Synthesis of Stable Isotope, Tritiated, and Carbon-14 Labeled Balcinrenone.

As part of a medicinal chemistry program aimed at discovering a mineralocorticoid receptor modulator for treatment of kidney and cardiovascular indications, multiple labeled versions of the lead compound, balcinrenone (AZD9977), were prepared. Four stable isotope labeled versions of the compound were prepared for clinical bioanalysis and biological investigations. Three of these stable isotope labeled compounds were tritiated as well as the parent for biology applications and DMPK investigations. They were prepared using a standard iodination-tritiodehalogentation approach. Finally, AZD9977 was prepared in carbon-14 labeled form for preclinical and clinical applications.

Efficient palladium-catalyzed electrocarboxylation enables late-stage carbon isotope labelling.

Carbon isotope labelling of bioactive molecules is essential for accessing the pharmacokinetic and pharmacodynamic properties of new drug entities. Aryl carboxylic acids represent an important class of structural motifs ubiquitous in pharmaceutically active molecules and are ideal targets for the installation of a radioactive tag employing isotopically labelled CO2. However, direct isotope incorporation via the reported catalytic reductive carboxylation (CRC) of aryl electrophiles relies on excess CO2, which is incompatible with carbon-14 isotope incorporation. Furthermore, the application of some CRC reactions for late-stage carboxylation is limited because of the low tolerance of molecular complexity by the catalysts. Herein, we report the development of a practical and affordable Pd-catalysed electrocarboxylation setup. This approach enables the use of near-stoichiometric 14CO2 generated from the primary carbon-14 source Ba14CO3, facilitating late-stage and single-step carbon-14 labelling of pharmaceuticals and representative precursors. The proposed isotope-labelling protocol holds significant promise for immediate impact on drug development programmes.

Synthesis and preclinical evaluation of [11C]EAI045 as a PET tracer for imaging tumors expressing mutated epidermal growth factor receptor.

BACKGROUND: Mutations in the epidermal growth factor receptor (EGFR) kinase domain are common in non-small cell lung cancer. Conventional tyrosine kinase inhibitors target the mutation site in the ATP binding pocket, thereby inhibiting the receptor's function. However, subsequent treatment resistance mutations in the ATP binding site are common. The EGFR allosteric inhibitor, EAI045, is proposed to have an alternative mechanism of action, disrupting receptor signaling independent of the ATP-binding site. The antibody cetuximab is hypothesized to increase the number of accessible allosteric pockets for EAI045, thus increasing the potency of the inhibitor. This work aimed to gain further knowledge on pharmacokinetics, the EGFR mutation-targeting potential, and the influence of cetuximab on the uptake by radiolabeling EAI045 with carbon-11 and tritium. RESULTS: 2-(5-fluoro-2-hydroxyphenyl)-2-((2-iodobenzyl)amino)-N-(thiazol-2-yl)acetamide and 2-(5-fluoro-2-hydroxyphenyl)-N-(5-iodothiazol-2-yl)-2-(1-oxoisoindolin-2-yl)acetamide were synthesized as precursors for the carbon-11 and tritium labeling of EAI045, respectively. [11C]EAI045 was synthesized using [11C]CO in a palladium-catalyzed ring closure in a 10 ± 1% radiochemical yield (decay corrected to end of [11C]CO2 production), > 97% radiochemical purity and 26 ± 1 GBq/µmol molar activity (determined at end of synthesis) in 51 min. [3H]EAI045 was synthesized by a tritium-halogen exchange in a 0.2% radiochemical yield, 98% radiochemical purity, and 763 kBq/nmol molar activity. The ability of [11C]EAI045 to differentiate between L858R/T790M mutated EGFR expressing H1975 xenografts and wild-type EGFR expressing A549 xenografts was evaluated in female nu/nu mice. The uptake was statistically significantly higher in H1975 xenografts compared to A549 xenografts (0.45 ± 0.07%ID/g vs. 0.31 ± 0.10%ID/g, P = 0.0166). The synergy in inhibition between EAI045 and cetuximab was evaluated in vivo and in vitro. While there was some indication that cetuximab influenced the uptake of [3H]EAI045 in vitro, this could not be confirmed in vivo when tumor-bearing mice were administered cetuximab (0.5 mg), 24 h prior to injection of [11C]EAI045. CONCLUSIONS: EAI045 was successfully labeled with tritium and carbon-11, and the in vivo results indicated [11C]EAI045 may be able to distinguish between mutated and non-mutated EGFR in non-small cell lung cancer mouse models. Cetuximab was hypothesized to increase EAI045 uptake; however, no significant effect was observed on the uptake of [11C]EAI045 in vivo or [3H]EAI045 in vitro in H1975 xenografts and cells.

Pharmacokinetic Evaluation of New Drugs Using a Multi-Labelling Approach and PET Imaging: Application to a Drug Candidate with Potential Application in Neuromuscular Disorders.

BACKGROUND AND OBJECTIVE: The determination of pharmacokinetic properties of new chemical entities is a key step in the process of drug development. Positron emission tomography (PET) is an ideal technique to obtain both biodistribution and pharmacokinetic parameters of new compounds over a wide range of chemical modalities. Here, we use a multi-radionuclide/multi-position labelling approach to investigate distribution, elimination, and metabolism of a triazole-based FKBP12 ligand (AHK2) with potential application in neuromuscular disorders. METHODS: Target engagement and stabilizing capacity of the drug candidate (AHK2) towards FKBP12-RyR was evaluated using competitive ligand binding and proximity ligation assays, respectively. Subsequently, AHK2 was labelled either with the positron emitter carbon-11 (11C) via 11C-methylation to yield both [11C]AHK2.1 and [11C]AHK2.2, or by palladium-catalysed reduction of the corresponding 5-iodotriazole derivative using 3H gas to yield [3H]AHK2. Metabolism was first investigated in vitro using liver microsomes. PET imaging studies in rats after intravenous (IV) administration at different doses (1 µg/Kg and 5 mg/Kg) were combined with determination of arterial blood time-activity curves (TACs) and analysis of plasma samples by high performance liquid chromatography (HPLC) to quantify radioactive metabolites. Arterial TACs were obtained in continuous mode by using an in-house developed system that enables extracorporeal blood circulation and continuous measurement of radioactivity in the blood. Pharmacokinetic parameters were determined by non-compartmental modelling of the TACs. RESULTS: In vitro studies indicate that AHK2 binds to FKBP12 at the rapamycin-binding pocket, presenting activity as a FKBP12/RyR stabilizer. [11C]AHK2.1, [11C]AHK2.2 and [3H]AHK2 could be obtained in overall non-decay corrected radiochemical yields of 14 ± 2%, 15 ± 2% and 0.05%, respectively. Molar activities were 60-110 GBq/µmol, 68-122 GBq/µmol and 0.4-0.5 GBq/µmol, respectively. In vitro results showed that oxidation of the thioether group into sulfoxide, demethylation of the CH3O-Ar residue and demethylation of -N(CH3)2 were the main metabolic pathways. Fast metabolism was observed in vivo. Pharmacokinetic parameters obtained from metabolite-corrected arterial blood TACs showed a short half-life (12.6 ± 3.3 min). Dynamic PET imaging showed elimination via urine when [11C]AHK2.2 was administered, probably reflecting the biodistribution of [11C]methanol as the major metabolite. Contrarily, accumulation in the gastrointestinal track was observed after administration of [11C]AKH2.1. CONCLUSIONS: AHK2 binds to FKBP12 at the rapamycin-binding pocket, presenting activity as a FKBP12/RyR stabilizer. Studies performed with the 3H- and 11C-labelled FKBP12/RyR stabilizer AHK2 confirm fast blood clearance, linear pharmacokinetics and rapid metabolism involving oxidation of the sulfide and amine moieties and oxidative demethylation of the CH3-O-Ar and tertiary amine groups as the main pathways. PET studies suggest that knowledge about metabolic pathways is paramount to interpret images.

Computationally-Guided Development of a Chelated NHC-P Iridium(I) Complex for the Directed Hydrogen Isotope Exchange of Aryl Sulfones.

Herein, we report the rational, computationally-guided design of an iridium(I) catalyst system capable of enabling directed hydrogen isotope exchange (HIE) with the challenging sulfone directing group. Substrate binding energy was used as a parameter to guide rational ligand design via an in silico catalyst screen, resulting in a lead series of chelated iridium(I) NHC-phosphine complexes. Subsequent preparative studies show that the optimal catalyst system displays high levels of activity in HIE, and we demonstrate the labeling of a broad scope of substituted aryl sulfones. We also show that the activity of the catalyst is maintained at low pressures of deuterium gas and apply these conditions to tritium radiolabeling, including the expedient synthesis of a tritium-labeled drug molecule.

Synthesis of C-14 labeled Rac-(3R,2S)-glycopyrronium bromide.

In an effort to better understand the drug metabolism and pharmacokinetics (DMPK) properties of glycopyrronium bromide (1), a muscarinic acetylcholine receptor antagonist, a C-14 labeled isotopologue was required. The compound was prepared in five synthetic steps and 5% overall radiochemical yield from Cu14 CN. During the synthesis, an unexpected decarboxylation of phenylglyoxylate resulted in the loss of much of the radiolabeled compound. Chiral chromatography was utilized to isolate and deliver the proper pair of enantiomers as [14 C]-1.

The synthesis of one H-2 labeled and two H-3 labeled leukotriene C4 synthase inhibitors.

As part of a medicinal chemistry program aimed at developing a leukotriene C4 synthase inhibitor for the treatment of asthma, two tritium-labeled and one stable isotope-labeled compounds were required. The synthesis of the tritium-labeled compounds used a standard bromination-tritiodehalogentation approach. One of the tritium-labeled compounds was observed to exchange its tritium label slowly with the solvent. The stable isotope-labeled compound was prepared in seven steps (3% overall yield) from [2 H6 ]acetone in a modification of the route used by medicinal chemistry.

Synthesis, 3 H-labelling and in vitro evaluation of a substituted dipiperidine alcohol as a potential ligand for chemokine receptor 2.

The immune system is implicated in the pathology of neurodegenerative disorders. The C-C chemokine receptor 2 (CCR2) is one of the key targets involved in the activation of the immune system. A suitable ligand for CCR2 could be a useful tool to study immune activation in central nervous system (CNS) disorders. Herein, we describe the synthesis, tritium radiolabelling, and preliminary in vitro evaluation in post-mortem human brain tissue of a known potent small molecule antagonist for CCR2. The preparation of a tritium-labelled analogue for the autoradiography (ARG) study gave rise to an intriguing and unexpected side reaction profile through a novel amination of ethanol and methanol in the presence of tritium. After successful preparation of the tritiated radioligand, in vitro ARG measurements on human brain sections revealed nonspecific binding properties of the selected antagonist in the CNS.

Differential Pharmacology and Binding of mGlu2 Receptor Allosteric Modulators.

Allosteric modulation of metabotropic glutamate receptor 2 (mGlu2) has demonstrated efficacy in preclinical rodent models of several brain disorders, leading to industry and academic drug discovery efforts. Although the pharmacology and binding sites of some mGlu2 allosteric modulators have been characterized previously, questions remain about the nature of the allosteric mechanism of cooperativity with glutamate and whether structurally diverse allosteric modulators bind in an identical manner to specific allosteric sites. To further investigate the in vitro pharmacology of mGlu2 allosteric modulators, we developed and characterized a novel mGlu2 positive allosteric modulator (PAM) radioligand in parallel with functional studies of a structurally diverse set of mGlu2 PAMs and negative allosteric modulators (NAMs). Using an operational model of allosterism to analyze the functional data, we found that PAMs affect both the affinity and efficacy of glutamate at mGlu2, whereas NAMs predominantly affect the efficacy of glutamate in our assay system. More importantly, we found that binding of a novel mGlu2 PAM radioligand was inhibited by multiple structurally diverse PAMs and NAMs, indicating that they may bind to the mGlu2 allosteric site labeled with the novel mGlu2 PAM radioligand; however, further studies suggested that these allosteric modulators do not all interact with the radioligand in an identical manner. Together, these findings provide new insights into the binding sites and modes of efficacy of different structurally and functionally distinct mGlu2 allosteric modulators and suggest that different ligands either interact with distinct sites or adapt different binding poses to shared allosteric site(s).

Synthesis of 2 C-14 labeled cathepsin C inhibitors: The use of a cyanide to displace a Benzotriazole.

In support of the development of a new treatment for COPD, 2 C-14 labeled compounds were required for in vitro animal studies. The synthesis of nitrile [14 C]-1 was completed in 3 steps from C-14 labeled 4-bromobenzonitrile in accord with the previously developed medicinal chemistry route. The second compound, 2, did not possess an arylnitrile as did 1, which made the synthetic design more complex. An advanced, unlabeled benzotriazole containing intermediate, 10, was synthesized in low yield over 3 steps and was subsequently reacted with K14 CN to give a mixture of diastereomers 12. Separation of the diastereomers followed by deprotection afforded [14 C]-2 in a 13% radiochemical yield.

Synthesis of 1ß-hydroxydeoxycholic acid in H-2 and unlabeled forms.

1ß-hydroxydeoxycholic acid in unlabeled and stable isotope labeled forms was required for use as a biomarker for Cytochrome P450 3A4/5 activity. A lengthy synthesis was undertaken to deliver the unlabeled compound and in the process, to develop a route to the deuterium labeled compound. The synthesis of the unlabeled compound was completed but in a very low yield. Concurrent with the synthetic approach, a biosynthetic route was pursued and this approach proved to be much more rapid and afforded the compound in both unlabeled and deuterium labeled forms in a 1-step oxidation from deoxycholic acid and [D4 ]deoxycholic acid, respectively.

Multiple compound-related adverse properties contribute to liver injury caused by endothelin receptor antagonists.

Drug-induced liver injury has been observed in patients treated with the endothelin receptor antagonists sitaxentan and bosentan, but not following treatment with ambrisentan. The aim of our studies was to assess the possible role of multiple contributory mechanisms in this clinically relevant toxicity. Inhibition of the bile salt export pump (BSEP) and multidrug resistance-associated protein 2 was quantified using membrane vesicle assays. Inhibition of mitochondrial respiration in human liver-derived HuH-7 cells was determined using a Seahorse XF(e96) analyzer. Cytochrome P450 (P450)-independent and P450-mediated cell toxicity was assessed using transfected SV40-T-antigen-immortalized human liver epithelial (THLE) cell lines. Exposure-adjusted assay ratios were calculated by dividing the maximum human drug plasma concentrations by the IC50 or EC50 values obtained in vitro. Covalent binding (CVB) of radiolabeled drugs to human hepatocytes was quantified, and CVB body burdens were calculated by adjusting CVB values for fractional drug turnover in vitro and daily therapeutic dose. Sitaxentan exhibited positive exposure-adjusted signals in all five in vitro assays and a high CVB body burden. Bosentan exhibited a positive exposure-adjusted signal in one assay (BSEP inhibition) and a moderate CVB body burden. Ambrisentan exhibited no positive exposure-adjusted assay signals and a low CVB body burden. These data indicate that multiple mechanisms contribute to the rare, but potentially severe liver injury caused by sitaxentan in humans; provide a plausible rationale for the markedly lower propensity of bosentan to cause liver injury; and highlight the relative safety of ambrisentan.

Design, synthesis and structure-activity relationships of zwitterionic spirocyclic compounds as potent CCR1 antagonists.

A series of zwitterionic spirocyclic compounds were synthesised. In vitro data revealed that these compounds were potent CCR1 antagonists. In particular, 2, 4, 11 and 20 inhibited CCR1 mediated chemotaxis of THP-1 cells in a functional assay.

Design, synthesis and structure activity relationships of spirocyclic compounds as potent CCR1 antagonists.

A series of CCR1 antagonists based upon spirocyclic compounds 1b and 2b were synthesised in which substituted aniline moiety was replaced with substituted benzamides. In vitro data revealed that CCR1 potency could be retained in such compounds.

Spirocyclic compounds, potent CCR1 antagonists.

Conformationally constrained spirocycles (17-23) and (31-36) were synthesised. In vitro data revealed that these compounds are CCR1 antagonists with sub-nanomolar potency. In a functional assay 22, 23 and 36 inhibited CCR1 mediated chemotaxis with an IC50 value of 2, 2.6 and 68nM, respectively.