Glucose derivative modified by L-proline and application thereof

Abstract

The invention relates to the technical field of radiopharmaceutical chemistry and clinical nuclear medicine, in particular to an L-proline modified glucose derivative and application thereof. The L-proline modified glucose derivative is a glucose derivative which contains different carbon chain lengths and has a structure as shown in a general formula (I), and a radioactive preparation obtained by labeling the L-proline modified glucose derivative with radionuclide is high in uptake in tumors and good in tumor / non-target ratio, and is a novel tumor radiopharmaceutical worthy of popularization. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to the technical fields of radiopharmaceutical chemistry and clinical medicine, and particularly relates to a glucose derivative modified with L-proline and its application. Technical Background

[0002] Cancer seriously threatens human health. Early detection and treatment of cancer can effectively improve the survival rate, reduce the pain of patients, and save treatment costs. Nuclear medicine imaging technology can non-invasively detect the molecular biological behavior and pathophysiological changes in the body and has become a major class of detection means for tumor diagnosis.

[0003] Currently, the most commonly used tumor imaging agent in clinical practice is the positron radionuclide 18 F-labeled tumor imaging agent 18 F-fluorodeoxyglucose ( 18 F-FDG). This drug is based on the principle that glucose is the main energy source for cell metabolism, and malignant tumor cells proliferate rapidly and have a much higher demand for glucose uptake than normal cells, and can be widely used for tumor imaging. However 18 The F radionuclide needs to be produced by an accelerator, and the cost of PET / CT examination is relatively expensive, which restricts its widespread use to a certain extent, especially in underdeveloped areas. Compared with PET, the number of SPECT instruments is larger, the diagnostic cost is lower, and with the development of technology, the resolution and sensitivity of the instruments are also constantly improving. And 99m Tc is the most commonly used single photon radionuclide in clinical practice and can be prepared by a 99 Mo- 99m Tc generator, and the source is convenient. In summary, preparing a 99m Tc-labeled glucose derivative with excellent performance, high tumor uptake, and good target / non-target ratio has important scientific significance and broad clinical application prospects.

[0004] In the research of 99m Tc-labeled glucose derivatives, 99m Tc-labeled glucose derivatives containing isonitrile have been successfully prepared as tumor imaging agents (Patent No.: ZL201710451094.8), among which 99m Tc-(CN5DG)6 + has a good target / non-target ratio, but the tumor uptake needs to be improved. Regarding 99m Tc-labeled glucose derivatives containing benzene rings (Patent No.: ZL202010032704.2), among which 99m Tc-CNPEDG has a high tumor uptake and good retention in tumors, but the blood clearance is slow and the tumor / blood ratio needs to be improved. In 99mTc-labeled glucose derivatives containing cyclohexane (Patent No.: ZL202111298353.0) involved in 99m Tc-CNMECHDG, which has a relatively high tumor / blood ratio in tumor-bearing mice, but poor tumor retention properties. To develop a new tumor imaging agent with high tumor uptake and good tumor / non-target ratio, the present invention synthesizes a glucose ligand containing L-proline (abbreviated as CNLPDG) by introducing L-proline between glucosamine and different carbon-chain isocyanides, and 99m Tc-label it in order to obtain a new glucose tumor imaging agent with excellent properties. Summary of the Invention

[0005] The present invention provides a glucose derivative modified with L-proline and its application. After being radiolabeled, this derivative has good in vitro stability, is simple to prepare, has high tumor uptake, and a high target-to-non-target ratio, and has broad clinical application prospects for tumor diagnosis.

[0006] Specifically, the present invention provides the following technical solutions:

[0007] A glucose derivative modified with L-proline, and the structural formula (I) is as follows:

[0008]

[0009] In the formula, n represents an integer from 2 to 15.

[0010] Preferably, in the above-mentioned glucose derivative modified with L-proline, when n = 5, the structural formula is one of the following, and the 99m Tc complex prepared from this compound has low uptake in non-target organs, high tumor uptake value, high tumor / blood and tumor / muscle ratios, and good tumor diagnosis and treatment effects.

[0011]

[0012] The present invention also provides a radioactive preparation, and the radioactive preparation contains the above-mentioned glucose derivative modified with L-proline that is radiolabeled.

[0013] Preferably, the above radioactive nuclide part is a metallic radioactive nuclide 99m Tc, 99 Tc, 94m Tc, 94 Tc, 52 Mn, 186 Re or 188 Re.

[0014] Preferably, the structural formula of the above radioactive preparation is (II):

[0015]

[0016] The present invention also provides the use of the above radioactive preparation in the preparation of tumor radioactive drugs.

[0017] The beneficial effects of the present invention are as follows: The present invention provides a glucose derivative modified with L-proline, and the radioactive preparation obtained by labeling it with a radionuclide has high uptake in tumors and a good tumor / non-target organ ratio, and is a novel glucose-based tumor radioactive drug with promotional significance. Specific implementation examples

[0018] The present invention provides a glucose derivative modified with L-proline and its application. In a preferred embodiment, the present invention provides a radioactive preparation with the structural general formula 99m Tc]Tc-CNLPDG:

[0019]

[0020] In the formula, n represents an integer from 2 to 15.

[0021] Its preparation steps are as follows:

[0022] (1) Synthesis of ligand CNLPDG

[0023] Synthesis of CN-LP: Weigh an appropriate amount of compound L-proline in a 50 mL round-bottom flask, add an appropriate amount of N,N-dimethylformamide (DMF) to dissolve it, then add Et3N, stir at room temperature for 30 min until the solid is completely dissolved, add the DMF solution of compound CN-TFP thereto, react overnight at room temperature, distill off the solvent under reduced pressure after the reaction, and purify by column chromatography (methylene chloride: methanol = 10:1) to obtain compound CN-LP.

[0024] Synthesis of CN-LP-TFP: Weigh an appropriate amount of compound CN-LP and 2,3,5,6-tetrafluorophenol (TFP) in a 50 mL round-bottom flask, dissolve with DMF, add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) after stirring at room temperature for 30 min, react overnight at room temperature, distill off the solvent under reduced pressure after the reaction ends, and purify by column chromatography (petroleum ether: ethyl acetate = 1:1) to obtain compound CN-LP-TFP.

[0025] Synthesis of CNLPDG: Weigh an appropriate amount of D-glucosamine hydrochloride (DGAH) and sodium hydroxide in a 50 mL round-bottom flask, add methanol to dissolve, after dissolving at room temperature, add compound CN-LP-TFP, react at room temperature for 24 h, distill off the solvent under reduced pressure after the reaction ends, and purify by column chromatography (methylene chloride: methanol = 5:1) to obtain ligand CNLPDG.

[0026]

[0027] In the formula, n represents an integer from 2 to 15.

[0028] (2) 99m Preparation of

[0029] Dissolve an appropriate amount of sodium citrate and L-cysteine in an appropriate amount of physiological saline, add an appropriate amount of SnCl2·2H2O thereto, adjust the pH of the solution to 5.8, and then successively add an appropriate amount of ligand CNLPDG and freshly eluted 99m Tc]NaTcO4, and react at 100 °C for 20 min to obtain the labeled product 99m Tc]Tc-CNLPDG.

[0030] The 99m Tc]Tc-CNLPDG complex prepared by the above method has a radiochemical purity greater than 95%, good in vitro stability, is a water-soluble substance, has high tumor uptake in tumor-bearing mice and a good target-to-non-target ratio, and is conducive to being clinically promoted and applied as a new type of tumor imaging agent.

[0031] Example 1: This example provides a glucose derivative containing L-proline (CN5LPDG), and its structural formula is as follows:

[0032]

[0033] Perform 99m Tc labeling on CN5LPDG to obtain a 99m Tc-labeled glucose derivative containing L-proline, simply referred to as 99m Tc]Tc-CN5LPDG.

[0034] The preparation steps are as follows:

[0035] 1. Synthesis of CN5LPDG

[0036]

[0037] Synthesis of CN5LP: Add 654 mg (5.70 mmol) of L-proline to a round-bottom flask, dissolve it with DMF, add 7.2 mL (51.9 mmol) of triethylamine, stir at room temperature until dissolved, and then add 1500 mg (5.19 mmol) of dissolved CN5TFP. React overnight at room temperature. After the reaction is completed, distill off the solvent under reduced pressure and purify by column chromatography (dichloromethane:methanol = 10:1) to obtain 1.094 g of a yellow oily product CN5LP with a yield of 88%. 11H NMR (400 MHz, Methanol-d4) δ 4.41 - 4.36 (m, 1H), 3.46 (tt, J = 6.7, 4.1, 2.0 Hz, 2H), 2.39 - 2.33 (m, 2H), 2.27 - 2.15 (m, 2H), 2.02 - 1.95 (m, 2H), 1.69 - 1.58 (m, 4H), 1.50 (dd, J = 11.0, 7.5 Hz, 2H), 1.43 - 1.32 (m, 2H).

[0038] Synthesis of CN5LP-TFP: Add 1.087 g (4.57 mmol) of CN5LP and 826 mg (4.98 mmol) of TFP to a round-bottom flask. After adding DMF, stir at room temperature for 30 min. Then add 795 mg (4.16 mmol) of EDCI in an ice-water bath and stir overnight at room temperature. After the reaction is completed, extract with dichloromethane, dry with anhydrous sodium sulfate, and purify by column chromatography (petroleum ether: ethyl acetate = 1:1) to obtain 948 mg of a yellow oily product with a yield of 59%. 1 1H NMR (600 MHz, Chloroform-d) δ 7.01 - 6.93 (m, 1H), 4.79 (dd, J = 8.8, 4.0 Hz, 1H), 3.71 - 3.65 (m, 1H), 3.56 (dt, J = 9.7, 7.2 Hz, 1H), 2.43 - 2.27 (m, 4H), 2.26 - 2.19 (m, 1H), 2.19 - 2.13 (m, 1H), 2.13 - 2.05 (m, 1H), 1.76 - 1.65 (m, 5H), 1.57 - 1.41 (m, 2H).

[0039] Synthesis of CN5LPDG: Add 23 mg of sodium hydroxide (0.57 mmol) to a round-bottom flask, add an appropriate amount of methanol and stir until dissolved. Then add 112 mg (0.52 mmol) of D-glucosamine hydrochloride and stir until dissolved at room temperature. Then add 200 mg (0.52 mmol) of CN5LP-TFP and react overnight at room temperature, monitored by TLC. After the reaction is completed, remove the solvent by rotary evaporation and purify by column chromatography to obtain 114 mg of a white solid product with a yield of 55%. 1 1H NMR (400 MHz, Methanol-d4) δ 5.09 (d, J = 3.4 Hz, 1H), 3.82 (d, J = 3.5 Hz, 1H), 3.81 -

[0040] 3.77 (m, 2H), 3.70 (t, J = 4.6 Hz, 1H), 3.67 (d, J = 1.8 Hz, 1H), 3.47 (tt, J = 6.4, 1.8 Hz, 4H), 2.44 - 2.35 (m, 2H), 2.21 - 2.11 (m, 2H), 2.07 - 1.98 (m, 2H), 1.70 - 1.58 (m, 6H), 1.47 (t, 2H). 13 C NMR (101 MHz, Methanol - d4) δ 173.79, 173.08, 153.97, 91.27, 71.94, 71.46, 71.12, 61.48, 60.21, 54.62, 41.02, 33.72, 32.04, 29.67, 28.71, 25.76, 24.41, 23.56. HR - MS (ESI) for C 18 H 30 N3O7 [M + H] + : found 400.2068, calcd 400.2078.

[0041] 2. 99m Preparation of [[Tc]]Tc - CN5LPDG

[0042] Dissolve 2.6 mg of sodium citrate and 1 mg of L - cysteine in an appropriate amount of physiological saline, add 0.1 mg of SnCl2·2H2O thereto, adjust the pH of the solution to 5.8, and then successively add 0.5 mg of the ligand CN5LPDG and freshly eluted 99m [[Tc]]NaTcO4, and react at 100 °C for 20 min to obtain [[Tc]]Tc - CN5LPDG of this example. 99m

[0043] Test Examples

[0044] 1. Chromatographic identification of the radioactive preparation provided in Example 1

[0045] (1) TLC method

[0046] The radiochemical yield and radiochemical purity of the labeled compound were determined by thin - layer chromatography (TLC). The developing system used was polyamide film - ammonium acetate (1 M) / methanol (volume ratio: 2 / 1). Under this system, the R f values of each radioactive component are shown in Table 1.

[0047] Table 1 R f values of radioactive components in the polyamide film - ammonium acetate (1 M) / methanol (volume ratio: 2 / 1) system

[0048] ​

[0049] As measured by the above chromatography identification 99m The radiochemical yield and radiochemical purity of the [Tc]Tc-CN5LPDG complex were both greater than 95%, and it was used in subsequent experiments without further purification.

[0050] (2) HPLC method

[0051] High performance liquid chromatography (HPLC) was used to identify the radiochemical purity of the labeled compound: SHIMADZU high performance liquid chromatograph (CL-20AVP), Kromasil C18 reversed-phase column (5 μm, 250×4.6 mm). The elution gradient was as shown in Table 2. Phase A was water (containing 0.1% trifluoroacetic acid), and phase B was acetonitrile (containing 0.1% trifluoroacetic acid). The flow rate was 1 mL / min.

[0052] Table 2 Gradient elution conditions of the complex

[0053]

[0054] The HPLC identification results showed that 99m the radiochemical purity of [Tc]Tc-CN5LPDG was greater than 95%, and the retention time was 10.03 min.

[0055] 2. Determination of lipid-water partition coefficient and stability

[0056] Take 100 μL of the labeled solution and place it in a 2 mL centrifuge tube. Then add 800 μL of n-octanol and 700 μL of PBS (0.025 M, pH 7.4) to it. Vortex and let it stand. After the solution is layered, centrifuge it in a centrifuge for 3 min (10000 rpm). Take out 3 portions of 100 μL from each of the two phases and measure their radioactivity counts in a γ-counter. The lipid-water partition coefficient P = radioactivity count in the organic phase / radioactivity count in the aqueous phase. Usually, the lipid-water partition coefficient is expressed as log P. After measurement, 99m the log P value of [Tc]Tc-CN5LPDG was -3.78 ± 0.11, and it was a water-soluble substance.

[0057] When 99m [Tc]Tc-CN5LPDG was placed at room temperature and in mouse serum at 37 °C for 4 h, the radiochemical purity was greater than 90%, indicating that 99m [Tc]Tc-CN5LPDG had good in vitro stability and could be used for subsequent research.

[0058] 3. Preliminary exploration of the uptake mechanism

[0059] In order to explore 99mWhether the uptake mechanism of Tc-CN5LPDG is related to glucose transporters was determined by measuring the effects of D-glucose, L-glucose, and bovine insulin on the 99m cellular uptake of Tc-CN5LPDG in the A549 cell line. A549 cells in the logarithmic growth phase were collected and diluted to 3×10 5 cells / mL with sugar-free complete medium. 1 mL of the cell suspension was added to each well of a 24-well plate and incubated overnight in starvation. After the cells adhered to the wall, they were washed once with 0.5 mL of sugar-free DMEM basal medium, and then 0.1 mL of D-glucose / media (20 mg / mL), 0.1 mL of L-glucose / media (20 mg / mL), and 0.1 mL of bovine insulin / media (20 units / mL) were added. After 30 min of incubation, 0.1 mL 99m of the Tc-CN5LPDG labeling solution (1.85 MBq / mL) was added, and the basal medium was added to make the total volume of each well 0.5 mL. After 4 h of incubation, the medium was aspirated, and the cells were washed twice with cold PBS (1 mL / well), then 1 mL of sodium hydroxide solution (1 M) was added to lyse the cells. The cells were transferred to a plastic tube, and their radioactivity counts were measured. Six wells were measured in parallel for each group. The cellular uptake was calculated according to the following formula:

[0060]

[0061] The results showed that after adding D-glucose, the cellular uptake was inhibited by 47% (p = 0.0005), there was no significant change after adding L-glucose, and after adding bovine insulin, the cellular uptake increased by 247% (p = 0.0021), indicating that 99m the pathway of Tc-CN5LPDG entering tumor cells is related to glucose transporters.

[0062] 4. Biodistribution experiment in tumor-bearing mice

[0063] The 99m Tc-CN5LPDG labeling solution was diluted to 1.85 MBq / mL. 0.1 mL of the diluted labeling solution was injected into the tail veins of S180 tumor-bearing and A549 tumor-bearing mice respectively. After 120 min, the mice were anesthetized and sacrificed. After dissection, tissues or organs such as the heart, liver, lung, kidney, spleen, stomach, bone, muscle, small intestine, tumor, and blood were taken out, and the radioactivity counts of each organ were measured with a γ-counter and divided by the mass of the corresponding organ to obtain the uptake value of each organ, expressed as %ID / g. The biodistribution of the labeled compound in tumor-bearing mice is shown in Table 3.

[0064] Table 3. Biodistribution results of markers in S180 tumor-bearing mice (n=5) and A549 tumor-bearing mice (n=4) after administration for 120 min (mean±SD, %ID / g)

[0065]

[0066] From the biodistribution results, it can be seen that [ 99m The uptake of Tc-CN5LPDG in tumors was significantly higher than that in other non-target organs, and the tumor / meat ratio was good. The blood was cleared quickly, and the tumor / blood ratios in mice bearing S180 tumors and mice bearing A549 tumors reached 84.87 and 151.7 respectively 120 minutes after injection, which was very beneficial for tumor imaging.

[0067] 5. SPECT / CT imaging experiments in tumor-bearing mice

[0068] The cells were injected into the tail vein of S180 tumor-bearing mice. 99m Tc]Tc-CN5LPDG labeling solution (about 18.5MBq) was added. The mice were anesthetized 120 minutes after administration, the parameters were set, the mice were fixed, and SPECT / CT imaging was performed. Finally, the scanning images were obtained by HiSPECT software and vivoquant 2.5 software.

[0069] From the SPECT / CT imaging results of mice bearing S180 tumors, [ 99m The uptake of Tc]Tc-CN5LPDG in non-target organs was low, the background was clean, and the radioactivity concentration in tumor lesions was obvious, which was consistent with the biodistribution results.

[0070] Although the present invention has been described in detail above with general description and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made on the basis of the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention, and the radioactive preparations obtained by labeling with radionuclides using ligands obtained by glucose modified with natural amino acids of L configuration other than L-proline, all fall within the scope of protection claimed by the present invention.

Claims

1. A glucose derivative modified with L-proline, characterized in that, The structural formula of the glucose derivative is (I): In the formula, n represents an integer from 2 to 15.

2. A radioactive preparation, characterized in that, The radioactive preparation contains the L-proline-modified glucose derivative described in any one of claims 1 labeled with a radionuclide.

3. The radioactive preparation according to claim 2, wherein, 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 preparation is: In the formula, n represents an integer from 2 to 15.

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

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