N-(4-[18F]fluorobenzyl)cholylglycine, a novel tracer for PET of enterohepatic circulation of bile acids: radiosynthesis and proof-of-concept studies in rats

Enterohepatic circulation (EHC) of conjugated bile acids is an important physiological process crucial for regulation of intracellular concentrations of bile acids and their function as detergents and signal carriers. Only few bile acid-derived imaging agents have been synthesized and hitherto none have been evaluated for studies of EHC. We hypothesized that N-(4-[F]fluorobenzyl)cholylglycine ([F]FBCGly), a novel fluorine-18 labeled derivative of endogenous cholylglycine, would be a suitable tracer for PET of the EHC of conjugated bile acids, and we report here a radiosynthesis of [F]FBCGly and a proof-of-concept study by PET/MR in rats.A radiosynthesis of [F]FBCGly was developed based on reductive alkylation of glycine with 4-[F]fluorobenzaldehyde followed by coupling to cholic acid. [F]FBCGly was investigated in vivo by dynamic PET/MR in anesthetized rats; untreated or treated with cholyltaurine or rifampicin. Possible in vivo metabolites of [F]FBCGly were investigated by analysis of blood and bile samples, and the stability of [F]FBCGly towards enzymatic de-conjugation by Cholylglycine Hydrolase was tested in vitro.[F]FBCGly was produced with a radiochemical purity of 96% ± 1% and a non-decay corrected radiochemical yield of 1.0% ± 0.3% (mean ± SD; n = 12). PET/MR studies showed that i.v.-administrated [F]FBCGly underwent EHC within 40-60 min with a rapid transhepatic transport from blood to bile. In untreated rats, the radioactivity concentration of [F]FBCGly was approximately 15 times higher in bile than in liver tissue. Cholyltaurine and rifampicin inhibited the biliary secretion of [F]FBCGly. No fluorine-18 metabolites of [F]FBCGly were observed.We have developed a radiosynthesis of a novel fluorine-18 labeled bile acid derivative, [F]FBCGly, and shown by PET/MR that [F]FBCGly undergoes continuous EHC in rats without metabolizing. This novel tracer may prove useful in PET studies on the effect of drugs or diseases on the EHC of conjugated bile acids

Synthesis, characterization and biological activities of semicarbazones and their copper complexes

Substituted semicarbazones/thiosemicarbazones and their copper complexes have been prepared and several single crystal structures examined. The copper complexes of these semicarbazone/thiosemicarbazones were prepared and several crystal structures examined. The single crystal X-ray structure of the pyridyl-substituted semicarbazone showed two types of copper complexes, a monomer and a dimer. We also found that the p-nitrophenyl semicarbazone formed a conventional 'magic lantern' acetate-bridged dimer. Electron Paramagnetic Resonance (EPR) of several of the copper complexes was consistent with the results of single crystal X-ray crystallography. The EPR spectra of the p-nitrophenyl semicarbazone copper complex in dimethylsulfoxide (DMSO) showed the presence of two species, confirming the structural information. Since thiosemicarbazones and semicarbazones have been reported to exhibit anticancer activity, we examined the anticancer activity of several of the derivatives reported in the present study and interestingly only the thiosemicarbazone showed activity while the semicarbazones were not active indicating that introduction of sulphur atom alters the biological profile of these thiosemicarbazones. (C) 2016 Elsevier Inc All rights reserved

Synthesis of small organic molecules and their therapeutic potential towards drug development

The thesis describes 20 years of research in the field of development of small organic molecules that are drug candidates for combating various diseases. In particular the thesis focuses on the use of small organic molecules as anti-HIV agents. In the preliminary studies, we synthesized two class of compounds, the first class comprising of nucleoside derivatives and the second class of compounds which comprising of non-nucleoside derivatives. Although nucleoside derivatives are well known to act as anti-HIV agents in the past, the development of drug-resistant viral strains hampered the use of these agents on a continuous basis for patients who is harbouring with AIDS. To circumvent this problem, we focused our attention in altering the structure of the nucleoside derivative with a hope that the vital enzyme reverse transcriptase will not recognize this change. Accordingly, we synthesized nucleoside analog with a phosphoramidate side chain since it is well known that the ordinary nucleoside derivatives gets phosphorylated with thymidine kinases present in the cells. Our goal was to avoid the first step of phosphorylation which may in turn result in the formation of the active metabolite d4T-Aia-Phosphate which subsequently converts in to the di- and triphosphate respectively. Thus we obtained our lead compound stampidine which showed extraordinary antiviral activity in normal HIV -strains in nanomolar concentrations. Based on the successful outcome, we examined the potential of this compound to act against other mutant viral strains which are resistance to multi-drug treatment. Exposure of RT-MDR strains to stampidine resulted in inhibition of HIV-replication demonstrating that our agent was powerful anti-HIV drug. Once we established this, we extended our investigations towards other mutant strains derived from AIDS patients. Remarkably stampidine inhibited the viral replication providing a land mark opportunity to further understand the mechanism of action of this novel agent. We further advanced our research in conducting a pharmacokinetic study in mouse model, rat model and proceeded with a dog and feline model. In all the cases, the viral replication was halted and this provided the basis for preparing the compound under cGMP conditons. Accordingly the cGMP product was prepared at Cardinal Health, North Carolina under the direction of myself and subsequently formulated to a gel capsule form at University of Iowa, Pharmaceutical department. Administration of the formulated capsules to the cats and dogs resulted in the total inhibition of HIV demonstrating the usefulness of this compound for further clinical studies. Thus we have achieved the first research goal in the development of an anti-HIV agent. On the similar ground, we also developed a non-nucleoside agent, namely a thiophene ring attached thiourea which also acted very similar to the stampidine profile. We further extended our investigations towards rodent and animal species and finally formulated this compound also under cGMP conditions. During the entire course of the research study, we gained the knowledge about the usefulness of these small molecules serving as potential drug candidates. We studied the mechanism of action of these agents using a systematic approach and the thesis represents the results obtained. All these research work was done initially at the Biotherapy Program of University of Minnesota, followed by Parker Hughes Cancer Center and at Paradigm Pharmaceuticals

Investigation of two- and three-bond carbon-hydrogen coupling constants in cinnamic acid based compounds

Two- and three-bond coupling constants (2 JHC and 3 JHC ) were determined for a series of 12 substituted cinnamic acids using a selective 2D inphase/antiphase (IPAP)-single quantum multiple bond correlation (HSQMBC) and 1D proton coupled 13 C NMR experiments. The coupling constants from two methods were compared and found to give very similar values. The results showed coupling constant values ranging from 1.7 to 9.7 Hz and 1.0 to 9.6 Hz for the IPAP-HSQMBC and the direct 13 C NMR experiments, respectively. The experimental values of the coupling constants were compared with discrete density functional theory (DFT) calculated values and were found to be in good agreement for the 3 JHC . However, the DFT method under estimated the 2 JHC coupling constants. Knowing the limitations of the measurement and calculation of these multibond coupling constants will add confidence to the assignment of conformation or stereochemical aspects of complex molecules like natural products. Copyright © 2016 John Wiley & Sons, Ltd

Investigation on the impact of three different quaternary methyl ammonium cartridges on the radiosynthetic yields of [18F]fluoromethyl tosylate

Our recent investigations for the radiosynthesis of [ F]fluoromethyl tosylate have highlighted that choice of quaternary methyl ammonium (QMA) cartridge used during the radiosynthesis can significantly impact the radiochemical yields. Often the details of the QMA cartridge used in fluourine-18 syntheses are not fully described. However, our studies demonstrate that the type, the size and nature (method by which it has been conditioned) of the QMA cartridge used during the radiosynthesis can make a significant impact in the labelling efficiency. This paper investigates the use of three QMA cartridges and demonstrates that radiochemical yield (decay corrected) of [ F]fluoromethyl tosylate can increase from 46 to 60% by simply changing the QMA cartridge (and leaving all other reagents and labelling conditions exactly the same). These learnings may be applied to improve the radiochemical yields of a number of [ F]-fluorinated tracers (and synthons) where the labelling step is base-sensitive to increase the radiochemical yield, thereby significantly benefiting the radiochemistry and nuclear medicine community. This paper also highlights the necessity of the radiochemistry community to ensure the details of QMA cartridges used in fluorine-18 chemistry are fully and accurately described, since this will improve the translation of radiochemical methods from one laboratory to another

Synthesis and characterisation of indium(iii) bis-thiosemicarbazone complexes: 18 F incorporation for PET imaging

Several structurally related indium chlorido complexes of bis-thiosemicarbazones were prepared, starting from the appropriately substituted bis-thiosemicarbazones, using sodium methoxide in methanol. Detailed NMR studies were conducted to assign the structure including COSY, HSQC, and HMBC techniques. The structures of all indium complexes were solved using single crystal X-ray diffraction. The chlorido ligand was present at the apex of the square pyramidal coordination sphere in all indium complexes. In some complexes, an intermolecular hydrogen bond was present between the chlorine atom and an NH group. Three different indium chlorido complexes were converted into the corresponding fluorido-derivative by a simple halide exchange method using K F. These novel complexes, containing the positron emitting isotope F, may have potential applications in positron emission tomography (PET)

Targeted and modular architectural polymers employing bioorthogonal chemistry for quantitative therapeutic delivery

There remain several key challenges to existing therapeutic systems for cancer therapy, such as quantitatively determining the true, tissue-specific drug release profile in vivo, as well as reducing side-effects for an increased standard of care. Hence, it is crucial to engineer new materials that allow for a better understanding of the in vivo pharmacokinetic/pharmacodynamic behaviours of therapeutics. We have expanded on recent "click-to-release" bioorthogonal pro-drug activation of antibody-drug conjugates (ADCs) to develop a modular and controlled theranostic system for quantitatively assessing site-specific drug activation and deposition from a nanocarrier molecule, by employing defined chemistries. The exploitation of quantitative imaging using positron emission tomography (PET) together with pre-targeted bioorthogonal chemistries in our system provided an effective means to assess in real-time the exact amount of active drug administered at precise sites in the animal; our methodology introduces flexibility in both the targeting and therapeutic components that is specific to nanomedicines and offers unique advantages over other technologies. In this approach, the in vivo click reaction facilitates pro-drug activation as well as provides a quantitative means to investigate the dynamic behaviour of the therapeutic agent