Design And Synthesis Of Fibroblast Activation Protein-targeting Small-molecule Probes For In Vivo Imaging

Malignant tumors seriously affect human life and health.If cancer can be diagnosed and treated at an early stage,the five-year survival rate of patients may be greatly improved.The current means of early diagnosis of cancer is mainly the imaging of tumor biomarkers using molecular imaging techniques.Cancer Associated Fibroblasts（CAF）are important components of the tumor microenvironment and play an important role in the growth,proliferation and migration of tumors.Fibroblast Activation Protein（FAP）is an important biomarker for CAF cells and it is highly expressed in more than 90%of tumor tissues and almost not expressed in healthy tissues.Therefore,FAP as an imaging target has become a hot topic in oncology diagnostic and therapeutic drug research.Based on this,this thesis explores the application of FAP inhibitors（FAPI）as a targeting moiety in tumor diagnosis.Chapter 1:A brief overview of the role of molecular imaging technology in the early and precise diagnosis of cancer.Using imaging technology to image cancer biomarkers can improve tumor detection rates.Tumor microenvironment is an important part of tumor,and imaging for abnormal physiological environment and biomarkers is an important tool for current cancer precision diagnosis.FAP,as a biomarker in tumor microenvironment,becomes an important target for current imaging.Chapter 2:a novel FAP ligand（DOTA-GPFAPI-04）was designed and synthesized through assembling three functional moieties:a quinoline-based FAP inhibitor that allowed specific FAP targeting,a FAP substrate Gly-Pro as a linker that might increase FAP protein interaction,and2,2’,2’’,2’’’-（1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrayl）tetraacetic acid（DOTA）chelator for radiolabeling with different radionuclides.The FAP targeting ability of DOTA-GPFAPI-04was investigated by molecular docking studies.DOTA-GPFAPI-04 was then radiolabeled with⁶⁸Ga to give ⁶⁸Ga-DOTA-GPFAPI-04 for positron emission tomography（PET）imaging of glioblastoma.⁶⁸Ga-DOTA-GPFAPI-04 exhibited purity of>98%as analyzed by radio-HPLC and high stability as observed in saline and mouse serum.Cell uptake studies demonstrated the targeting specificity of the probe.Further in vivo pharmacokinetic studies in normal mice demonstrated the quick clearance of the probe.Moreover,compared with the widely studied⁶⁸Ga-FAPI-04,⁶⁸Ga-DOTA-GPFAPI-04 showed much higher U87MG tumor uptake values(4.467±0.379%ID/g for ⁶⁸Ga-DOTA-GPFAPI-04 and 1.267±0.208%ID/g for ⁶⁸Ga-FAPI-04at 0.5 h post-injection,respectively).The area under the curve（AUCs）based on time–activity curves（TACs）analysis for tumor radioactivity in small animal models,was 422.5 for ⁶⁸Ga-DOTA-GPFAPI-04 and 98.14 for ⁶⁸Ga-FAPI-04,respectively,demonstrating that the former had longer tumor retention time.The tumor to muscle（T/M）ratio for ⁶⁸Ga-DOTA-GPFAPI-04reached 9.15 in a U87MG xenograft animal model.PET imaging studies and blocking assays showed that ⁶⁸Ga-DOTA-GPFAPI-04 had specific tumor uptake.In summary,this study demonstrates the successful synthesis and evaluation of a new generation of FAPI targeting probe,⁶⁸Ga-DOTA-GPFAPI-04 with a Gly-Pro sequence.It shows favorable in vivo glioblastoma imaging properties and relatively long tumor retention,highlighting DOTA-GPFAPI-04 as a promising molecular scaffold for developing FAP-targeting tumor theroanstic agents.Chapter 3:To verify the potential of FAP for optical imaging applications,two second-near infrared（NIR-Ⅱ）cyanine dyes D1 and D2 were synthesized and coupled with FAPI to afford NIR-Ⅱ small-molecule probes FAPI-D1 and FAPI-D2.The maximum absorption and emission wavelengths of FAPI-D1 were 780 nm and 912 nm,respectively,and the maximum absorption and emission wavelengths of FAPI-D2 were 780 nm and 899 nm,respectively.The molar absorbance coefficients of FAPI-D1 and FAPI-D2 were 1.082×10⁵ M^-1cm^-1 and4.437×10⁴ M^-1cm^-1,respectively,and the probes showed certain photostability under continuous laser irradiation.Using HCT-116 tumor-bearing mice as a model,FAPI-D2 showed much higher tumor-to-background ratio（2.766±0.087 for FAPI-D2 and 1.313±0.078 for FAPI-D1at 4 h post-injection,respectively）.The specific uptake of FAPI-D2 in tumors is expected to be used in fluorescence-guided surgical navigation studies.In conclusion,this study demonstrates the successful synthesis and evaluation of a new generation of FAPI targeting probe,⁶⁸Ga-DOTA-GPFAPI-04 with a Gly-Pro sequence.It shows favorable in vivo glioblastoma imaging properties and relatively long tumor retention,highlighting DOTA-GPFAPI-04 as a promising molecular scaffold for developing FAP-targeting tumor theroanstic agents.Two NIR-Ⅱ probes have been successfully designed and synthesised.The studies on cells and in vivo experiments indicate that the new compounds have ability to specifically image tumors.The present findings may provide new ideas for the diagnosis of tumors.