DESIGN, SYNTHESIS AND CHARACTERIZATION OF NOVEL HETEROCYCLIC LIGANDS FOR BIOMEDICAL IMAGING

Abstract

The translocator protein (TSPO, 18 kDa) and highly conserved protein during evolution, has been studied for its importance in various life essential functions. TSPO is mainly found on the outer surface of mitochondria in central nervous system and in peripheral tissues. It is an intracellular protein complexed with voltage dependent anion channel (VCAD) having molecular weight 32 kDa as well as adenine nucleotide translocator (ANT) having molecular weight 30 kDa in mitochondria. TSPO has been investigated as biomarker for inflammatory conditions of lungs, liver, kidneys and brain etc. Its role has been studied in other disorders such as depression, anxiety, and neurodegenerative disorders. Therefore, high affinity TSPO ligands can found applications for non-invasive disease evaluations and for the drug delivery to the pathologically affected tissues. This led to the development of TSPO ligands as diagnostic ligands. There are few classical ligands available such as PK11195, Ro5-4864 and FGIN-127 which are isoquinoline carboxamide, benzodiazepine and indole derivatives, respectively. All of them showed compatibility to tetrapyrrole protoporphyrin IX (PPIX) structure which is a well-known endogenous TSPO ligand. 11C(R) PK11195 ligand is most widely studied positron emission tomography (PET) TSPO ligand from first generation which has limitation of high lipophilicity and low in vivo specific binding. These limitations were overcome by second generation TSPO ligands for improved imaging. Some of the TSPO ligands used in clinical human studies are [18F]FEDAA, [11C]DAA, [11C]PBR28, [18F]DPA714 and [11C]AC-5216. These second-generation ligands have the problem of intersubject variability. A new skeleton acetamidobenzoxazolone (ABO), which has been derived from Ro5-4864 by opening the diazepine ring and reorganizing it, that can overcome the problems of intersubject variability and non-specific binding. In the present study, acetamidobenzoxazolone (ABO) was chosen as lead skeleton for modification and investigation as Technetium-99m (99mTc) complexed TSPO ligands for single photon emission computed tomography (SPECT) application. This skeleton was chosen as it has recently shown to overcome non-specificity and polymorphism issues, the limitations of TSPO ligands from first two generations. Recently, our group has developed acetamidobenzoxazolone (ABO) moiety which has been reported for PET application with improved properties. Firstly, pharmacophore modelling was performed taking 51 acetamidobenzoxazolone based TSPO derivatives from literature. The best four featured pharmacophore hypothesis AAAR (three acceptors and one aromatic group) was used for designing the TSPO ligands for diagnostic application. Besides that, computational ligand protein interactions (PDB used: 4RYQ, 2MGY and 4UCI) and ADME (absorption, distribution, metabolism, and excretion) properties have also been compared to known TSPO ligands to get more insight into the complete pharmacokinetics of 99mTc ABEO-Br/Cl skeleton for SPECT application. Six new halogenated acetamidobenzoxazolone-amino acid methyl esters (ABEO Br/Cl) falling in three categories on the basis of amino acids chosen (tryptophan, phenylalanine and methionine) were synthesized and tested for their ability to bind with 99mTc. The stability of radiocomplexes were >93% and >87% in saline and human serum, respectively over 24 h. ABO was modified to ABEO-Br/Cl for SPECT application using tryptophan methyl ester, phenylalanine methyl ester and methionine methyl ester to provide skeleton for 99mTc and improve pharmacokinetics. Amino acid esters were used for modification as they are biocompatible and/or provide pharmacophoric features. As per literature, halogen substitution at 5th position of acetamindobenzoxazolone was important for TSPO affinity, therefore bromo and chloro analogs were synthesized. The radiocomplexes were evaluated as SPECT imaging agents for targeting TSPO through in vitro, ex vivo and in vivo means. PK11195 was used for proving the specificity of the radioligands towards TSPO in normal mice. These studies demonstrated significant decrease in uptakes of radioligands in TSPO rich organs when PK11195 was injected 10 min prior to radioligand injection in mice. Thereafter, these novel TSPO radioligands were compared with each other in terms of ex vivo biodistribution and in vivo radioactivity uptake in TSPO enriched organs. All the radiocomplexes demonstrated uptakes majorly in organs rich in TSPO, such as kidneys, lungs, heart, livers, spleen and brain. The uptakes in brain were minimal in normal mice and rabbits as TSPO is present in minimal concentration in normal brain. All the analogs were able to cross blood brain barrier. The blood kinetics were fast leading to good contrast as seen under in vivo studies of mice and rabbits. As per biodistribution pattern, there is preference of 99mTc-ABTO analogs for lungs, 99mTc-ABPO analogs for spleen and 99mTc-ABMO analogs for brain. The release kinetics of these tracers were compared to see their retention under in vivo conditions. Both ex vivo studies in mice and in vivo studies in rabbits demonstrated fast release still providing significantly long window for diagnostic applications in case of all the radioligands. The 99mTc-ABTOs, being most potential candidates for lungs, were further studied for cell uptake in A549 human lung tumor cell lines, A549 xenograft nude mice model and lung inflammation model with and without PK11195. These studies further proved their potential as TSPO imaging agents for SPECT.