A derivative of deferasirox, a radionuclide label and a preparation method and application thereof

Abstract

The application discloses a deferasirox derivative, a nuclide marker and a preparation method and application thereof. The deferasirox derivative is connected with a deferasirox residue, a linker and a labeling group in a specific chemical form. The application further provides a nuclide marker based on the structure of the compound. The compound and the nuclide marker can be enriched in inflammatory or tumor lesion tissues, and can be specifically recognized by bacteria, thereby accurately diagnosing and positioning early bacterial infection. The application further provides a use of the nuclide marker in living animals for distinguishing bacterial infection and sterile inflammation. The nuclide marker has the advantages of simple labeling, good stability, high specificity and the like, and is suitable for industrial production and clinical promotion.

Description

Technical Field

[0001] This invention relates to the field of bacterial targeting compounds and radiopharmaceutical technology, specifically to a derarosi derivative, a radionuclide marker, its preparation method, and its application. Background Technology

[0002] Infections caused by pathogenic bacteria pose a significant threat to human health, resulting in high morbidity and mortality rates. This situation is exacerbated by rising antimicrobial resistance, rendering existing treatments ineffective, while the pipeline for developing new antibiotics capable of overcoming resistance remains scarce. Timely and accurate detection and localization of the infecting pathogen are crucial for avoiding unnecessary antibiotic use and finding appropriate, potentially life-saving treatments.

[0003] Positive bacterial cultures from suspected sites (tissue and / or blood) are currently the gold standard for diagnosing specific bacterial infections. This type of method has several limitations: (a) it can be invasive; (b) it cannot determine the extent of infection dissemination; (c) it lacks sensitivity, often leading to negative results; (d) it is usually only identified when the infection has reached the systemic stage; and (e) the time required to obtain results often leads to complications, including death. Molecular imaging can achieve specific, sensitive, and location-specific imaging of infections, especially single-light computed tomography (SPECT) and positron emission tomography (PET) based on radiotracers. Many nonspecific radiotracers are already used clinically for infection imaging. These probes (including […]) 18 F]Fluorodeoxyglucose([ 18 F]FDG),[ 68 Ga] Citric acid salt ([ 68 Ga's citric acid primarily targets secondary effects of infection, such as increased blood flow and vascular permeability, endothelial cell activation, or polymorphonuclear cell migration. However, it cannot differentiate between bacterial infection and aseptic inflammation.

[0004] Therefore, developing bacterial-specific targeting molecular probes to differentiate between bacterial infections and aseptic inflammation is a pressing scientific problem. Recent development of pathogen-specific tracers has focused on specific bacterial pathways, including carbohydrate metabolism, bacterial folic acid biosynthesis and unique bacterial iron transport systems, bacterial cell wall components and selective binders, substrates for intracellular bacterial proteins, and antimicrobial peptides. However, no bacterial-specific targeting molecular probe has yet been approved for clinical use, and there is still room for improvement in probe sensitivity and pharmacokinetic properties.

[0005] Ferric ions (Fe) 3+Fe is a micronutrient essential for the survival and replication of bacteria and fungi, involved in bacterial growth and biofilm formation. Bacteria and fungi secrete specific chelating agents called siderophores to capture Fe from the surrounding environment. 3+ The Fe produced as a result 3+ The complex is very stable and can be recognized, absorbed, and internalized by active transport proteins in bacteria, resulting in a high concentration of iron ions at the site of bacterial infection. 4-[3,5-di(2-hydroxyphenyl)-1,2,4-triazol-1-yl]benzoic acid (delararosse) is an FDA-approved Fe... 3+ Complexing groups are used to treat iron overload. Recent studies have demonstrated that deferasirox has significant antibacterial and antifungal activity, primarily through its mechanism of action by competing for Fe in bacterial siderophores. 3+ This is to inhibit the formation of bacterial cell walls and biofilms. Among existing bacterial-targeted radioactive imaging agents, there is currently no molecular imaging probe constructed using derarosi as the parent structure and used for in vivo imaging. Summary of the Invention

[0006] To overcome the above problems, this invention uses deferrosi as a lead compound and obtains deferrosi derivatives and their radionuclide markers through structural optimization. These derivatives provide support for the diagnosis of bacterial infections through medical imaging, which helps to differentiate between bacterial infections and aseptic inflammation in clinical practice, further reduces the overuse of antibiotics, and achieves the goal of precision medicine.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] This invention provides a derafloxacin derivative with the structural formula: R1-R2-R3;

[0009] Where R1 is

[0010] R2 is a linking agent, which is selected from linking group 1 or linking group 2;

[0011] The linking group 1 is selected from any of the following structures:

[0012]

[0013] Where n and m are independent integers between 0 and 10; in the above structure, the left wavy line connects to R1, and the right wavy line connects to R3.

[0014] The linking group 2 is selected from any of the following structures:

[0015]

[0016] Where x and y are independent integers between 0 and 10; in the above structural formula, the left wavy line connects to R1, and the right wavy line connects to R3.

[0017] The L1 and L2 groups in linking groups 1 and 2 can be independently selected to be present or absent. For ease of understanding, an example is provided here: when L1 is absent, the -NH- group connected to L1 is directly connected to R1; when L2 is absent, -CO- is directly connected to -OH to form a carboxyl group.

[0018] When L1 is present, L1 can be independently selected from any one or a combination of the following amino acid fragments:

[0019]

[0020] Where a, b, and t are independent integers between 0 and 10; in the above structural formula, the imino terminus (-NH-) is connected to R1. When L1 is a combination of several segments among the above amino acid fragments, the specific combination method is as follows: the carbonyl terminus (-CO-) of one amino acid fragment is connected to the imino terminus (-NH-) of another amino acid fragment, and so on, forming a multi-segment L1. The groups that need to be linked at both ends of L1 are the imino terminus (-NH-) and the carbonyl terminus (-CO-), respectively, wherein the imino terminus (-NH-) is connected to R1.

[0021] When L2 is present, L2 can be independently selected from any one or a combination of the following amino acid fragments:

[0022]

[0023] Where a, b, and t are independent integers between 0 and 10; in the above structural formula, the carbonyl end (-CO-) is connected to the hydroxyl end (-OH) in the first structural formula of linking group 1 and linking group 2. When L2 is a combination of several fragments among the above amino acid fragments, the specific combination method is as follows: the carbonyl end (-CO-) of one amino acid fragment is connected to the imino end (-NH-) of another amino acid fragment, and so on, forming a multi-fragment L2. The groups that need to be linked at both ends of L2 are the imino end (-NH-) and the carbonyl end (-CO-), where the carbonyl end (-CO-) is connected to the hydroxyl end (-OH) in the first structural formula of linking group 1 and linking group 2.

[0024] R3 is a labeling group. When R2 is a linking group 1, R3 is selected from either chelating structure 1 or a fluorescent group.

[0025] The chelation structure 1 includes:

[0026]

[0027]

[0028] The fluorescent group includes:

[0029]

[0030] When R2 is a linking group 2, R3 is selected from any one of the following chelate structures 2:

[0031]

[0032] As one possible implementation, further, when R1 in equation I is When R3 represents different chelating structures and R2 represents different linking agents, the deferasirox derivatives include the following structures:

[0033]

[0034]

[0035]

[0036]

[0037]

[0038] Based on the above, the present invention provides a method for preparing a deferasirox derivative, as detailed below:

[0039] When R2 is a linking group 1, the method for preparing the delarosi derivative is either method 1 or method 2.

[0040] Method 1 includes the following steps:

[0041] The carboxyl group of compound 1 is activated to form compound 2, which is an ester with a carboxyl group activation.

[0042] Compound 2 was subjected to a first substitution reaction with compound 3, which has an A1 protecting group, to give compound 4;

[0043] Compound 4 was subjected to a deprotection reaction to generate compound 5;

[0044] The derarosi derivative was obtained by reacting compound 5 with an active compound R3 containing an activated ester group, an anhydride group, or an isothiocyanate group in a second substitution reaction.

[0045] The structural formulas of compounds 1 to 5 are as follows:

[0046]

[0047] Method 2 includes the following steps:

[0048] Compound 7 is obtained by reacting the R3 active compound with the A2 protecting group with the compound 6 via a third substitution reaction.

[0049] Compound 7 was subjected to a deprotection reaction to generate compound 8;

[0050] Compound 2 and compound 8 were subjected to a fourth substitution reaction to obtain the derarosi derivative;

[0051] The structural formulas of compounds 6 to 8 are as follows:

[0052]

[0053] The structure of the R3 active compound containing an activated ester group, an anhydride group, or an isothiocyanate group includes:

[0054]

[0055]

[0056] When R2 is a linking group 2, the method for preparing the derafloxacin derivative includes the following steps:

[0057] Compound 10 was obtained by reacting the active compound R3 containing a maleimide fragment with compound 9 containing a thiol group;

[0058] Compound 2 was reacted with compound 10 to obtain the derafloxacin derivative;

[0059] The structural formulas of compounds 9 to 10 are as follows:

[0060]

[0061] The structure of the R3 active compound with the maleimide fragment is shown below:

[0062]

[0063] The A1 protecting group and the A2 protecting group are selected from any one of the Boc protecting group, DDE protecting group or Fmoc protecting group.

[0064] Based on the above, the present invention also provides a radionuclide-labeled derarosi derivative, which has the structural formula: R1-R2-R4, wherein R4 is a radionuclide labeling group;

[0065] The radionuclide-labeled delarosine derivative R1-R2-R4 is obtained by coordinating the R3 chelate structure of the aforementioned delarosine derivative R1-R2-R3 with a radionuclide.

[0066] Preferably, the nuclide includes 18 F, 43 Sc、 44 Sc、 47 Sc、 51 Cr 55 Co、 57 Co、 62 Cu、 64 Cu、 67 Cu、 67 Ga、 68 Ga、 72 As、 72 Se、 89 Zr、 86 Y、 89 Sr, 86 Y、 90 Y、 97 Ru、 99m Tc, 105 Rh、 101m Rh、 109 Pd, 111 In、 119 Sb, 128 Ba、 139 La、 140 La、 142 Pr、 149 Pm, 149 Tb, 151 Tb, 151 Eu、 153 Eu、 169 Eu、 153 Sm、 152 Gd, 153 Gd, 157 Gd, 159 Gd, 161 Tb, 165 Dy、 166 Ho、 169 Er、 175 Yb、 177 Lu、 186 Re、 188 Re、 197 Hg, 198 Au、 201 Tl、 203 Pb, 211 At、 212 Pb, 212 Bi、 213 Bi、 223 Ra、 227 Th and 225 At least one of Ac.

[0067] Based on the above, the present invention also provides a method for preparing a radionuclide-labeled derarosi derivative, which is prepared by wet method or freeze-drying method;

[0068] The wet preparation process includes the following steps:

[0069] The deferasirox derivative and / or its pharmaceutically acceptable form are dissolved in a solvent; then a solution containing a radionuclide is added, and the mixture is reacted at room temperature to 110°C for 10 to 30 minutes to obtain a radionuclide-labeled deferasirox derivative solution.

[0070] The freeze-drying preparation steps are as follows:

[0071] The deferasirox derivative and / or its pharmaceutically acceptable form are dissolved in a solvent, dispensed into lyophilized containers, freeze-dried, and then sealed into lyophilized kits. A solvent is added to the lyophilized kits to dissolve the derivatives, followed by the addition of a solution containing a radionuclide, and the mixture is reacted at room temperature to 110°C for 10 to 30 minutes to obtain a radionuclide-labeled deferasirox derivative solution.

[0072] Furthermore, the pharmaceutically acceptable forms of the deferasirox derivatives include salts, stereoisomers, racemates, hydrates, and solvates.

[0073] The present invention also provides a pharmaceutical composition comprising an active ingredient and pharmaceutically acceptable excipients;

[0074] The active ingredient includes the deferasirox derivative and its pharmaceutically acceptable form, as well as the radionuclide-labeled deferasirox derivative and its pharmaceutically acceptable form.

[0075] Furthermore, the pharmaceutically acceptable forms of the deferasirox derivatives include salts, stereoisomers, racemates, hydrates, and solvates;

[0076] The pharmaceutically acceptable forms of the radionuclide-labeled derarosi derivatives include salts, stereoisomers, racemates, hydrates, and solvates.

[0077] This invention also proposes the use of the above-mentioned deferasirox derivatives and their pharmaceutically acceptable forms, the above-mentioned radionuclide-labeled deferasirox derivatives and their pharmaceutically acceptable forms, and the use of the above-mentioned pharmaceutical compositions in the preparation of diagnostic reagents or therapeutic drugs for human or animal diseases. Further, the diseases include tumors and their metastases, inflammation, and bacterial infections.

[0078] The diagnostic reagents or drugs can be used to differentiate between bacterial infections and aseptic inflammatory sites; the diagnostic reagents or drugs are prepared as injectables and administered via intravenous injection; and medical imaging provides support for the diagnosis of bacterial infections.

[0079] The beneficial effects of this invention are as follows:

[0080] 1) This invention creatively proposes to use 4-[3,5-di(2-hydroxyphenyl)-1,2,4-triazol-1-yl]benzoic acid (derarosi) as a clinically used iron chelator, as a broad-spectrum bacterial-specific recognition molecule. Based on this, the structure is modified, optimized and labeled with radionuclides to obtain a molecular imaging probe with excellent performance and suitable in vivo pharmacokinetic properties.

[0081] 2) This invention proposes a class of radionuclide-labeled derarosi derivatives, whose ligands have the characteristics of strong labeling ability, short labeling time and high labeling yield, which is conducive to commercial application and clinical promotion.

[0082] 3) The radionuclide-labeled derarosi derivative provided by this invention can effectively detect Escherichia coli and Staphylococcus aureus infection areas in mammalian models of bacterial infection, and effectively distinguish between live bacterial infection and aseptic inflammation, providing an effective reference for clinical bacterial inflammation diagnosis, which is conducive to reducing the overuse of antibiotics and meets the needs of today's precision medicine.

[0083] 4) The deferasirox derivative designed in this invention introduces linking units such as amino acids between the targeting group and the coordination structure, which can increase the distance between the targeting group and the coordination structure, avoiding mutual interference. Simultaneously, the linking structure described in this invention provides a good basis for regulating the lipophilicity of the molecule, improving the pharmacokinetic properties of the complex, accelerating its clearance rate in non-target tissues, increasing the target / non-target ratio, enhancing the affinity between the ligand and the receptor, and improving the lesion uptake of the radionuclide-labeled deferasirox derivative, resulting in clearer imaging and better diagnostic effects. This effectively expands the application scope of the radionuclide-labeled deferasirox derivative described in this invention. Attached Figure Description

[0084] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0085] Figure 1 This is the mass spectrum of intermediate 1 of the compound in this invention.

[0086] Figure 2 This is the mass spectrum of intermediate 2 of the compound in this invention.

[0087] Figure 3 This is the mass spectrum of intermediate 3 of the compound in this invention.

[0088] Figure 4 This is the NMR spectrum of compound intermediate 3 in this invention.

[0089] Figure 5 This is the mass spectrum of compound DFC2D in this invention.

[0090] Figure 6 This is the mass spectrum of compound DFC2F in this invention.

[0091] Figure 7 This is the mass spectrum of compound DFY1D in this invention.

[0092] Figure 8 This is the mass spectrum of compound DFA2D in this invention.

[0093] Figure 9 The HPLC spectra of DFC2D, DFY1D, and DFA2D are shown.

[0094] Figure 10 for[ 68 Ga]Ga-DFC2D、[ 68 Ga]Ga-DFY1D and [ 68 HPLC spectrum of Ga]Ga-DFA2D.

[0095] Figure 11 DFX, DFC2D, DFY1D and DFA2D with different concentrations of Fe 3+ Trend of UV absorption after complexation.

[0096] Figure 12 for[ 68 Ga]Ga-DFC2D、[ 68 Ga]Ga-DFY1D and [ 68 HPLC identification chromatogram of the stability of Ga]Ga-DFA2D in physiological saline and serum.

[0097] Figure 13 for[ 68 Ga]Ga-DFC2D、[ 68 Ga]Ga-DFY1D and [ 68 The uptake of Ga]Ga-DFA2D by Escherichia coli, Staphylococcus aureus, and inactivated bacteria.

[0098] Figure 14 DFC2F was generated in E. coli and the transporter knockout E. coli (E. coli Δ).

[0099] EntA / B) bacterial fluorescence image.

[0100] Figure 15for[ 68 Ga]Ga-DFC2D、[ 68 Ga]Ga-DFY1D and [ 68 60-minute MicroPET imaging of Ga]Ga-DFA2D in Staphylococcus aureus-induced acute muscle inflammation (right upper limb) and LPS-induced aseptic inflammation (left upper limb) mouse models, dynamic time-uptake curves of infection sites and major organs.

[0101] Figure 16 for[ 68 Static MicroPET imaging of Ga]Ga-DFY1D in mouse models of acute muscle inflammation (right upper limb) induced by Staphylococcus aureus or Escherichia coli and LPS-induced aseptic inflammation (left upper limb), uptake of infection sites and major organs, and HE staining of ex vivo tissues.

[0102] Figure 17 for[ 68 Ga]Ga-DFC2D、[ 68 Ga]Ga-DFY1D and [ 68 Biodistribution of Ga-DFA3D at 30-minute time points and target / non-target ratio in Staphylococcus aureus-induced acute muscle inflammation (right upper limb) and LPS-induced aseptic inflammation (left upper limb) models in mice. Detailed Implementation

[0103] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are some embodiments of the present invention, but not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0104] Preparation Example 1: Preparation of derafloxacin derivative DFC2D

[0105] The DFC2D compositing route is as follows:

[0106]

[0107] Specifically, the following steps are included:

[0108] 1) Synthesis of intermediate 1

[0109] 4-[3,5-bis(2-hydroxyphenyl)-1,2,4-triazol-1-yl]benzoic acid, dicyclohexylcarbodiimide (DCC), and N-hydroxysuccinimide (NHS) were dissolved in an appropriate amount of N,N-dimethylformamide (DMF) solution at a molar ratio of 1:2.5:2. The reaction was carried out overnight at room temperature, and the reaction progress was monitored by TLC. After the reaction was completed, the solvent was evaporated, and the crude product was purified by silica gel column chromatography to obtain intermediate 1. The mass spectrum of intermediate 1 is attached. Figure 1 As shown: C 25 H 18 N4O6, theoretical molecular weight 470.12, mass-to-charge ratio (M / Z): [M+H] + 471.07.

[0110] 2) Synthesis of intermediate 2

[0111] Intermediate 1, along with compounds N-tert-butoxycarbonyl-1,2-ethylenediamine and N,N-diisopropylethylamine (DIPEA), were dissolved in DMF solution at a molar ratio of 1:2:2. The reaction was carried out overnight at room temperature, and the reaction progress was monitored by TLC. After the reaction was completed, the solvent was evaporated, and the crude product was purified by silica gel column chromatography to obtain intermediate 2. The mass spectrum of intermediate 2 is attached. Figure 2 As shown: C 28 H 29 N5O5, theoretical molecular weight 515.22, mass-to-charge ratio (M / Z): [M+H] + 516.23.

[0112] 3) Synthesis of intermediate 3

[0113] An appropriate amount of intermediate 2 was dissolved in methanol in a reaction flask. Under ice bath conditions, an appropriate amount of hydrochloric acid / dioxane mixture was slowly added dropwise to the reaction flask. The reaction solution was subjected to the removal of the amino protecting group at room temperature. The mixture was stirred until the reaction was complete, the solvent was removed under reduced pressure, and the product intermediate 3 was obtained by silica gel column chromatography. The mass spectrum and NMR spectrum of intermediate 3 are attached. Figure 3 and attached Figure 4 As shown: C 23 H 21 N5O3, theoretical molecular weight 415.16, mass-to-charge ratio (M / Z): [M+H] + 416.17.

[0114] 4) Synthesis of DFC2D

[0115] Intermediate 3 and DOTA-NHS were dissolved in an appropriate amount of DMF at a molar ratio of 1:1.5, and then triethylamine was added. The mixture was reacted at 50°C for 4 hours. After the reaction was completed, the mixture was acidified with an appropriate amount of hydrochloric acid and purified by high-performance liquid chromatography (HPLC). The mobile phase conditions were as follows: semi-preparative column (250×10mm, 5μm, Thermo); Phase A: water + 0.1% trifluoroacetic acid (TFA); Phase B: acetonitrile + 0.1% TFA. Gradient elution conditions: 0-5 min: 20%-28% Phase B; 5-15 min: 36% Phase B; 15-20 min: 36%-49% Phase B; 20-25 min: 49%-95% Phase B; 25-40 min: 20% Phase B; flow rate: 3 mL / min; UV wavelength: 254 nm. The purified solution was dried under vacuum to obtain the target product. The mass spectrum of DFC2D is attached. Figure 5 As shown: C 39 H 47 N9O 10 Theoretical molecular weight: 801.34; mass-to-charge ratio (M / Z): [M+H] + :802.27. HPLC identification chromatogram is attached. Figure 9 As shown in A, the chemical purity is greater than 95%.

[0116] Preparation Example 2: Preparation of derafloxacin derivative DFC2F

[0117] The synthesis route for DFC2F is as follows:

[0118]

[0119] Following the preparation method of DFC2D, the corresponding derafloxacin derivative DFC2F was obtained by replacing DOTA-NHS with FITC-SCN. The mass spectrum of DFC2F is attached. Figure 6 As shown:

[0120] C 44 H 32 The theoretical molecular weight of N6O8S is 804.20, and the mass-to-charge ratio (M / Z): [M+H]: 805.17.

[0121] Preparation Example 3: Preparation of derafloxacin derivative DFY1D

[0122] The DFY1D synthesis route is shown below:

[0123]

[0124] DFX-NHS and peptide fragment Y1D were dissolved in an appropriate amount of DMF solution at a molar ratio of 1:1.5, and then triethylamine was added. The mixture was reacted at 50°C for 4 hours. After the reaction was completed, the mixture was acidified with an appropriate amount of hydrochloric acid and then purified by high performance liquid chromatography (HPLC). The mobile phase conditions were as follows: semi-preparative column (250×10mm, 5μm, Thermo), phase A: water + 0.1% trifluoroacetic acid (TFA), phase B: acetonitrile + 0.1% TFA.

[0125] Gradient elution conditions: 0-25 min: 20%-95% B phase; 20.1-25 min: 95% B; 25.1-30 min: 20% B phase; flow rate: 3 mL / min; UV wavelength: 254 nm. The purified solution was vacuum dried to obtain the target product. The mass spectrum of DFY1D is attached. Figure 7 As shown: DFY1D: C 49 H 61 N 11 O 16 Theoretical molecular weight: 1059.43; mass-to-charge ratio (M / Z): [M+H] 1060.10. HPLC identification chromatogram is attached. Figure 9 As shown in B, the chemical purity is greater than 95%.

[0126] Preparation Example 4: Preparation of delarosi derivative DFA2D

[0127] The DFA2D composition route is shown below:

[0128]

[0129] Following the preparation method of DFY1D, the corresponding deferasirox derivative DFA2D was obtained by replacing the peptide fragment Y1D with A2D. The mass spectrum of DFA2D is attached. Figure 8 As shown: DFA2D: C 55 H 12 N 68 O 21 Theoretical molecular weight: 1232.46; mass-to-charge ratio (M / Z): [M+H] + 1233.44. HPLC identification chromatogram is attached. Figure 9 As shown in C, the chemical purity is greater than 95%.

[0130] Preparation Example 5: 68 Preparation of Ga-labeled delaroxy derivatives

[0131] 68 The Ga nuclide labeling process is as follows:

[0132] Wet method: approximately 37-3700 MBq 68GaCl3 hydrochloric acid solution (rinsed from the germanium-gallium generator) is added to a centrifuge tube containing 0.5–2 mL of acetic acid-acetate solution of DFC2D, DFY1D, or DFA2D (20–200 μg). The mixture is placed at room temperature to 100°C and reacted for 20 minutes. After cooling to room temperature, it is diluted with physiological saline or water for injection and sterile filtered to obtain the labeled compound injection solution.

[0133] Lyophilization method: A certain amount of buffer solution and approximately 37–3700 MBq of... 68 GaCl3 hydrochloric acid solution (rinsed from the germanium-gallium generator) is added to a lyophilized kit containing DFC2D, DFY1D or DFA2D (20-200 μg), mixed and dissolved, and then reacted at room temperature to 100°C for 20 minutes. After cooling to room temperature, it is diluted with physiological saline or water for injection and sterile filtered to obtain the labeled compound injection solution.

[0134] If the radiochemical purity is below 95%, purification is required. The purification steps are as follows: Take a Sep-Pak C18 separation column and activate and rinse it successively with 10 mL of anhydrous ethanol and 10 mL of water. Dilute the labeled solution with 10 mL of water and load the sample onto the separation column. Rinse the separation column with water to remove unreacted components. 68 Ga ions were obtained by rinsing with ethanol solution. 68 Ga-labeled complexes were prepared. The organic solvent was removed by nitrogen purging, and the mixture was diluted with physiological saline and then sterile filtered to obtain the labeled compound injection solution.

[0135] As attached Figure 10 As shown, for [ 68 Ga]Ga-DFC2D、[ 68 Ga]Ga-DFY1D or [ 68 Ga]Ga-DFA2D samples were analyzed and identified by HPLC. The HPLC system was as follows: reversed-phase C18 analytical column (4.6×250mm), elution gradient: 0-15 min: 20% acetonitrile (0.1% TFA) and 80% water (0.1% TFA) increased to 85% acetonitrile (0.1% TFA) and 15% water (0.1% TFA), 15-20 min: 85% acetonitrile (0.1% TFA) and 15% water (0.1% TFA) changed to 20% acetonitrile (0.1% TFA) and 80% water (0.1% TFA), flow rate was 1 mL / min. The retention time of the radiolabeled target complex was approximately 11.91 min. 68 Ga]Ga-DFC2D), 10.47 minutes ([ 68 Ga]Ga-DFY1D), 11.02 minutes ([ 68 Ga]Ga-DFA2D), with a radiochemical purity greater than 95%.

[0136] Example 1: Fe3+ Complexation experiment

[0137] Deferrasirox (DFX), DFC2D, DFY1D, and DFA2D compounds were dissolved in an appropriate amount of DMSO to prepare a 1 mM concentration, which was then diluted with deionized water to 15 μM. FeCl3 was diluted with water to prepare concentrations of 15, 10, 5, and 2.5 μM. Equal volumes of the compounds were incubated with FeCl3 solutions at 37 °C for 3 hours. After incubation, the UV absorption was measured.

[0138] As attached Figure 11 As shown, the UV absorption peaks and trends of DFC2D, DFY1D, and DFA2D after complexing with iron ions are similar to those of DFX, proving that these compounds can all complex with Fe. 3+ Effective complexation.

[0139] Example 2: In vitro stability test

[0140] Will[ 68 Ga]Ga-DFC2D、[ 68 Ga]Ga-DFY1D or [ 68 Ga]Ga-DFA2D was placed in physiological saline medium and left at room temperature for different times. Samples were then analyzed by HPLC.

[0141] Will[ 68 Ga]Ga-DFC2D、[ 68 Ga]Ga-DFY1D or [ 68 Ga]Ga-DFA2D was co-incubated with serum at room temperature for different times, acetonitrile was added to remove protein, the supernatant was collected by centrifugation, and samples were analyzed by HPLC. 68 Ga]Ga-DFC2D、[ 68 Ga]Ga-DFY1D and [ 68 The HPLC results for the stability of Ga]Ga-DFA2D in physiological saline and in vitro serum are attached. Figure 12 As shown, this indicates that it maintains high stability (>95%) in physiological saline or in vitro serum systems up to 2 hours.

[0142] Example 3: Determination of lipid-water distribution coefficient (logP)

[0143] 100 μL of the diluted radiolabeled derarosi derivative was added to a centrifuge tube containing a mixture of 0.9 mL PBS and 1 mL n-octanol. The mixture was vortexed for 3 minutes, then centrifuged at 9000 rpm for 5 minutes. 100 μL of each phase was collected from the aqueous and n-octanol phases and counted using a γ-counter. The experiment was repeated three times, and the average value was taken. The formula for calculating logP is:

[0144] P = I有机相 / I 水相

[0145] Among them, I 有机相 Represents the radioactivity count measured in the organic phase, I 水相 The radioactivity counts measured in the aqueous phase are represented. The lipid-water distribution coefficients of each radiolabeled target probe were calculated. The results are shown in Table 1 below; the labeled compounds exhibit hydrophilicity.

[0146] Table 1

[0147] Labeled compounds LogP <![CDATA[[ 68 Ga]Ga-DFC2D]]> -1.39 <![CDATA[[ 68 Ga]Ga-DFY1D]]> -2.06 <![CDATA[[ 68 Ga]Ga-DFA2D]]> -2.69

[0148] Example 4: Validation of bacterial targeting specificity

[0149] The concentration is approximately 37 kBq / mL. 68 Ga]Ga-DFC2D、[ 68 Ga]Ga-DFY1D or [ 68 Ga-DFA2D was co-incubated with Escherichia coli, Staphylococcus aureus, and inactivated bacteria (HK), respectively. Incubation was stopped at different time points (15, 30, 60, and 90 minutes), free probe solution was removed, and the sample was washed three times with pre-cooled physiological saline. Finally, the count was determined using a gamma counter. (See attached image) Figure 13 As shown, the probe can be specifically recognized by live bacteria, while inactivated bacteria cannot take up the probe, with a statistically significant difference (P<0.001).

[0150] The deferasirox analogue (DFC2F) coupled with the fluorescent dye FITC was co-incubated with normal E. coli and E. coli with the iron transporter knocked out (ΔENA / B) receptor. Figure 14 It is known that wild-type E. coli emits green fluorescence due to the uptake of DFC2F, but E. coli with the transporter knocked out lacks the ability to uptake DFC2F. This further demonstrates that deferasirox is a bacterial-specific recognition molecule.

[0151] Example 5: MicroPET / CT Imaging of a Mouse Model of Acute Muscle Inflammation

[0152] Select female Balb / c mice (approximately 6 weeks old and weighing about 20g), and inject 100μL of bacteria (10 μg) into their right upper limb intramuscularly 24 hours prior to the treatment. -7 CFU), and an appropriate amount of LPS was injected into the left upper limb. Approximately 9.25 MBq was injected via the tail vein of the mouse. 68 Ga]Ga-DFC2D、[ 68 Ga]Ga-DFY1D or [ 68Ga]Ga-DFA2D was used to perform MicroPET imaging at different time points, and regions of interest (ROIs) were delineated in the whole-body decay-corrected images obtained from the scans, and probe distribution values ​​were calculated.

[0153] As attached Figure 15 As shown, the present invention provides 68 Ga-labeled delarose derivatives [ 68 Ga]Ga-DFC2D、[ 68 Ga]Ga-DFY1D and [ 68 Ga]Ga-DFA2D exhibits different metabolic pathways in mice. 68 Ga-DFC2D is metabolized by the liver and kidneys, and has a high radioactivity level in the kidneys. 68 Ga-DFA2D is primarily metabolized by the kidneys, but it exhibits radioactive signals at sites of bacterial infection. 68 Ga-DFY1D showed significant uptake at bacterial infection sites, but less uptake at sterile testing sites or other normal tissues, exhibiting a high target-to-non-target ratio. At 30 minutes post-injection of the radioactive probe, […]. 68 Ga]Ga-DFC2D、[ 68 Ga]Ga-DFY1D and [ 68 The uptake of Ga-DFA2D at the site of Staphylococcus aureus infection was 3.40±0.13, 4.45±0.28, and 3.20±0.07% ID / g, respectively.

[0154] As attached Figure 16 As shown, in [ 68 At the 30-minute time point of Ga-DFY1D imaging, the probe uptake at Staphylococcus aureus and Escherichia coli infection sites was 4.75% ID / g and 2.83% ID / g, respectively; muscle uptake was 0.61% ID / g; and aseptic inflammation site uptake was 1.22% ID / g. The ratio of bacterial infection sites to aseptic inflammation sites was 3.89 (Staphylococcus aureus / aseptic myositis) and 2.32 (Escherichia coli / aseptic myositis), respectively. The ratio of bacterial infection sites to normal muscle was 7.79 (Staphylococcus aureus / normal muscle) and 4.64 (Escherichia coli / normal muscle).

[0155] Comprehensive Appendix Figure 15 and Figure 16 As shown, [ 68 Ga]Ga-DFY1D exhibits excellent pharmacokinetic properties and a high target-to-non-target ratio, enabling it to diagnose bacterial infections in vivo and effectively differentiate between bacterial infections and aseptic inflammation.

[0156] Example 6: Biodistribution Experiment of Muscle Inflammation Model in Mice

[0157] Select female Balb / c mice (approximately 6 weeks old and weighing about 20g), and inject them intramuscularly with 100μL of bacteria (10) in their right upper limb 24 hours prior to the treatment. -7 CFU), and an appropriate amount of Freund's adjuvant LPS was injected into the left upper limb. Approximately 0.74 MBq of radionuclide-labeled derarosipro derivative was injected into the tail vein of mice. Forty-five minutes after injection, the mice (3 mice) were euthanized by decapitation. Blood, brain, heart, liver, lungs, kidneys, intestines, spleen, muscles, and bones (or other organs and tissues of interest) were collected, weighed, and their radioactivity counts were measured. The results were expressed as percentage uptake dose per gram of tissue or organ (%ID / g). See attached... Figure 17 As shown, similar to the results of PET imaging, the probe [ 68 Ga-DFY1D is most uptaken at the site of bacterial infection, with a high target-to-non-target ratio, and low uptake in non-target organs.

[0158] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A delarosus derivative, characterized in that, The delarosi derivatives include: DFC2D, DFC2F, DFY1D, and DFA2D; The structural formula of DFC2D is as follows: The structural formula of the DFC2F is as follows: The structural formula of the DFY1D is as follows: The structural formula of DFA2D is as follows: 。 2. A method for preparing the delarosi derivative as described in claim 1, characterized in that, The DFC2D is prepared as follows: 4-[3,5-bis(2-hydroxyphenyl)-1,2,4-triazol-1-yl]benzoic acid, dicyclohexylcarbodiimide (DCC), and N-hydroxysuccinimide (NHS) were dissolved in an appropriate amount of N,N-dimethylformamide (DMF) solution at a molar ratio of 1:2.5:

2. The reaction was carried out overnight at room temperature, and the reaction progress was monitored by TLC. After the reaction was completed, the solvent was evaporated, and the crude product was purified by silica gel column chromatography to obtain intermediate 1. Intermediate 1 was dissolved in DMF solution with N-tert-butoxycarbonyl-1,2-ethylenediamine and N,N-diisopropylethylamine (DIPEA) in a molar ratio of 1:2:

2. The reaction was carried out overnight at room temperature. The reaction progress was monitored by TLC. After the reaction was completed, the solvent was evaporated and the crude product was purified by silica gel column chromatography to obtain intermediate 2. An appropriate amount of intermediate 2 was dissolved in methanol in a reaction flask. Under ice bath conditions, an appropriate amount of hydrochloric acid / dioxane mixture was slowly added dropwise to the reaction flask. The reaction solution was subjected to the removal of amino protecting groups at room temperature. The mixture was stirred until the reaction was complete. The solvent was removed under reduced pressure and purified by silica gel column chromatography to obtain intermediate 3 of the target product compound. Intermediate 3 and DOTA-NHS were dissolved in an appropriate amount of DMF at a molar ratio of 1:1.5, and then triethylamine was added. The mixture was reacted at 50°C for 4 hours. After the reaction was completed, an appropriate amount of hydrochloric acid was added for acidification, and the mixture was purified by high performance liquid chromatography. The purified solution was dried under vacuum to obtain the target product DFC2D. The preparation method of DFC2F is as follows: it is prepared according to the preparation method of DFC2D, and the corresponding derarosi derivative DFC2F is obtained by replacing DOTA-NHS with FITC-SCN; The DFY1D is prepared as follows: DFX-NHS and polypeptide fragment Y1D were dissolved in an appropriate amount of DMF solution at a molar ratio of 1:1.5, and then triethylamine was added. The mixture was reacted at 50°C for 4 hours. After the reaction was completed, an appropriate amount of hydrochloric acid was added for acidification, and the mixture was purified by high performance liquid chromatography. The purified solution was dried under vacuum to obtain the target product DFY1D. The DFA2D is prepared as follows: The corresponding derafloxacin derivative DFA2D was obtained by replacing Y1D with A2D, following the preparation method of DFY1D. The structural formula of intermediate 1 is as follows: The structural formula of intermediate 2 is as follows: The structural formula of intermediate 3 is as follows: The structural formula of DOTA-NHS is as follows: The structural formula of the FITC-SCN is as follows: The structural formula of the DFX-NHS is as follows: The structural formula of Y1D is as follows: The structural formula of A2D is as follows: 。 3. A radionuclide-labeled delarosus derivative, characterized in that, It is obtained by coordinating the derarosixin derivative as described in claim 1 with a nuclide; The nuclides include 18 F, 43 Sc、 44 Sc、 47 Sc、 51 Cr 55 Co、 57 Co、 62 Cu、 64 Cu、 67 Cu、 67 Ga、 68 Ga、 72 As、 72 Se、 89 Zr、 86 Y、 89 Sr、 90 Y、 97 Ru、 99m Tc, 105 Rh、 101m Rh、 109 Pd, 111 In、 119 Sb, 128 Ba、 139 La、 140 La、 142 Pr、 149 Pm, 149 Tb, 151 Tb, 151 Eu、 153 Eu、 169 Eu、 153 Sm、 152 Gd, 153 Gd, 157 Gd, 159 Gd, 161 Tb, 165 Dy、 166 Ho、 169 Er、 175 Yb、 177 Lu、 186 Re、 188 Re、 197 Hg, 198 Au、 201 Tl、 203 Pb, 211 At、 212 Pb, 212 Bi、 213 Bi、 223 Ra、 227 Th and 225 At least one of Ac.

4. A method for preparing a radionuclide-labeled delarosus derivative as described in claim 3, characterized in that, Prepared using either a wet method or a freeze-drying method; The wet preparation process includes the following steps: The derarosi derivative as described in claim 1 and / or its pharmaceutically acceptable salt are dissolved in a solvent; then a solution containing a radionuclide is added, and the mixture is reacted at room temperature to 110°C for 10 to 30 minutes to obtain a radionuclide-labeled derarosi derivative solution. The freeze-drying preparation steps are as follows: The deferasirox derivative as described in claim 1 and / or its pharmaceutically acceptable salt are dissolved in a solvent, dispensed into lyophilization containers, and then freeze-dried and sealed to form a lyophilized drug case. A solvent is added to the lyophilized drug case to dissolve the deferasirox derivative, and then a solution containing a radionuclide is added. The mixture is reacted at room temperature to 110°C for 10 to 30 minutes to obtain a radionuclide-labeled deferasirox derivative solution.

5. A pharmaceutical composition, characterized in that, Including active ingredients and pharmaceutically acceptable excipients; The active ingredient includes the deferasirox derivative as described in claim 1 and its pharmaceutically acceptable salt, and the radiolabeled deferasirox derivative as described in claim 3 and its pharmaceutically acceptable salt.

6. The use of the deferasirox derivative of claim 1 and its pharmaceutically acceptable salt, the radiolabeled deferasirox derivative of claim 3 and its pharmaceutically acceptable salt, and the pharmaceutical composition of claim 5 in the preparation of a medicament for diagnosing bacterial infections.

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