Technetium-99m labeled D-proline modified alendronic acid derivative as well as preparation method and application thereof

Abstract

The invention relates to the technical field of radiopharmaceutical chemistry and clinical nuclear medicine, in particular to a D-proline modified alendronic acid derivative and application thereof. A radioactive preparation obtained by labeling the D-proline modified alendronate derivative with radionuclide can be prepared through a medicine box, is high in radiochemical purity, easy in chemical structure determination, good in stability, relatively high in uptake and retention at bones of normal mice, low in non-target organ or tissue uptake and good in target / non-target ratio, and can be applied to preparation of a medicine box. The bone imaging effect is good, the requirement of further clinical bone imaging can be met, and the bone imaging agent can be popularized and applied as a novel bone imaging agent.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of radiopharmaceuticals and nuclear medicine, and particularly relates to technetium-99m labeled alendronate derivatives containing D-proline modification, and preparation methods and applications thereof. BACKGROUND

[0002] Cancer, as a major disease that seriously endangers human health, has attracted extensive attention worldwide due to its high incidence and mortality. Among various cancer types, malignant tumors such as prostate cancer, breast cancer, and lung cancer often develop bone metastasis when reaching the advanced stage. Bone metastasis refers to the process in which tumor cells from the primary lesion spread to the skeletal system through blood circulation or lymphatic system, and form secondary tumor lesions in the bone. Bone metastasis of malignant tumors can destroy the bone structure, leading to complications such as pathological fractures and spinal cord compression, and is often accompanied by severe bone pain, which seriously affects the quality of life of patients. More importantly, the occurrence of bone metastasis often indicates that the disease has entered the advanced stage, significantly affecting the treatment options and prognosis of patients. Therefore, early diagnosis of bone metastasis has important clinical significance.

[0003] Conventional imaging relies on structural changes and can only detect lesions when bone destruction is obvious. Nuclear medicine, on the other hand, uses radioactive tracers combined with molecular imaging to achieve non-invasive and high-sensitivity early diagnosis of bone metastasis. Bone metastasis lesions significantly disrupt local bone metabolism balance, leading to abnormal enhancement of osteogenic or osteolytic activity. Nuclear medicine takes advantage of this biological characteristic by targeting abnormal metabolic areas with specific tracers to achieve early imaging. For example, technetium-99m labeled methylene diphosphonate ([99m]Tc-MDP) is widely used in clinical practice. Its phosphate group can bind to hydroxyapatite crystals, concentrating in areas of active bone formation, and thus revealing bone metastasis lesions through SPECT imaging. 99m Tc]Tc-MDP), its phosphate group can bind to hydroxyapatite crystals, concentrating in areas of active bone formation, and thus revealing bone metastasis lesions through SPECT imaging.

[0004] However, [ 99m Tc]Tc-MDP still has certain limitations. On the one hand, [ 99m Tc]Tc-MDP cannot be clearly chemically structured, and is a mixture composed of various valence 99m Tc complexes, and different components have different affinities for bone, which may lead to differences in the quality of imaging agents prepared by different manufacturers' kits ( Handeland, M. W. Lindegaard, D. E. Heggli. Biodistribution of anionic separated MDP complexes from different MDP preparations [J], European Journal of Nuclear Medicine, 1989, 15: 609-611); on the other hand, its pharmacokinetic properties need to be improved, 99m Tc]Tc-MDP has a slow clearance rate in soft tissues, and clinical imaging is generally performed 2-4 hours after injection. Therefore, it is urgent to develop a new generation of bone imaging agents to meet the new needs of the clinic. New bone imaging agents should meet the following requirements: clear chemical structure, excellent pharmacokinetic properties and in vivo distribution properties, etc., in order to improve the accuracy of diagnosis and patient comfort.

[0005] The linker is a key chemical structure that connects the radionuclide and the targeting group, and is crucial for regulating the pharmacokinetic properties of radiopharmaceuticals. Studies have shown that proline and its analogs have broad prospects in optimizing drug properties (Vladimir Kubyshkin, Pavel K. Mykhailiuk. Proline analogues in drug design: current trends and future prospects [J]. Journal of Medicinal Chemistry, 2024, 67: 20022-20055). Therefore, the present application uses D-proline as a linker in order to improve the pharmacokinetic properties of the drug, increase the target / non-target ratio, and optimize the signal-to-noise ratio. Hydrazinonicotinamide (HYNIC) is 99m a bifunctional linker commonly used in Tc-labeled radiopharmaceuticals. The HYNIC group contains only one coordination atom, and other co-ligands are needed to form a stable complex with 99m Tc. Different co-ligands play an important role in regulating the water solubility and pharmacokinetic properties of the complex. Based on the above research background, the present application uses a commercial new generation of bisphosphonate drug, alendronate sodium, as the raw material, retains its bone-seeking pharmacophore structure, and modifies its structure to synthesize alendronate derivatives containing D-proline and HYNIC. Subsequently, different co-ligands are used to label them with 99m Tc to explore new bone imaging agents with clear chemical structure, good targeting, and excellent pharmacokinetic properties. SUMMARY

[0006] The present application aims to provide a technetium-99m labeled D-proline modified alendronate derivative with high radiochemical purity and good stability for bone imaging, and a preparation method thereof.

[0007] The present application provides the following technical solutions: 99m A Tc labeled D-proline modified alendronate derivative has the following structural formula (I):

[0008]

[0009] In the formula, n=2, 3.

[0010] A corresponding Tc complex is prepared from the derivative. 99m The Tc complex has high bone affinity, low uptake of non-target organs and tissues, excellent target / non-target ratio, and good bone imaging effect.

[0011] The present application also provides a radioactive preparation containing the above-mentioned D-proline modified alendronate derivative labeled with a radionuclide.

[0012] Preferably, in the radioactive preparation, the radionuclide is a metal radionuclide.

[0013] Preferably, in the radioactive preparation, the metal radionuclide is 99m Tc, 99 Tc, 94m Tc, 94 Tc, 52 Mn, 186 Re or 188 Re.

[0014] Most preferably, in the radioactive preparation, the radionuclide is 99m Tc, and the radioactive preparation has the following structural formula (II):

[0015]

[0016] Wherein: n=2, 3; L is N-tris (hydroxymethyl) methyl glycine (Tricine) and triphenylphosphine tris-m-sulfonic acid sodium (TPPTS), N-tris (hydroxymethyl) methyl glycine (Tricine) and diphenylphosphine phenyl-3-sulfonic acid sodium (TPPMS), N-tris (hydroxymethyl) methyl glycine (Tricine) and nicotinic acid (NIC), N-tris (hydroxymethyl) methyl glycine (Tricine) and isonicotinic acid (ISONIC), N-tris (hydroxymethyl) methyl glycine (Tricine) and 3, 5-pyridine dicarboxylic acid (PDA), N-tris (hydroxymethyl) methyl glycine (Tricine) and 3-pyridine sulfonic acid (PSA), N-tris (hydroxymethyl) methyl glycine (Tricine) and ethylenediamine-N, N'-diacetic acid (EDDA), N-tris (hydroxymethyl) methyl glycine (Tricine) and 2- (pyridine-4-yl) acetic acid (PA).

[0017] The present application also provides the use of the above radioactive preparation in the diagnosis and / or treatment of bone metastasis.

[0018] The present application has the advantages that the present application provides a D-proline modified alendronate derivative, a preparation method and use thereof, and a radioactive preparation obtained by labeling the same with a radionuclide, which has high uptake in bone, good target / non-target ratio, and low uptake in non-target organs and tissues, and is a safe and effective new type of bone imaging radioactive drug with promotional significance. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 The normal Kunming white mice were injected with 99m SPECT / CT imaging of the mice one hour after injection of DETAILED DESCRIPTION

[0020] The present application provides a preparation method and use of a technetium-99m labeled D-proline modified alendronate derivative, and in a preferred embodiment, the present application provides a radioactive preparation of Tc-DPALN-L, wherein the structure is as shown in the formula: 99m Tc]Tc-DPALN-L:

[0021]

[0022] Wherein: n=3; L is a co-ligand component for forming a stable complex with 99m Tc, which is N-tris (hydroxymethyl) methyl glycine (Tricine) and triphenylphosphine tris-m-sulfonic acid sodium (TPPTS), N-tris (hydroxymethyl) methyl glycine (Tricine) and nicotinic acid (NIC), N-tris (hydroxymethyl) methyl glycine (Tricine) and 3, 5-pyridine dicarboxylic acid (PDA), etc.

[0023] The preparation steps are as follows:

[0024] a. Synthesis of ligand DPALN:

[0025] An appropriate amount of alendronate sodium is weighed into a reaction vessel, dissolved with an aqueous solution of triethylamine, and then an appropriate amount of compound 1 is added, and the reaction is carried out at room temperature overnight. After the reaction is completed, recrystallization is carried out with ethanol, and after vacuum drying, the ligand DPALN is obtained.

[0026] The synthesis route is as follows:

[0027]

[0028] b. Preparation of [ 99m Tc]Tc-DPALN-L

[0029] DPALN is dissolved in pure water, Tricine is dissolved in physiological saline, TPPTS or NIC or PDA is added, SnCl2·2H2O is added, the pH of the solution is adjusted to 5.0, and then a freshly eluted [ 99m Tc]Na 99m TcO4 solution is added, and after 30 min of reaction at 100℃, the [ 99m Tc]Tc-DPALN-L complex is obtained.

[0030] The [ 99m Tc]Tc-DPALN-L complex prepared by the above method has a radiochemical purity of more than 90%, is a hydrophilic substance, and has good in vitro stability. The biodistribution results show that the [ 99m Tc]Tc-DPALN-L has high bone uptake, high target / non-target ratio, low non-target organ and tissue uptake, and the SPECT / CT imaging results show that there is obvious radioactive concentration in the spine and bone joints, and the signal-to-noise ratio is good, so it is worth popularizing and applying as a new type of bone imaging agent.

[0031] The following examples are used to illustrate the present application, but are not used to limit the scope of the present application. If specific techniques or conditions are not indicated in the examples, the techniques or conditions described in the literature in the art, or according to the product instructions are used.

[0032] Example 1

[0033] This example provides a 99m Tc-labeled alendronate derivative containing D-proline modification, referred to as [ 99m Tc]Tc-DPALN-L, and the structural formula is as follows:

[0034]

[0035] wherein: n = 3; L is 99m Tc forms stable 99m The co-ligand components in the Tc complex are N-tris(hydroxymethyl)methylglycine (Tricine) and triphenylphosphine tris- sodium sulfonate (TPPTS), N-tris(hydroxymethyl)methylglycine (Tricine) and nicotinic acid (NIC), N-tris(hydroxymethyl)methylglycine (Tricine) and 3,5-pyridinedicarboxylic acid (PDA), etc.

[0036] The preparation method is as follows, but is not limited to the exemplified complex:

[0037] a. Synthesis of ligand DPALN

[0038] Take 50 mg of alendronate sodium (0.15 mmol) in a 10 mL vial, dissolve in 2 mL of purified water, then add triethylamine to adjust the pH to 8-9, add 100 mg of compound 1 (0.19 mmol) in batches, and react overnight at room temperature. After the reaction is completed, stepwise recrystallization is carried out with ethanol, and after vacuum drying, 5.9 mg of ligand DPALN is obtained.

[0039] 1 H NMR (400 MHz, Deuterium Oxide) δ 8.81 (d, J = 2.6 Hz, 1H), 8.39 (d, J = 2.3 Hz, 1H), 8.22 (d, J = 7.8 Hz, 1H), 7.99 - 7.86 (m, 2H), 7.62 (t, J = 7.3 Hz, 1H), 7.51 (t, J = 7.7 Hz, 1H), 7.23 (d, J = 8.7 Hz, 1H), 4.51 (t, J = 7.3 Hz, 1H), 3.82 - 3.69 (m, 2H), 3.26 (s, 2H), 2.41 (d, J = 6.9 Hz, 1H), 2.06 - 1.88 (m, 7H).

[0040] 31 P NMR (162 MHz, Deuterium Oxide) δ 19.12.

[0041] HRMS (m / z): found 672.0906, calcd 672.09 for C 22 H 29 N5O 12 NaP2S[M+H] + .

[0042] b. [ 99m Preparation of Tc]Tc-DPALN-TPPTS complex:

[0043] Take 200 μg ligand DPALN dissolved in the appropriate amount of purified water, 2 mg N-tris (hydroxymethyl) methyl glycine (Tricine), 2 mg triphenylphosphine triisulfonate (TPPTS) dissolved in the appropriate amount of normal saline, and then add 50 μg SnCl2·2H2O and 0.20 mL succinic acid buffer (pH = 5.0), and then add fresh eluted[ 99m Tc]Na 99m TcO4, 100 ℃ for 30 min, the target complex[ 99m Tc]Tc-DPALN-TPPTS, using TLC to determine the radiochemical purity of more than 90%.

[0044]

[0045] c. 99m Preparation of the complex of[

[0046] Take 200 μg ligand DPALN dissolved in the appropriate amount of purified water, 2 mg N-tris (hydroxymethyl) methyl glycine (Tricine), 2 mg nicotinic acid (NIC) dissolved in the appropriate amount of normal saline, and then add 50 μg SnCl2·2H2O and 0.20 mL succinic acid buffer (pH = 5.0), and then add fresh eluted[ 99m Tc]Na 99m TcO4, 100 ℃ for 30 min, the target complex[ 99m Tc]Tc-DPALN-NIC, using TLC to determine the radiochemical purity of more than 90%.

[0047]

[0048] d. 99m Preparation of the complex of[

[0049] Take 200 μg ligand DPALN dissolved in the appropriate amount of purified water, 2 mg N-tris (hydroxymethyl) methyl glycine (Tricine), 2 mg 3,5-pyridine dicarboxylic acid (PDA) dissolved in the appropriate amount of normal saline, and then add 50 μg SnCl2·2H2O and 0.20 mL succinic acid buffer (pH = 5.0), and then add fresh eluted[ 99m Tc]Na 99m TcO4, 100 ℃ for 30 min, the target complex[ 99m Tc]Tc-DPALN-PDA, using TLC to determine the radiochemical purity of more than 90%.

[0050]

[0051] Experiments show that [ 99m The properties of the Tc]Tc-DPALN-L complex are as follows:

[0052] 1. Identification of complexes:

[0053] Thin layer chromatography (TLC) was used to determine the radiochemical yield and radiochemical purity of the labeled substance. The system used was: Whatman filter paper as the support, acetone: physiological saline = 5:1 as the developing solvent. Under this system, the R f See Table 1 for values.

[0054] Table 1 Chromatographic results of each component (R f value)

[0055]

[0056] The [ 99m The radiochemical yield and radiochemical purity of the [Tc]Tc-DPALN-L complex were both greater than 90%, and it could be used in subsequent experiments without purification.

[0057] 2. Determination of lipid-water partition coefficient of complex

[0058] Take 1.0mL of n-octanol and 0.90mL of pH=7.4 (0.025mol / L) phosphate buffer in a 5mL centrifuge tube, add 0.10mL of [ 99m The Tc]Tc-DPALN-L complex solution was stoppered, vortexed for 3 minutes, and centrifuged for 5 minutes (1600 rpm). Then, 3 × 0.10 mL of the solution was taken from each of the organic and aqueous phases. The radioactivity counts in the two phases were measured, and the partition coefficient D (D = radioactivity count in the organic phase / radioactivity count in the aqueous phase) was calculated. This was repeated three times. The lipid-water partition coefficient of the complex is shown in the following table:

[0059] Table 2 Results of lipid-water partition coefficients of complexes

[0060]

[0061] The results of lipid-water partition coefficients showed that the complexes were all hydrophilic substances.

[0062] 3. In vitro stability determination of the complex

[0063] The labeled complexes [ 99mTc]Tc-DPALN-L respectively at room temperature and in 37℃ mouse serum for 4 hours, the results showed that the radiochemical purity of the complex was more than 90% after being placed at room temperature and in 37℃ mouse serum for 4 hours, which indicated that the complex had good in vitro stability.

[0064] 4. The biodistribution experiment of the complex in normal mice:

[0065] From 18 to 22 g normal mice, 0.10 mL[ 99m Tc]Tc-DPALN-L complex solution (about 7.4×10 5 Bq) was injected through the tail vein, and the mice were killed after 4 hours. The blood, heart, liver, lung, kidney, muscle, bone and other related tissues and organs were taken, cleaned and weighed, and the radioactivity count was measured on the γ-Counter, with 5 mice in each group. The percentage of injected dose per gram of each tissue (%ID / g) was calculated. The MDP injection was prepared using the MDP kit produced by Beijing Shihong Pharmaceutical Co., Ltd., and the biodistribution experiment in normal mice was carried out by the same method as above. 99m The biodistribution results are shown in Table 3:

[0066] Table 3 99m Tc]Tc-DPALN-L and[ 99m Tc]Tc-MDP in mice after 4 hours of injection (%ID / g, n=5)

[0067]

[0068] From Table 3, it can be seen that[ 99m Tc]Tc-DPALN-TPPTS has a bone uptake of more than[ 99m Tc]Tc-MDP after 4 hours of injection, 99m Tc]Tc-DPALN-NIC and[ 99m Tc]Tc-DPALN-PDA have bone uptakes almost equal to[ 99m Tc]Tc-MDP. In addition, 99m Tc]Tc-DPALN-TPPTS, 99m Tc]Tc-DPALN-NIC, 99m Tc]Tc-DPALN-PDA all show lower uptakes in the heart, liver, spleen, lung and other non-target organs compared with[ 99m Tc]Tc-MDP, so[ 99m Tc]Tc-DPALN-TPPTS, 99m Tc]Tc-DPALN-NIC, 99mTc]Tc-DPALN-PDA target / non-target ratio is good, which can be used as a new bone imaging agent.

[0069] 5.[ 99m Tc]Tc-DPALN-TPPTS complex in normal mice in vivo SPECT / CT imaging experiments:

[0070] Because[ 99m Tc]Tc-DPALN-TPPTS in biodistribution experiments show more excellent bone uptake and target / non-target ratio, so select it for SPECT / CT imaging experiments. The labeled[ 99m Tc]Tc-DPALN-TPPTS (about 18.5 MBq) from the tail vein injection into normal mice, 1h after administration, the mice were anesthetized with isoflurane concentration of 1.5%, set the scanning parameters, SPECT scan 15 min, CT scan 4 min, and finally through the HiSPECT software and vivoquant2.5 software to obtain the scanning image. SPECT / CT imaging results show that[ 99m Tc]Tc-DPALN-TPPTS bone imaging effect is good, especially the uptake of spine, bone joint position is obvious, clean background, good signal-to-noise ratio, indicating that[ 99m Tc]Tc-DPALN-TPPTS complex can be used as a new type of bone imaging agent with excellent performance.

[0071] Although the present application has been described in detail with general description and specific embodiments above, some modifications or improvements can be made on the basis of the present application, which is obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present application, except for the D-proline modified alendronate derivatives, D-proline modified pamidronate derivatives, L-proline modified alendronate derivatives, L-proline modified pamidronate derivatives, and the corresponding co-ligand M is N-tris (hydroxymethyl) methyl glycine (Tricine) and ethylenediamine-N, N'-diacetic acid (EDDA), N-tris (hydroxymethyl) methyl glycine (Tricine) and triphenylphosphine sodium 3-sulfonate (TPPTS), N-tris (hydroxymethyl) methyl glycine (Tricine) and diphenylphosphine sodium phenyl-3-sulfonate (TPPMS), N-tris (hydroxymethyl) methyl glycine (Tricine) and 2- (pyridine-4-yl) acetic acid (PA), N-tris (hydroxymethyl) methyl glycine (Tricine) and nicotinic acid (NIC), N-tris (hydroxymethyl) methyl glycine (Tricine) and isonicotinic acid (ISONIC), N-tris (hydroxymethyl) methyl glycine (Tricine) and 3, 5-pyridine dicarboxylic acid (PDA), N-tris (hydroxymethyl) methyl glycine (Tricine) and 3-pyridine sulfonic acid (PSA) of the present application, the radioactive preparations obtained after radionuclide labeling are within the scope of the present application. In addition, the radioactive preparations obtained after radionuclide labeling of the D-proline or L-proline modified alendronate derivatives or pamidronate derivatives and the co-ligand M is N-tris (hydroxymethyl) methyl glycine (Tricine) and 3, 3'- (phenylphosphine diyl) bis (benzene-1-sulfonic acid) disodium (TPPDS), N-tris (hydroxymethyl) methyl glycine (Tricine) and glucoheptonate, N-tris (hydroxymethyl) methyl glycine (Tricine) and glucosamine, N-tris (hydroxymethyl) methyl glycine (Tricine) and mannitol, N-tris (hydroxymethyl) methyl glycine (Tricine) and diphenylphosphine benzoic acid also belong to the scope of the present application.

Claims

1. A D-proline-modified alendronic acid derivative, characterized in that: The compound structural formula is as follows (I): Where: n = 2, 3.

2. A radioactive preparation, characterized in that The radioactive preparation comprises the alendronic acid derivative containing D-proline modification according to claim 1 labeled with a radionuclide.

3. The radioactive preparation according to claim 2, characterized in that The radionuclide is 99m Tc, 99 Tc, 94m Tc, 94 Tc, 52 Mn, 186 Re or 188 Re.

4. The radioactive preparation according to claim 3, characterized in that The structural formula of the radioactive agent is (II): Wherein: n=2,3; L is N-tris(hydroxymethyl)methylglycine and triphenylphosphine tris-sulfonate, N-tris(hydroxymethyl) Methylglycine and sodium diphenylphosphine-3-sulfonate, N-tris(hydroxymethyl)methylglycine and nicotinic acid, N-tris(hydroxymethyl) Methylglycine and isonicotinic acid, N-tricine and 3,5-pyridinedicarboxylic acid, N-tricine and 3-pyridinesulfonic acid, N-tricine and ethylenediamine-N,N'-diacetic acid, N-tricine and 2-(pyridin-4-yl)acetic acid.

5. Use of the radioactive preparation according to any one of claims 2 to 4 in the preparation of a bone imaging agent.

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