| BackgroundPositron emission tomography (PET) is the most advanced imaging techniques, and the in vivo visualization and monitoring of biological processes within the living system. Labeling tumor-targeting moleculars with radioisotopes can be apply for tumor imaging to assist diagnosis and guide therapy.The widely used tracer in clinic is 18F-Fluorodeoxyglucose (18F-FDG). It is closely correlated with tissue glucose metabolism. Since increased uptake is seen both in tumor and inflammation, differential diagnosis between them is not satisfactory.Researchers designed a hairpin-shaped activatable cell penetrating peptide (ACPP) to enhanced the targeting effect of tracers. The hairpin is made of matrix metal loproteinase (MMP) substrate, which can be hydrolyzed by MMP. MMP is highly expressed around the surface of malignant tumors. Therefore, the hairpin of ACPP is hydrolyzed by MMP when travel to the tumor cells. Carrying the radioisotopes, the active cell penetrating peptide gets into tumor cell. And it can be used for tumor-targeting PET imaging.Apart from ACPP, receptor imaging can also improve the specificity of tumor-imaging.PET receptor imaging is using the distinctivet combined of radio-labeling ligand and highly specify receptor. It offers high specificity of ligand-receptor and excellent sensitivity of radio-detection. Receptor imaging can be divided into two groups. One is neurotransmitter receptors including dopamine receptor, and the other is tumor receptors including estrogen, androgen and somatostatin receptors (SSTR). Among all, SSTR is the most well investigated and widely used tumor-imaging target.SSTR is a glycoprotein located in the surface of cell membrane, which has 5 types. In addition to central nervous system and peripheral tissues, SSTR is highly expressed in numbers of tumor cells (including neuroendocrine tumors, nervous system tumors and other cancers, such as breast cancer, liver cancer, stomach cancer, colon cancer, prostate cancer, ovarian cancer, etc.). The quantity and density of receptor in tumor are larger than in normal tissues. When labeled with radioisoptoes, somatostatin and its analogues can be exploited for tumor-imaging.This research is divided into two parts. Part one is to discuss the feasibility of synthesis of 18F-FEA labeled ACPP by click chemistry. Part two is the fast and efficient preparation of 18F-TOCA by click chemistry, which is a PET tracer for neuroendocrine tumors.Objective1. Synthesize 2-azidoethyl-4-toluenesulfonate for the next step.2. Synthesize 18F-FEA as precursor for the next step of click reaction. 3. Synthesize 18F-ACPP by click chemistry.4. Adjust the reaction conditions of 18F-ACPP preparation, and check failure reasons.5. Synthesize 18F-TEGay as precursor for the next step of click reaction.6. Synthesize 18F-TOCA by click chemistry.7. Optimize the temperature of click reaction. Obtain the best reaction temperature through the test of radiochemical yield (RCY) under different reaction temperature.8. Optimize the synthesis time of click reaction. Obtain the best synthesis time through the test of RCY under different synthesis time.Methods1. Bromine ethanol 1.25g (10mmol dissolved in 5ml water) and azide sodium 0.78g(12mmol) was heated at 100℃ and stirring reacted for 12 hours. The mixture was extracted 3 times with dichloromethane 25mL and water 2mL. The organic phase solution 25mL was taken and dry. Triethylamine 1.417g (2ml,14mmol) and p-toluene sulfonyl chloride 1.91g (10mmol) were added and stirred for 4 hours at room temperature. After that, glycine was added and stirred for 2 hours. The organic phase solution was taken and cleaned with sodium hydroxide 2×50mL (lmol/L), and dry again. The solvent was heated at 40℃ and evaporated. After silica gel column chromatography separation, filtered liquids were collected and taken nuclear magnetic resonance detection.2. Synthetize of’"F-FEA. Through the nuclear reaction 18O(p,n)18F, no-carrier-added [18F]fluoride was obtained on the PET trace cyclotron. Passed through a QMA cartridge, [18F]fluoride was trapped and then eluted off to a reaction vessel with a solution of K2.2.2./K2CO3. After the azeotropic water removing,2-azidoethyl-4-toluenesulfonate 5mg(4.14nmol) in 400μL ACN was added to the above dried [K/K2.2.2.]+18F- complex, and heated at 85℃ for 10min to produce 18F-FEA. The mixture was distilled at 85°C under a slow flow of nitrogen into a trapping vial surrounded by salty ice water.3. Synthetize of 18F-ACPP. Sodium L-ascobate (ASC) 100u L(1.5mol/L), CuSO4 100 u L (0.45mol/L) and ACPP 500μL (0.28nmol/L) were added subsequently, and reacted for 15min with above performed 18F-TEGay under the temperature of 60℃.4. (1) ACPP was heated at 60℃ and stirred for 15min. The reaction was monitored by radio-HPLC to test whether ACPP will decompose after heated. (2) Expanded the reaction time from 15min to 75,110 and 145min. (3) Condensated solvent volume and increased concentration, changed to ASC 50μL (3mol/L), CuSO450μL (0.9mol/L), ACPP 100μL (1.4nmol/L), and reaction volume reduced from 950 μL to 450μL. (4) Reduced the dosage of Cu and ASC, changed to ASC 40μL (0.3mol/L), CuSO4 20μL (0.096mol/L),ACPP 100μL (1.4nmol/L). (5) Added Copper(I)-Stabilizing Ligand, changed to ASC 14μL (0.3mol/L), CuSO42μL(0.09mol/L), ACPP 100μL (1.4nmol/L) and THPTA 2μL (0.45mol/L).5. Synthetize of 18F-TEGay. Through the nuclear reaction 180 (p, n) 18F, no-carrier-added [18F]fluoride was obtained on the PET trace cyclotron. Passed through a QMA cartridge, [18F]fluoride was trapped and then eluted off to a reaction vessel with a solution of K2.2.2./K2C03. After the azeotropic water removing, TsOTEGay 0.16mg (0.455μmol) in 280μL ACN was added to the above dried [K/K2.2.2.]+18F-complex, and heated at 110℃ for 15min to produce 18F-TEGay.6. Synthetize of 18F-TOCA. ASC 100μL (1.05mol/L), CuSO4 100μL (0.14mol/L), THPTA 14μL (0.125mol/L) and TOCA 200μL (1.75nmol/L) were added subsequently, and reacted for 15min with above performed 18F-TEGay under the temperature of 60℃. And the 18F-TOCA was obtained.7. Optimization of reaction temperature:performed click reaction using different temperature as 50℃、60℃、70℃, and remained the other conditions the same, to investigate the influence of temperature to the RCY.8. Optimization of synthesis time:performed click reaction using different time as 15min、30min、45min、60min, the temperature was same as 70℃, and remained the other conditions the same, to investigate the influence of synthesize time to the RCY.Results1. Crude product was separated by silica gel column chromatography, and filtered liquid were collected and underwent the hydrogen spectrum of nuclear magnetic resonance detection. Filtration liquid No.3 was confirmed to be 2-azidoethyl-4-toluenesulfonate.2. Labeling conditions of 18F-FEA:after the azeotropic drying of 18F, 2-azidoethyl-4-toluenesulfonate was added. The reaction mixture was incubated at 95℃ for 10 minutes. The RCY of 18F-TEGay was 98.4%.3. Labeling conditions of 18F-ACPP:after ASC, CuSO4 and ACPP were mixed, and 18F-FEA was added. The reaction mixture was incubated at 60℃ for 15 minutes, and HPLC showed the reaction was failed.4. (1) ACPP would not decompose after heated at 60℃ and stirred for 15min. (2) Expanded the reaction time from 15min to 75,110 and 145min, and the radio-labeling was failed. (3) Condensated solvent volume and increased concentration, and the radio-labeling was failed. (4) Reduced the dosage of Cu and ASC, and the radio-labeling was failed. (5) Added Copper(Ⅰ)-Stabilizing Ligand THPTA, and the radio-labeling was failed.5. Labeling conditions of 18F-TEGay:after the azeotropic drying of 18F, TsOTEGay 0.16mg (0.455μmol) in 280μL ACN was added. The reaction mixture was incubated at 110℃ for 15 minutes. The RCY of 18F-TEGay was 70%.6. Labeling conditions of 18F-TOCA:After ASC 100μL(1.05mol/L), CuSO4 100 μ L(0.14mol/L), THPTA 14μL(0.125mol/L) and TOCA 200μL(1.75nmol/L) were mixed, and 18F-TEGay was added. The reaction mixture was incubated at 60℃ for 15 minutes. The RCY of 18-TOCA was 30%. Overall RCY was 21%.7. Optimization of reaction temperature:the RCY were 17.9%,30.9%and 19.2% respectively under different temperature as 50℃,60℃,70℃. Thus,60℃ was the optimized temperature.8. Optimization of synthesis time:the RCY were 19.2%,17.7%,12.9%and 9.6% respectively under different time as 15mi、30min,45min,60min. Thus, 15min was the optimized time.ConclusionIn part one,2-azidoethyl-4-toluenesulfonate and 18F-FEA were synthesized, but the radio-labeling of 18F-ACPP was failed. All the above-mentioned improved methods were failed. The reason is contributed to the large scale of ACPP, which lead to the reaction failure.In part two, using click chemistry, a new SSTR PET tracer was synthesized in a two-steps one-pot reaction. Through the exploitation of click chemistry RCY in different reaction temperature and synthesis time, the optimized reaction conditions was obtained. In the synthesis of 18F-T0CA, shorter synthesis duration and higher RCY can be achieved using click chemistry. The lipid water partition coefficient, in vitro stability, biodistribution in tumor-bearing nude mice and Micro-PET scanning are to be further studied. 18F-T0CA is a potential radiotracer for PET imaging of SSTR positive tumors in the future. |
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