Preparation and application of isonitrile mannose cuprous complex salt derivative

Abstract

The invention relates to the technical field of radiopharmaceutical chemistry and clinical nuclear medicine, in particular to preparation and application of isonitrile mannose cuprous complex salt derivatives. The structure of the isonitrile mannose cuprous complex salt derivative is shown in (I), the isonitrile mannose cuprous complex salt derivative is good in stability, a marking method is simple when a freeze-drying medicine box prepared from the isonitrile mannose cuprous complex salt derivative is used for preparing < 99m > Tc tumor marking radiopharmaceuticals, the marking rate of a final product is larger than 90%, the tumor affinity performance is good, and clinical application and popularization are convenient. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to the technical fields of radiopharmaceutical chemistry and clinical medicine, and particularly relates to the preparation and application of an isocyanide mannose cuprous complex salt derivative. Background Art

[0002] Cancer is one of the important factors causing human death and seriously endangers human health. Early diagnosis of cancer is an important way to improve the cure rate and survival rate of patients. At present, the diagnostic methods for malignant tumors mainly include two categories: tissue biopsy method and imaging method, among which the imaging method can non-invasively detect the location of tumors and lesions. Among them, nuclear medicine imaging can visualize the mechanism process at the molecular and cellular levels and achieve qualitative and quantitative analysis, with the advantages of being safe, non-invasive, and highly sensitive.

[0003] Nuclear medicine imaging includes single photon emission computed tomography (SPECT) and positron emission tomography (PET), which belong to functional imaging. Its fusion with structural imaging such as CT and MRI further strengthens the advantages of nuclear medicine imaging technology. Radiopharmaceutical diagnostic agents are the backbone of nuclear medicine and play a key role in tumor diagnosis. Technetium-99m, as the most widely used single photon radionuclide in clinical practice, can be prepared by a 99 Mo- 99m Tc generator, with convenient sources and ideal radionuclide properties; and compared with PET, there are more SPECT instruments and they are cheaper, enabling more patients to benefit.

[0004] Malignant tumor cells have the characteristic of rapid and abnormal proliferation. To meet this demand, tumor cells need to take up more sugar to provide energy. Mannose, as the C-2 epimer of glucose, can enter tumor cells through glucose transporters like glucose. Based on this, we previously developed a 99m Tc-labeled isocyanide mannose derivative ( 99m Tc]Tc-CN7DM) for tumor imaging (Patent No.: ZL 20211 0839524.X). This imaging agent has a high tumor uptake value in tumor-bearing mice, low uptake in non-target tissues, and fast blood clearance, showing potential for clinical translation and worthy of in-depth study. The labeling method of 99m Tc]Tc-CN7DM reported in this patent is as follows: Dissolve 2.6 mg of sodium citrate and 1 mg of L -cysteine in an appropriate amount of physiological saline, add 0.10 mg of SnCl2·2H2O thereto, adjust the pH of the solution to 6.0, and then sequentially add 0.5 mg of CN7DM and 1 mL of freshly eluted 99m Tc]NaTcO4 eluate, and react at 100 °C for 20 min to obtain 99mTc]Tc-CN7DM. This labeling method is applicable to the scientific research stage and has limitations in promoting its application in clinical settings. To facilitate clinical translation and promotion, we developed a freeze-dried kit according to the formula of the above labeling method for the preparation of 99m Tc]Tc-CN7DM. However, when the prepared freeze-dried kit was labeled with radioactive 99m Tc, unknown radioactive impurities were generated. The reason was analyzed to be that CN7DM was unstable during the freeze-drying process of the kit, resulting in unknown components, and ultimately leading to a labeling rate of the final product 99m Tc]Tc-CN7DM lower than 90%, which was not conducive to the subsequent clinical promotion and application of the product.

[0005] 99m Tc]Tc-methoxyisobutyl isonitrile ( 99m Tc]Tc-MIBI) has been widely used clinically as a myocardial perfusion imaging agent. It is prepared by the method of direct 99m Tc labeling using the MIBI freeze-dried kit. In the formula of the MIBI freeze-dried kit, instead of using the methoxyisobutyl isonitrile (MIBI) ligand, a more stable copper complex [Cu(MIBI)4]BF4 is used to replace the MIBI ligand. Based on the above background, the present invention prepares a copper tetrafluoroborate complex salt of the CN7DM ligand (i.e., [Cu(CN7DM)4]BF4), which can be used for the preparation of freeze-dried kits and facilitate the clinical translation of the tumor radioactive drug 99m Tc]Tc-CN7DM, having important scientific significance and clinical promotion value, and also being an important task faced by this field. Summary of the Invention

[0006] The present invention provides a preparation and application of an isonitrile mannosyl copper complex salt derivative. The prepared copper complex salt derivative has good stability. The kit prepared with it has a simple labeling method when preparing 99m Tc]Tc-CN7DM, the labeling rate of the final product is greater than 90%, and it has excellent tumor affinity, facilitating clinical promotion and application.

[0007] Specifically, the present invention provides the following technical solutions:

[0008] An isonitrile mannosyl copper complex salt derivative [Cu(CN7DM)4]BF4, the structural formula (I):

[0009]

[0010] The kit prepared from the above copper complex salt derivative has good stability. The 99m ​The labeling rate of the Tc-Tc-CN7DM labeling solution is high, with low uptake in non-target organs and high uptake in tumors, facilitating the subsequent clinical research and transformation of 99m Tc-Tc-CN7DM.

[0011] The present invention also provides the application of a radioactive preparation prepared from the above-mentioned cuprous complex salt derivative in the field of tumor diagnosis and treatment.

[0012] The beneficial effects of the present invention are as follows: The present invention provides an isocyanide-based mannose cuprous complex salt derivative, which is used for the preparation of a freeze-dried kit and facilitates the clinical transformation of the tumor radioactive drug 99m Tc-Tc-CN7DM, and is a compound with great promotion value. Detailed implementation manners

[0013] The present invention provides an isocyanide-based mannose cuprous complex salt derivative and its application, and the structure is shown in (I).

[0014] The preparation steps are as follows:

[0015] 1. Synthesis of the compound [Cu(CN7DM)4]BF4

[0016]

[0017] Synthesis of compound 2: Weigh compound 1 (10 g, 63 mmol), dissolve it in DMF, add 5 mL of formic acid, heat under reflux at 110 °C for 4 h. After the reaction is completed, let it stand overnight at room temperature. White solid precipitates. Filter, wash the filter cake with ethyl acetate, and dry it under vacuum to obtain 9.73 g of white solid product, with a yield of 83%. 1 1H NMR (400 MHz, DMSO-d6) δ 11.92 (s, 1H), 8.21–7.41 (m, 2H), 3.01 (t, J = 5.3 Hz, 2H), 2.19–2.11 (m, 2H), 1.52–1.39 (m, 2H), 1.37–1.32 (m, 2H), 1.24–1.19 (m, 6H).

[0018] Synthesis of compound 3: Weigh compound 2 (9.73 g, 52 mmol) and 2,3,5,6-tetrafluorophenol (TFP, 7.85 g, 47 mmol) into a round-bottom flask, add an appropriate amount of DCM to dissolve, stir at room temperature for 30 min, then add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI, 10.8 g, 56 mmol), and react overnight at room temperature. After the reaction is completed, wash it twice with saturated NaHCO3 solution and deionized water respectively, collect the organic phase, dry it with anhydrous Na2SO4, filter it by rotary evaporation, and remove the organic phase by rotary evaporation to obtain 14.56 g of pale yellow solid product, with a yield of 92%.1 1H NMR (400 MHz, CDCl3) δ 8.16 (s, 1H), 7.16–6.86 (m, 1H), 5.55 (s, 1H), 3.35–3.26 (m, 2H), 2.66 (td, J = 7.3, 2.1 Hz, 2H), 1.76 (p, J = 7.2 Hz, 2H), 1.53 (p, J = 7.2 Hz, 2H), 1.45–1.30 (m, 6H).

[0019] Synthesis of Compound 4: Weigh Compound 3 (14.56 g, 43 mmol). After dissolving it with an appropriate amount of DCM, add Burgess reagent (12.4 g, 52 mmol). React at room temperature overnight. After the reaction is completed, purify by column chromatography (PE:EA = 10:1, v / v). After vacuum drying, obtain 7.84 g of a yellow oily product with a yield of 58%. 1 1H NMR (400 MHz, CDCl3) δ 7.07–6.78 (m, 1H), 3.38 (tt, J = 6.7, 1.9 Hz, 2H), 2.67 (t, J = 7.3 Hz, 2H), 1.78 (p, J = 7.3 Hz, 2H), 1.73–1.61 (m, 2H), 1.53–1.31 (m, 6H).

[0020] Synthesis of Compound CN7DM: Weigh D -mannosamine hydrochloride (DMAH, 1.0 g, 4.6 mmol) and sodium hydroxide (200 mg, 5.0 mmol) into a round-bottom flask. Add an appropriate amount of methanol to it and stir at room temperature for 30 min until the solid dissolves. Then add a methanol solution of Compound 4 (1.74 g, 5.5 mmol) and react at room temperature overnight. After the reaction is completed, rotary evaporate to remove the solvent. Purify by column chromatography (DCM:MeOH = 5:1, v / v). After vacuum drying, obtain 989 mg of a yellow solid product with a yield of 65%. 1 1H NMR (400 MHz, CD3OD) δ 4.96 (d, J = 1.6 Hz, 1H), 4.26 (dd, J = 4.7, 1.6 Hz, 1H), 4.03–3.93 (m, 1H), 3.82–3.77 (m, 1H), 3.74 (d, J = 9.6 Hz, 2H), 3.59–3.51 (m, 1H), 2.29–2.22 (m, 2H), 1.69–1.57 (m, 4H), 1.45–1.32 (m, 8H). 1313C NMR(101MHz,CD3OD)δ175.66,153.87,93.68,72.12,69.27,67.16,60.94,53.68,41.10,35.55,28.81,28.74,28.21,25.96,25.52.HR-MS(ESI)for C 15 H 27 N2O6 + [M+H] + found 331.1869,calcd 331.1863.

[0021] Synthesis of [Cu(CN7DM)4]BF4: [Cu(CH3CN)4]BF4 (190 mg, 0.61 mmol) and compound CN7DM (800 mg, 2.43 mmol) were successively added into a 50 mL round-bottom flask, and then an appropriate amount of methanol was added to dissolve them. After reacting at room temperature for 3 h, a large amount of solid precipitated upon standing at room temperature. The mixture was filtered, and the filter cake was washed with methanol. The filter cake was dried under vacuum to obtain 876 mg of a yellow powder product with a yield of 98%. 1 1H NMR(600MHz,CD3OD)δ4.97(s,4H),4.26(d,J=4.6Hz,4H),3.98(dd,J=9.7,4.6Hz,4H),3.82–3.80(m,4H),3.77–3.73(m,8H),3.56(t,J=9.7Hz,4H),2.25(p,J=6.9Hz,8H),1.73(p,J=7.4Hz,8H),1.68–1.55(m,16H),1.40–1.34(m,24H).MS(ESI)for[C 60 H 104 CuN8O 24 + [M] + ,found 1383.6551,calcd 1383.6459.

[0022] 2. Preparation of the freeze-dried cartridge

[0023] Weigh 1 g of mannitol, 260 mg of sodium citrate dihydrate, 100 mg L ​- cysteine was added to a beaker, dissolved in 50 mL of water for injection, then 10 mg of SnCl2·2H2O was added, the pH of the solution was adjusted to 5.8, 100 mg of the compound [Cu(CN7DM)4]BF4 was added, and the volume was made up to 100 mL. It was dispensed into clean penicillin vials at 1 mL each and immediately placed in a medical freeze dryer for freeze drying. Each vial contained 1.0 mg of [Cu(CN7DM)4]BF4 and 0.10 mg of SnCl2·2H2O and other components.

[0024] Test Example

[0025] 1. Radioactively label and identify the freeze-dried drug kit provided in the example

[0026] (1) Radioactive labeling

[0027] Freshly eluted 99m Tc]NaTcO4 eluent was added to the prepared freeze-dried drug kit, and the reaction was carried out at 100 °C for 20 min to obtain 99m Tc]Tc-CN7DM labeling solution.

[0028] (2) TLC identification

[0029] Thin layer chromatography (TLC) was used to determine the radiochemical purity of the labeled product. The developing system used was polyamide film - ammonium acetate (1 M) / methanol (volume ratio: 2 / 1). Under this system, the R f values are shown in Table 1.

[0030] Table 1 R f values of radioactive components in the polyamide film - ammonium acetate (1 M) / methanol (volume ratio: 2 / 1) system

[0031]

[0032] It was identified by the above chromatography system that no unknown radioactive impurities were generated. The labeling rate and radiochemical purity of the 99m Tc]Tc-CN7DM complex were both greater than 90%, and it could be used in subsequent experiments without purification.

[0033] (3) HPLC identification

[0034] HPLC identification used gradient elution. Phase A was an aqueous solution containing 0.1% trifluoroacetic acid (TFA), and phase B was an acetonitrile solution containing 0.1% TFA. The flow rate was 1 mL / min, and the elution gradient is shown in Table 2. The results showed that: 99m The identification result of

[0035] Table 2 99m Elution gradient of Tc]Tc-CN7DM

[0036]

[0037]

[0038] 2. Biodistribution experiment in tumor-bearing mice

[0039] The 99m Tc]Tc-CN7DM labeling solution (0.1 mL, 1.85 MBq / mL) prepared from the freeze-dried kit was injected into the S180 tumor-bearing mice via the tail vein. The mice were anesthetized and sacrificed at 30 min and 120 min after injection, respectively. Organs or tissues such as heart, liver, lung, kidney, spleen, stomach, bone, muscle, small intestine, tumor, blood, muscle, and thyroid were taken, weighed, and the radioactivity counts of each organ or tissue were measured. The final results were expressed as (radioactivity count of the organ or tissue / total radioactivity count corrected by the tail) / mass of the organ or tissue, that is, %ID / g, and the final results were expressed as %ID / g ± SD.

[0040] Table 3 Biodistribution results of 99m Tc]Tc-CN7DM prepared from the freeze-dried kit in S180 tumor-bearing mice (n = 5, %ID / g, x ± SD)

[0041]

[0042] The biodistribution results are shown in Table 3. 99m Tc]Tc-CN7DM has a high tumor uptake value and good retention in tumor-bearing mice. The uptake in non-target organs is lower than that in the tumor, and the tumor-to-non-target ratio is high, especially the tumor / blood ratio, which reaches 69.28 ± 9.03 at 120 min after injection, indicating that 99m Tc]Tc-CN7DM prepared from the freeze-dried kit has excellent tumor affinity and is worthy of being vigorously promoted and applied as a new type of tumor radioactive drug.

[0043] Although the present invention has been described in detail above with general descriptions and specific embodiments, based on the present invention, some modifications or improvements can be made, which are obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention, other isonitrile mannose cuprous complex derivatives, the freeze-dried kits prepared therefrom, and the radioactive preparations obtained after being labeled with radionuclides also fall within the scope of the present invention claimed.

Claims

1. An isonitrile mannose cuprous complex salt derivative, characterized in that: The structural formula of the derivative is (I) 2. A freeze-dried drug kit for preparing tumor radioactive drugs, characterized in that: The freeze-dried drug kit comprises the isonitrile mannose cuprous complex salt derivative according to claim 1.

3. Use of the isonitrile mannose cuprous complex salt derivative as claimed in claim 1 in the preparation of tumor radiopharmaceuticals.

4. Use of the freeze-dried medicine kit as claimed in claim 2 in the preparation of tumor radioactive drugs.

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