0054] Single-component nanoformulations with size aggregation effects after activation in the tumor microenvironment (TME) provide a new approach to solve the problems in the prior art. First, the single-component self-assembly ensures that the structure of the nanocontrast agent is single, clear and reproducible. Second, due to the adjustable size, nanoICAs can maintain a small size before activation, which ensures that they have good penetration depth, and can grow rapidly after activation, thereby extending the imaging window period. In addition, due to the increase in the mass ratio of iodine after the reaction, the required amount of contrast agent can be reduced, thereby reducing potential biological toxicity.

[0055] However, few nano-ICAs are based on this strategy because: 1) the inherent small molecular weight of ICAs makes them susceptible to rapid metabolism and clearance from the body; 2) achieving in situ aggregation usually requires the accumulation of monomers to a specific threshold concentration, which is hindered by current activation systems due to their long reaction times and high stimulant concentrations; and 3) the efficiency of monomer-to-aggregate formation becomes critical to ensure that aggregation occurs before monomers are cleared from the system.

[0056] Aggregation Induced Emission (AIE)

[0057] Aggregation-induced emission (AIE) is a unique photophysical phenomenon first reported in 2001. Propeller-shaped fluorescent substances are non-emissive when the molecules are dissolved in solution, but emit efficiently when they form aggregates. AIE luminogens (AIEgens) can quickly form nanoaggregates in aqueous solution and have been widely used in the modification of contrast agents. Among AIEgens, tetraphenylethylene (TPE) is a commonly used monomer for constructing aggregates due to its fast aggregation properties and ease of synthesis.

[0058] This patent uses a combination of TPE and ICAs to prepare a monomer with efficient aggregation performance. The monomer is then integrated with an activated response platform to create an AIEgen-based nano-ICA response aggregation system. As shown in Formula I, this nano-ICA (Formula I) consists of a biotin ether chain as a hydrophilic and active targeting part, a bridging group (2-amino-1,3-phenylene) dimethanol group modified with a response group, and an iodine-containing compound modified with TPE. The amphiphilicity of Formula I enables them to self-assemble into single-component nanoparticles in aqueous solution.

[0059] Organ targeting properties

[0060] In order to grow rapidly, tumor cells need more nutrients and oxygen, so they secrete growth factors related to tumor angiogenesis, such as vascular endothelial growth factor. Especially when the tumor reaches 150-200 microns in size, it will be highly dependent on the nutrient and oxygen supply of tumor blood vessels. At this time, the newly generated tumor blood vessels are very different from normal blood vessels in structure and morphology. The gaps between its endothelial cells are larger, the smooth muscle layer of the vascular wall is lacking, and the angiotensin receptor function is lost. In addition, the tumor tissue also lacks lymphatic vessels, which causes the return of lymph to be blocked. These two factors make it possible for large molecules to easily pass through the blood vessel wall and accumulate in the tumor tissue, and not be carried away by the return of lymph and can remain in the tumor tissue for a long time, so it is called the "high permeability and long retention effect" (EPR) of solid tumors.

[0061] Compound of formula (I)

[0062] According to one aspect of the present application, the present application provides a method for constructing a self-assembled nano-CT contrast agent compound and a pharmaceutically acceptable salt thereof by utilizing a size aggregation effect strategy, wherein the compound improves in vivo tumor CT imaging.

[0063] According to certain embodiments of the present application, the pharmaceutically acceptable salt includes sodium salt, potassium salt, ammonium salt, and calcium salt.

[0064] According to certain embodiments of the present application, the compound is the compound of formula (I) BioDHU-CT.

[0065] According to certain embodiments of the present application, the compound has a structure shown in formula (I):

[0066]

[0067] Where: (1) the value of n is between 12 and 24;

[0068] (2) R can be a stimuli-responsive group.

[0069] According to certain embodiments of the present application, the value of n in the compound of formula (I) BioDHU-CT is between 12-24, for example, 12-20, 12-16, 16-24, 16-20 or 20-24. According to certain embodiments of the present application, the value of n in the compound of formula (I) BioDHU-CT is 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24.

[0070] According to certain embodiments of the present application, the stimulus responsive group is selected from a hydrogen peroxide responsive group, an ·OH responsive group, a t-BuOO· responsive group, a hypochlorous acid responsive group, a peroxynitrite responsive group, a cysteine ​​responsive group, a glutathione responsive group, a hydrogen sulfide responsive group, a nitroreductase responsive group, and a phosphatase responsive group.

[0071] The hypochlorous acid responsive group refers to a group that can react with hypochlorous acid, such as methylene blue or its derivatives in compound 1. According to certain embodiments of the present application, the hypochlorous acid responsive group includes a derivative of methylene blue.

[0072] The hydrogen peroxide responsive group refers to a group that can react with hydrogen peroxide, such as the boronate structure in compound 2. According to certain embodiments of the present application, the hydrogen peroxide responsive group includes a boronate structure.

[0073] The glutathione responsive group refers to a group that can react with glutathione, such as nitrobenzene sulfonic acid in compound 3. According to certain embodiments of the present application, the glutathione responsive group includes nitrobenzene sulfonic acid.

[0074] The hydrogen sulfide responsive group refers to a group that can react with hydrogen sulfide, such as the azide group in compound 4. According to certain embodiments of the present application, the hydrogen sulfide responsive group includes an azide group.

[0075] The peroxynitrite responsive group refers to a group that can react with peroxynitrite, such as the thiobenzylidene in compound 5. According to certain embodiments of the present application, the peroxynitrite responsive group includes thiobenzylidene.

[0076] The cysteine ​​responsive group refers to a group that can react with cysteine, such as the disulfide bond structure and acrylate structure in compounds 6 and 9. According to certain embodiments of the present application, the cysteine ​​responsive group includes a disulfide bond.

[0077] The phosphatase responsive group refers to a group that can react with phosphatase, such as the phosphate in compound 7. According to certain embodiments of the present application, the phosphatase responsive group includes a phosphate structure and an acrylate structure.

[0078] The nitroreductase responsive group refers to a group that can react with nitroreductase, such as nitrobenzene in compound 8. According to certain embodiments of the present application, the nitroreductase responsive group includes nitrobenzene.

[0079] According to certain embodiments of the present application, the compound represented by formula (I) is selected from:

[0080]

[0081]

[0082]

[0083] The amphiphilicity of the compound BioDHU-CT of formula (I)

[0084] As shown in formula (I), the PEG ether chain on the left is water-soluble, while the tetraphenylethylene and triiodobenzoic acid parts on the right are fat-soluble, so the BioDHU-CT compound of formula (I) exhibits amphiphilic characteristics.

[0085] In aqueous solution, amphiphilic substances can easily self-assemble into nanoparticles with uniform particle size. After forming nanoparticles, the contrast agent can be enriched in the tumor area through the EPR effect.

[0086] In the prior art, most nano CT contrast agents are multi-component, and have the disadvantages of complex preparation procedures and difficulty in reproducing, which makes them difficult to clinically transform. The present application discloses that the compound BioDHU-CT of formula (I) can constitute a single-component nano CT contrast agent, which solves this problem.

[0087] The response mechanism of the compound of formula (I) to active oxygen

[0088] The present invention relates to a compound BioDHU-CT having a structure of formula (I) and a response mechanism with active oxygen as shown below:

[0089]

[0090] The compound BioDHU-CT having the structure of the formula (I) reacts with a stimulus in an aqueous solution, causing the system to dissociate, and the water-soluble ether chain portion and the tetraphenylethylene-modified iodinated contrast agent portion to separate.

[0091] According to certain embodiments of the present application, the stimulants are all compounds present in living organisms, and are all endogenous substances highly expressed in tumors. According to certain embodiments of the present application, the stimulants include reactive oxygen, glutathione, cysteine, hydrogen sulfide, nitroreductase, and phosphatase.

[0092] Preparation method of the compound of formula (I)

[0093] According to one aspect of the present application, the present application provides a method for preparing the compound of formula (I), comprising:

[0094] 1) Dissolve the precursor compound 1 aminotetraphenylethylene and the precursor compound 2 N-Fmoc-N'-Boc-L-ornithine, the condensation reagent HOBT, the condensation reagent HBTU and N,N-diisopropylethylamine in N,N-dimethylformamide (10-30 mL), stir at room temperature for 1-3 hours, and then quickly add 1-(4-aminophenyl)-1,2,2-triphenylethylene. Stir the resulting mixture at room temperature until TLC analysis shows that the reaction is complete; evaporate the reaction mixture on a rotary evaporator to obtain a solid residue, which is purified by column chromatography (petroleum ether / ethyl acetate = 5 / 1-1 / 1) to obtain a white solid precursor compound 1;

[0095] 2) Precursor compound 3 was dissolved in a mixture of dichloromethane (5-15 mL) and piperidine (1-3 mL) to remove Fmoc at 0°C for 20-60 minutes, and the mixture was washed with water, the organic layer was separated and eluted with anhydrous Na 2 SO 4 After drying, spin dry; 2,3,5-triiodobenzoic acid, HOBT, and HBTU were dissolved in N,N-dimethylformamide (10-30 mL), reacted in an ice bath for 10-30 min, and then the spin-dried system was added, and the resulting mixed system was stirred at room temperature until the reaction was completed. The solvent dichloromethane was removed on a rotary evaporator to obtain a solid residue, which was purified by column chromatography (dichloromethane / methanol = 300 / 1-100 / 1) to obtain a white solid precursor compound 5;

[0096] 3) Precursor compound 5 was dissolved in a mixture of dichloromethane (5-15 mL) and trifluoroacetic acid (1-5 mL), reacted at room temperature for 5-15 minutes to remove the Boc protecting group, and then the solvent dichloromethane was removed on a rotary evaporator to obtain a white solid mixture. Precursor compound 6 of compound A, triethylamine and 4-dimethylaminopyridine were then dissolved in dichloromethane (10-30 mL), and the above mixture was added, and the resulting mixture was stirred at room temperature until the reaction was completed, and then the mixture was washed with water, the organic layer was separated and the reaction mixture was washed with anhydrous Na 2 SO 4 Drying; Next, adding tetrabutylammonium fluoride to the obtained mixture, stirring at room temperature for 0.5-2 hours, and evaporating on a rotary evaporator to obtain a solid residue, which is purified by column chromatography (dichloromethane / ethyl acetate = 5 / 1-1 / 1) to obtain a precursor of compound A 7 as a light blue solid;

[0097] 4) Precursor compound 8, precursor compound 7, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine were dissolved in dichloromethane (1-10 mL). The reaction mixture was stirred at room temperature for 2-10 hours. After the reaction was complete, the reaction mixture was washed with 1M HCl solution (5-15 mL), saturated NaHCO 3 (5-15mL) and saturated NaCl (5-15mmL); the organic phase was dried over anhydrous sodium sulfate, filtered and concentrated to give a crude product, which was purified by column chromatography (dichloromethane / methanol=15 / 1-5 / 1) to give a light green solid compound of formula (I).

[0098] According to certain embodiments of the present application, in step 1), the amount of N,N-dimethylformamide is 10-30 mL, such as 10-20 or 20-30 mL. According to certain embodiments of the present application, in step 1), the amount of N,N-dimethylformamide is 10 mL, 11 mL, 12 mL, 13 mL, 14 mL, 15 mL, 16 mL, 17 mL, 18 mL, 19 mL, 20 mL, 21 mL, 22 mL, 23 mL, 24 mL, 25 mL, 26 mL, 27 mL, 28 mL, 29 mL or 30 mL.

[0099] According to certain embodiments of the present application, in step 1), stirring at room temperature is performed for 1-3 hours, such as 1-2 hours or 2-3 hours. According to certain embodiments of the present application, stirring at room temperature is performed for 1 hour, 1.5 hours, 2 hours, 2.5 hours or 3 hours.

[0100] According to certain embodiments of the present application, in the step 1) column chromatography purification, petroleum ether / ethyl acetate is 5 / 1-1 / 1, such as 5 / 1-3 / 1 or 3 / 1-1 / 1. According to certain embodiments of the present application, in the step 1) column chromatography purification, petroleum ether / ethyl acetate is 5 / 1, 4 / 1, 3 / 1, 2 / 1 or 1 / 1.

[0101] According to certain embodiments of the present application, in step 2), the amount of dichloromethane is 5-15 mL, such as 5-10 mL or 10-15 mL. According to certain embodiments of the present application, in step 2), the amount of dichloromethane is 5 mL, 6 mL, 7 mL, 8 mL, 9 mL, 10 mL, 11 mL, 12 mL, 13 mL, 14 mL or 15 mL.

[0102] According to certain embodiments of the present application, in step 2), the piperidine is 1-3 mL, such as 1-2 mL or 2-3 mL. According to certain embodiments of the present application, in step 2), the piperidine is 1 mL, 1.5 mL, 2 mL, 2.5 mL or 3 mL.

[0103] According to certain embodiments of the present application, in step 2), Fmoc is removed for 20-60 minutes, for example, 20-40 minutes or 40-60 minutes. According to certain embodiments of the present application, in step 2), Fmoc is removed for 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes or 60 minutes.

[0104] According to certain embodiments of the present application, in step 2), N,N-dimethylformamide is 10-30 mL, such as 10-20 or 20-30 mL. According to certain embodiments of the present application, in step 2), N,N-dimethylformamide is 10 mL, 11 mL, 12 mL, 13 mL, 14 mL, 15 mL, 16 mL, 17 mL, 18 mL, 19 mL, 20 mL, 21 mL, 22 mL, 23 mL, 24 mL, 25 mL, 26 mL, 27 mL, 28 mL, 29 mL or 30 mL.

[0105] According to certain embodiments of the present application, the step 2) is reacted in an ice bath for 10-30 min, for example, 10-20 min or 20-30 min. According to certain embodiments of the present application, the step 2) is reacted in an ice bath for 10 min, 11 min, 12 min, 13 min, 14 min, 15 min, 16 min, 17 min, 18 min, 19 min, 20 min, 21 min, 22 min, 23 min, 24 min, 25 min, 26 min, 27 min, 28 min, 29 min or 30 min.

[0106] According to certain embodiments of the present application, in the step 2) column chromatography purification, dichloromethane / methanol is 300 / 1-100 / 1, for example, 300 / 1-200 / 1 or 200 / 1-100 / 1. According to certain embodiments of the present application, in the step 2) column chromatography purification, dichloromethane / methanol is 300 / 1, 250 / 1, 200 / 1, 150 / 1 or 100 / 1.

[0107] According to certain embodiments of the present application, in step 3), the amount of dichloromethane is 5-15 mL, such as 5-10 mL or 10-15 mL. According to certain embodiments of the present application, in step 3), the amount of dichloromethane is 5 mL, 6 mL, 7 mL, 8 mL, 9 mL, 10 mL, 11 mL, 12 mL, 13 mL, 14 mL or 15 mL.

[0108] According to certain embodiments of the present application, in step 3), the amount of trifluoroacetic acid is 1-5 mL, such as 1-3 mL or 2-5 mL. According to certain embodiments of the present application, in step 3), the amount of trifluoroacetic acid is 1 mL, 1.5 mL, 2 mL, 2.5 mL, 3 mL, 3.5 mL, 4 mL, 4.5 mL or 5 mL.

[0109] According to certain embodiments of the present application, the step 3) is reacted at room temperature for 5-15 minutes. For example, 5-10 minutes or 10-15 minutes. According to certain embodiments of the present application, the step 3) is reacted at room temperature for 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes or 15 minutes.

[0110] According to certain embodiments of the present application, in the step 3), the amount of dichloromethane is 10-30 mL, for example, 10-20 mL or 20-30 mL. According to certain embodiments of the present application, in the step 3), the amount of dichloromethane is 10 mL, 11 mL, 12 mL, 13 mL, 14 mL, 15 mL, 16 mL, 17 mL, 18 mL, 19 mL, 20 mL, 21 mL, 22 mL, 23 mL, 24 mL, 25 mL, 26 mL, 27 mL, 28 mL, 29 mL or 30 mL.

[0111] According to certain embodiments of the present application, in step 3), stirring is performed at room temperature for 0.5-2 hours, such as 0.5-1 hour or 1-2 hours. According to certain embodiments of the present application, in step 3), stirring is performed at room temperature for 0.5 hour, 1 hour, 1.5 hours or 2 hours.

[0112] According to certain embodiments of the present application, in the step 3) column chromatography, dichloromethane / ethyl acetate is 5 / 1-1 / 1, such as 5 / 1-3 / 1 or 3 / 1-1 / 1. According to certain embodiments of the present application, in the step 3) column chromatography, dichloromethane / ethyl acetate is 5 / 1, 4 / 1, 3 / 1, 2 / 1 or 1 / 1.

[0113] According to certain embodiments of the present application, in step 4), the amount of dichloromethane is 1-10 mL, such as 1-5 mL or 5-10 mL. According to certain embodiments of the present application, in step 4), the amount of dichloromethane is 1 mL, 2 mL, 3 mL, 4 mL, 5 mL, 6 mL, 7 mL, 8 mL, 9 mL or 10 mL.

[0114] According to certain embodiments of the present application, in step 4), stirring is performed at room temperature for 2-10 hours, such as 2-5 hours or 5-10 hours. According to certain embodiments of the present application, in step 4), stirring is performed at room temperature for 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours or 10 hours.

[0115] According to certain embodiments of the present application, in step 4), saturated NaHCO is 5-15 mL, such as 5-10 mL or 10-15 mL. According to certain embodiments of the present application, in step 4), saturated NaHCO is 5 mL, 6 mL, 7 mL, 8 mL, 9 mL, 10 mL, 11 mL, 12 mL, 13 mL, 14 mL or 15 mL.

[0116] According to certain embodiments of the present application, in step 4), saturated NaCl is 5-15mL, such as 5-10mL or 10-15mL. According to certain embodiments of the present application, in step 4), saturated NaCl is 5mL, 6mL, 7mL, 8mL, 9mL, 10mL, 11mL, 12mL, 13mL, 14mL or 15mL.

[0117] According to certain embodiments of the present application, in the step 3) column chromatography, dichloromethane / methanol is 15 / 1-5 / 1, for example, 15 / 1-10 / 1 or 10 / 1-5 / 1. According to certain embodiments of the present application, in the step 3) column chromatography, dichloromethane / methanol is 15 / 1, 14 / 1, 13 / 1, 12 / 1, 11 / 1, 10 / 1, 9 / 1, 8 / 1, 7 / 1, 6 / 1 or 5 / 1.

[0118] According to certain embodiments of the present application, the compound of formula (I) is prepared from triiodobenzoic acid that provides a CT signal, an active oxygen responsive group, an ether chain that improves water solubility, and an AIE fluorophore tetraphenylethylene that induces rapid aggregation.

[0119] According to certain embodiments of the present application, the method for preparing the compound of formula (I) comprises:

[0120]

[0121] Composition of CT contrast agents

[0122] According to one aspect of the present application, the present application provides a CT contrast agent, which comprises the compound of formula (I) and a pharmaceutically acceptable salt thereof.

[0123] According to certain embodiments of the present application, the iodine content of the CT contrast agent is 12%-40%, for example, 12%-36%, 12%-32%, 12%-28%, 12%-24%, 12%-20%, 12%-16%, 16%-40%, 16%-36%, 16%-32%, 16%-28%, 16%-24%, 16%-20%, 20%-40%, 20%-36%, 20%-32%, 20%-28%, 20%-24%, 24%-40%, 24%-36%, 24%-32%, 24%-28%, 28%-40%, 28%-36%, 28%-32%, 32%-40%, 32%-36% or 36%-40%.

[0124] According to certain embodiments of the present application, the iodine content of the CT contrast agent is 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39% or 40%.

[0125] Preparation of CT contrast agent for detection solution

[0126] According to certain embodiments of the present application, the preparation steps of the CT contrast agent are as follows: dissolving the compound BioDHU-CT of formula (I) and its pharmaceutically acceptable salt in an organic solvent, and then adding it to a PBS phosphate buffer solution with a pH of 7-8.

[0127] According to certain embodiments of the present application, the organic solvent is selected from N,N-dimethylformamide, ethanol, dimethyl sulfoxide, acetonitrile, and tetrahydrofuran.

[0128] According to certain embodiments of the present application, the pH of the PBS phosphate buffer solution is 7-8, such as 7-7.5 or 7.5-8. According to certain embodiments of the present application, the pH of the PBS phosphate buffer solution is 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9 or 8.0.

[0129] Detection methods and uses

[0130] According to one aspect of the present application, the present application provides a method for using the compound of formula (I) BioDHU-CT and its pharmaceutically acceptable salt or the CT contrast agent for CT imaging to detect tumors, the method comprising using the compound of formula (I) BioDHU-CT and its pharmaceutically acceptable salt or the CT contrast agent to perform CT detection of in vivo tumor imaging on a patient.

[0131] According to certain embodiments of the present application, the method for detecting in vivo tumor imaging by CT comprises injecting the compound of formula (I) BioDHU-CT and its pharmaceutically acceptable salt or the CT contrast agent into a living animal model for evaluating the CT imaging effect and tumor targeting properties of the system.

[0132] According to certain embodiments of the present application, the CT includes CT imaging for small animals and CT imaging for humans.

[0133] According to certain embodiments of the present application, the in vivo tumor includes breast cancer, ovarian cancer, liver cancer, lung cancer, colon cancer, and pancreatic cancer.

[0134] According to one aspect of the present application, the present application provides the use of the compound of formula (I) BioDHU-CT and its pharmaceutically acceptable salt or the CT contrast agent in the preparation of a drug for in vivo tumor CT detection imaging.

[0135] According to certain embodiments of the present application, the use in in vivo tumor CT detection imaging includes injecting the compound of formula (I) BioDHU-CT and its pharmaceutically acceptable salt or the CT contrast agent into a living animal model for evaluating the CT imaging effect and tumor targeting properties of the system.

[0136] According to certain embodiments of the present application, the CT includes CT imaging for small animals and CT imaging for humans.

[0137] According to certain embodiments of the present application, the in vivo tumor includes breast cancer, ovarian cancer, liver cancer, lung cancer, colon cancer, and pancreatic cancer.

[0138] Example

[0139] Example 1: Precursor compound 3 and its preparation

[0140] This example discloses a precursor compound 3 and a method for preparing the same.

[0141] The structure of the precursor compound 3 is as follows:

[0142]

[0143] Precursor 1 aminotetraphenylethylene and precursor compound 2 N-Fmoc-N'-Boc-L-ornithine (1.200 g, 2.64 mmol, 1 eq), condensation reagent HOBT (0.430 g, 3.19 mmol, 1.2 eq), condensation reagent HBTU (1.200 g, 3.17 mmol, 1.2 eq) and N, N-diisopropylethylamine (0.410 g, 3.18 mmol, 1.2 eq) were dissolved in N, N-dimethylformamide (20 mL), stirred at room temperature for 2 h, and then 1-(4-aminophenyl)-1,2,2-triphenylethylene (1.100 g, 3.17 mmol, 1.2 eq) was quickly added. The resulting mixture was stirred at room temperature until TLC analysis showed that the reaction was complete. The reaction mixture was evaporated on a rotary evaporator to obtain a solid residue, which was purified by column chromatography (petroleum ether / ethyl acetate = 3 / 1) to obtain a white solid precursor compound 1. Yield: 1.0 g, 48%.

[0144] The NMR hydrogen spectrum and carbon spectrum of the precursor compound 3 are as follows Figure 1 and 2 shown.

[0145] 1 H NMR (400 MHz, DMSO-d 6 )δ9.95(s,1H),7.89(d,J=7.6Hz,2H),7.73(t,J=6.8Hz,2H),7.64(d,J= 7.6Hz,1H),7.43-7.29(m,6H),7.18-7.08(m,9H),6.99-6.94(m,6H),6.8 8(d,J=8.4Hz,2H),6.81(t,J=5.2Hz,1H),4.27-4.16(m,3H),4.07(dd,J= 13.6Hz, 8.4Hz, 1H), 2.92 (d, J = 6Hz, 2H), 1.69-1.45 (m, 4H), 1.36 (s, 9H).

[0146] 13 C NMR (100 MHz, DMSO-d 6 )δ171.0,156.1,155.6,143.8,143.2,140.7,140.1,138.1,137.3,131.1,130.7,1 27.8,127.1,126.5,125.3,120.1,118.4,77.4,65.7,55.2,46.6,29.2,28.3,26.3.

[0147] HR-MS(ESI):[M+H] + Calculated: 784.3745; Measured: 784.3751.

[0148] Example 2: Precursor compound 5 and its preparation

[0149] This example discloses a precursor compound 5 and a method for preparing the same.

[0150] The structure of the precursor compound 5 is as follows:

[0151]

[0152] Precursor compound 3 (1.000 g, 1.28 mmol, 1 eq) was dissolved in a mixture of dichloromethane (10 mL) and piperidine (2 mL) to remove Fmoc at 0 °C for 30 min, and the mixture was washed with water, the organic layer was separated and eluted with anhydrous Na 2 SO 4 After drying, spin dry. 2,3,5-triiodobenzoic acid (0.638 g, 1.28 mmol, 1 eq), HOBT (0.207 g, 1.53 mmol, 1.2 equivalents), HBTU (0.580 g, 1.53 mmol, 1.2 eq) were dissolved in N,N-dimethylformamide (20 mL), reacted for 30 min under ice bath, and then the spin-dried system was added, and the resulting mixed system was stirred at room temperature until the reaction was completed. The solvent dichloromethane was removed on a rotary evaporator to obtain a solid residue, which was purified by column chromatography (dichloromethane / methanol=200 / 1) to obtain a white solid precursor compound 5. The yield was 0.900 g, 67%.

[0153] The NMR hydrogen spectrum and carbon spectrum of the precursor compound 5 are as follows Figure 3 and 4 shown.

[0154] 1 H NMR (400 MHz, DMSO-d 6 )δ10.05(s,1H),8.76(d,J=7.6Hz,1H),8.26(dd,J=8.8Hz,2Hz,2H),7.47(s,1H),7.38(d,J=8.4Hz,2H),7.19-7.08(m,9H),7.01-6.9 5(m,6H),6.89(d,J=8.4Hz,2H),6.80(t,J=5.6Hz,1H),4.42(d,J=5.6Hz,1H),2.94(d,J=5.6Hz,2H),1.71-1.47(m,4H),1.36(s,9H).

[0155] 13C NMR (100 MHz, DMSO-d 6 )δ170.1,167.9,155.6,147.0,143.2,140.1,138.2,137.2,134.7,131.1,130. 7,127.8,126.5,118.6,113.4,107.6,95.1,77.5,53.7,29.2,28.3,26.3,22.5.

[0156] HR-MS(ESI):[M+H] + Calculated: 1044.0226; Measured: 1044.0231.

[0157] Example 3: Precursor compound 7 of compound A and its preparation

[0158] This embodiment discloses compound A precursor compound 7 and its preparation method.

[0159] The structure of compound A precursor compound 7 is as follows:

[0160]

[0161] Precursor compound 5 (2.000 g, 1.92 mmol, 1 eq) was dissolved in a mixture of dichloromethane (10 mL) and trifluoroacetic acid (2 mL), reacted at room temperature for 10 minutes to remove the Boc protecting group, and then the solvent dichloromethane was spun off on a rotary evaporator to obtain a white solid mixture. Precursor compound 6 of compound A (1.427 g, 1.92 mmol, 1 eq), triethylamine (0.582 g, 5.76 mmol, 3 eq) and 4-dimethylaminopyridine (0.234 g, 1.92 mmmol, 1 eq) were then dissolved in dichloromethane (20 mL), and the above mixture was added, and the resulting mixture was stirred at room temperature until the reaction was complete, and then the mixture was washed with water, the organic layer was separated and the reaction mixture was washed with anhydrous Na 2 SO 4 Next, p-tetrabutylammonium fluoride (0.330 g, 1.92 mmol, 1 eq) was added to the obtained mixture, stirred at room temperature for 1 hour, and evaporated on a rotary evaporator to obtain a solid residue, which was purified by column chromatography (dichloromethane / ethyl acetate=2 / 1) to obtain a precursor 7 of compound A as a light blue solid. Yield 0.950 g, 35%.

[0162] The NMR hydrogen spectrum and carbon spectrum of the precursor compound 7 of compound A are as follows Figure 5 and 6 shown.

[0163] 1 H NMR (400 MHz, DMSO-d6 )δ10.06(s,1H),8.77(s,1H),8.27(s,1H),7.84(s,1H),7.47-7.31(m,7H),7.24-7.11(m,13H),6.97-6.89(m,6H),6. 77(s,2H),6.67(d,J=6Hz,2H),5.13(s,1H),4.97(s,2H),4.42(s,3H),3.02(s,2H),2.89(s,12H),1.74-1.51(m,4H).

[0164] 13 C NMR (100 MHz, DMSO-d 6 )δ170.0,167.9,156.1,153.8,148.7,147.0,146.0,143.2,140.2,138.2,137.2,134.7,133.9,133.6,131.2,1 30.7,128.1,127.8,127.5,126.5,118.6,113.4,111.2,110.4,107.6,95.1,62.1,60.3,53.7,40.2,29.1,26.2.

[0165] HR-MS(ESI):[M+H] + Calculated: 1406.1427; Measured: 1046.1432.

[0166] Example 4: BioDHU-CT and its preparation method

[0167] This example discloses a compound BioDHU-CT compound A and a preparation method thereof.

[0168] The structure of BioDHU-CT compound A disclosed in this example is as follows:

[0169]

[0170] The preparation method of the compound BioDHU-CT compound A having the structure of formula (I) is as follows:

[0171]

[0172] The precursor compound 6 of compound A was synthesized according to the literature method (Chem. Sci., 2022, 13, 10815-10823), which is incorporated into this application by reference in its entirety.

[0173] Precursor compound 1, precursor compound 2, precursor compound 4, and precursor compound 8 were purchased commercially. Precursor compound 1 was purchased from Bidex Pharmaceuticals, product number BD01080402; Precursor compound 2 was purchased from Bidex Pharmaceuticals, product number BD1772; Precursor compound 4 was purchased from Bidex Pharmaceuticals, product number BD4099; Precursor compound 8 was purchased from Hangzhou Hanlai Biochemical Technology Co., Ltd., product number P171004.

[0174] Precursor compound 8 (0.100 g, 0.07 mmol, 1 eq), precursor compound 7 obtained in Example 3 (0.125 g, 0.09 mmol, 1.2 eq), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.025 g, 0.13 mmol and 1.5 eq) and 4-dimethylaminopyridine (0.009 g, 0.07 mmol, 1.0 eq) were dissolved in dichloromethane (5 mL). The reaction mixture was stirred at room temperature for 6 hours. After the reaction was complete, the reaction mixture was washed with 1 M HCl solution (10 mL), saturated NaHCO 3 (10 ml) and saturated NaCl (10 mm L). The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated to obtain a crude product, which was purified by column chromatography (dichloromethane / methanol=10 / 1) to obtain BioDHU-CT as a light green solid. Yield: 0.400 g, 20%.

[0175] The H NMR spectrum and C NMR spectrum of the compound BioDHU-CT compound A of formula (I) are as follows: Figure 7 and 8 shown.

[0176] 1 H NMR (400 MHz, DMSO-d 6)δ10.08(s,1H),8.79(d,J=7.2Hz,1H),8.27(d,J=1.6Hz,1H),7.85(t,J=5.2Hz,1H ),7.71(s,1H),7.46(d,J=1.6Hz,1H),7.37(t,J=7.2Hz,4H),7.33-7.30(m,3H),7. 24(t,J=7.6Hz,1H),7.18-7.08(m,9H),6.99-6.94(m,6H),6.89(d,J=8.4Hz,2H),6 .76(d,J=2.8Hz,2H),6.67(dd,J=8.8Hz,2.4Hz,2H),6.4(d,J=27.2Hz,2H),5.01(d ,J=22Hz,4H),4.45-4.40(m,1H),4.31-4.28(m,1H),4.13-4.10(m,1H),3.61(t,J= 6Hz,2H),3.50(s,96H),3.38(t,J=6Hz,2H),3.20-3.15(m,2H),3.11-3.06(m,1H), 3.01(t,J=6Hz,2H),2.89(s,12H),2.81(dd,J=12.4Hz,4.8Hz,1H),2.59-2.53(m,2 H),2.06(t,J=7.6Hz,2H),1.65-1.56(m,2H),1.53-141(m,4H),1.33-1.23(m,2H).

[0177] 13 C NMR (100 MHz, DMSO-d 6 )δ172.1,170.9,167.9,162.7,153.9,148.8,143.3,140.1,137.3,133.7,128.0,127.8,127.5,126.6,118. 6,111.2,110.4,107.6,95.2,69.8,69.2,66.0,62.6,61.0,59.2,55.5,40.2,38.5,35.1,34.5,28.1,25.3.

[0178] HR-MS(ESI):[M+2Na] 2+ Calculated: 1416.9280; Measured: 1416.9280.

[0179] Example 5: Formation of BioDHU-CT Compound A Single Component Nanoparticles (NPs)

[0180] Examples 5-8 used compound A synthesized in Examples 1-4.

[0181] This example discloses the formation of BioDHU-CT Compound A single component nanoparticles (NPs).

[0182] Most of the existing technologies are multi-component nano CT contrast agents, which have the disadvantages of complex preparation procedures and difficulty in reproducing, making it difficult to achieve clinical transformation. However, single-component nano CT contrast agents have a clear structure and can be prepared repeatedly, which solves this problem in the existing technology.

[0183] 4.3 mg of BioDHU-CT Compound A was weighed and dissolved in 0.5 mL of DMSO and fully dissolved under ultrasound. The mixture was then added dropwise to 4.5 mL of deionized water and stirred slightly at room temperature for 10 minutes. Subsequently, the solution was dialyzed at room temperature for 48 hours (molecular weight cutoff = 1000 Da) to obtain single-component BioDHU-CT Compound A nanoparticles. Transmission electron microscopy and dynamic light scattering were used to characterize the morphology and size of the nanoparticles.

[0184] like Fig. 9 As shown in (a), the transmission electron microscopy (TEM) image shows that the NPs are monodispersed with a uniform spherical structure and a diameter of approximately 30 nm.

[0185] like Fig. 9 As shown in b, the average hydrodynamic diameter of the NPs determined by dynamic light scattering (DLS) was 35 nm.

[0186] The results of this example show that BioDHU-CT compound A can form excipient-free nanoformulations through simple self-assembly.

[0187] Example 6: Selectivity of BioDHU-CT Compound A

[0188] This example discloses the selectivity of BioDHU-CT Compound A.

[0189] Fig.10 The response behavior of the BioDHU-CT compound A described in the present invention to ROS. From A to J, pure BioDHU-CT, H 2 O 2 , O 2 -, TBHP, ROO·, NO, t-BuOO·, ·OH, ONOO-, HOCl. ·OH, t-BuOO·, ONOO- and HOCl are all active oxygen species, which can react with BioDHU-CT compound A to dissociate the nanoparticles and release the fluorescent methylene blue fluorophore. The concentration of BioDHU-CT compound A is 10 μM, and the concentration of active oxygen is 40 μM. The fluorescence intensity at 686 nm is collected, and the excitation wavelength is 620 nm.

[0190] like Fig.10 As shown, BioDHU-CT was prepared at a concentration of 10 μM in 2 mL of pH 7.4 PBS phosphate buffer solution, and 40 μM H 2 O 2 ,TBHP,NO,O 2 - ,ROO·,H 2 O 2 ,·OH,t-BuOO·,ONOO - and HOCl, the excitation wavelength was selected as 620nm, and the fluorescence was measured, and TBHP, NO, O 2 - , ROO· (all concentrations were 40 μM) could not cause changes in the fluorescence intensity of BioDHU-CT (concentration was 10 μM) at 686 nm.

[0191] While 40 μM ·OH, t-BuOO·, ONOO - and HOCl resulted in a significant change in the fluorescence intensity of BioDHU-CT compound A (concentration of 10 μM) at 686 nm, indicating that BioDHU-CT compound A could be catalyzed by ·OH, t-BuOO·, ONOO - In addition, it was found that BioDHU-CT compound A was more active against HOCl than the other three reactive oxygen species.

[0192] Fig.11 The fluorescence spectrum of the reaction of the BioDHU-CT compound A of the present invention with ·OH, t-BuOO·, ONOO- and HOCl. The concentration of the BioDHU-CT compound A is 10 μM, the concentration of active oxygen is 40 μM, and the excitation wavelength is 620 nm.

[0193] like Fig.11 As shown, BioDHU-CT was prepared at a concentration of 10 μM in 2 mL of pH 7.4 PBS phosphate buffer solution, and 40 μM of ·OH, t-BuOO·, and ONOO - and HOCl, 10 μM BioDHU-CT was used as the control, the excitation wavelength was selected as 620 nm, and its fluorescence was measured. It was found that BioDHU-CT (concentration of 10 μM) had no obvious fluorescence, while when 40 μM ·OH, t-BuOO·, ONOO - After addition of HOCl, there was an obvious fluorescence enhancement.

[0194] The results of this example show that BioDHU-CT compound A can be activated to release methylene blue fluorophore in the presence of ROS (·OH, t-BuOO·, ONOO- and HOCl). In addition, it also shows that BioDHU-CT compound A can be activated by a variety of reactive oxygen species.

[0195] The results of this example show that, since tumors release a variety of ROS, the selectivity of the compound BioDHU-CT of formula (I) increases the probability of the compound BioDHU-CT of formula (I) being activated in the tumor area, thereby prompting more BioDHU-CT to release more tetraphenylethylene-modified triiodobenzoic acid in the tumor, which can aggregate rapidly. Therefore, BioDHU-CT can be activated by a variety of reactive oxygen species, which is more conducive to the retention of more CT contrast agents in the tumor area.

[0196] Example 7: BioDHU-CT Compound A increases in size after in vitro activation

[0197] This example discloses that BioDHU-CT Compound A increases in size upon in vitro activation.

[0198] Fig.12 The following are verification diagrams of the size increase of the BioDHU-CT compound A described in the present invention after the reaction with hypochlorous acid. Figure a is a transmission electron microscope image of the BioDHU-CT compound A after the reaction with hypochlorous acid. Figure b is a maldi-tof image of the BioDHU-CT compound A after the reaction with hypochlorous acid. Figure c is the fluorescence of tetraphenylethylene in a system with different water contents (ethanol: water) after the BioDHU-CT compound A responds to hypochlorous acid, with the water content ranging from 10% to 80%, the concentration of the BioDHU-CT compound A being 10 μM, the concentration of hypochlorous acid being 40 μM, and the excitation wavelength being 360 nm. Figure d shows the fluorescence and CT signals of tetraphenylethylene in the filtrate tested after the BioDHU-CT compound A responded to different concentrations of hypochlorous acid and was filtered with a filter membrane with a pore size of 220 nm. The concentration of the BioDHU-CT was 50 μM, and the concentrations of hypochlorous acid were 0 μM, 5 μM, 10 μM, 15 μM, 20 μM, 30 μM, and 50 μM, respectively, and the excitation wavelength was 360 nm.

[0199] like Fig.12 As shown in a, b, BioDHU-CT compound A (concentration of 10 μM) was reacted with hypochlorous acid (concentration of 40 μM) in pH 7.4 PBS phosphate buffer solution. TEM images showed that BioDHU-CT compound A was converted into larger aggregates of micron size after reacting with HOCl. This transformation was due to the release of the AIE fluorophore tetraphenylethylene, which rapidly formed larger nanoaggregates.

[0200] like Fig.12 As shown in Figure c, in order to further verify the above conclusion, BioDHU-CT compound A was added to a mixed solvent of ethanol and water after fully reacting with HOCl. As the proportion of water increased, the fluorescence of tetraphenylethylene gradually increased.

[0201] In addition, the solution was treated with different concentrations of HOCl (ranging from 0-50 μM) and then filtered using a membrane with a pore size of 220 nm to measure the fluorescence of tetraphenylethylene and CT signals in the filtrate.

[0202] like Fig.12 As shown in Figure d, with the increase of HOCl concentration, the fluorescence intensity of tetraphenylethylene and CT signals in the filtrate gradually decreased, indicating that after being activated by HOCl, the tetraphenylethylene produced was partially intercepted by the filter membrane due to the formation of large-sized aggregates, resulting in a gradual decrease in fluorescence and CT signals.

[0203] The results of this example show that BioDHU-CT compound A can quickly release the triiodobenzoic acid part modified with AIE fluorophore tetraphenylethylene after activation. Driven by the AIE performance, large-sized nanoaggregates are quickly formed in aqueous solution. The large-sized nanoaggregates formed are difficult to be eliminated by the tumor. Compared with small molecule contrast agents, they can stay in the tumor area for a longer time, thereby extending the window period of CT imaging.

[0204] Example 8: BioDHU-CT Compound A extends the in vivo imaging window

[0205] This example discloses that BioDHU-CT compound A can extend the in vivo imaging window.

[0206] Fig.13 The CT images of the intratumoral injection of the BioDHU-CT compound A of the present invention in vivo. Subcutaneous tumors of Hela cells were planted in the left and right hind legs of mice, and iohexol, a commonly used CT contrast agent in clinical practice, was selected as a control. The BioDHU-CT compound A was injected into the left tumor of the mouse, and iohexol was injected into the right tumor of the mouse, and then CT imaging was performed at a certain interval. The concentration of the BioDHU-CT compound A was 8 mg / mL, and the injection volume was 20 μL.

[0207] like Fig.13As shown, 0.1 mL (5 million cells) of Hela cell solution was injected into the left and right sides of the back of three-week-old BALB / c mice to construct a subcutaneous Hela tumor model. After the tumor grew to 0.8 cm (about 14 days), BioDHU-CT compound A NPs (arrow, 8 mg I / mL, 20 μL) and the control iohexol (circle, 8 mg I / mL, 20 μL) were injected into the tumor, and the CT signal of the tumor was monitored at different times. Among them, iohexol is one of the commonly used CT contrast agents in clinical practice.

[0208] After injection of BioDHU-CT compound A NP, the CT signal in the tumor area was significantly enhanced and remained unchanged within 1 hour. After 1.5 hours of injection, the CT signal began to slowly decrease, and after 12 hours, the CT signal in the tumor area was still higher than before injection. After 24 hours of injection, there was still a strong CT signal.

[0209] In contrast, for tumors injected with iohexol, the CT signal of the tumor area was only slightly enhanced 1 hour after injection compared with that before injection because iohexol is easily metabolized.

[0210] The results of this example show that after activation, BioDHU-CT compound A NP can quickly form larger nano-aggregates in tumor tissue, improve the retention efficiency of CT contrast agents, and thus extend the imaging window of contrast agents. The larger-sized nano-aggregates formed are difficult to be eliminated by the tumor, and compared with small molecule contrast agents, they can stay in the tumor area for a longer time, thereby extending the CT imaging window.

[0211] The present invention constructs a single-component nano CT contrast agent BioDHU-CT using the AIE fluorophore tetraphenylethylene. After activation, the BioDHU-CT nanoparticles exhibit a unique size aggregation effect. Both in vitro and in vivo studies have shown that the BioDHU-CT nanoparticles exhibit excellent responsiveness and rapidly form larger aggregates within the tumor area after activation. This aggregation phenomenon improves the retention efficiency of the CT contrast agent and extends the imaging time window. In addition, activation leads to a significant increase in the iodine content in the nanoparticles, thereby improving the overall performance of CT imaging in the tumor area. Specifically, the iodine content increases from 14% to 40% after activation, further improving the imaging quality of the tumor.

[0212] The embodiments disclosed in the present invention are only for detailed description of the present invention, but are not sufficient to limit the present invention. A person skilled in the art may replace the preparations described herein with obvious similar substitutes or modifications, or replace the preparations with certain chemically or biologically structurally related preparations, or make changes to the relevant contents of the present invention, but they shall not exceed the spirit, scope and idea of ​​the present invention, and shall fall within the scope of protection claimed by the present invention.

Claims

1. A compound and a pharmaceutically acceptable salt thereof, wherein the compound has a structure shown in formula (I): in: (1) The value of n can be between 12 and 24; (2) R can be a stimuli-responsive group.

2. The compound according to claim 1, characterized in that The stimulus-responsive group is selected from a hydrogen peroxide responsive group, a hypochlorous acid responsive group, an ·OH responsive group, a t-BuOO· responsive group, a peroxynitrite responsive group, a cysteine ​​responsive group, a glutathione responsive group, a hydrogen sulfide responsive group, a nitroreductase responsive group, and a phosphatase responsive group; wherein the pharmaceutically acceptable salt includes a sodium salt, a potassium salt, an ammonium salt, and a calcium salt.

3. The compound according to any one of claims 1 to 2 and a pharmaceutically acceptable salt thereof, characterized in that: The compound of formula (I) is selected from:

4. A method for preparing the compound according to any one of claims 1 to 3, characterized in that: The method comprises: 1) Dissolve the precursor compound 1 aminotetraphenylethylene and the precursor compound 2 N-Fmoc-N'-Boc-L-ornithine, the condensation reagent HOBT, the condensation reagent HBTU and N,N-diisopropylethylamine in N,N-dimethylformamide (10-30 mL), stir at room temperature for 1-3 hours, and then quickly add 1-(4-aminophenyl)-1,2,2-triphenylethylene. Stir the resulting mixture at room temperature until TLC analysis shows that the reaction is complete; evaporate the reaction mixture on a rotary evaporator to obtain a solid residue, which is purified by column chromatography (petroleum ether / ethyl acetate = 5 / 1-1 / 1) to obtain a white solid precursor compound 1; 2) Precursor compound 3 was dissolved in a mixture of dichloromethane (5-15 mL) and piperidine (1-3 mL) to remove Fmoc at 0°C for 20-60 minutes, and the mixture was washed with water, the organic layer was separated and dried with anhydrous Na2SO4 and then spin-dried; 2,3,5-triiodobenzoic acid, HOBT, and HBTU were dissolved in N,N-dimethylformamide (10-30 mL), reacted under ice bath for 10-30 minutes, and then the spin-dried system was added, and the resulting mixed system was stirred at room temperature until the reaction was completed. The solvent dichloromethane was spun off on a rotary evaporator to obtain a solid residue, which was purified by column chromatography (dichloromethane / methanol=300 / 1-100 / 1) to obtain a white solid precursor compound 5; 3) The precursor compound 5 was dissolved in a mixture of dichloromethane (5-15 mL) and trifluoroacetic acid (1-5 mL), reacted at room temperature for 5-15 minutes to remove the Boc protecting group, and then the solvent dichloromethane was removed on a rotary evaporator to obtain a white solid mixture. Then, the precursor compound 6 of compound A, triethylamine and 4-dimethylaminopyridine are dissolved in dichloromethane (10-30 mL), and the above mixture is added, and the resulting mixture is stirred at room temperature until the reaction is completed, and then the mixture is washed with water, and the organic layer is separated and dried over anhydrous Na2SO4; Next, p-tetrabutylammonium fluoride is added to the obtained mixture, and stirred at room temperature for 0.5-2 hours, and evaporated on a rotary evaporator to obtain a solid residue, which is purified by column chromatography (dichloromethane / ethyl acetate=5 / 1-1 / 1) to obtain a light blue solid precursor compound 7 of compound A; 4) Precursor compound 8, precursor compound 7, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine were dissolved in dichloromethane (1-10 mL). The reaction mixture was stirred at room temperature for 2-10 hours. After the reaction was complete, the reaction mixture was washed with 1M HCl solution (5-15 mL), saturated NaHCO3 (5-15 mL) and saturated NaCl (5-15 mmL), respectively; the organic phase was dried over anhydrous sodium sulfate, filtered and concentrated to obtain a crude product, which was purified by column chromatography (dichloromethane / methanol=15 / 1-5 / 1) to obtain a light green solid compound of formula (I).

5. The method according to claim 4, characterized in that The method comprises:

6. A CT contrast agent, characterized in that: The CT contrast agent comprises the compound according to any one of claims 1 to 3 and a pharmaceutically acceptable salt thereof.

7. The CT contrast agent according to claim 6, wherein the iodine content of the CT contrast agent is 12%-40%.

8. A method for preparing the CT contrast agent according to claims 6-7, comprising dissolving the compound of formula (I) BioDHU-CT and a pharmaceutically acceptable salt thereof in an organic solvent, and then adding the solution to a PBS phosphate buffer solution at pH 7-8.

9. The preparation method according to claim 8, wherein the organic solvent is selected from N,N-dimethylformamide, ethanol, dimethyl sulfoxide, acetonitrile, and tetrahydrofuran.

10. Use of the compound according to any one of claims 1 to 3 and a pharmaceutically acceptable salt thereof or the CT contrast agent according to claims 6 to 7 in the preparation of a medicament for in vivo tumor CT detection and imaging.

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