Synthesis of Bifunctional Chelating Agents to label monoclonal antibodies for radioimmunodiagnosis of cancer

The invasive biopsy techniques ‘Radiodiagnosis’ utilize radiation emitted by radioactive metal to diagnose the location of infection using radiopharmaceuticals. Bifunctional chelating agents (BCA) which can hold the metals strongly in biological systems, are used to attach radioactive metal to targeting molecule (biomolecule, peptides, proteins and steroids). In this study macrocyclic BCAs with increase in rigidity for complex stability besides requisite flexibility for complexation along with variation in ring size and donor ligands group were synthesized and evaluated for radiodiagnostic applications. The Bifunctional chelating agents {4,10-diamino-1,13-dithia-[2:3; 6:8 (3’-amino); 11:12] tribenzocyclopentadecane (5a), 4,10-diaino-1,13-dithia-[2:3; 6:8 (3’-amino); 11:12]tribenzo-cyclohexadecane (5b) and 4,10-diamino-16-oxa-1,13-ditha-[2:3; 6:8 (3’-amino); 11:12] tribenzocyclooctadecane (5c)} based on the Bis(2-aminophenyl thio) alkane moiety having benzyl group in backbone were synthesized by macrocyclization of bis (2-aminophenyl thio) alkanes with 3,5-bis-bromomethylnitrobenzene followed by reduction of nitro group using Zn/NH2NH2. Chelating agents 5a-c formed radiocomplexes with radioactive 99mTc in 82-98% yield. Among the three chelating agents used, 5c showed maximum complexation with 98% purity followed by 5b (92-94%) with lowest being of 5a (82%). 99mTc-5b and 99mTc-5c are stable in serum under physiological conditions. The in-vivo imaging of tumours was evaluated by bioconjugating BCA 5b to monoclonal antibody anti-EGFr  and by using Avidin-Biotin pretargeting strategy against the EAT (Eherlic Ascite Tumor) developed in right thigh of mice by gamma imaging. Infection imaging radiopharmaceutical was synthesized from p-aminosalicylic acid (PAS) and DTPA dianhydride to yield BPAS-DTPA (N1,N3-bis{N-(3-hydroxy-4-carboxyphenyl) methylenecarboxamide} diethylenetriamine-N1,N2,N3-triacetic acid) and evaluated for diagnosis of P. aureoginosa infection. A varied general transformation observed during the cyclization of bis(2-aminophenylthio)propane (and other 2-aminophenylthioalkanes) with bromoacetyl- bromide where the product obtained was 3-oxo-3,4-dihydro-2H-1,4-benzothiazine (1) instead of expected macrocycle (4,7-diamino-1,10-dithia-5-oxo-[2:3; 8:9]dibenzo-cyclotridecane was studied as a general synthesis for benzothiazine.