Radioactive halogen labeled compound based on rotaxane protection as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of pharmaceutical chemistry, radiopharmaceutical chemistry and clinical nuclear medicine, and particularly relates to a rotaxane protection-based radioactive halogen labeled compound as well as a preparation method and application thereof. A rotaxane protection-based radioactive halogen labeled compound comprises: a macrocyclic compound having a cavity, the macrocyclic compound comprising one of cyclodextrin, cucurbituril, calixarene and pillararene; a radioactive halogen-labeled compound: the radioactive halogen-labeled position is located in the cavity of the macrocyclic compound; and the end-capping group is positioned at the opening end of the cavity, is connected to two ends of the radioactive halogen-labeled compound and is used for preventing the radioactive halogen-labeled compound from sliding out of the cavity. The rotaxane protection-based radioactive halogen labeled compound provided by the invention has good in-vivo stability, and solves the problem of poor in-vivo stability caused by dissociation of radioactive halogen and a tracer agent in the existing radioactive halogen labeled compound.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of pharmaceutical chemistry, radiopharmaceutical chemistry and clinical nuclear medicine, and particularly relates to a radiohalogen labeled compound based on rotaxane protection, and a preparation method and application thereof. BACKGROUND

[0002] Radiohalogen has become the most commonly used radionuclide in clinical and research due to its relatively suitable half-life, excellent nuclear properties and easy chemical insertion. However, in practical application, the dissociation of radiohalogen and tracer leads to unnecessary accumulation of radiohalogen in thyroid, stomach and salivary glands, which reduces the signal-to-noise ratio of the image. In the treatment process, the non-target accumulation of radiohalogen reduces the radiation dose of the target tissue and increases the dose of the non-target tissue, causing unnecessary damage to the non-target tissue.

[0003] Therefore, how to prevent or minimize the dehalogenation of the radiohalogen labeled compound without affecting the clearance and metabolism of the radiohalogen labeled drug in the blood is crucial. SUMMARY

[0004] The technical purpose of the present application is to at least solve the problem of poor in-vivo stability of the radiohalogen labeled compound due to the dissociation of radiohalogen and tracer in the prior art. The present application reduces the decrease of the radiation dose of the target tissue, the increase of the dose of the non-target tissue, and the unnecessary damage to the non-target tissue. The radiohalogen labeled compound provided by the present application has good in-vivo stability.

[0005] The first aspect of the present application is to provide a radiohalogen labeled compound based on rotaxane protection, comprising: a macrocyclic compound having a cavity, wherein the macrocyclic compound comprises one of a cyclodextrin, a cucurbituril, a calixarene and a pillararene; a radiohalogen labeled compound, wherein the position of the radiohalogen labeled compound is located in the cavity of the macrocyclic compound; and end-capping groups, wherein the end-capping groups are located at the opening end of the cavity and are connected to both ends of the radiohalogen labeled compound to prevent the radiohalogen labeled compound from sliding out of the cavity. The radiohalogen labeled compound prevents or minimizes dehalogenation without affecting the clearance and metabolism of the radiohalogen labeled drug in the blood. The problem of poor in-vivo stability of the radiohalogen labeled compound due to the dissociation of radiohalogen and tracer is solved. The problems of the decrease of the radiation dose of the target tissue, the increase of the dose of the non-target tissue and the unnecessary damage to the non-target tissue are reduced.

[0006] In one embodiment, the radiohalogen labeled compound comprises an aromatic ring or an aromatic heterocycle.

[0007] In one embodiment, the aromatic ring comprises one of a benzene ring, a biphenyl and a naphthalene ring; and the aromatic heterocycle comprises one of a triazole, a quinoline and a pyridine.

[0008] In one embodiment, the position of the radiohalogen label is at any vacant position on the aromatic ring or the aromatic heterocyclic ring of the aromatic ring.

[0009] In one embodiment, the radiohalogen comprises 123 I, 124 I, 125 I, 131 I and 211 At one of.

[0010] In one embodiment, the capping group comprises one of a trityl group, an adamantyl group, a tert-butyl group, a 3,5-dimethylphenyl group, a diisopropylphenyl group, and an anthryl group.

[0011] In one embodiment, further comprising a linker connected between the radiohalogen labeled compound and the capping group; the linker comprises one of an alkyl chain, an amino acid chain, a PEG (polyethylene glycol) chain, a triazole, an amide bond, and a maleimide.

[0012] The second aspect of the present application is to provide a preparation method of a radiohalogen labeled compound based on wheel-alkane protection, mixing a radiohalogen labeled compound, a macrocyclic compound, and a capping group, stirring at 60-80°C, cooling to room temperature after the reaction is completed, filtering, separating and purifying to obtain a radiohalogen labeled compound based on wheel-alkane protection. By using the host-guest interaction of supramolecules to "imprison" the radiohalogen labeled small molecule in the cavity of the macrocyclic compound, the small molecule is protected from external interference. The use of large steric hindrance groups at both ends forms a capping, effectively preventing the radiohalogen labeled part from sliding out of the cavity. The preparation method provided by the present application is a new method for inhibiting dehalogenation which is safe and effective and has promotional significance.

[0013] In one embodiment, the mass ratio of the radiohalogen labeled compound, the macrocyclic compound, and the capping group is 1:1.1-1.3:2.2-2.6.

[0014] The third aspect of the present application is to provide an application of a radiohalogen labeled compound, an application in a tumor radio-pharmaceutical.

[0015] The beneficial effects of the present application include at least one of the following: The radiohalogen labeled compound based on the wheel-alkane protection provided in the application has good in-vivo stability, and prevents hydrolysis, enzymatic hydrolysis and protein adsorption. The radiohalogen labeled compound is prevented from dehalogenation or minimized dehalogenation, and does not affect the clearance and metabolism of the radiohalogen labeled drug in blood. The problem of poor in-vivo stability due to the dissociation of radiohalogen from the tracer in the radiohalogen labeled compound is solved. The problems of reduced radiation dose of target tissue and increased dose of non-target tissue are reduced, and unnecessary damage to non-target tissue is reduced.

[0016] The preparation method of the radiohalogen labeled compound based on the wheel-alkane protection provided in the application is to "cage" the radiohalogen labeled small molecule in the cavity of the macrocycle compound by the host-guest interaction of the supramolecule, so as to protect the small molecule from external interference. The end-capping is formed by using large steric hindrance groups at both ends, which effectively prevents the radiohalogen labeled part from sliding out of the cavity. The preparation method provided in the application is a new method for inhibiting dehalogenation which is safe and effective and has promotion significance. BRIEF DESCRIPTION OF DRAWINGS

[0017] Various other advantages and benefits will become apparent to those of ordinary skill in the art upon reading the following detailed description of the preferred embodiments. The accompanying drawings are included only to illustrate preferred embodiments and are not to be considered as limiting of the present application. In the drawings: Figure 1 Exemplary structure schematic diagram of the radiohalogen labeled compound based on the wheel-alkane protection provided in the application is shown; Figure 2 In (a), (b), (c), respectively, are the 1 H NMR chart, 13 C NMR chart and ESI-HRMS chart of the radio-labeled precursor compound 1 used in an embodiment of the application; Figure 3 In (a), (b), (c), respectively, are the 131 I]I-tria and wheel-alkane[ 131 I]I-tBuR radio-high performance liquid chromatography (radio-HPLC) detection chart; Figure 4 In (a), (b), (c), respectively, are the 1 H NMR chart, 13 C NMR chart and ESI-HRMS chart of the I-tBuR cold product provided in an embodiment of the application; Figure 5 In (a), (b), respectively, are the 131 I]I-tBuR radio-HPLC chart after incubation in PBS and FBS for different time; Figure 6 for[ 131 Radio-HPLC chromatograms of I-tBuR after incubation in GSH for different times; Figure 7 In the middle (a), (b), and (c), respectively, the cold products of I-tria are... 1 H NMR spectrum, 13 C NMR and ESI-HRMS plots; Figure 8 for[ 131 I]I-tBuR radioactivity accumulation in various tissues and organs of normal mice; Figure 9 for[ 131 I]I-tria radioactivity accumulation in various tissues and organs of normal mice; Figure 10 for[ 131 I]I-tBuR and [ 131 Radioactive accumulation in organs of normal mice with I-tria, *(p<0.05), **(p<0.01), ***(p<0.001); Figure 11 This is a molecular structure diagram of the existing technology for inhibiting deiodination. Detailed Implementation

[0018] To make the objectives, technical solutions, and advantages of this application clearer, the application will be further described below with reference to the accompanying drawings and specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the application. All other embodiments obtained by those skilled in the art based on the embodiments in this application without inventive effort are within the scope of protection of this application.

[0019] Unless otherwise specified in the examples, all procedures were performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0020] Radioactive iodine has a long history in nuclear medicine. Due to its relatively suitable half-life, excellent nuclear properties, and ease of chemical insertion, it has become one of the most commonly used radionuclides in clinical practice and research. Currently, the most commonly used radioactive iodine isotopes in clinical practice and research include... 123 I, 124 I, 125 I, and 131 I. Due to the different properties of their nuclides and preparation methods, they also have different focuses in scientific research and clinical applications. 123 I can be used for SPECT imaging. 125I can be used for brachytherapy, 131 I can be used for SPECT imaging and radiotherapy, 124 I is used for PET imaging. The dehalogenation mechanism in vivo is complex, and nucleophiles (such as glutathione), cytochrome P450 enzymes and deiodinases can all cause dehalogenation. Since the C-I bond is less stable in vivo, the deiodination of radioiodine-labeled drugs in vivo is more serious, which limits their application in radionuclide imaging and therapy.

[0021] During the treatment, the dissociation of radiohalogen from the tracer leads to unnecessary accumulation of radiohalogen in the thyroid, stomach and salivary glands. Non-targeted accumulation of radiohalogen reduces the radiation dose to the target tissue and reduces the signal-to-noise ratio of the image. The increase in radiation dose to non-target tissues causes damage to non-target tissues. Many promising radiohalogen-labeled drugs are affected by in vivo dehalogenation, which affects the therapeutic effect. Therefore, how to prevent or minimize dehalogenation in the design of radiopharmaceuticals is crucial.

[0022] In the prior art, methods for inhibiting in vivo dehalogenation of radiohalogen-labeled drugs still have high thyroid radioaccumulation, or have the disadvantages of slow blood clearance or excessive metabolism. Therefore, the present application provides an effective method for inhibiting in vivo dehalogenation of radiohalogen-labeled drugs.

[0023] In a first aspect, the present application provides a radiohalogen-labeled compound based on wheel protection, which "traps" the radiohalogen-labeled moiety in the cavity of the macrocycle compound. The macrocycle compound provides physical protection for the radiohalogen-labeled moiety, so that it is protected from external interference, thereby improving the in vivo stability of the radiohalogen-labeled drug.

[0024] The radiohalogen-labeled compound based on wheel protection provided by the present application labels the radiohalogen on the aromatic ring or aromatic heterocycle in the cavity of the macrocycle compound, one structure of which is shown as Figure 1 wherein the meanings of the symbols are as follows: Y is an aromatic ring, including benzene rings, biphenyl, naphthalene rings and other benzene series aromatic rings, as well as triazoles, quinolines, pyridines and other aromatic heterocycles.

[0025] X includes radiohalogen, and X is located at any vacant site of Y.

[0026] Linker includes alkyl chain, amino acid chain, PEG (polyethylene glycol) chain, triazole, amide bond, maleimide and other commonly used linkers in medicinal chemistry.

[0027] R includes a bulky group, a bulky group formed by a cucurbituril and other groups together, or a bulky group connected to a targeting molecule. Specifically, the bulky group includes a trityl group, an adamantyl group, a tert-butyl group, a 3,5-dimethylphenyl group, a diisopropylphenyl group, an anthracenyl group, and other common bulky groups.

[0028] The targeting molecule includes a monoclonal antibody with targeting properties, an RGD peptide, a prostate-specific membrane antigen (PSMA) inhibitor, an epidermal growth factor receptor (EGFR) inhibitor, a fibroblast activation protein inhibitor (FAPI), and other commonly used targeting groups for radiopharmaceuticals.

[0029] Z includes a macrocyclic compound such as a cyclodextrin, a cucurbituril, a calixarene, and a pillararene.

[0030] The cucurbituril can be cucurbituril[6], cucurbituril[7], or cucurbituril[8].

[0031] The cyclodextrin can be α-cyclodextrin, β-cyclodextrin, or γ-cyclodextrin.

[0032] Figure 1 The [4] rotaxane structure can also be a [2] rotaxane structure, a [3] rotaxane structure, or a [5] rotaxane structure.

[0033] In one example, the aromatic ring labeled with a radioactive halogen is located in the cavity of the macrocyclic compound cyclodextrin to form a [4] rotaxane with the cyclodextrin and the cucurbituril, and X is a radioactive halogen. The radioactive halogen can be labeled at other positions of the aromatic ring, including but not limited to the following structures:

[0034] CB[6] and β-CD are cucurbituril[6] and β-cyclodextrin, respectively, 4HCl is a rotaxane combined with 4HCl to form a hydrochloride salt.

[0035] The present application provides a radiohalogen labeled compound based on the protection of a wheel-shaped cavity. The hydrophobic cavity of the macrocycle compound can effectively bind to a hydrophobic small molecule to form a host-guest complex, which can enhance the stability of the small molecule in vivo. In one example, the macrocycle compound is preferably a cyclodextrin, which can enhance the stability of the small molecule in vivo due to the physical protection of the cyclodextrin shell, effectively reducing the effects of hydrolysis, enzymatic hydrolysis and protein adsorption. The formation of such host-guest complex is reversible. Based on the wheel-shaped structure, the present application can "imprison" the guest small molecule in the cyclodextrin cavity irreversibly, protecting the small molecule from external interference. The macrocycle itself acts as a "molecular shield" to physically block the active site of deiodinase or the approach of nucleophilic molecules (such as glutathione GSH) to the easily broken C-I bond. Further functionalization of the "molecular shield" outer wall and the "molecular plug" of the wheel-shaped structure is used to covalently connect additional functional units (such as targeting groups, fluorescent reporter groups, cell-penetrating peptides, etc.), achieving true multifunctional integration.

[0036] In some examples of the present application, the structure of the radiohalogen labeled precursor compound can include the following structure:

[0037]

[0038] In some examples, the radiohalogen can be 123 I, 124 I, 125 I, 131 I and 211 At.

[0039] It should be noted that the meanings of the above symbols are only exemplary and are not limited to the radiohalogen labeled compound based on the protection of the wheel-shaped cavity in the present application. Those skilled in the art can select the specific structure of the above symbols according to the actual needs, "imprison" the radiohalogen labeled part in the cavity of the macrocyclic compound to inhibit its dehalogenation in vivo, which can achieve the principle of the present application.

[0040] In a second aspect, the present application provides a preparation method of a radiohalogen labeled compound based on the protection of a wheel-shaped cavity. The macrocycle compound and the radiohalogen labeled small molecule are connected through the host-guest interaction of a supramolecule, and the radiohalogen labeled small molecule enters the cavity of the macrocycle compound to form a host-guest complex. The ends of the host-guest complex are connected with large steric hindrance groups to complete the end-capping and form a wheel-shaped cavity, which can effectively prevent the radiohalogen labeled part from sliding out of the cavity and inhibit its dehalogenation in vivo. The preparation method includes the following steps: Step S01: selecting a radiohalogen labeled precursor compound, or preparing a radiohalogen labeled precursor compound.

[0041] Step S02: radiolabeling the radiolabeled precursor compound using a radioactive halogen, or selecting a labeled radiolabeled precursor compound (labeled radiolabeled precursor compound, also described as a radiolabeled compound).

[0042] Step S03: selecting a macrocycle compound and a capping group, and performing rotaxane assembly on the radiolabeled compound, the macrocycle compound, and the capping group to obtain a rotaxane-protected radiolabeled compound.

[0043] Step S03 of the present application includes: mixing the radiolabeled compound, the macrocycle compound, and the capping group, stirring at 60-80°C for 30-60 min, cooling to room temperature after the reaction is completed, filtering, and separating and purifying to obtain the rotaxane-protected radiolabeled compound. The mass ratio of the radiolabeled compound, the macrocycle compound, and the capping group is 1:1.1-1.3:2.2-2.6.

[0044] The macrocycle compound includes a series of macrocycle compounds such as cyclodextrin, cucurbituril, calixarene, and pillararene; the capping group includes a bulky group, a bulky group composed of cucurbituril and other groups, or a bulky group connected to a targeting molecule. The bulky group includes common bulky groups such as trityl, adamantyl, t-butyl, 3,5-dimethylphenyl, diisopropylphenyl, and anthracene.

[0045] The targeting molecule includes a targeting monoclonal antibody, an RGD peptide, a prostate-specific membrane antigen (PSMA) inhibitor, an epidermal growth factor receptor (EGFR) inhibitor, a fibroblast activation protein inhibitor (FAPI), and other commonly used targeting groups of radiopharmaceuticals.

[0046] In some examples, the reaction solution is filtered using a 0.22 µm water-based needle filter.

[0047] In some examples, the Sep-pak C18 solid-phase extraction column is used for separation and purification. First, the remaining 131 I]I - The labeled product is eluted into a centrifuge tube using an appropriate concentration of ethanol solution.

[0048] It should be noted that the above steps S01 and S02 are only exemplary and do not limit the preparation method provided by the present application. Those skilled in the art can prepare corresponding compounds according to actual needs, or directly select a labeled radiolabeled precursor compound for step S03.

[0049] In one example, step S01 is shown in the following reaction formula:

[0050] Specific step S01 includes: selecting compound 1.1 required for preparation of radiohalogen labeled precursor compound, adding dilute hydrochloric acid at 0℃, stirring for 30 min. Dropwise add NaNO2 solution, strictly control temperature at 0-5℃ during dropwise addition, stir for 30 min after completion of addition. Finally add ice KI solution, stir overnight at room temperature. After completion of reaction, dilute with appropriate amount of ethyl acetate, separate. Decolorize organic phase with saturated sodium thiosulfate solution, extract aqueous phase with ethyl acetate. Combine organic phases, dry over anhydrous sodium sulfate, filter, concentrate under vacuum. Purify by flash chromatography to obtain crude product, compound 1.2. Dissolve compound 1.2 in super-dry DMF, then add propargylamine, stir overnight at 80℃ under N2 protection. After completion of reaction, cool to room temperature, spin off DMF until just precipitate. Add ether, a large amount of precipitate appears, filter, wash solid with ether to obtain white precipitate. Purify by semi-preparative HPLC, spin dry to collect liquid, then dissolve in HCl methanol solution (4M), add a large amount of ether to produce precipitate, centrifuge to obtain radiohalogen labeled precursor compound, denoted as compound 1.

[0051] In one example, specific step S02 includes: dissolve cuprous chloride (CuCl) in 0.1N HCl solution. Add compound 1 in a pressure tube, then sequentially add appropriate amount of water, prepared CuCl solution, [ 131 I]NaI solution, vortex for 20 s, react at 150℃ for 1 h. Finally slowly reduce to room temperature. Filter reaction liquid with 0.22µm water-based needle filter and determine RCP and RCY by radio-HPLC.

[0052] The radiohalogen labeled compound based on rotaxane protection prepared by the preparation method provided in the present application has high radiochemical purity, and good in vitro and in vivo stability.

[0053] In a third aspect, the present application provides an application of the radiohalogen labeled compound based on rotaxane protection, and the application of the radiohalogen labeled compound based on rotaxane protection provided in the first aspect or prepared by the preparation method provided in the second aspect in preparation of a tumor radiopharmaceutical.

[0054] Example One In the present example, the structural formula of the radiohalogen labeled compound based on rotaxane protection is as follows: ; Wherein, CB[6] and β-CD are cucurbit[6] and β-cyclodextrin respectively, 4HCl is 4HCl combined with rotaxane to form hydrochloride.

[0055] In the present embodiment, the preparation method adopted comprises the following steps: Step S01: Preparation of a radioactive halogen-labeled precursor compound (Compound 1) 1) In a 100 mL round-bottom flask, compound 1.1 (1.56 g, 5.2 mmol) was added. Dilute hydrochloric acid (8 mL of concentrated hydrochloric acid in 25 mL of water) was added at 0 °C and stirred for 30 min. 5 mL of NaNO2 (0.538 g, 7.8 mmol) solution was added dropwise, and the temperature was strictly controlled at 0-5 °C during the dropwise addition. After the addition was completed, it was stirred for 30 min. Finally, 5 mL of ice KI (1.726 g, 10.4 mmol) solution was added, and it was stirred at room temperature overnight. After the reaction was completed, 50 mL of ethyl acetate was added for dilution, and the liquid was separated. The organic phase was decolorized with saturated sodium thiosulfate solution, and the aqueous phase was extracted with ethyl acetate (30 mL x 3). The combined organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The crude product compound 1.2 (yield 51%) was obtained as a colorless transparent oil after purification by flash chromatography. ESI-MS (C 14 H 11 Cl2I): [M+Na] + Calcd: 398.92, Found: 398.92; [M+K] + Calcd: 414.89, Found: 414.90.

[0056] 2) Compound 1.2 (5.5 mmol) was dissolved in 135 mL of super dry DMF, then propargylamine (55 mmol) was added, and N2 protection was performed. It was stirred at 80 °C overnight. After the reaction was completed, it was cooled to room temperature, and the DMF was removed by rotary evaporation until just precipitated. Ethyl ether was added, and a large amount of precipitate appeared. The solid was washed with ethyl ether to obtain a white precipitate. Finally, it was purified by semi-preparative HPLC, the liquid was collected by rotary evaporation, and then dissolved in 10 mL of HCl methanol solution (4M). A large amount of ethyl ether was added to produce a precipitate, which was centrifuged to obtain a white solid as compound 1, with a yield of 50%.

[0057]

[0058] Figure 2 a, b, and c are the 1 H NMR chart, 13 C NMR chart and ESI-HRMS chart. Among them, 1H NMR (400 MHz, D2O) δ 8.08 (d, J = 1.7 Hz, 1H), 7.53 (dd, J = 8.0, 1.8 Hz, 3H), 7.43 – 7.33 (m, 3H), 4.37 (s, 2H), 4.31 (s, 2H), 3.90 (dd, J = 6.0, 2.6 Hz, 4H), 3.04 (q, J = 2.4 Hz, 2H). 13 C NMR (400 MHz, D2O) δ 146.67, 144.49, 140.82,131.19, 130.49, 130.04, 130.03, 129.88, 129.80, 98.06, 78.67, 78.57, 72.99,72.86, 49.48, 48.49, 35.65, 35.62. ESI-HRMS(C 20 H 19 IN2): [M+H + Calculated value: 415.0666, measured value: 415.0664; [M+Na]+ calculated value: 437.0485, measured value: 437.0494.

[0059] Step S02: Radiolabel the radiolabeled precursor compound with radioactive halogen (radioactive halogen). 131 (I mark) CuCl (5 mg) was dissolved in 0.1 N HCl solution (5 mL). 2.0 mg of compound 2.8 was added to a 4 mL pressure-resistant tube, followed by 0.6 mL of water, 0.4 mL of the prepared CuCl solution, and 45 MBq. 131 In a solution of NaI, the mixture was vortexed for 20 seconds and reacted at 150°C for 1 hour. The mixture was then slowly cooled to room temperature. The reaction solution was filtered through a 0.22 µm aqueous syringe filter, and RCP and RCY were determined by radio-HPLC.

[0060] Step S03: Preparation of radiolabeled halogen compounds protected by rotaxane (denoted as rotaxane […]) 131 I]I-tBuR) In this embodiment, the macrocyclic compound was selected as β-cyclodextrin, and the end-capping group was cucurbituril [6]. The labeled radio-halogenated precursor compound 1, the macrocyclic compound, and the end-capping group were assembled in rotaxane to obtain a radio-halogenated compound protected by rotaxane. The reaction process is shown in the following formula:

[0061] Add compound 9.1 (1.7 mg), β-cyclodextrin (5.9 mg), and cucurbituril [6] (9.2 mg) to a 5 mL round-bottom flask. Then add the compound from the previous step [ 131 I] - The entire solution of compound 1 was added to this flask and stirred at 60°C for 30 min. After the reaction was complete, the mixture was cooled to room temperature, filtered through a 0.22 µm aqueous syringe filter, and RCP and RCY were determined by radio-HPLC. Separation and purification were performed using a Sep-pak C18 solid-phase extraction column. The remaining [[] were first purified by diluting the column with 2 mL of physiological saline.] 131 I]I - After rinsing to remove the label, the labeled product was eluted into a centrifuge tube with 3% ethanol solution (radio-HPLC determination of RCP).

[0062] Performance of radiohalogen-labeled compounds protected by rotaxane 1. Results of radio-high performance liquid chromatography (radio-HPLC) test The radio-HPLC system was equipped with a radiodetector, a dual-wavelength UV absorption detector, and a C18 reversed-phase column (4.6 × 150 mm, 5 µm, Agilent). Phase A was water (containing 0.1% trifluoroacetic acid), and phase B was methanol. The gradient was as follows: 0-5 min, phase B changed from 20% to 60%; 5-15 min, phase B changed from 60% to 100%; 15-17 min, phase B was 100%; and 17-20 min, phase B changed from 100% to 20%. The injection volume was 20 µL, and the flow rate was 1 mL / min. The radio-HPLC results are as follows: Figure 3 As shown in Figure b, the determination of radioactive iodine-labeled rotaxane [ 131 The retention time of I]I-tBuR was 6.26 min, corresponding to a retention time of 6.00 min for the cold product. Figure 4 In the diagram, a, b, and c represent the cold product I-tBuR. 1 H NMR spectrum, 13 (C NMR and ESI-HRMS plots). The small difference in retention time indicates that the structure of the radiolabeled compound is consistent with that of the non-radioactive cold product.

[0063] Rotaxane was identified by radio-HPLC. 131 The radiochemical purity of I]I-tBuR is 98%.

[0064] 2. In vitro stability test rotaxane [ 131 I]I-tBuR was added to PBS and FBS, and after incubation at 37°C for 24 h, 48 h, and 72 h, radio-HPLC was performed. The results are as follows:Figure 5 As shown. Under the same radio-HPLC elution conditions, [ 131 I]I - The retention time was 2.0 min. Radio-HPLC identification showed that the labeled compound [ 131 I]I-tBuR maintained a 98% RCP after incubation in PBS and FBS for 24h, 48h, and 72h, with no free [ 131 I]I- is produced. Stability experiments with PBS and FBS show [ 131 I]I-tBuR exhibits good in vitro stability and can be used for subsequent experiments.

[0065] Iodine is a good leaving group in nucleophilic substitution reactions. The reaction of radiolabeled iodine drugs with nucleophilic groups is also a significant cause of deiodination. GSH has a high intracellular concentration and is a representative nucleophile in vivo. Therefore, […]. 131 I]I-tBuR was added to 12.5 mM GSH-PBS solution and incubated for 24 h, 48 h, and 72 h to assess the stability of radiolabeled rotaxane against nucleophilic group attack. Radio-HPLC results are shown below. Figure 6 As shown. 131 I]I-tBuR maintained a high RCP of 97% after incubation in GSH for 24h, 48h, and 72h, releasing only a very small amount of free [ 131 I]I - This indicates that the cyclodextrin shell of the radioiodine-labeled moiety can provide physical protection for the unstable CI, preventing the nucleophile GSH from approaching the CI bond and improving the stability of the radioiodine-labeled compound for the nucleophilic group.

[0066] Applications of radiolabeled halogen compounds protected by rotaxane According to the experimental requirements for radioactivity, the radiolabeled halogen compound protected by rotaxane was diluted. If used for animal experiments, NaCl was added to prepare a 0.9% NaCl solution.

[0067] To better study the effect of rotaxane protection on inhibiting in vivo dehalogenation, radiolabeled iodine molecules without rotaxane protection were selected. 131 I]I-tria (compound 6) was used as a control, and the control molecule structure is as follows ( Figure 3 In the middle, 'a' represents the control molecule. 131 Radio-HPLC detection chromatogram of I]I-tria Figure 7 a, b, and c in the text represent the cold product I-tria, respectively. 1 H NMR spectrum, 13 (C NMR and ESI-HRMS plots):

[0068] Normal Kunming male mice were injected with 100 μL of 131 I-labeled molecule solution (1 MBq / mL) through the tail vein. Blood was collected from the mice at 0.5 h, 2 h, 8 h, 24 h, 48 h, and 72 h after administration, and the mice were sacrificed and dissected to obtain the brain, heart, liver, spleen, lung, kidney, stomach, intestine, bone, muscle, and thyroid, etc. The organs and tissues were washed with clean water, wiped dry, and weighed. The radioactivity of each tissue and organ was measured using an FH-463B intelligent scaler. Five mice were used for parallel experiments at each time point, and the uptake percentage (%ID / g) of each organ was calculated as (%ID / g) = (radioactivity of the tissue or organ / 1% of the total radioactive dose injected into the mouse body) / mass of the corresponding tissue or organ. The average value was taken, and the results were expressed as the average value ± deviation (%ID / g ± SD). Due to the small size of the thyroid, it was not possible to accurately weigh it, so the thyroid uptake was expressed as %ID ± SD.

[0069] Rotaxane 131 I]The radioactivity of each tissue and organ of the I-tBuR group was lower than that of the control molecule 131 I]The results of the tissue distribution experiment of the I-tria group of mice were Figure 8 and Figure 9 Rotaxane 131 I]The radioactivity of each tissue and organ of the I-tBuR group of mice was lower than that of the control molecule 131 I]I-tria group. Further comparison of the radioactivity accumulation in the thyroid and stomach of the two groups of mice Figure 10 ) showed that, due to the slower in vivo metabolism of the control molecule 131 I]I-tria than the rotaxane 131 I]I-tBuR, the thyroid radioactivity accumulation of the rotaxane 131 I]I-tBuR was higher than that of the control group 131 I]I-tria at 0.5 h after administration. However, at 8 h, the thyroid radioactivity accumulation of the control molecule 131 I]I-tria (1.02 ± 0.11 %ID) was about 7 times that of the rotaxane 131 I]I-tBuR (0.17 ± 0.02 %ID), and at 24 h, 48 h, and 72 h, the difference in thyroid radioactivity accumulation between the two was even greater, with the control molecule 131 I]I-tria being about 10 times that of the rotaxane 131 I]I-tBuR. There was also a huge difference in the radioactivity accumulation in the stomach between the two. From 0.5 h to 72 h, the radioactivity accumulation in the stomach of the control molecule 131 I]I-tria was higher than that of the rotaxane 131The radioactive accumulation of I]I-tBuR in the stomach increased from approximately a 2-fold to a 46-fold difference. More importantly, the control molecule [ 131 I]I-tria showed the least radioactive accumulation in the stomach over 72 hours, approximately 1.84% ID / g. This is lower than that of rotaxane. 131 The maximum radioactive accumulation of I]I-tBuR in the stomach after 0.5 hours was approximately 1.32%ID / g. These data indicate that the presence or absence of rotaxane protection on the radioiodine-labeled portion of the compound significantly affects the degree of deiodination in vivo. Rotaxane protection can significantly inhibit the in vivo dehalogenation of radiohalogenated compounds, suggesting it could serve as a novel method for inhibiting dehalogenation in the diagnosis and treatment of tumors using radiohalogenated drugs.

[0070] Comparative Example Will[ 131 I]I-tBuR Thyroid radioactive accumulation and molecules that inhibit deiodination in existing technologies [ 125 I]I-10、[ 125 I]I-11、[ 131 I]I-12 and [ 125 I]I-13( Figure 11 The results of thyroid radioactivity accumulation were compared. Table 1 shows the results of thyroid radioactivity accumulation.

[0071] Table 1 Comparison of Thyroid Radioactivity Accumulation

[0072] As can be seen from Table 1, [ 131 Compared to the maximum thyroid radioactive accumulation of I]I-tBuR, [ 125 I]I-10 and [ 131 The maximum value of I]I-12 is [ 131 The effect of I]I-tBuR is 16 times greater. This indicates that the rotaxane-protected radiohalogen-labeled compound provided in this application has a more significant inhibitory effect on in vivo deiodination. Although [ 131 The thyroid radioactivity accumulation of I]I-tBuR is slightly higher than [ 125 I]I-2.11 and [ 125 I]I-2.13, but due to [ 125 I]I-11 has a strong binding affinity to albumin, resulting in very slow blood clearance. Blood retention levels remain high at 11.71±0.83 and 7.42±0.50% ID / g at 8h and 24h, respectively, leading to an increase in the systemic effective dose received by the patient. [ 125 I]I-13 is a hippuric acid derivative that is excreted from the body with almost no further metabolic transformation. 125I]I-13 is mainly metabolized by kidney and the metabolism is very fast, almost no retention in mice after 4h injection. The fast metabolism will significantly reduce the therapeutic effect of the drug.

[0073] It is to be understood that the terminology used herein is for the purpose of describing particular example embodiments only and is not intended to be limiting. It is further understood that additional or alternative steps can be utilized. The above description is merely illustrative of the application and not restrictive.

[0074] The above describes the embodiments of the present disclosure. However, these embodiments are only for the purpose of illustration, and are not intended to limit the scope of the present disclosure. The scope of the present disclosure is defined by the appended claims and their equivalents. Without departing from the scope of the present disclosure, those skilled in the art can make various substitutions and modifications, which should all fall within the scope of the present disclosure.

Claims

1. A radiohalogen labeled compound based on wheel-alkane protection, characterized in that, Comprise: A macrocycle: having a cavity, the macrocycle comprising one of a cyclodextrin, a cucurbituril, a calixarene, and a pillararene; A radiohalogen-labeled compound: a radiohalogen-labeled position is located within the cavity of the macrocycle; An end-capping group: located at the open end of the cavity, connected to both ends of the radiohalogen-labeled compound, for preventing the radiohalogen-labeled compound from sliding out of the cavity.

2. The wheel-encapsulation based radiohalogen labeling compound according to claim 1, wherein, The radiohalogen-labeled compound comprises an aromatic ring or an aromatic heterocycle.

3. The wheel-encapsulation based radiohalogen labeling compound according to claim 2, wherein, The aromatic ring comprises one of a benzene ring, a biphenyl, and a naphthalene ring; The aromatic heterocycle comprises one of a triazole, a quinoline, and a pyridine.

4. The wheel-encapsulation based radiohalogen labeling compound according to claim 2, wherein, The radiohalogen-labeled position is at any vacant position on the aromatic ring or the aromatic heterocycle.

5. The wheel-encapsulation-based radiohalogen labeling compound according to claim 1, wherein, The radioactive halogen comprises 123 I, 124 I, 125 I, 131 I and 211 At one of.

6. The wheel-encapsulation-based radiohalogen labeling compound according to claim 1, wherein, The end-capping group comprises one of a trityl group, an adamantyl group, a tert-butyl group, a 3,5-dimethylphenyl group, a diisopropylphenyl group, and an anthryl group.

7. The wheel-encapsulation-based radiohalogen labeling compound according to claim 1, wherein, Also comprising a linker connected between the radiohalogen-labeled compound and the end-capping group; The linker comprises one of an alkyl chain, an amino acid chain, a PEG (polyethylene glycol) chain, a triazole, an amide bond, and a maleimide.

8. A method for the preparation of a radiohalogen labeled compound based on wheel-alkane protection according to any one of claims 1 to 7, characterized in that, Mixing the radiohalogen-labeled compound, the macrocycle, and the end-capping group, stirring at 60-80°C, cooling to room temperature after the reaction is completed, filtering, separating and purifying to obtain a radiohalogen-labeled compound based on a rotaxane-protected.

9. The preparation method according to claim 8, characterized in that, The mass ratio of the radiohalogen-labeled compound, the macrocycle, and the end-capping group is 1:1.1-1.3:2.2-2.

6.

10. Use of a radiohalogen labelled compound, characterized in that, Application in tumor radio-pharmaceuticals.

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