Ligand, metal ion residual quantity detection method and application

Abstract

The invention discloses a ligand, a metal ion residual quantity detection method and application. The ligand is used for being subjected to a complexation reaction with metal ions to generate a coordination compound, and under the same chromatographic condition, the retention time of the ligand is different from that of the coordination compound. By adopting the ligand provided by the invention, metal ion residues in carrier molecules for preparing radiopharmaceuticals can be rapidly and effectively detected.

Description

Technical Field

[0001] This application relates to the field of radiopharmaceutical preparation technology, specifically to a method and application for detecting ligands and residual metal ions. Background Technology

[0002] Radiopharmaceuticals are medical preparations containing radioactive nuclides used for diagnosis or treatment. They are characterized by achieving medical purposes through the release of radiation via radioactive decay, possessing both pharmacological and radiation properties. Radiopharmaceuticals consist of a carrier molecule and a radioactive nuclide bound to the carrier molecule via a complexation reaction. The preparation process and storage environment of the carrier molecule can introduce metallic impurities. These impurities react with the complexing groups on the carrier molecule, thereby reducing or even completely eliminating the carrier molecule's ability to complex other radioactive nuclides, ultimately leading to the failure of radiopharmaceutical preparation.

[0003] In related technologies, ICP-MS (inductively coupled plasma mass spectrometry) or ICP-OES (inductively coupled plasma optical emission spectrometry) can be used to detect the residual amount of metal ions in the carrier molecules. However, these detection methods are complicated in sample processing, expensive, and time-consuming. Summary of the Invention

[0004] The purpose of this application is to provide a method and application for detecting ligands and residual metal ions. The side chain region has a weak affinity for polynucleotide-binding proteins, which avoids the side chain region from binding to polynucleotide-binding proteins, thereby increasing the binding rate of polynucleotide-binding proteins to the main chain and improving the efficiency of biomolecule characterization.

[0005] The technical solution of this application is as follows: Firstly, a ligand is provided, as shown in either formula (1) or formula (2):

[0006] Equation (1)

[0007] Equation (2) R1 is selected from any one of H, a hydrocarbon group with 1 to 20 substituted or unsubstituted carbon atoms, an aromatic group with 6 to 14 substituted or unsubstituted cyclic atoms, and a heteroaromatic group with 5 to 14 substituted or unsubstituted cyclic atoms, and n is 1 to 12. Ligands are used to undergo complexation reactions with metal ions to form coordination compounds. Under the same chromatographic conditions, the retention times of ligands and coordination compounds are different.

[0008] In some embodiments, R1 is selected from any one of the following: alkane group with 1 to 15 substituted or unsubstituted carbon atoms, alkene group with 2 to 15 substituted or unsubstituted carbon atoms, alkyne group with 2 to 15 substituted or unsubstituted carbon atoms, and cycloalkyl group with 3 to 10 substituted or unsubstituted carbon atoms.

[0009] In some embodiments, R1 is selected from any one of an alkane group having 1 to 10 carbon atoms, substituted or unsubstituted, or an aromatic group having 6 to 10 cyclic atoms, substituted or unsubstituted.

[0010] In some embodiments, R1 is selected from any one of substituted or unsubstituted alkane groups having 1 to 6 carbon atoms, or substituted or unsubstituted phenyl groups.

[0011] In some embodiments, R1 is as shown in equation (3):

[0012] Equation (3) Secondly, the application of the ligands provided in the first aspect in detecting the residual amount of metal ions in the carrier molecules of radiopharmaceuticals is provided.

[0013] Thirdly, a method for detecting residual metal ions is provided, including: The test solution containing the carrier molecule is mixed and reacted with a salt solution containing metal ions, and then a detection solution containing the ligand provided in the first aspect is added and mixed and reacted to obtain a reaction solution. The reaction solution was analyzed by liquid chromatography to obtain a liquid chromatogram; The results of residual metal ions in the test solution are obtained based on the difference between the chromatographic peaks of the coordination compound and the chromatographic peaks of the ligand in the liquid chromatogram.

[0014] In some embodiments, the residual metal ion result in the test solution is obtained based on the difference between the chromatographic peak of the coordination compound and the chromatographic peak of the ligand in the liquid chromatogram, including: The residual amount of metal ions in the test solution can be obtained from the peak area of ​​the coordination compound in the liquid chromatogram.

[0015] In some embodiments, a test solution including a carrier molecule is mixed and reacted with a salt solution including metal ions, and then a detection solution including the ligand provided in the first aspect is added and mixed and reacted to obtain a reaction solution, comprising: Mix the test solution with the salt solution, adjust the pH to 4.5~6.0, and react at 37℃~95℃ for 30min~45min to obtain the mixed solution; Mix the mixed solution with the test solution, adjust the pH to 4.5~6.0, and react at 37℃~95℃ for 30min~45min to obtain the reaction solution.

[0016] In some embodiments, the pH of the mixture of the test solution and the detection solution is adjusted using sodium acetate-glacial acetic acid buffer and sodium acetate solution.

[0017] The technical solutions provided by the embodiments of this application bring at least the following beneficial effects: In the methods and applications for detecting residual ligands and metal ions provided in this application, the ligands can effectively undergo complexation reactions with metal ions to generate coordination compounds. By utilizing the fact that the chromatographic peak positions of the ligands and coordination compounds are different in the liquid chromatograms, it is possible to determine whether there are residual metal ions in the test solution by observing the chromatographic peaks in the liquid chromatograms.

[0018] It should be understood that the above general description and the following detailed description are exemplary and explanatory only, and do not limit this application. Attached Figure Description

[0019] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments consistent with this application and, together with the description, serve to explain the principles of this application, and do not constitute an undue limitation of this application.

[0020] Figure 1 This is a schematic diagram of the chemical structure of a coordination compound provided in an embodiment of this application; Figure 2 This is a superimposed liquid chromatogram of experimental solution 1 and control solution 1 provided in the embodiments of this application; Figure 3 This is the liquid chromatogram of control solution 2 provided in the embodiments of this application; Figure 4 This is the liquid chromatogram of Experimental Example 2 provided in the embodiments of this application; Figure 5 This is the liquid chromatogram of Experimental Example 3 provided in the embodiments of this application; Figure 6 This is the liquid chromatogram of Experimental Example 4 provided in the embodiments of this application; Figure 7 This is the liquid chromatogram of Experimental Example 5 provided in the embodiments of this application; Figure 8 This is the liquid chromatogram of Experimental Example 6 provided in the embodiments of this application; Figure 9 This is the liquid chromatogram of Experimental Example 7 provided in the embodiments of this application; Figure 10 These are the liquid chromatograms of Experimental Examples 8 to 16 provided in the embodiments of this application; Figure 11 The ligand solution complexation provided in the embodiments of this application 175 Lu gradient standard curve. Detailed Implementation

[0021] To enable those skilled in the art to better understand the technical solutions of this application, the technical solutions in the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. It should be understood that the specific embodiments described herein are merely intended to explain this application and not to limit it. For those skilled in the art, this application can be implemented without some of these specific details. The following description of the embodiments is merely to provide a better understanding of this application by illustrating examples.

[0022] It should be noted that the terms "first," "second," etc., used in the specification, claims, and accompanying drawings of this application are used to distinguish similar objects and are not necessarily used to describe a specific order or sequence. It should be understood that such data can be interchanged where appropriate so that the embodiments of this application described herein can be implemented in orders other than those illustrated or described herein. The embodiments described in the following exemplary embodiments do not represent all embodiments consistent with this application. Rather, they are merely examples consistent with some aspects of this application as detailed in the appended claims.

[0023] In related technologies, ICP-MS (inductively coupled plasma mass spectrometry) or ICP-OES (inductively coupled plasma optical emission spectrometry) can be used to detect the residual amount of metal ions in the carrier molecules. However, these detection methods are complicated in sample processing, expensive, and time-consuming.

[0024] To address the aforementioned issues, this application provides a connector, as detailed in the following embodiments.

[0025] This application provides a ligand as shown in either formula (1) or formula (2):

[0026] Equation (1)

[0027] Equation (2) R1 is selected from any one of H, a hydrocarbon group with 1 to 20 substituted or unsubstituted carbon atoms, an aromatic group with 6 to 14 substituted or unsubstituted cyclic atoms, or a heteroaromatic group with 5 to 14 substituted or unsubstituted cyclic atoms. Ligands are used to undergo complexation reactions with metal ions to form coordination compounds. Under the same chromatographic conditions, the retention times of ligands and coordination compounds are different.

[0028] The ligand provided in this application can be used to detect residual metal ions in a test solution containing a carrier molecule, which is used to prepare a radiopharmaceutical. The carrier molecule can be a monoclonal antibody, peptide, small molecule compound, etc. A radiopharmaceutical consisting of a carrier molecule and a metal radionuclide is prepared through a complexation reaction. The metal radionuclide can be... 177 Lu、 90 Y、 99m Tc, 225 Ac、 212 Pb and other elements can release gamma rays and beta rays through radioactive decay. - Ion rays and other radiation.

[0029] Please see Figure 1 The provided ligand can undergo a complexation reaction with the metal ion M to form a coordination compound. The R1 group has an inert chemical structure and does not undergo a complexation reaction with the metal ion. In the coordination compound, the metal ion M is the central atom, and -COO For ligands, 3 ligands -COO The ligand forms a coordination host with the central atom M. If the number of carboxyl groups (-COOH) in the ligand for coordination with the metal ion is too small, the resulting coordination compound will have poor stability, and the retention times of the coordination compound and the ligand will overlap or be poorly distinguishable. If the number of carboxyl groups (-COOH) in the ligand for coordination with the metal ion is too large, the resulting coordination compound will have poor solubility in the mobile phase and a long liquid chromatography analysis time. The ligand provided in this application has three carboxyl groups for coordination with the metal ion, such that the three ligands have three -COOH groups. The central atom M forms a stable coordination matrix, reducing the dissociation of the coordination compound during liquid chromatography. The three carboxyl groups (-COOH) in the ligand, which are used to coordinate with the metal ion, are symmetrically distributed on the heterocyclic decane, which is conducive to forming a stable and single coordination compound with the metal ion M, thus presenting a sharp and symmetrical single chromatographic peak in the liquid chromatogram.

[0030] A liquid chromatogram is a graph showing the change of a detector signal over time in high-performance liquid chromatography (HPLC) or ultra-high-performance liquid chromatography (UPLC) analysis. Under the same chromatographic conditions, the retention times of a single ligand detected by the detector will differ from those of a single coordinating compound detected by the detector. These same chromatographic conditions include, but are not limited to, the composition, ratio, pH, and flow rate of the mobile phase; the type, column length, and particle size of the stationary phase; and the ambient temperature.

[0031] Under the same chromatographic conditions, the retention times of a single ligand detected by the detector are different from those of a single coordination compound detected by the detector. That is, the positions of the chromatographic peaks in the liquid chromatograms of a single ligand and a single coordination compound are different. Therefore, when a known equivalent of metal ion M is added to the test solution, the carrier molecules in the test solution react with the metal ion. If no metal ions remain in the test solution during production or transportation, the added metal ion M will complex with the carrier molecules. If metal ions remain in the test solution during production or transportation, some carrier molecules will complex with the remaining metal ions, while the remaining metal ion M will not complex with the carrier molecules. When a ligand is added, if all metal ion M complexes with the carrier molecules, no metal ion M will complex with the ligand; if the remaining metal ion M does not complex with the carrier molecules, the remaining metal ion M will complex with the ligand. By irradiating a mixture of carrier molecules, metal ion M, and ligands with ultraviolet light using liquid chromatography, a liquid chromatogram of the mixture is obtained. The presence of a chromatographic peak corresponding to the retention time of the metal ion M and the ligand is observed. If a chromatographic peak corresponding to the retention time of the ligand is observed, it indicates that metal ions remain in the carrier molecules; if no chromatographic peak corresponding to the retention time of the ligand is observed, it indicates that metal ions do not remain in the carrier molecules.

[0032] In the ligands provided in this application embodiment, the ligands can effectively undergo complexation reactions with metal ions to generate coordination compounds. Under the same chromatographic conditions, the retention times of the ligands and coordination compounds are different, and it can be determined whether there are residual metal ions in the test solution by observing the chromatographic peaks in the liquid chromatogram.

[0033] Optionally, the ligand can be formulated as a detection solution with water, sodium acetate-glacial acetic acid buffer, or the like as the solvent.

[0034] In some embodiments, R1 is selected from any one of the following: alkane group with 1 to 15 substituted or unsubstituted carbon atoms, alkene group with 2 to 15 substituted or unsubstituted carbon atoms, alkyne group with 2 to 15 substituted or unsubstituted carbon atoms, and cycloalkyl group with 3 to 15 substituted or unsubstituted carbon atoms.

[0035] In some embodiments, R1 is selected from any one of an alkane group having 1 to 10 carbon atoms, substituted or unsubstituted, or an aromatic group having 6 to 10 cyclic atoms, substituted or unsubstituted.

[0036] In some embodiments, R1 is selected from any one of substituted or unsubstituted alkane groups having 1 to 6 carbon atoms, or substituted or unsubstituted phenyl groups. A reduced number of carbon atoms or cyclic atoms in R1 is advantageous for synthetic preparation.

[0037] In some embodiments, R1 is as shown in equation (3):

[0038] Equation (3) The two Br atoms are strong electron-withdrawing groups, which significantly reduces the electron cloud density of the double bond, making it difficult for the structure of formula (3) to coordinate with metal ions, thus improving the stability of the structure of formula (3). In the liquid chromatography process, the detector generates a liquid chromatogram by irradiation with ultraviolet light. The Br atoms absorb ultraviolet light under a specific wavelength of ultraviolet light, which improves the sensitivity of liquid chromatography detection. The amide bond (-CON-) is chemically stable, which is conducive to the stability of the R1 group. The carbonyl group (C=O) and the amino group (N) stabilize the electron delocalization of the ring structure, reducing the degradation reaction of the R1 group caused by conformational changes.

[0039] In some embodiments, the chromatographic conditions are as follows: C18 column, 150 x 4.6 mm in size, particle size 2.7 μm; mobile phase: 0.1% formic acid-acetonitrile; gradient: 0.1% formic acid:acetonitrile = 90:10, changing to 10:90 after 10 minutes; detection environment: column temperature 30℃; detection wavelength 254 nm; flow rate 1 mL / min; detection time 10 min. The difference between the retention time of the ligand and the retention time of the coordinating compound is defined as H, where H ≥ 0.1 min.

[0040] This application also provides an application of ligands in detecting residual metal ions in carrier molecules of radiopharmaceuticals.

[0041] The carrier molecule can be a monoclonal antibody, peptide, small molecule compound, etc. The metal ion can be Cu²⁺. Fe² Fe³ Ca² Mg² At least one of them. Metal ions can also be 177 Lu、 90 Y、 99m Tc, 225 Ac、 212 At least one of Pb.

[0042] This application also provides a method for detecting residual metal ions, including: S1, the test solution including the carrier molecule is mixed and reacted with a salt solution including metal ions, and then a detection solution including the ligand of any one of claims 1 to 5 is added and mixed and reacted to obtain a reaction solution; S2, perform liquid chromatography analysis on the reaction solution to obtain a liquid chromatogram; S3. Based on the chromatographic peaks of the coordination compound and the chromatographic peaks of the ligand in the liquid chromatogram, the results of the residual metal ions in the test solution are obtained.

[0043] The test solution is a pre-prepared solution with a known number of carrier molecules. The number of moles of metal ion M added is determined based on the number of carrier molecules in the test solution, so that all carrier molecules in the test solution can react completely with metal ion M.

[0044] After the reaction between the carrier molecule in the test solution and the metal ion M in the salt solution is completed, the number of moles of ligand to be added is determined according to the number of moles of metal ion M added, so that the metal ion M in the mixed solution that has not complexed with the carrier molecule can form a coordination compound with the ligand.

[0045] If no metal ions remain in the test solution during production and transportation, the added metal ion M will undergo a complexation reaction with the carrier molecule, resulting in a mixed solution without residual metal ion M. Upon addition of the ligand, no metal ion M will undergo a complexation reaction with the ligand. The liquid chromatogram of this reaction solution will only show the chromatographic peak corresponding to the ligand.

[0046] If residual metal ions remain in the test solution during production and transportation, some carrier molecules will undergo a complexation reaction with the residual metal ions. After adding metal ion M, the remaining metal ion M will not undergo a complexation reaction with the carrier molecules. Upon adding a ligand, the residual metal ion M will undergo a complexation reaction with the ligand. A chromatographic peak corresponding to the coordination compound will appear in the liquid chromatogram of the solution reacting with this solution.

[0047] Therefore, by observing whether there is a chromatographic peak of a corresponding coordination compound in the liquid chromatogram, we can obtain the detection result regarding whether there are residual metal ions in the test solution.

[0048] In some embodiments, the molar ratio of metal ions added to the test solution to ligands added to the mixed solution is 1:1. When the molar ratio is 1:1, the chromatographic peak of the coordination compound in the liquid chromatogram shows a greater variation compared to the chromatographic peak of the ligand, which is beneficial for identifying the chromatographic peak of the coordination compound and calculating the peak area of ​​the coordination compound.

[0049] In some embodiments, S3 includes: The residual amount of metal ions in the test solution can be obtained from the peak area of ​​the coordination compound in the liquid chromatogram.

[0050] The inventors discovered that in the liquid chromatogram of the coordination compound formed by the ligand and metal ion, the peak area of ​​the coordination compound is positively correlated with the molar ratio of metal ion M to ligand. Therefore, given the number of moles of the ligand, the number of moles of metal ion complexed with the ligand can be calculated by measuring the peak area of ​​the coordination compound. Furthermore, since the number of moles of added metal ion M is known, the number of moles of carrier molecules that have complexed with residual metal ions in the test solution can be calculated based on the number of moles of added metal ion M and the number of moles of metal ion complexed with the ligand, thus allowing evaluation of the chelating ability of the carrier molecules in the test solution. Optionally, the residue results include whether there are residual metal ions in the test solution, the chelating ability of the carrier molecules in the test solution, and the number of moles of residual metal ions in the test solution.

[0051] In some embodiments, S1 includes: Mix the test solution with the salt solution, adjust the pH to 4.5~6.0, and react at 37℃~95℃ for 30min~45min to obtain the mixed solution; Mix the mixed solution with the test solution, adjust the pH to 4.5~6.0, and react at 37℃~95℃ for 30min~45min to obtain the reaction solution.

[0052] The pH of the mixture of the test solution and the detection solution is adjusted using sodium acetate-glacial acetic acid buffer and sodium acetate solution. Optionally, the reaction time after mixing the test solution with the salt solution, and the reaction time after mixing the mixture with the detection solution, are inversely proportional to the reaction temperature.

[0053] To verify the effects achievable by the above embodiments, the following examples and comparative examples are provided for illustration: Experimental Example 1: Mix 20 μL of 5 mg / mL ligand solution with 180 μL of ultrapure water to prepare a 1 mg / mL ligand solution; Take 40 μL of 1 mg / mL ligand solution, 16 μL of 0.1 mol / L sodium acetate-glacial acetic acid buffer, 130 μL of 0.25 mol / L sodium acetate solution, and 0.5 mL of 144 μL of 10 mg / mL ligand solution. 175 The LuCl3 solution was mixed thoroughly to prepare experimental solution 1; 0.5 μL of experimental solution 1 was taken and the pH was measured to be 5.0. Experimental solution 1 was placed in a constant temperature mixer and reacted at 37°C for 60 min. After the reaction was completed, 100 μL was transferred to a liquid phase tube and 5 μg was taken for HPLC detection.

[0054] Comparative Example 1: Take 20 μL of 5 mg / mL ligand solution and mix it with 80 μL of ultrapure water, 98 μL of 0.1 mol / L sodium acetate-glacial acetic acid buffer, and 10 μL of 0.25 mol / L sodium acetate solution in a liquid chromatography tube to prepare a control solution 1 with approximately 0.5 mg / mL ligand. Take 0.5 μL of the solution to check the pH at 5.0, and take 5 μg for HPLC detection.

[0055] Unless otherwise specified, the examples and comparative examples in this application used an Agilent 1100 series liquid chromatograph for HPLC detection. The chromatographic column was a GOWON™ C18 150x4.6mm 2.7μm; the mobile phase was 0.1% formic acid-acetonitrile; the gradient was 0.1% formic acid:acetonitrile = 90:10, changing to 10:90 after 10 minutes; the detection conditions were: column temperature 30℃; detection wavelength 254nm; flow rate 1mL / min; and detection time 10min.

[0056] Unless otherwise specified, the pH value of the sodium acetate-glacial acetic acid buffer solution used in the examples and comparative examples of this application is 5.5, and the pH value of the sodium acetate solution is 8.2.

[0057] Unless otherwise specified, the molecular formulas of the ligands in the following examples and comparative examples are as follows: .

[0058] Please see Figure 2 The image shows the superimposed liquid chromatograms of experimental solution 1 and control solution 1. Compared to control solution 1, which includes a ligand, experimental solution 1 contains a coordination compound of ligand -Lu. The peak area of ​​experimental solution 1 at 6.139(0) min is significantly increased, and the peak at 5.965 min completely disappears. Control solution 1 contains a ligand, and the peak area of ​​control solution 1 at 5.965 min is significantly increased. This indicates that the ligand and... 175 Following the Lu complexation reaction, the retention times of the complexed and uncomplexed ligands on HPLC showed distinct differences (Δt>0.1). The complexation compound: ligand- 175 The chromatographic peak area of ​​Lu changed significantly. The changes in chromatographic peak shift and peak area can be used to determine whether the ligand has undergone a complexation reaction with the radioactive metal ion.

[0059] Experimental Example 2-7: Take 0.0362 g of ferric chloride hexahydrate and dissolve it in 3.62 mL of 0.1 mol / L hydrochloric acid to prepare a 10 mg / mL ferric chloride hexahydrate stock solution (M=270.30); take 10 μL of the 10 mg / mL ferric chloride hexahydrate stock solution and mix it with 190 μL of 0.1 mol / L hydrochloric acid solution to prepare a 0.5 mg / mL ferric chloride hexahydrate solution; Dissolve 0.0590 mg of zinc chloride in 5.9 mL of 0.1 mol / L hydrochloric acid to prepare a 10 mg / mL anhydrous zinc chloride stock solution (M=136.32); mix 10 μL of the 10 mg / mL zinc chloride stock solution with 190 μL of 0.1 mol / L hydrochloric acid solution to prepare a 0.5 mg / mL zinc chloride solution. Take 50 μL of 1 mg / mL lead standard solution and mix it with 50 μL of 0.1 mol / L hydrochloric acid to prepare a 0.5 mg / mL lead standard solution.

[0060] Referring to Table 1, mix the test solutions and place them in a constant temperature mixer at 37°C for 1 hour. After the reaction is complete, transfer the mixture to a liquid chromatography tube and take 50 μL for HPLC detection.

[0061] Table 1

[0062] Comparative Example 2: Take 20 μL of 5 mg / mL ligand solution, 60 μL of 0.1 mol / L sodium acetate-glacial acetic acid buffer, and 20 μL of 0.25 mol / L sodium acetate, mix them, and measure the pH value to 5.0 to prepare a 1 mg / mL ligand solution; take 50 μL of 1 mg / mL ligand solution and 50 μL of 0.1 mol / L sodium acetate-glacial acetic acid buffer in a liquid chromatography tube and mix well to prepare control solution 2. After mixing, take 0.5 μL and measure the pH to 5.0.

[0063] Please see Figure 3 , Figure 4 and Figure 5 , Figure 3 This is the HPLC chromatogram of control solution 2. Figure 4 The HPLC chromatogram for Example 2 is shown below. Figure 5 This is the HPLC chromatogram for Example 3. From... Figures 3 to 5 It can be seen that the ligand can interact with Pb 2+ A complexation reaction occurs, and with the addition of Pb 2+ With the increase in proportion, the peak area near 6.4 min changes significantly.

[0064] Please see Figure 3 , Figure 6 and Figure 7 , Figure 6 The HPLC chromatogram for Example 4 is shown below. Figure 7 This is the HPLC chromatogram for Example 5. From... Figure 3 , Figure 6 and Figure 7 It can be seen that the ligand can interact with Fe. 3+ A complexation reaction occurs, and with the addition of Fe... 3+With the increase in proportion, the peak area near 6.4 min changes significantly.

[0065] Please see Figure 3 , Figure 8 and Figure 9 , Figure 8 The HPLC chromatogram for Example 6 is shown below. Figure 9 This is the HPLC chromatogram for Example 7. From... Figure 3 , Figure 8 and Figure 9 It can be seen that the ligand can interact with Zn 2+ A complexation reaction occurs, and with the addition of Zn 2+ With the increase in proportion, the peak area near 6.2 min changes significantly.

[0066] Explain the relationship between ligands and Fe 3+ Zn 2+ and Pb 2+ After complexation, the corresponding peak areas in the HPLC chromatogram of the coordination compound will change significantly, and the chromatographic peaks will change significantly when the molar ratio of metal ion to ligand is 1.

[0067] Experimental Example 8-16 Mix 20 μL of 5 mg / mL ligand solution with 80 μL of 0.1 mol / L sodium acetate-glacial acetic acid buffer to prepare a 1 mg / mL ligand solution; Take 10 μL of 5 mg / mL ligand solution, 90 μL of 0.1 mol / L sodium acetate-glacial acetic acid buffer, and 20 μL of 0.25 mol / L sodium acetate solution in a liquid chromatography tube to prepare control solution 2. After mixing, take 0.5 μL to test the pH, which is 5.0. Take 20 μL of 10 mg / mL 175 Lu was mixed with 60 μL of 0.1 mol / L hydrochloric acid to prepare a solution of 2.5 mg / mL. 175 Lu ion salt solution; take 10 μL of 2.5 mg / mL 175 Lu was mixed with 40 μL of 0.1 mol / L hydrochloric acid to prepare a solution of 0.5 mg / mL. 175 Lu ion salt solution; take 10 μL of 0.5 mg / mL 175 Lu was mixed with 40 μL of 0.1 mol / L hydrochloric acid to prepare a solution of 0.1 mg / mL. 175 Lu ion salt solution; Add each test solution according to Table 2, place in a constant temperature mixer and react at 37°C for 1 hour, then transfer all to a liquid phase tube and take 50 μL for HPLC detection.

[0068] Measure the area of ​​the corresponding chromatographic peak in the liquid chromatogram of each coordination compound, and compare the peak area with...175 Lu 3+ Linear fitting was performed on the ligand molar ratio to evaluate the dependence of the peak area of ​​the coordination compound on the reaction molar ratio and the linear range.

[0069] Table 2

[0070] Please see Figure 10 ,in Figure 10 (1) and (2) are the HPLC chromatograms of control solution 2. Figure 10 (3) to Figure 10 (11) The HPLC chromatograms of Experimental Examples 8 to 16 are shown in sequence.

[0071] Table 3

[0072] from Figure 10 As can be seen from the addition of ligand solution, 175 With increasing Lu, the chromatographic peak area of ​​the coordination compound gradually increases; the chromatographic peak at 6.4 min shows no significant change before reaching a multiple of 0.62 equivalents; at 0.62 equivalents, the chromatographic peak area of ​​the ligand decreases significantly, indicating that most of the ligands have been chelated. 175 Lu ions were present, with only trace amounts remaining. The retention time difference between the chromatographic peaks of the ligands and the chromatographic peaks of the coordination compounds was >0.15 min.

[0073] Please see Figure 11 , for ligand solution complexation 175 Lu gradient standard curve, where the horizontal axis is 175 Lu 3+ The molar ratio of the ligands is shown on the left, and the vertical axis represents the peak area of ​​the coordination compound. This shows that in the liquid chromatogram of the coordination compound formed by the ligands and metal ions, the peak area of ​​the coordination compound is related to the molar ratio of the ligands to the metal ions. 175 Lu 3+ The molar ratio of the ligands is positively correlated. Given the number of moles of the ligands, the number of moles of metal ions complexed with the ligands can be calculated by measuring the peak area of ​​the coordination compound.

[0074] Experimental Example 17-32 Prepare ligand solutions corresponding to the following structural formulas (5) to (20); Take 40 μL of 1 mg / mL ligand solution, 16 μL of 0.1 mol / L sodium acetate-glacial acetic acid buffer, and 130 μL of 0.1 mol / L sodium acetate solution and add them to 0.5 mL of 144 μL of 10 mg / mL ligand solution. 175The LuCl3 solution was mixed thoroughly to prepare experimental solution 1; 0.5 μL of experimental solution 1 was taken and the pH was measured to be 5.0. Experimental solution 1 was placed in a constant temperature mixer and reacted at 37°C for 60 min. After the reaction was completed, 100 μL was transferred to a liquid phase tube and 5 μg was taken for HPLC detection.

[0075] Equation (5) Equation (6) Equation (7) Equation (8) Equation (9) Equation (10) Equation (11) Equation (12) Equation (13)

[0076] Equation (14) Equation (15) Equation (16) Equation (17) Equation (18) Equation (19) Equation (20) Table 4

[0077] Please refer to Table 4. As can be seen from Table 4, the ligands with different structural formulas provided in this application can effectively complex metal ions to form coordination compounds, and obvious chromatographic peaks can be observed in the liquid chromatograms of the coordination compounds.

[0078] It should also be noted that the exemplary embodiments mentioned in this application describe methods, complexes, or kits based on a series of steps. However, this application is not limited to the order of the above steps; that is, the steps can be performed in the order mentioned in the embodiments, or in a different order, or several steps can be performed simultaneously.

[0079] The above are merely specific embodiments of this application. Those skilled in the art will clearly understand that, for the sake of convenience and brevity, the methods, complexes, and kits described above can be referred to the corresponding descriptions in the foregoing connective body embodiments, and will not be repeated here. It should be understood that the scope of protection of this application is not limited thereto. Any person skilled in the art can easily conceive of various equivalent modifications or substitutions within the scope of the technology disclosed in this application, and these modifications or substitutions should all be covered within the scope of protection of this application.

Claims

1. A ligand, characterized in that, The ligands are shown in either formula (1) or formula (2): Equation (1) Equation (2) R1 is selected from any one of H, a hydrocarbon group with 1 to 20 substituted or unsubstituted carbon atoms, an aromatic group with 6 to 14 substituted or unsubstituted cyclic atoms, or a heteroaromatic group with 5 to 14 substituted or unsubstituted cyclic atoms, and n is 1 to 12. The ligand is used to undergo a complexation reaction with metal ions to generate a coordination compound. Under the same chromatographic conditions, the retention time of the ligand and the retention time of the coordination compound are different.

2. The ligand according to claim 1, characterized in that, R1 is selected from any one of the following: alkane group with 1 to 15 substituted or unsubstituted carbon atoms, alkene group with 2 to 15 substituted or unsubstituted carbon atoms, alkyne group with 2 to 15 substituted or unsubstituted carbon atoms, and cycloalkyl group with 3 to 10 substituted or unsubstituted carbon atoms.

3. The ligand according to claim 1, characterized in that, R1 is selected from any one of an alkane group with 1 to 10 carbon atoms (substituted or unsubstituted) or an aromatic group with 6 to 10 cyclic atoms (substituted or unsubstituted).

4. The ligand according to claim 1, characterized in that, R1 is selected from any one of substituted or unsubstituted alkane groups having 1 to 6 carbon atoms, or substituted or unsubstituted phenyl groups.

5. The ligand according to claim 1, characterized in that, R1 is as shown in equation (3): Equation (3).

6. The application of the ligand as described in any one of claims 1 to 5 in detecting the residual amount of metal ions in the carrier molecule of a radiopharmaceutical.

7. A method for detecting residual metal ions, characterized in that, include: The test solution containing the carrier molecule is mixed and reacted with a salt solution containing metal ions, and then a detection solution containing the ligand of any one of claims 1 to 5 is added and mixed and reacted to obtain a reaction solution; The reaction solution was subjected to liquid chromatography analysis to obtain a liquid chromatogram; The results of metal ion residues in the test solution are obtained based on the difference between the chromatographic peaks of the coordination compound and the chromatographic peaks of the ligand in the liquid chromatogram.

8. The method for detecting residual metal ions according to claim 7, characterized in that, The step of obtaining the metal ion residue result in the test solution based on the difference between the chromatographic peak of the coordination compound and the chromatographic peak of the ligand in the liquid chromatogram includes: The residual amount of metal ions in the test solution is obtained based on the peak area of ​​the coordination compound in the liquid chromatogram.

9. The method for detecting residual metal ions according to claim 7, characterized in that, The step of mixing and reacting the test solution containing the carrier molecule with a salt solution containing metal ions, and then adding a detection solution containing the ligand as described in any one of claims 1 to 5 to react further, to obtain a reaction solution, comprises: The test solution is mixed with the salt solution, the pH is adjusted to 4.5~6.0, and the mixture is reacted at 37℃~95℃ for 30min~45min to obtain a mixed solution; The mixed solution is mixed with the detection solution, the pH is adjusted to 4.5~6.0, and the mixture is reacted at 37℃~95℃ for 30min~45min to obtain the reaction solution.

10. The method for detecting residual metal ions according to claim 9, characterized in that, The pH of the mixture of the test solution and the detection solution was adjusted using sodium acetate-glacial acetic acid buffer and sodium acetate solution.

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