Lipid shape and packing are key for optimal design of pH-sensitive mRNA lipid nanoparticles.

The ionizable-lipid component of RNA-containing nanoparticles controls the pH-dependent behavior necessary for an efficient delivery of the cargo-the so-called endosomal escape. However, it is still an empirical exercise to identify optimally performing lipids. Here, we study two well-known ionizable lipids, DLin-MC3-DMA and DLin-DMA using a combination of experiments, multiscale computer simulations, and electrostatic theory. All-atom molecular dynamics simulations, and experimentally measured polar headgroup pKa values, are used to develop a coarse-grained representation of the lipids, which enables the investigation of the pH-dependent behavior of lipid nanoparticles (LNPs) through Monte Carlo simulations, in the absence and presence of RNA molecules. Our results show that the charge state of the lipids is determined by the interplay between lipid shape and headgroup chemistry, providing an explanation for the similar pH-dependent ionization state observed for lipids with headgroup pKa values about one-pH-unit apart. The pH dependence of lipid ionization is significantly influenced by the presence of RNA, whereby charge neutrality is achieved by imparting a finite and constant charge per lipid at intermediate pH values. The simulation results are experimentally supported by measurements of α-carbon 13C-NMR chemical shifts for eGFP mRNA LNPs of both DLin-MC3-DMA and DLin-DMA at various pH conditions. Further, we evaluate the applicability of a mean-field Poisson-Boltzmann theory to capture these phenomena.

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.

Impact of Excited-State Antiaromaticity Relief in a Fundamental Benzene Photoreaction Leading to Substituted Bicyclo[3.1.0]hexenes.

Benzene exhibits a rich photochemistry which can provide access to complex molecular scaffolds that are difficult to access with reactions in the electronic ground state. While benzene is aromatic in its ground state, it is antiaromatic in its lowest ππ* excited states. Herein, we clarify to what extent relief of excited-state antiaromaticity (ESAA) triggers a fundamental benzene photoreaction: the photoinitiated nucleophilic addition of solvent to benzene in acidic media leading to substituted bicyclo[3.1.0]hex-2-enes. The reaction scope was probed experimentally, and it was found that silyl-substituted benzenes provide the most rapid access to bicyclo[3.1.0]hexene derivatives, formed as single isomers with three stereogenic centers in yields up to 75% in one step. Two major mechanism hypotheses, both involving ESAA relief, were explored through quantum chemical calculations and experiments. The first mechanism involves protonation of excited-state benzene and subsequent rearrangement to bicyclo[3.1.0]hexenium cation, trapped by a nucleophile, while the second involves photorearrangement of benzene to benzvalene followed by protonation and nucleophilic addition. Our studies reveal that the second mechanism is operative. We also clarify that similar ESAA relief leads to puckering of S1-state silabenzene and pyridinium ion, where the photorearrangement of the latter is of established synthetic utility. Finally, we identified causes for the limitations of the reaction, information that should be valuable in explorations of similar photoreactions. Taken together, we reveal how the ESAA in benzene and 6π-electron heterocycles trigger photochemical distortions that provide access to complex three-dimensional molecular scaffolds from simple reactants.

Metabolism of Strained Rings: Glutathione S-transferase-Catalyzed Formation of a Glutathione-Conjugated Spiro-azetidine without Prior Bioactivation.

AZD1979 [(3-(4-(2-oxa-6-azaspiro[3.3]heptan-6-ylmethyl)phenoxy)azetidin-1-yl)(5-(4-methoxyphenyl)-1,3,4-oxadiazol-2-yl)methanone] is a melanin-concentrating hormone receptor 1 antagonist designed for the treatment of obesity. In this study, metabolite profiles of AZD1979 in human hepatocytes revealed a series of glutathione-related metabolites, including the glutathionyl, cysteinyl, cysteinylglycinyl, and mercapturic acid conjugates. The formation of these metabolites was not inhibited by coincubation with the cytochrome P450 (P450) inhibitor 1-aminobenzotriazole. In efforts to identify the mechanistic features of this pathway, investigations were performed to characterize the structure of the glutathionyl conjugate M12 of AZD1979 and to identify the enzyme system catalyzing its formation. Studies with various human liver subcellular fractions established that the formation of M12 was NAD(P)H-independent and proceeded in cytosol and S9 fractions but not in microsomal or mitochondrial fractions. The formation of M12 was inhibited by ethacrynic acid, an inhibitor of glutathione S-transferases (GSTs). Several human recombinant GSTs, including GSTA1, A2-2, M1a, M2-2, T1-1, and GST from human placenta, were incubated with AZD1979. All GSTs tested catalyzed the formation of M12, with GSTA2-2 being the most efficient. Metabolite M12 was purified from rat liver S9 incubations and its structure elucidated by NMR. These results establish that M12 is the product of the GST-catalyzed glutathione attack on the carbon atom α to the nitrogen atom of the strained spiro-azetidinyl moiety to give, after ring opening, the corresponding amino-thioether conjugate product, a direct conjugation pathway that occurs without the prior substrate bioactivation by P450. SIGNIFICANCE STATEMENT: The investigated compound, AZD1979, contains a 6-substituted-2-oxa-6-azaspiro[3.3]heptanyl derivative that is an example of strained heterocycles, including spiro-fused ring systems, that are widely used in synthetic organic chemistry. An unusual azetidinyl ring-opening reaction involving a nucleophilic attack by glutathione, which does not involve prior cytochrome P450-catalyzed bioactivation of the substrate and which is catalyzed by glutathione transferases, is reported. We propose a mechanism involving the protonated cyclic aminyl intermediate that undergoes nucleophilic attack by glutathione thiolate anion in this reaction, catalyzed by glutathione transferases.

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.

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.

Discovery of a Novel Microsomal Epoxide Hydrolase-Catalyzed Hydration of a Spiro Oxetane.

Oxetane moieties are increasingly being used by the pharmaceutical industry as building blocks in drug candidates because of their pronounced ability to improve physicochemical parameters and metabolic stability of drug candidates. The enzymes that catalyze the biotransformation of the oxetane moiety are, however, not well studied. The in vitro metabolism of a spiro oxetane-containing compound AZD1979 [(3-(4-(2-oxa-6-azaspiro[3.3]heptan-6-ylmethyl)phenoxy)azetidin-1-yl)(5-(4-ethoxyphenyl)-1,3,4-oxadiazol-2-yl)methanone] was studied and one of its metabolites, M1, attracted our interest because its formation was NAD(P)H independent. The focus of this work was to elucidate the structure of M1 and to understand the mechanism(s) of its formation. We established that M1 was formed via hydration and ring opening of the oxetanyl moiety of AZD1979. Incubations of AZD1979 using various human liver subcellular fractions revealed that the hydration reaction leading to M1 occurred mainly in the microsomal fraction. The underlying mechanism as a hydration, rather than an oxidation reaction, was supported by the incorporation of (18)O from H2 (18)O into M1. Enzyme kinetics were performed probing the formation of M1 in human liver microsomes. The formation of M1 was substantially inhibited by progabide, a microsomal epoxide hydrolase inhibitor, but not by trans-4-[4-(1-adamantylcarbamoylamino)cyclohexyloxy]benzoic acid, a soluble epoxide hydrolase inhibitor. On the basis of these results, we propose that microsomal epoxide hydrolase catalyzes the formation of M1. The substrate specificity of microsomal epoxide hydrolase should therefore be expanded to include not only epoxides but also the oxetanyl ring system present in AZD1979.

CYP3A Specifically Catalyzes 1ß-Hydroxylation of Deoxycholic Acid: Characterization and Enzymatic Synthesis of a Potential Novel Urinary Biomarker for CYP3A Activity.

The endogenous bile acid metabolite 1ß-hydroxy-deoxycholic acid (1ß-OH-DCA) excreted in human urine may be used as a sensitive CYP3A biomarker in drug development reflecting in vivo CYP3A activity. An efficient and stereospecific enzymatic synthesis of 1ß-OH-DCA was developed using a Bacillus megaterium (BM3) cytochrome P450 (P450) mutant, and its structure was confirmed by nuclear magnetic resonance (NMR) spectroscopy. A [(2)H4]-labeled analog of 1ß-OH-DCA was also prepared. The major hydroxylated metabolite of deoxycholic acid (DCA) in human liver microsomal incubations was identified as 1ß-OH-DCA by comparison with the synthesized reference analyzed by UPLC-HRMS. Its formation was strongly inhibited by CYP3A inhibitor ketoconazole. Screening of 21 recombinant human cytochrome P450 (P450) enzymes showed that, with the exception of extrahepatic CYP46A1, the most abundant liver P450 subfamily CYP3A, including CYP3A4, 3A5, and 3A7, specifically catalyzed 1ß-OH-DCA formation. This indicated that 1ß-hydroxylation of DCA may be a useful marker reaction for CYP3A activity in vitro. The metabolic pathways of DCA and 1ß-OH-DCA in human hepatocytes were predominantly via glycine and, to a lesser extent, via taurine and sulfate conjugation. The potential utility of 1ß-hydroxylation of DCA as a urinary CYP3A biomarker was illustrated by comparing the ratio of 1ß-OH-DCA:DCA in a pooled spot urine sample from six healthy control subjects to a sample from one patient treated with carbamazepine, a potent CYP3A inducer; 1ß-OH-DCA:DCA was considerably higher in the patient versus controls (ratio 2.8 vs. 0.4). Our results highlight the potential of 1ß-OH-DCA as a urinary biomarker in clinical CYP3A DDI studies.

Synthesis of [(3) H] and [(2) H6 ]AZD6642, an inhibitor of 5-lipoxygenase activating protein (FLAP).

An AstraZeneca effort to identify a 5-lipoxygenase activating protein inhibitor with good drug-like properties resulted in the identification of AZD6642. To further understand its drug metabolism and pharmacokinetic properties, it was required labeled with tritium. The tritiation of AZD6642 was effected by Ir-catalyzed exchange chemistry to give an average of one tritium per molecule. Additionally, a stable isotope labeled version of AZD6642 was required to support bioanalytical studies. The synthesis originated from [(2) H6 ]acetone which was converted to the trimethylsilyl cyanide adduct and subsequently reduced to give 2-(aminomethyl)-[1,1,1,3,3,3-(2) H6 ]propan-2-ol in good yield. Carbonylation to give an amide adduct resulted in an intermediate that was converted to the final compound in four steps.

A P450 fusion library of heme domains from Rhodococcus jostii RHA1 and its evaluation for the biotransformation of drug molecules.

The actinomycete Rhodococcus jostii RHA1 contains a multitude of oxygenase enzymes, consonant with its remarkable activities in the catabolism of hydrophobic xenobiotic compounds. In the interests of identifying activities for the transformation of drug molecules, we have cloned genes encoding 23 cytochrome P450 heme domains from R. jostii and expressed them as fusions with the P450 reductase domain (RhfRED) of cytochrome P450Rhf from Rhodococcus sp. NCIMB 9784. Fifteen of the fusions were expressed in the soluble fraction of Escherichia coli Rosetta (DE3) cells. Strains expressing the fusions of RhfRED with genes ro02604, ro04667, ro11069, ro11320, ro11277, ro08984 and ro04671 were challenged with 48 commercially available drugs revealing many different activities commensurate with P450-catalyzed hydroxylation and demethylation reactions. One recombinant strain, expressing the fusion of P450 gene ro11069 (CYP257A1) with RhfRED, and named Ro07-RhfRED, catalyzed the N-demethylation of diltiazem and imipramine. This observation was in accord with previous reports of this enzyme's activity as a demethylase of alkaloid substrates. Ro07-RhfRED was purified and characterised, and applied in cell-free biotransformations of imipramine (7 µM) giving a 63% conversion to the N-desmethyl product.

Characterization and demonstration of drug compound ring-chain tautomer formation and its impacts on quality control.

Unknown chromatographic peaks, potential impurities, were observed in a series of related compounds. This led to the identification and characterization of tautomeric equilibria. Structural elucidation was required to understand the potential impurity profile, thus impacting method development for quality control. In this work, characterization of the chemical structures, AZ13581258 and AZD5718, and equilibria of the tautomeric forms was performed using a range of advanced analytical techniques such as preparative chromatography, nuclear magnetic resonance (NMR), chromatographic detection by mass spectrometry (MS), MSMS, and ultraviolet spectroscopy (UV). Predictions using density functional theory (DFT) further explains and confirms the tautomer equilibria through predictions of reaction barrier energies, UV-spectra and NMR data. These investigations led to fully understand the impurity profile and to the development of a quality control method for AZD5718 drug substance and drug product. In conclusion, ring-chain tautomeric structures are predominately formed under acidic conditions, and the additional peaks observed in LC during organic impurity determination were found to originate from ring-chain closed tautomers in equilibria with the parent open form compound. Hence, the closed and open tautomer forms should all be considered as the same compound.

4,4,16-Trifluoropalmitate: Design, Synthesis, Tritiation, Radiofluorination and Preclinical PET Imaging Studies on Myocardial Fatty Acid Oxidation.

Fatty acid oxidation (FAO) produces most of the ATP used to sustain the cardiac contractile work, although glycolysis is a secondary source of ATP under normal physiological conditions. FAO impairment has been reported in the advanced stages of heart failure (HF) and is strongly linked to disease progression and severity. Thus, from a clinical perspective, FAO dysregulation provides prognostic value for HF progression, the assessment of which could be used to improve patient monitoring and the effectiveness of therapy. Positron emission tomography (PET) imaging represents a powerful tool for the assessment and quantification of metabolic pathways in vivo. Several FAO PET tracers have been reported in the literature, but none of them is in routine clinical use yet. Metabolically trapped tracers are particularly interesting because they undergo FAO to generate a radioactive metabolite that is subsequently trapped in the mitochondria, thus providing a quantitative means of measuring FAO in vivo. Herein, we describe the design, synthesis, tritium labelling and radiofluorination of 4,4,16-trifluoro-palmitate (1) as a novel potential metabolically trapped FAO tracer. Preliminary PET-CT studies on [18 F]1 in rats showed rapid blood clearance, good metabolic stability - confirmed by using [3 H]1 in vitro - and resistance towards defluorination. However, cardiac uptake in rats was modest (0.24±0.04 % ID/g), and kinetic analysis showed reversible uptake, thus indicating that [18 F]1 is not irreversibly trapped.

Novel metabolites of amodiaquine formed by CYP1A1 and CYP1B1: structure elucidation using electrochemistry, mass spectrometry, and NMR.

An aldehyde metabolite of amodiaquine and desethylamodiaquine has been identified. The aldehyde was the major metabolite formed in incubations with two recombinantly expressed human cytochromes P450 (rP450s), namely, CYP1A1 and CYP1B1. The aldehyde metabolite was also formed, to a lesser extent, in both human and rat liver microsomes. When comparing results from incubations with liver microsomes from 3-methylcholanthrene-treated rats (inducing CYP1A1 and CYP1B1) with those from noninduced rats, a 6-fold increase of the aldehyde metabolite was observed in the rat liver microsomes after 3-methylcholanthrene treatment. The metabolic oxidation was mimicked by the electrochemical system, and the electrochemical oxidation product was matched with the metabolite from the in vitro incubations. The electrochemical generation of the aldehyde metabolite was repeated on a preparative scale, and the proposed structure was confirmed by NMR. Trapping of the aldehyde metabolite was done with methoxyl amine. Trapping experiments with N-acetyl cysteine revealed that the aldehyde was further oxidized to an aldehyde quinoneimine species, both in the rP450 incubations and in the electrochemical system. Three additional new metabolites of amodiaquine and desethylamodiaquine were formed via rCYP1A1 and rCYP1B1. Trace amounts of these metabolites were also observed in incubations with liver microsomes from 3-methylcholanthrene-treated rats. Tentative structures of the metabolites and adducts were assigned based on liquid chromatography/tandem mass spectrometry in combination with accurate mass measurements.

Electrochemical oxidation of troglitazone: identification and characterization of the major reactive metabolite in liver microsomes.

Troglitazone (TGZ) was developed for the treatment of type 2 diabetes but was withdrawn from the market due to hepatotoxicity. The formation of reactive metabolites has been associated with the observed hepatotoxicity. Such reactive metabolites have been proposed to be formed via three different mechanisms. One of the proposed mechanisms involves the oxidation of the chromane moiety of TGZ to a reactive o-quinone methide. The two other mechanisms involve metabolic activation of the thiazolidinedione moiety of TGZ. In the present study, it is shown that electrochemical oxidations can be used to generate a reactive metabolite of TGZ, which can be trapped by GSH or N-acetylcysteine. From incubations of TGZ with rat and human liver microsomes in the presence of either GSH or N-acetylcysteine, it was shown that similar conjugates were formed in vitro as formed from electrochemical oxidations of TGZ. One- and two-dimensional NMR studies of the troglitazone- S-( N-acetyl)cysteine conjugate revealed that N-acetylcysteine was attached to a benzylic carbon in the chromane moiety, showing that the conjugate was formed via a reaction between the o-quinone methide of TGZ and N-acetylcysteine. From electrochemical oxidations of rosiglitazone, pioglitazone, and ciglitazone in the presence of GSH, no GSH conjugates could be identified. These three compounds all contain a thiazolidinedione moiety. In conclusion, it has been shown that the primary reactive metabolite of TGZ formed from electrochemical oxidation was the o-quinone methide, and this metabolite was similar to what was observed to be the primary reaction product in human and rat liver microsomes.

Phenotypic Screen for Cardiac Regeneration Identifies Molecules with Differential Activity in Human Epicardium-Derived Cells versus Cardiac Fibroblasts.

Activation and proliferation of resident cardiac progenitor cells has therapeutic potential to repair the heart after injury. However, research has been impeded by a lack of well-defined and characterized cell sources and difficulties in translation to screening platforms. Here, we describe the development, validation, and use of a 384-well phenotypic assay in primary human epicardium-derived cells (EPDCs) to identify compounds that induce proliferation while maintaining the progenitor phenotype. Using this assay, we screened 7400 structurally diverse compounds where greater than 90% are biologically annotated and known to modulate a broad range of biological targets. From the primary screen, we identified and validated hits and expanded upon the lead molecules of interest. A counterscreen was developed in human cardiac fibroblasts to filter out compounds with a general proliferative effect, after which the activity of selected molecules was confirmed across multiple EPDC donors. To further examine the mechanism of action of compounds with annotated targets, we performed knockdown experiments to understand whether a single known target was responsible for the proliferative effect, confirming results with protein expression and activity assays. Here, we were able to show that the annotated targets of compounds of interest were not responsible for the proliferative effect, which highlights potential differences in cell types and signaling pathways and possible polypharmacology. These studies demonstrate the feasibility of using relevant human primary cells in a phenotypic screen to identify compounds as novel biological tools and starting points for drug discovery projects, and we disclose the first small molecules to proliferate human primary EPDCs.

Macrocyclic Prodrugs of a Selective Nonpeptidic Direct Thrombin Inhibitor Display High Permeability, Efficient Bioconversion but Low Bioavailability.

The only oral direct thrombin inhibitors that have reached the market, ximelagatran and dabigatran etexilat, are double prodrugs with low bioavailability in humans. We have evaluated an alternative strategy: the preparation of a nonpeptidic, polar direct thrombin inhibitor as a single, macrocyclic esterase-cleavable (acyloxy)alkoxy prodrug. Two homologous prodrugs were synthesized and displayed high solubilities and Caco-2 cell permeabilities, suggesting high absorption from the intestine. In addition, they were rapidly and completely converted to the active zwitterionic thrombin inhibitor in human hepatocytes. Unexpectedly, the most promising prodrug displayed only moderately higher oral bioavailability in rat than the polar direct thrombin inhibitor, most likely due to rapid metabolism in the intestine or the intestinal wall. To the best of our knowledge, this is the first in vivo ADME study of macrocyclic (acyloxy)alkoxy prodrugs, and it remains to be established if the modest increase in bioavailability is a general feature of this category of prodrugs or not.

Impact of stereospecific intramolecular hydrogen bonding on cell permeability and physicochemical properties.

Profiling of eight stereoisomeric T. cruzi growth inhibitors revealed vastly different in vitro properties such as solubility, lipophilicity, pKa, and cell permeability for two sets of four stereoisomers. Using computational chemistry and NMR spectroscopy, we identified the formation of an intramolecular NH→NR3 hydrogen bond in the set of stereoisomers displaying lower solubility, higher lipophilicity, and higher cell permeability. The intramolecular hydrogen bond resulted in a significant pKa difference that accounts for the other structure-property relationships. Application of this knowledge could be of particular value to maintain the delicate balance of size, solubility, and lipophilicity required for cell penetration and oral administration for chemical probes or therapeutics with properties at, or beyond, Lipinski's rule of 5.

Electrochemical generation of electrophilic drug metabolites: characterization of amodiaquine quinoneimine and cysteinyl conjugates by MS, IR, and NMR.

The chemical reactivity of electrophilic metabolites usually prevents their detection in vivo since, by definition, they are relatively short-lived and are likely to undergo one or more structural modifications to form more stable final products. Electrochemical oxidation provides a means to generate reactive metabolites in an environment without the presence of such nucleophiles. This paper describes the results of our MS, MS/MS, NMR, IR, and computational studies on oxidation products (and conjugates) that have been generated electrochemically from the antimalarial agent amodiaquine. The electrophilic quinoneimine metabolite of amodiaquine was the major oxidation product following electrochemical oxidation at +600 mV. The absence of biological nucleophiles in the electrochemical experiment facilitated (i) the acquisition of a clean IR spectrum of the amodiaquine quinoneimine and (ii) the addition of biologically relevant nucleophiles under controlled conditions. The addition of cysteine gave four cysteinyl conjugates, while the addition of glutathione gave four glutathionyl conjugates. The product ion spectra of the conjugates formed in the electrochemical experiment were used to identify suitable fragments for selected reaction monitoring (SRM) to selectively search for these conjugates in human liver microsomal (HLM) incubations. The four cysteinyl conjugates, as well as the four glutathionyl conjugates, were also detected as metabolites in HLM. The experiment with cysteine was repeated on a preparative scale that allowed characterization of the major conjugates by (1)H NMR. Desethylamodiaquine, the major metabolite formed in human liver microsomes, was also generated electrochemically by oxidation of amodiaquine at +1200 mV followed by reduction at -800 mV. In conclusion, the EC-ESI/MS technique provides the unique opportunity to generate reactive metabolites in the absence of biological nucleophiles, which enables studies that can give insight into the nature of these reactive intermediates. Such knowledge is valuable for risk assessment of new compound classes and can be complementary to computer-based structure-activity relationships of carcinogenicity, mutagenicity, and teratogenicity.