Structural Modification Of Lys-urea-Glu-based PET Imaging Agents And Their In Vitro And In Vivo Properties

Objective:Prostate specific membrane antigen（PSMA）is expressed thousands of times higher in prostate cancer cells than in normal tissue and is an important target for prostate cancer imaging and treatment.Small molecule inhibitors of PSMA based on the lysine-urea-glutamate（Lys-urea-Glu）backbone are currently dominating clinical studies of radiopharmaceuticals for the treatment of prostate cancer.At present,there are few studies on lysine terminal tertiary amination modifications in the Lys-urea-Glu backbone and their effects on biological properties,while no conformational studies on tertiary amination halogen substitution have been reported in the literature.Previously,the understanding of halogen substitution was limited to template molecules,and there were no studies on the structure-activity relationship to prove the reliability of the results.The purpose of this thesis is to investigate the role of the ureido structure in targeting PSMA,establish a structure-activity relationship between halogen substituents and PSMA,and provide a scientific basis for the design of new PSMA inhibitors.Methods:（1）Based on the backbone of Lys-urea-Glu,one urea derivative（PSMA-CO）was obtained by double carbonyl modification of the urea group;seven tertiary aminated halogen-derived structures（DOTA-SC691-R）were obtained by substituting the lysine terminal amino group with p-halogenated phenyl.（2）Radionuclide ⁶⁸Ga labeling of the above eight derivatives to obtain⁶⁸Ga-labeled compounds with radiochemical purity greater than or equal to95%(⁶⁸Ga-PSMA-CO and ⁶⁸Ga-DOTA-SC691-R).（3）The ⁶⁸Ga markers were added to phosphate buffer solution（PBS）and fetal bovine serum（FBS）systems,respectively,and the radiochemical purity of the markers was determined by HPLC with a radioactivity monitor after a certain period of time to determine their stability.（4）To study the in vitro cellular uptake,internalization and half-inhibitory concentration(IC₅₀)of the marker to determine its affinity for PSMA at the cellular level based on human prostate cancer（LNCa P）cells.（5）Non-obese diabetic/severe combined immunodeficient（NOD/SCID）mice were selected to construct LNCa P-loaded mice by subcutaneous inoculation,and the specificity,biodistribution and small animal PET/CT（Micro-PET/CT）imaging characteristics were studied at different time points after tail vein injection of the marker.Results:Although ⁶⁸Ga-PSMA-CO with a bis-carbonyl substituent performed well in radiochemical purity,radiochemical yield,and in vitro stability experiments.However,in the Micro-PET/CT imaging study,the LNCa P tumors of hormonal mice still did not take up ⁶⁸Ga-PSMA-CO after 30min of tail vein injection,and the affinity for PSMA was too poor and will not be studied in subsequent experiments.The halogen substituent⁶⁸Ga-DOTA-SC691-R has high putative yield with putative purity（98-99%）.Compared with the other four compounds with similar hydrophilicity and good in vitro stability,the more hydrophobic ⁶⁸Ga-DOTA-SC691-3FM,the more hydrophilic ⁶⁸Ga-DOTA-SC691-6KL and ⁶⁸Ga-DOTA-SC691-7KL were less stable in vitro,and the retention rate in FBS after 1 h was less than 50%.The conditions for in vivo studies were not met and no further in vivo experimental studies will be conducted.The results of in vitro competitive inhibition assay showed that the affinity of ^natGa-DOTA-SC691-R for PSMA showed a trend of H<F<Cl<Br<I,which was generally consistent with the trend of LNCa P cells internalization values.The IC₅₀ value of ⁶⁸Ga-DOTA-SC691-I with the best affinity was 148.90±1.87 n M.Biodistribution data showed that⁶⁸Ga-DOTA-SC691-R was metabolized by the kidney and the accumulation of LNCa P tumors at 60 min followed the trend of H<F≈Cl<I<Br,with the highest uptake value of ⁶⁸Ga-DOTA-SC691-Br at LNCa P tumors of 20.39±4.38%ID/g.The results of Micro-PET/CT imaging study showed that⁶⁸Ga-DOTA-SC691-R had clear uptake at LNCa P tumors with very low background uptake,high contrast,rapid targeting of LNCa P tumors,and specificity for PSMA.Conclusion:We obtained the urea-based derivative ⁶⁸Ga-PSMA-CO after the bis-carbonyl modification of the urea group in the Lys-urea-Glu backbone showing poor PSMA affinity.The lower affinity of the bis-carbonyl compounds may be explained by the increased chain length,the increased distance between the P1 structure and the P1’structure of the compound PSMA-CO,and the reduced fitness between the S1 pocket and S1’pocket of the two recognition sites of PSMA.It is suggested that the chain length of the urea-based structure of the Lys-urea-Glu backbone may affect the binding between the P1 structure and the P1’structure of the compound and the S1 and S1’pockets of PSMA.The lysine terminal amino group was substituted by p-halogenated phenyl groups to obtain seven tertiary aminated halogen-derivatized structures.With similar hydrophobicity,the size of the halogen atom volume affects its binding to PSMA.Compounds possessing bulkier bromine and iodine atoms have higher affinity and,accordingly,exhibit superior targeting,drug metabolism and imaging characteristics.