Oxidation-responsive macrocycles, macrocyclic complexes and uses thereof

Abstract

The application discloses an oxidation-responsive macrocycle compound, a macrocycle complex and application thereof. The macrocycle compound has the following characteristics: 1) more than one macrocycle nucleus, the macrocycle nucleus has a ring structure containing carbon atoms and at least one nitrogen atom; 2) at least one nitrogen-containing aromatic ring group, the nitrogen-containing aromatic ring group exists as a part of the macrocycle nucleus or as an auxiliary pendant group; 3) at least one oxygen atom or oxygen-containing group, the oxygen atom or oxygen-containing group exists as a part of the macrocycle nucleus or an auxiliary pendant group; 4) at least one auxiliary pendant group, the auxiliary pendant group is coordinated with or not coordinated with an iron ion; and 5) the auxiliary pendant group is covalently attached to a nitrogen atom of the macrocycle nucleus. The iron complex formed by coordination of the macrocycle compound with a divalent or trivalent iron ion is the macrocycle complex according to the application. The macrocycle compound according to the application can be used as a T1 MRI contrast agent. The macrocycle complex provided by the application can also be used as a T2 MRI contrast agent.

Description

Technical Field

[0001] This invention belongs to the field of biomaterials technology. Specifically, this invention relates to a class of oxidation-responsive macrocyclic compounds, macrocyclic complexes and their applications. Background Technology

[0002] Since the 20th century, organometallic coordination chemistry has greatly promoted the development and application of magnetic resonance imaging (MRI) contrast agents. Gadolinium (Gd) has long been the preferred metal for paramagnetic contrast agents. Recent studies have increasingly shown that Gd… 3+ Inadequate renal clearance of gadolinium-based contrast agents in patients with severe renal insufficiency can lead to symptoms of nephrogenic systemic fibrosis. In patients receiving repeated doses of gadolinium contrast agents, enhanced MR imaging signals in deep brain tissue are dose-dependent. Subsequent studies in human and animal tissues have confirmed gadolinium deposition in brain tissue, raising concerns about long-term tolerance to gadolinium contrast agents. In recent years, with the development of high-field MRI imaging technology, the development of iron-based contrast agents composed of iron ions and ligand molecules has become a research direction for next-generation MRI contrast agents.

[0003] With the development of diagnostic technologies, medical imaging techniques must provide increasingly molecularly specific information. Biochemically specific MRI contrast agents modulate MR signals in the presence of specific biochemical features, enabling non-invasive detection and quantification of pathological changes at the molecular level. The ability to visualize biochemical changes through MRI allows for differential diagnosis, treatment planning, and recovery monitoring without the need for invasive biopsies or ionizing radiation imaging. Ideally, a biochemically specific contrast agent will activate and generate a signal in the presence of a specific biochemical target, but will produce almost no signal in surrounding tissues where the biochemical target is less abundant.

[0004] Redox balance is crucial for maintaining normal life activities in various organisms. Once biological tissues lose their redox buffering mechanisms, biochemical damage occurs to cells, exacerbating disease progression. Redox imbalance is characteristic of many diseases. For example, oxidative stress caused by elevated reactive oxygen species (ROS) levels is associated with a wide range of diseases, including ischemia / reperfusion injury, cardiovascular disease, atherosclerosis, diabetes, neurodegenerative diseases, aging, and inflammation; hypoxic environments are more common in malignant tumors and acute and chronic liver diseases. Therefore, abnormal redox states in tissues can be considered a valuable diagnostic marker for diseases.

[0005] Redox-active metal ion complexes hold great promise as biochemically specific contrast agents targeting tissue redox states. Iron (Fe) is a multivalent paramagnetic metal element that exhibits different magnetic properties in different oxidation states. When used as an MRI contrast agent, its ability to alter 1H relaxation signals—specifically, its relaxation degree—is significantly different. Therefore, Fe contrast agents show promising applications in responding to tissue redox states. Specifically, Fe... 2+ It can respond to oxidizing environments and be oxidized to Fe with high relaxation. 3+ This allows for the acquisition of MRI signals that are several times stronger. However, reported Fe complexes have limitations in terms of stability, biosafety, response efficiency, and rate, and to date, no metal complexes targeting tissue redox responses have entered clinical application. Summary of the Invention

[0006] To address the shortcomings and deficiencies of existing technologies, this invention provides a class of oxidation-responsive iron complexes that have the potential to be used in imaging specific to the oxidation state of biological tissues.

[0007] This invention provides a macrocyclic compound having the following characteristics:

[0008] 1) The macrocyclic compound has one or more macrocyclic nuclei, which have a ring structure containing a carbon atom and at least one heteroatom, and the ring structure is at least a nine-membered ring, and at least one of the heteroatoms is a nitrogen atom;

[0009] 2) The macrocyclic compound contains at least one nitrogen-containing aromatic ring group, which may be part of the macrocyclic core or exist as an auxiliary suspensory group; the number of nitrogen atoms in these nitrogen-containing aromatic ring groups may be 1 to 4, for example, the nitrogen-containing aromatic ring group may be one or more of pyrrole, oxazole, thiazole, imidazole, pyrazole, triazole, pyridine, pyrimidine, indole, quinoline, isoquinoline, purine, or derivatives of these groups;

[0010] 3) The macrocyclic compound contains at least one oxygen atom or oxygen-containing group, which may be part of the macrocyclic core or exist as an auxiliary suspensory group; when the oxygen-containing group is an auxiliary suspensory group, it may be one of hydroxyl and carboxyl groups; when there is more than one oxygen-containing auxiliary suspensory group, it may be one or more combinations of hydroxyl and carboxyl groups; when the macrocyclic compound is coordinated with iron (II / III) ions, the oxygen-containing groups may be deprotonated.

[0011] 4) According to points 1) to 3), the macrocyclic compound contains at least one auxiliary suspensory group, which is selected from one or more nitrogen-containing aromatic ring groups, oxygen-containing groups (hydroxyl, carboxyl or their protonated analogs), and these auxiliary suspensory groups may or may not be coordinated with iron (II / III) ions;

[0012] 5) The auxiliary suspensory group is covalently attached to the nitrogen atom of the macrocyclic nucleus.

[0013] Preferably, the macrocyclic core is a nine- to fifteen-membered ring; the macrocyclic core contains two or more heteroatoms (more preferably three to five), and these heteroatoms are separated by at least two carbon atoms.

[0014] Preferably, when the nitrogen-containing aromatic ring group is present as part of the macrocyclic core, the nitrogen-containing aromatic ring group is one or more of pyridine, pyrimidine, quinoline, isoquinoline or their derivatives.

[0015] Preferably, when a nitrogen-containing aromatic ring group is present as an auxiliary suspensory group, the nitrogen-containing aromatic ring group may be one or more of pyrrole, oxazole, thiazole, imidazole, pyrazole, triazole, pyridine, pyrimidine, indole, quinoline, isoquinoline, purine, or derivatives of these groups, and the macrocyclic core is a triazacycloalkane.

[0016] Preferably, when the oxygen-containing group serves as an auxiliary suspending group, each oxygen-containing suspending arm can independently be one of the following structures:

[0017]

[0018] Wherein, R is a substituted or unsubstituted alkyl group with a straight-chain or branched structure of C1 to C12, or a substituted or unsubstituted aryl group with 5 to 6 carbon atoms, or a PEG group (-CH2CH2O-). n (n = 1–12, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12), or naturally occurring (e.g., glycine) or synthetic amino acids or their analogues. When macrocyclic compounds are complexed with Fe(II / III) or at pH 7–14, some of the side-suspension donors in the oxygen-assisted side suspension, such as carboxylic acids or alcohols, can be deprotonated, and such protonation and deprotonation are within the scope of this invention.

[0019] Preferably, in some embodiments, the macrocyclic compound has the structure shown in formula (1) or formula (2):

[0020]

[0021] R1 is a substituted or unsubstituted nitrogen-containing aromatic ring group. ，The nitrogen-containing aromatic ring group is selected from pyrrole, oxazole, thiazole, imidazole, pyrazole, triazole, pyridine, pyrimidine, indole, quinoline, isoquinoline, purine, and their derivatives; n1, n2, and n3 are each independently 1 or 2; m1, m2, and m3 are each independently 1 or 2; Y1 and Y2 are each independently oxygen-containing auxiliary dangling groups; X1 is an oxygen atom or a nitrogen atom; L1, L2, and L3 are each independently a hydrogen atom or a substituent, and the substituent is selected from substituted or unsubstituted alkyl groups with straight or branched structures from C1 to C12, or PEG groups (-CH2CH2O-). n (n = 1–12, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12), or naturally occurring (e.g., glycine) or synthetic amino acids or their analogues.

[0022] Preferably, the macrocyclic compound may have more than one macrocyclic core, i.e., one or more macrocyclic cores are covalently linked together via a linker group (e.g., alkyl, aromatic, polypeptide, PEG (polyethylene glycol) derivative, etc.); the linker may be directly connected to the nitrogen atom of the macrocyclic core (e.g., as shown for 2PC2A-Car), or may have an intermediate methylene group (e.g., as shown for 2PC2A-B), or may be connected to a dangling group of the macrocyclic compound (e.g., as shown for 2NO2A-BP); the linker may have one or more coordinating groups or may be non-coordinating.

[0023] Preferably, in some embodiments, the macrocyclic compound has the following structure:

[0024]

[0025] Preferably, when a chiral carbon is present in the macrocyclic compound structure, the chiral carbon can have an R or S configuration.

[0026] Preferably, when the macrocyclic compound is coordinated with iron (II / III) ions, the macrocyclic compound can be referred to as a ligand;

[0027] When a macrocyclic compound coordinates with an iron (II / III) ion, at least one nitrogen atom on the nitrogen-containing aromatic ring group is coordinated with the iron (II / III) ion;

[0028] When a macrocyclic compound coordinates with an iron ion to form an iron complex, the ligand has 5 to 7 heteroatoms (one or more combinations of N, O, and S) that coordinate with the iron ion (e.g., for Fe-PyNO2A).

[0029] The preparation method of the macrocyclic compound described in this invention:

[0030] 1) When a nitrogen-containing aromatic ring group exists as an auxiliary suspending group, the synthesis of macrocyclic compounds generally uses a nitrogen heterocycle as a starting material, including but not limited to 1,4,7-triazacyclononane. First, the two secondary amines on the nitrogen heterocycle are protected with a tert-butoxycarbonyl group or other protecting group, leaving one secondary amine. Then, the nitrogen-containing aromatic ring group is modified on the remaining secondary amine through a reductive amination reaction or a nucleophilic substitution reaction. Next, the tert-butoxycarbonyl group or other protecting group is removed by a suitable method to obtain a monosubstituted nitrogen heterocycle structure. Finally, the oxygen-containing group is modified on the remaining secondary amine through a nucleophilic substitution reaction to obtain the target macrocyclic compound.

[0031] 2) When a nitrogen-containing aromatic ring group exists as part of a macrocyclic core, the synthesis of macrocyclic compounds generally uses nitrogen-containing aromatic ring compounds modified with halogen or aldehyde groups as raw materials. First, the compounds are cyclically formed with an alkane chain containing at least two secondary or primary amines through a reductive amination reaction or a nucleophilic substitution reaction. Then, oxygen-containing groups are modified on the amine to obtain the target macrocyclic compound.

[0032] A macrocyclic complex, wherein the macrocyclic complex is an iron complex formed by coordinating an iron (II / III) ion with a macrocyclic compound as a ligand described in this invention; the ligand has 5 to 7 heteroatoms (one or more combinations of N, O, and S) coordinated with the iron (II / III) ion; and at least one nitrogen atom in the nitrogen-containing aromatic ring group of the ligand is coordinated with the iron (II / III) ion.

[0033] Preferably, in some embodiments, the macrocyclic complex has the following structure:

[0034]

[0035] Preferably, the macrocyclic complex exhibits an oxidation peak potential of 0.2–0.5 V (relative to the standard hydrogen electrode (NHE)) in an aqueous medium with a pH of 6.5–7.5.

[0036] This invention provides imaging methods using the macrocyclic complexes and macrocyclic compounds described herein, utilizing magnetic resonance imaging (MRI). Non-limiting examples of such methods include magnetic resonance imaging (MRI). Specifically, the macrocyclic compounds of this invention can be used as T1 MRI contrast agents. The imaging methods of this invention can be used to image cells, tissues, organs, vascular systems, or portions thereof. The cells, tissues, organs, and vascular systems may be part of an individual. The macrocyclic complexes provided in this invention can also be used as T2 MRI contrast agents.

[0037] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0038] The macrocyclic complexes formed by the macrocyclic compounds of the present invention and iron ions have the characteristics of oxidation response. That is, when the complexes are dissolved in water or serum, the iron ions contained therein can be stabilized in the +2 oxidation state, while when oxidizing substances are present in the solution, such as hydrogen peroxide, oxygen free radicals, superoxide anions, peroxidase and other oxidants commonly found in animal tissues, the iron ions can be oxidized to the +3 oxidation state.

[0039] Certain macrocyclic complexes of this invention, including Fe-PyNO2A, Fe-CPyNO2A, and Fe-BPyNO2A, have had their binding constants (logK) determined. The results showed that, under conditions of pH 7.4 and containing 0.1 M KNO3, the logK of these macrocyclic complexes before oxidation was >17, and the logK after oxidation was >24, indicating that these macrocyclic complexes possess high stability. In preliminary toxicity studies, mice were injected with Fe-BPyNO2A via tail vein at a dose of 0.03 μmol (complex) / g (mice). No significant changes in mouse behavior were observed after injection, and the weight changes over two weeks were not significantly different compared to the control group (injected with physiological saline), indicating that Fe-BPyNO2A has high biocompatibility. Attached Figure Description

[0040] Figure 1 This is a flowchart illustrating the synthesis process of the Fe-PyNO2A complex of the present invention.

[0041] Figure 2 This is a flowchart illustrating the synthesis process of the Fe-CPyNO2A complex of the present invention.

[0042] Figure 3 This is a flowchart illustrating the synthesis of the Fe-BPyNO2A complex of the present invention.

[0043] Figure 4 PyNO2A ligand 1 1H NMR spectrum (400MHz, D2O as solvent).

[0044] Figure 5 PyNO2A ligand 13 C10 NMR spectrum (400 MHz, D2O as solvent).

[0045] Figure 6 This is the mass spectrum of the Fe-PyNO2A complex.

[0046] Figure 7 CPyNO2A ligand 1 1H NMR spectrum (400MHz, D2O as solvent).

[0047] Figure 8 CPyNO2A ligand 13C10 NMR spectrum (400 MHz, D2O as solvent).

[0048] Figure 9 This is the mass spectrum of the Fe-CPyNO2A complex.

[0049] Figure 10 BPyNO2A ligand 1 1H NMR spectrum (400MHz, D2O as solvent).

[0050] Figure 11 BPyNO2A ligand 13 C10 NMR spectrum (400 MHz, D2O as solvent).

[0051] Figure 12 This is the mass spectrum of the Fe-BPyNO2A complex.

[0052] Figure 13 The relaxation degree before and after the Fe-PyNO2A oxidation response.

[0053] Figure 14 The relaxation degree before and after the Fe-CPyNO2A oxidation response.

[0054] Figure 15 The relaxation degree before and after the Fe-BPyNO2A oxidation response.

[0055] Figure 16 The oxidation response curve of Fe-PyNO2A is shown.

[0056] Figure 17 The oxidation response curve of Fe-CPyNO2A is shown.

[0057] Figure 18 The oxidation response curve of Fe-BPyNO2A is shown.

[0058] Figure 19 MRI images of mice injected with the complex, (A) after injection of Fe 2+ -BPyNO2A, (B) Fe injection 3+ -BPyNO2A. Detailed Implementation

[0059] The present invention will be further described in detail below with reference to embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto. All raw materials involved in the present invention can be purchased directly from the market. For process parameters not specifically specified, conventional techniques can be referred to.

[0060] Example 1: Preparation of Fe-PyNO2A

[0061] The preparation of the complex Fe-PyNO2A was carried out according to... Figure 1The process shown is as follows, with specific steps as follows:

[0062] (1) Synthesis of ligand 2,2'-(7-(pyridin-2-ylmethyl)-1,4,7-triaza-1,4-diyl)diacetic acid

[0063] Add 3 mL of saturated KOH solution and 2 mL of water to a 100 mL round-bottom flask containing 1.00 g of 1,4,7-triazacyclononane trihydrochloride, then add 30 mL of dichloromethane and stir the mixture vigorously. Next, add 1.80 g of N-succinimide tert-butyl carbonate dissolved in 15 mL of dichloromethane dropwise. After the addition is complete, pour the mixture into a separatory funnel, wash three times with 30 mL of 2 mol / L sodium dihydrogen phosphate solution, collect the organic phase, and extract three times with 30 mL of 5 mol / L citric acid solution. Collect the aqueous phase, add an appropriate amount of saturated KOH solution to the aqueous phase to adjust the alkali, then add 50 mL of dichloromethane for extraction, collect the organic phase, add 2 g of anhydrous Na₂SO₄ to the organic phase for drying, filter, and concentrate under reduced pressure to obtain di-tert-butyl 1,4,7-triazacyclononane-1,4-dicarboxylic acid.

[0064] 0.66 g of 1,4,7-triaza-1,4-dicarboxylic acid di-tert-butyl ester and 0.66 g of 2-chloromethylpyridine were added to 20 mL of acetonitrile, followed by 1 mL of triethylamine solution. The mixture was stirred at room temperature for 24 h, and then the solvent was evaporated. 20 mL of dichloromethane was added to dissolve the solvent, and the mixture was poured into a separatory funnel. The solution was washed three times with 30 mL of saturated sodium bicarbonate solution and then twice with saturated brine. The organic phase was collected, and 2 g of anhydrous Na2SO4 was added to the organic phase for drying. After filtration, the solution was evaporated to dryness to obtain 7-(pyridin-2-ylmethyl)-1,4,7-triaza-1,4-dicarboxylic acid di-tert-butyl ester.

[0065] 0.63 g of 7-(pyridin-2-ylmethyl)-1,4,7-triazaalkyl-1,4-dicarboxylic acid ditert-butyl ester was dissolved in 5 mL of ethyl acetate, and then 0.5 mL of concentrated hydrochloric acid was added dropwise. The mixture was stirred at room temperature for 3 h, and a solid precipitated out. The solid was collected by centrifugation, and then washed three times with 5 mL of ethyl acetate. The solid was dried under vacuum to obtain 1-(pyridin-2-ylmethyl)-1,4,7-triazacyclononane hydrochloride.

[0066] 0.36 g of 1-(pyridin-2-ylmethyl)-1,4,7-triazacyclononane hydrochloride and 1 mL of triethylamine were added to 20 mL of acetonitrile, followed by the addition of 200 μL of tert-butyl bromoacetate solution. The mixture was stirred at room temperature for 6 h, and the solvent was removed by rotary evaporation. 20 mL of dichloromethane was then added to dissolve the solvent. The mixture was poured into a separatory funnel, washed three times with 30 mL of saturated sodium bicarbonate solution, and then washed twice with saturated brine. The organic phase was collected, and 2 g of anhydrous Na2SO4 was added to the organic phase for drying. After filtration, the crude product was obtained by rotary evaporation and purified by column chromatography to give di-tert-butyl 2,2'-(7-(pyridin-2-ylmethyl)-1,4,7-triazacyclo-1,4-diyl)diacetate.

[0067] 0.36 g of di-tert-butyl 2,2'-(7-(pyridin-2-ylmethyl)-1,4,7-triazapan-1,4-diyl)diacetate was dissolved in 5 mL of ethyl acetate, and 0.5 mL of concentrated hydrochloric acid was added dropwise. The mixture was stirred at room temperature for 3 h, and a solid precipitated. The solid was collected by centrifugation, washed three times with 5 mL of ethyl acetate, and dried under vacuum to obtain 2,2'-(7-(pyridin-2-ylmethyl)-1,4,7-triazapan-1,4-diyl)diacetate hydrochloride. The obtained ligands 1 H-NMR spectrum as follows Figure 4 As shown, 13 C-NMR spectrum as follows Figure 5 As shown.

[0068] (2) Synthesis of Fe-PyNO2A complex

[0069] 0.38 g of 2,2'-(7-(pyridin-2-ylmethyl)-1,4,7-triaza-1,4-diyl)diacetic acid hydrochloride was dissolved in 2 mL of water. Under N2 protection, sufficient ferrous hydroxide or ferric hydroxide solid was added, and the mixture was stirred at room temperature until the solid was completely dissolved. The pH was then adjusted to 7-8 with 1 mol / L NaOH solution. The mixture was filtered, and the filtrate was collected. This filtrate contained a Fe-PyNO2A complex. ESI-MS: m / z = 393 [M+H] + 415[M+Na] + ,like Figure 6 As shown.

[0070] Example 2: Preparation of Fe-CPyNO2A

[0071] The preparation of the complex Fe-CPyNO2A was carried out according to... Figure 2 The process shown is as follows, with specific steps as follows:

[0072] (1) Synthesis of ligand 2,2'-(7-(pyridin-2-ylmethyl)-1,4,7-triaza-1,4-diyl)diacetic acid

[0073] A 100 mL round-bottom flask was placed in an ice-water bath. 0.924 g of dimethyl pyridine-2,5-dicarboxylate was added, followed by 0.447 g of sodium borohydride, 2.079 g of calcium chloride, 9.9 mL of tetrahydrofuran, and 20.1 mL of anhydrous ethanol. The mixture was stirred at 0 °C for 1 h, then quenched with 15 mL of water, and stirring continued for 0.5 h. The mixture was poured into a separatory funnel and extracted three times with 50 mL of dichloromethane. The organic phase was collected, dried over 2 g of anhydrous Na₂SO₄, filtered, and concentrated by rotary evaporation to approximately 50 mL. 1.5 mL of thionyl chloride was added, and the mixture was stirred overnight at room temperature. 15 mL of saturated brine was added, and the mixture was stirred for 2 hours. An appropriate amount of Na₂CO₃ was added to adjust the solution to alkaline, and the mixture was separated. The organic phase was washed three times with saturated brine and evaporated to dryness to obtain methyl 6-(chloromethyl)nicotinic acid.

[0074] 0.66 g of 1,4,7-triaza-1,4-dicarboxylic acid di-tert-butyl ester (prepared according to the method described in Example 1) was added to 30 mL of acetonitrile, followed by 1 mL of triethylamine and 0.55 g of 6-(chloromethyl)nicotinic acid methyl ester. The mixture was stirred at room temperature for 8 h, then poured into a separatory funnel, washed three times with saturated sodium bicarbonate solution, extracted with 50 mL of dichloromethane, separated and collected, dried with 2 g of anhydrous Na₂SO₄, and filtered. The filtrate was then evaporated to obtain 7-((5-(methoxycarbonyl)pyridin-2-ylmethyl)-1,4,7-triazacyclononane-1,4-dicarboxylic acid ditert-butyl ester. This ester was then dissolved in 5 mL of ethyl acetate, and 0.5 mL of concentrated hydrochloric acid was added dropwise. The mixture was stirred at room temperature for 3 h, and a solid precipitated out. The solid was collected by centrifugation, washed three times with 5 mL of ethyl acetate, and dried under vacuum to obtain 6-((1,4,7-triazacyclononane-1-yl)methyl)nicotinic acid methyl ester hydrochloride.

[0075] 0.40 g of methyl 6-((1,4,7-triazacyclononan-1-yl)methyl)nicotinic acid hydrochloride and 1 mL of triethylamine were added to 20 mL of acetonitrile, followed by the addition of 200 μL of tert-butyl bromoacetate solution. The mixture was stirred at room temperature for 6 h, and the solvent was removed by rotary evaporation. 20 mL of dichloromethane was then added to dissolve the solvent. The mixture was poured into a separatory funnel, washed three times with 30 mL of saturated sodium bicarbonate solution, and then washed twice with saturated brine. The organic phase was collected, and 2 g of anhydrous Na2SO4 was added to the organic phase for drying. After filtration, the crude product was obtained by rotary evaporation and purified by column chromatography to give di-tert-butyl 2,2'-(7-((5-(methoxycarbonyl)pyridin-2-ylmethyl)-1,4,7-triazacyclo-1,4-diyl)diacetic acid.

[0076] 0.40 g of 2,2'-(7-((5-(methoxycarbonyl)pyridin-2-ylmethyl)-1,4,7-triazapan-1,4-diyl)diacetic acid ditert-butyl ester was dissolved in 5 mL of ethyl acetate. 0.5 mL of concentrated hydrochloric acid was added dropwise, and the mixture was stirred at room temperature for 3 h. A solid precipitated, which was collected by centrifugation. The solid was then washed three times with 5 mL of ethyl acetate and dried under vacuum to obtain 2,2'-(7-(5-(methoxycarbonyl)pyridin-2-ylmethyl)-1,4,7-triazapan-1,4-diyl)diacetic acid hydrochloride. The obtained ligand... 1 H-NMR spectrum as follows Figure 7 As shown, 13 C-NMR spectrum as follows Figure 8 As shown.

[0077] (2) Synthesis of Fe-CPyNO2A complex

[0078] 0.40 g of 2,2'-(7-(5-(methoxycarbonyl)pyridin-2-ylmethyl)-1,4,7-triazaalkyl-1,4-diyl)diacetic acid hydrochloride was dissolved in 2 mL of water. Under N2 protection, sufficient ferrous hydroxide or ferric hydroxide solid was added, and the mixture was stirred at room temperature until the solid was completely dissolved. The pH was then adjusted to 7-8 with 1 mol / L NaOH solution. The solution was filtered to obtain a filtrate containing the Fe-CPyNO2A complex. ESI-MS: m / z = 449 [M+H] + ,like Figure 9 As shown.

[0079] Example 3: Preparation of Fe-BPyNO2A Complex

[0080] The preparation of the complex Fe-BPyNO2A was carried out according to... Figure 3 The process shown is as follows, with specific steps as follows:

[0081] (1) Synthesis of ligand 2,2'-(7-(pyridin-2-ylmethyl)-1,4,7-triaza-1,4-diyl)diacetic acid

[0082] 1 g of 2-hydroxymethylpyridine-3-ol was dissolved in 20 mL of anhydrous ethanol, and then 0.7 g of potassium hydroxide and 1.06 g of benzyl bromide were added. The mixture was refluxed at 90 °C for 24 hours, then cooled to room temperature, poured into a separatory funnel, and 50 mL of saturated saline solution was added. Then, 50 mL of dichloromethane was added for extraction. The dichloromethane layer was washed twice with saturated saline solution. The dichloromethane layer was collected, dried with 2 g of anhydrous Na₂SO₄, filtered, and concentrated to 10 mL. 1 mL of thionyl chloride was added, and the mixture was reacted at room temperature for 12 hours. Then, 10 mL of saturated saline solution was added, and stirring was continued for 0.5 h. The mixture was then poured into a separatory funnel for separation. The organic layer was washed twice with saturated saline solution and once with saturated sodium carbonate solution. The organic layer was collected, dried with 2 g of anhydrous Na₂SO₄, evaporated to dryness, and purified by column chromatography to obtain 3-benzyloxy-2-chloromethylpyridine.

[0083] 0.66 g of 1,4,7-triaza-1,4-dicarboxylic acid di-tert-butyl ester (prepared according to the method described in Example 1) was added to 30 mL of acetonitrile, followed by 1 mL of triethylamine and 0.70 g of 3-benzyloxy-2-chloromethylpyridine. The mixture was stirred at room temperature for 8 h, then poured into a separatory funnel, washed with saturated sodium bicarbonate solution, extracted with 50 mL of dichloromethane, separated and collected, dried with 2 g of anhydrous Na₂SO₄, filtered, and then rotated. The filtrate was dried to obtain 7-((3-(benzyloxy)pyridin-2-ylmethyl)-1,4,7-triazaalkyl-1,4-dicarboxylic acid ditert-butyl ester. This product was dissolved in 5 mL of ethyl acetate, and 0.5 mL of concentrated hydrochloric acid was added dropwise. The mixture was stirred at room temperature for 3 h, and a solid precipitated. The solid was collected by centrifugation, washed three times with 5 mL of ethyl acetate, and dried under vacuum to obtain 1-{[3-(benzyloxy)pyridin-2-yl]methyl}-1,4,7-triazacyclononane hydrochloride.

[0084] 0.45 g of 1-{[3-(benzyloxy)pyridin-2-yl]methyl}-1,4,7-triazacyclononane hydrochloride and 1 mL of triethylamine were added to 20 mL of acetonitrile, followed by the addition of 200 μL of tert-butyl bromoacetate solution. The mixture was stirred at room temperature for 6 h, and the solvent was removed by rotary evaporation. 20 mL of dichloromethane was then added to dissolve the solvent. The mixture was poured into a separatory funnel, washed three times with 30 mL of saturated sodium bicarbonate solution, and then washed twice with saturated brine. The organic phase was collected, and 2 g of anhydrous Na2SO4 was added to the organic phase for drying. After filtration, the crude product was obtained by rotary evaporation and purified by column chromatography to obtain 2,2'-(7-((3-(benzyloxy)pyridin-2-ylmethyl)-1,4,7-triazaalkyl-1,4-diyl)ditert-butyl diacetate.

[0085] 0.40 g of 2,2'-(7-((3-(benzyloxy)pyridin-2-methyl)-1,4,7-triazapan-1,4-diyl)diacetic acid ditert-butyl ester was added to 5 mL of ethyl acetate solution, and 0.5 mL of concentrated hydrochloric acid was added dropwise. The mixture was stirred at room temperature for 3 h, and a solid precipitated. The solid was collected by centrifugation, washed three times with 5 mL of ethyl acetate, and dried under vacuum to obtain 2,2'-(7-(3-(benzyloxy)pyridin-2-methyl)-1,4,7-triazapan-1,4-diyl)diacetic acid hydrochloride. The obtained ligand 1 H-NMR spectrum as follows Figure 10 As shown, 13 C-NMR spectrum as follows Figure 11 As shown.

[0086] (2) Synthesis of Fe-BPyNO2A complex

[0087] 0.40 g of 2,2'-(7-(3-(benzyloxy)pyridin-2-methyl)-1,4,7-triaza-1,4-diyl)diacetic acid hydrochloride was dissolved in 2 mL of water. Under N2 protection, sufficient ferrous hydroxide or ferric hydroxide solid was added, and the mixture was stirred at room temperature until the solid was completely dissolved. The pH was then adjusted to 7-8 with 1 mol / L NaOH solution. The solution was filtered to obtain a filtrate containing the Fe-CPyNO2A complex. ESI-MS: m / z = 496 [M+H] + ,519[M+H+Na] + ,like Figure 12 As shown.

[0088] Example 4: Response of the complex to H2O2

[0089] In this embodiment, the relaxation time was measured using a relaxation analyzer with a 0.5T field strength. The complex (Fe-PyNO2A, Fe-CPyNO2A, or Fe-BPyNO2A) was dissolved in water to prepare a 1.5 mmol / L solution with a volume of 10 mL. The initial relaxation time of the solution was measured using the relaxation analyzer, where the T1 relaxation time was determined using inversion recovery sequencing and the T2 relaxation time was determined using CPMG sequencing. Then, 100 μL of H2O2 solution was added to the complex solution to bring the final concentration to 5 mmol / L. The T2 relaxation time of the complex solution at different time points after the addition of H2O2 solution was measured. The relaxation rate (R) was defined as the reciprocal of the relaxation time. The R2 values ​​of the solution at different time points after the addition of H2O2 are shown below. Figure 16 , 17 As shown in Figure 18. Additionally, the unoxidized complex (Fe) 2+ -PyNO2A or Fe 2+ -CPyNO2A or Fe 2+-BPyNO2A) and the oxidized complex (Fe) 3+ -PyNO2A or Fe 3+ -CPyNO2A or Fe 3+ -BPyNO2A) was prepared into aqueous solutions of different concentrations, and its T1 was measured to obtain the relationship between the relaxation rate (R1) of the solution and the concentration of the complex, as shown in the figure. Figure 13 , 14 As shown in Figure 15.

[0090] Example 5: Application in mouse MRI imaging

[0091] The MRI images in this embodiment were obtained using a 1.0T MRI scanner, and the sequence was a spin echo (SE) sequence with the following parameters: 32℃, TR / TE = 150ms / 18.2ms, NS = 4, and field of view (FOV) = 100×100mm. 2 Layer thickness = 1.0 mm, resolution 256×256. Complex (Fe) 2+ -BPyNO2A or Fe 3+ -BPyNO2A) was dissolved in physiological saline to prepare a 5 mmol / L solution. 40g mice were anesthetized, and then 150 μL of the complex solution was injected via the tail vein. The injected mice were then placed in the imaging chamber of an MRI scanner for imaging, with images taken every 15 minutes. Because Fe... 2+ -BPyNO2A and Fe 3 + -BPyNO2A has liver-targeting characteristics, so after the complex is injected into mice, it mainly accumulates in the liver tissue and is gradually excreted into the gallbladder. Figure 19 This is a coronal MRI image of mice 1 hour after injection of the complex. The image shows the injection of Fe... 2+ -BPyNO2A after ( Figure 19 In mice A), the changes in liver and gallbladder brightness were not significant, while those injected with Fe... 3+ -BPyNO2A after ( Figure 19 The MRI signals of the liver and gallbladder of mice in group B) were significantly enhanced, indicating that the imaging ability of the complex before and after oxidation was significantly different.

[0092] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A macrocyclic complex, characterized in that, The macrocyclic complex has the following structure: 。 2. The use of the macrocyclic complex according to claim 1 in the preparation of MRI contrast agents.

Images