A mannose derivative modified with (D)-α-imino acid and its application

Abstract

The present invention relates to the fields of radiopharmaceutical chemistry and clinical nuclear medicine technology, and specifically to a mannose derivative modified with a (D)-α-imino acid and its application. The mannose derivative modified with a (D)-α-imino acid is a mannose derivative having a structure represented by general formula (I) with varying carbon chain lengths. The radioactive preparation obtained by labeling the mannose derivative with a radionuclide has high tumor uptake and a good tumor / non-target ratio, making it a novel tumor radiopharmaceutical worthy of promotion.

Description

Technical Field

[0001] The present invention relates to the technical field of radiopharmaceutical chemistry and clinical medicine, and in particular to a mannose derivative modified with (D)-α-imino acid and application thereof. Background Art

[0002] It is well known that cancer is one of the major killers of human health in the 21st century and seriously endangers human health. Studies have shown that early diagnosis of cancer is of great practical significance for saving patients' lives and prolonging their survival. Currently, there are many clinical diagnostic methods for cancer, mainly including tissue biopsy and imaging methods. However, tissue biopsy requires live sections and is invasive. Imaging methods, especially molecular imaging, can non-invasively measure the location of tumors and lesions. Among them, nuclear medicine imaging can visualize, quantify and characterize mechanistic processes at the molecular and cellular levels, and has the advantages of being non-invasive and highly sensitive. Nuclear medicine imaging, including single-photon emission computed tomography (SPECT) and positron emission tomography (PET), is a functional imaging method. Its combination with imaging technologies such as CT and MRI further enhances the advantages of nuclear medicine imaging.

[0003] Mannose is a monosaccharide and a hexose. Similar to glucose, it enters cells through the glucose transporter (GLUT). During metabolism, it is also phosphorylated by hexokinase to form mannose-6-phosphate. Mannose can modulate the immune system, inhibit tumor growth and metastasis, and increase cancer survival rates. Based on these properties, mannose can be radiolabeled with radionuclides for tumor imaging in conjunction with nuclear medicine imaging. 99m Tc nuclides are widely used in radioactive diagnostic drugs due to their excellent nuclide properties and convenient sources. 99m Tc(I) forms a regular octahedral six-coordination compound [ 99m Tc]Tc-(CNR)6 + Among them, isocyanate (-NC) is used as a bifunctional linker to 99m Tc nuclides are linked to mannose molecules to obtain mannose derivatives with excellent properties as tumor imaging agents. The linker connects the targeting group and the chelating group connected to the radionuclide, and plays an important role in regulating the efficacy and pharmacokinetics of radiopharmaceuticals. Based on the above background, the present invention intends to use (D)-α-imino acid modified fragments as linkers to synthesize (D)-α-imino acid modified mannose derivatives, and then 99m Tc labeling, by improving 99mThe pharmacokinetic properties of Tc-labeled complexes, on the one hand, maintain high uptake of the complex in tumors, and on the other hand, reduce the uptake of the complex in non-target organs, thereby improving the tumor / non-target ratio. This opens a new path for exploring new tumor radioactive drugs, has important scientific significance and broad clinical application prospects, and is also a major task facing this field. Summary of the Invention

[0004] The present invention provides a mannose derivative modified with a (D)-α-imino acid and its application. The derivative has good in vitro stability, is simple to prepare, and, after radiolabeling, is used for tumor diagnosis. It exhibits high tumor uptake and a good target-to-nontarget ratio, showing promising application prospects. Specifically, the present invention provides the following technical solutions:

[0005] A mannose derivative modified with a (D)-α-imino acid, wherein the structural formula (I) is:

[0006]

[0007] In the formula, n represents an integer of 2 or more.

[0008] Preferably, in the above-mentioned mannose derivative containing (D)-α-imino acid modification, when n=5, the structural formula is as follows, and the compound prepared from the compound is 99m Tc complexes have low levels in non-target organs, high tumor uptake values, high tumor / blood and tumor / muscle ratios, and good diagnostic and therapeutic effects on tumors.

[0009]

[0010] The present invention also provides a radioactive preparation comprising the radioactively labeled (D)-α-imino acid-modified mannose derivative.

[0011] Preferably, the radioactive nuclide part is a metal radionuclide 99m Tc, 99 Tc, 94m Tc, 94 Tc, 52 Mn, 186 Re or 188 Re. Preferably, the structural formula of the radioactive agent is (II):

[0012]

[0013] The present invention also provides the use of the radioactive preparation in preparing tumor radiopharmaceuticals.

[0014] The beneficial effects of the present invention are as follows: the present invention provides a mannose derivative modified with (D)-α-imino acid, the radioactive preparation obtained by radionuclide labeling thereof has high uptake in tumors and a good tumor / non-target organ ratio, and is a new tumor radiopharmaceutical with promotion value. DETAILED DESCRIPTION

[0015] The present invention provides a mannose derivative containing (D)-α-imino acid modification and its application. In a preferred embodiment, the present invention provides a mannose derivative having a general structural formula of [ 99m Radioactive preparations of Tc]Tc-CNIADM:

[0016]

[0017] In the formula, n represents an integer of 2 or greater.

[0018] The preparation steps are as follows:

[0019] (1) Synthesis of ligand CNIADM

[0020] Synthesis of 1c: An appropriate amount of compound 1b was weighed into a 50 mL round-bottom flask and dissolved in an appropriate amount of N,N-dimethylformamide (DMF). Et3N was then added and stirred at room temperature for 30 min until the solid was completely dissolved. A DMF solution of compound 1a was then added and the mixture was allowed to react overnight at room temperature. After the reaction, the solvent was removed by distillation under reduced pressure and purified by column chromatography (dichloromethane:methanol = 10:1) to obtain compound 1c.

[0021] Synthesis of 1d: An appropriate amount of compound 1c and 2,3,5,6-tetrafluorophenol (TFP) were weighed into a 50 mL round-bottom flask and dissolved in DMF. After stirring at room temperature for 30 min, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) was added and the mixture was allowed to react at room temperature overnight. After completion of the reaction, the solvent was removed by distillation under reduced pressure and purified by column chromatography (petroleum ether:ethyl acetate = 1:1) to obtain compound 1d.

[0022] Synthesis of CNIADM: Appropriate amounts of D-mannosamine hydrochloride (DMAH) and sodium hydroxide were weighed into a 50-mL round-bottom flask and dissolved in anhydrous methanol. After stirring at room temperature for 30 min, compound 1d was added and reacted at room temperature for 24 h. After completion of the reaction, the solvent was removed by distillation under reduced pressure and purified by column chromatography (dichloromethane:methanol = 5:1) to obtain the ligand CNIADM.

[0023] The specific synthetic route is as follows:

[0024]

[0025] In the formula, n represents an integer of 2 or more.

[0026] (2)[ 99m Preparation of Tc]Tc-CNIADM

[0027] Dissolve appropriate amount of sodium citrate and L-cysteine ​​in appropriate amount of physiological saline, add appropriate amount of SnCl2·2H2O to adjust the solution pH to 6.0, then add appropriate amount of ligand CNIADM and freshly washed Na 99m TcO4, react at 100℃ for 20min to obtain the marker [ 99m Tc]Tc-CNIADM.

[0028] The [ 99m The radiochemical purity of the [Tc]Tc-CNIADM complex is greater than 95%, and it has good in vitro stability and is water-soluble. It has high tumor uptake in tumor-bearing mice and a good target to non-target ratio, which is conducive to its promotion and application as a new tumor imaging agent.

[0029] Example 1

[0030] This example provides a (D)-proline-modified mannose derivative (CN5DPDM), the structural formula of which is as follows:

[0031]

[0032] CN5DPDM 99m Tc labeled 99m Tc-labeled (D)-proline-modified mannose derivative, referred to as [ 99m Tc]Tc-CN5DPDM.

[0033] The preparation steps are as follows:

[0034] 1. Synthesis of CN5DPDM

[0035]

[0036] Synthesis of 2c: To a 50 mL round-bottom flask, add 219 mg (1.90 mmol) of compound 2b and dissolve in DMF. Then, add 2.4 mL (17.3 mmol) of Et3N. Stir at room temperature for 30 minutes, then add 500 mg (1.73 mmol) of compound 2a. The mixture reacts overnight at room temperature. After completion of the reaction, the solvent is removed by distillation under reduced pressure. Purification by column chromatography (dichloromethane:methanol = 10:1) affords 309 mg of product 2c as a yellow oil in a 75% yield. 1H NMR(400MHz, Methanol-d4)δ4.45-4.29(m,1H),3.46(tt,J=6.7,2.0Hz,2H),2.43-2.31(m,2H), 2.27-2.11(m,2H),2.03-1.94(m,2H),1.74-1.57(m,4H),1.53-1.46(m,2H),1.43-1.34(m,2H).

[0037] Synthesis of 2d: To a 50 mL round-bottom flask, compound 2c (300 mg, 1.26 mmol) and 227 mg, 1.37 mmol, of 2,3,5,6-tetrafluorophenol were added. DMF was added for dissolution, and the mixture was stirred at room temperature for 30 minutes before the addition of EDCI (220 mg, 1.15 mmol). The reaction was allowed to proceed overnight at room temperature. After completion of the reaction, the solvent was removed by distillation under reduced pressure. The product was purified by column chromatography (petroleum ether:ethyl acetate = 1:1) to afford 150 mg of the product as a yellow oil in a 34% yield. 1 H NMR(400MHz,Chloroform-d)δ7.05-6.74(m,1H),4.80(dd,J=8.8,3.8Hz,1H),3.74-

[0038] 3.50(m,2H),3.42-3.33(m,2H),2.38(t,J=7.5Hz,2H),2.28-2.14(m,2H),2.16-1.94(m,2H),1.77-1.66(m,4H),1.56-1.44(m,2H).

[0039] Synthesis of CN5DPDM: To a 25 mL round-bottom flask were added 14 mg (0.36 mmol) of sodium hydroxide and 69 mg (0.36 mmol) of D-mannosamine hydrochloride, dissolved in anhydrous methanol, and allowed to react at room temperature for 30 min. Then, 150 mg (0.39 mmol) of compound 2d was added and allowed to react at room temperature overnight. After completion of the reaction, the solvent was removed by distillation under reduced pressure and purified by column chromatography (dichloromethane:methanol = 5:1) to obtain 39 mg of the product as a yellow oil in a 31% yield. 1H NMR (400MHz, Methanol-d4) δ5.05(d,J=1.6Hz,1H),4.24(dd,J=4.6,1.6Hz,1H),3.98(dd,J=9.7,4.6Hz,1H),3.83-3.77(m,2H),3.76(t,J=2.1Hz,1H),3.73 (t,J=2.9Hz,1H),3.65-3.61(m,1H),3.58-3.55(m,1H),3.50-3.48(m,1H),2. 38(t,J=6.5Hz,2H),1.99-1.88(m,2H),1.65-1.57(m,6H),1.52-1.41(m,4H). 13 CNMR(101MHz,Methanol-d4)δ173.99,173.05,158.03,92.22,72.12,69.76,67.22,61.41 ,59.12,53.79,40.90,33.70,29.83,28.71,25.74,24.44,23.51,22.35.HR-MS(ESI)forC 18 H 30 N3O7[M+H] + :found 400.2086,calcd 400.2078.

[0040] 2.[ 99m Preparation of Tc]Tc-CN5DPDM

[0041] 2.6 mg sodium citrate and 1 mg L-cysteine ​​were dissolved in an appropriate amount of normal saline, 0.1 mg SnCl2·2H2O was added thereto, and the pH of the solution was adjusted to 6.0. Then 0.5 mg CN5DPDM ligand and freshly washed Na 99m TcO4, react at 100 ° C for 20 minutes to obtain the [ 99m Tc]Tc-CN5DPDM.

[0042] Test example

[0043] 1. Chromatographic Identification of the Radioactive Preparation Provided in Example 1

[0044] (1) TLC method

[0045] Thin layer chromatography (TLC) was used to determine the radiochemical yield and radiochemical purity of the labeled substance. The developing system used was polyamide film-ammonium acetate (1M) / methanol (volume ratio: 2 / 1). Under this system, the R f The values ​​are shown in Table 1.

[0046] Table 1 R of radioactive components in polyamide film-ammonium acetate (1M) / methanol (volume ratio: 2 / 1) system f value

[0047]

[0048] The [ 99m The radiochemical yield and radiochemical purity of the [Tc]Tc-CN5DPDM complex were both greater than 90% and it was used in subsequent experiments without further purification.

[0049] (2) HPLC method

[0050] The radiochemical purity of the labeled compound was determined using high-performance liquid chromatography (HPLC) using a SHIMADZU HPLC (CL-20AVP) with a Kromasil C18 reverse-phase column (5 μm, 250 × 4.6 mm) and a Gabi raytest radioactivity detector. The elution gradient was as shown in Table 2, with a flow rate of 1 mL / min. Phase A consisted of pure water containing 0.1% trifluoroacetic acid, and phase B consisted of acetonitrile containing 0.1% trifluoroacetic acid.

[0051] Table 2 Gradient elution conditions of the complexes

[0052]

[0053] HPLC identification results showed that 99m The retention time of Tc]Tc-CN5DPDM is 9.85 min.

[0054] 2. Determination of lipid-water partition coefficient

[0055] 100 μL of the labeling solution was placed in a 4 mL centrifuge tube, and then 1 mL of n-octanol and 900 μL of PBS (0.025 M,

[0056] pH 7.4), vortex, and allow the solution to stand for separation. Centrifuge for 5 minutes (10,000 rpm). Take three 100 μL aliquots from each phase and measure the radioactivity count in a γ-counter. The lipid-water partition coefficient P = organic phase radioactivity count / aqueous phase radioactivity count. The lipid-water partition coefficient is usually expressed as log P. 99m The log P value of Tc]Tc-CN5DPDM is -4.23±0.14, indicating that it is a water-soluble substance.

[0057] 3. Stability Determination

[0058] Will[ 99mThe radiochemical purity of Tc]Tc-CN5DPDM was determined by HPLC after incubation in normal saline at room temperature and in mouse serum at 37°C for 4 hours. The experimental results showed that the radiochemical purity of Tc]Tc-CN5DPDM was greater than 95% after incubation in normal saline at room temperature and in mouse serum at 37°C for 4 hours, indicating good in vitro stability.

[0059] 4. Biodistribution Determination in Tumor-bearing Mice

[0060] (0.1mL, 185kBq)[ 99m Tc]Tc-CN5DPDM labeling solution is similar to that reported previously [ 99m [Tc]Tc-CN7DM labeling solution (Patent No.: ZL202110839524X) was injected into A549 tumor-bearing mice via the tail vein. 120 minutes later, the mice were anesthetized and sacrificed. After dissection, tissues or organs such as the heart, liver, lungs, kidneys, spleen, stomach, bones, muscles, small intestine, blood, and tumor were removed. The radioactivity count of each organ was measured using a γ-counter, and the uptake value for each organ (in %ID / g) was obtained by conversion based on the mass of each organ. The biodistribution results of the label in tumor-bearing mice are shown in Table 3.

[0061] Table 3. Biodistribution results of markers in A549 tumor-bearing mice 120 min after administration (n=4, mean±SD, %ID / g)

[0062]

[0063] From the results, it can be seen that [ 99m Tc]Tc-CN5DPDM maintains high uptake in tumors, low uptake in other non-target organs, and rapid blood clearance. 2 hours after administration, the background uptake in blood is only 0.05% ID / g, and the tumor / blood ratio is 100.20. Its tumor / blood ratio and tumor / muscle ratio are better than [ 99m Tc]Tc-CN7DM.

[0064] 5.[ 99m SPECT / CT Imaging Experiment of Tc]Tc-CN5DPDM in Tumor-Bearing Mice

[0065] Injected into the tail vein of A549 tumor-bearing mice 99m Tc]Tc-CN5DPDM (about 18.5 MBq). 2 h after administration, the mice were anesthetized, the parameters were set, the mice were fixed, and SPECT / CT imaging was performed. Finally, the scan images were obtained using HiSPECT software and vivoquant 2.5 software.

[0066] From the results of SPECT / CT imaging of mice bearing A549 tumors, [99m The radioactivity of Tc]Tc-CN5DPDM was significantly concentrated in the tumor of mice, while the uptake in other non-target organs was low and the background was clean, which was consistent with the biodistribution results.

[0067] While the present invention has been described in detail above using general descriptions and specific embodiments, modifications and improvements based on the present invention are readily apparent to those skilled in the art. Therefore, such modifications and improvements, without departing from the spirit of the present invention, include radioactive preparations obtained by radionuclide labeling of ligands modified with (D)-α-imido acids using monosaccharides other than mannose. Furthermore, radioactive preparations obtained by radionuclide labeling of monosaccharide ligands modified with (L)-α-imido acids also fall within the scope of the present invention.

Claims

1. A mannose derivative containing a (D)-α-imino acid, characterized in that: The structural formula of the mannose derivative is (I): Here, n represents an integer of 2 or greater.

2. A radioactive preparation, characterized in that The radioactive preparation comprises the mannose derivative containing a (D)-α-imino acid according to any one of claims 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): In the formula, n represents an integer of 2 or greater.

5. Use of the radioactive preparation according to any one of claims 2 to 4 in the preparation of tumor radiopharmaceuticals.

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