Identification of a Potent, Selective, and Brain-Penetrant Rho Kinase Inhibitor and its Activity in a Mouse Model of Huntington's Disease.

The Rho kinase (ROCK) pathway is implicated in the pathogenesis of several conditions, including neurological diseases. In Huntington's disease (HD), ROCK is implicated in mutant huntingtin (HTT) aggregation and neurotoxicity, and members of the ROCK pathway are increased in HD mouse models and patients. To validate this mode of action as a potential treatment for HD, we sought a potent, selective, central nervous system (CNS)-penetrant ROCK inhibitor. Identifying a compound that could be dosed orally in mice with selectivity against other AGC kinases, including protein kinase G (PKG), whose inhibition could potentially activate the ROCK pathway, was paramount for the program. We describe the optimization of published ligands to identify a novel series of ROCK inhibitors based on a piperazine core. Morphing of the early series developed in-house by scaffold hopping enabled the identification of a compound exhibiting high potency and desired selectivity and demonstrating a robust pharmacodynamic (PD) effect by the inhibition of ROCK-mediated substrate (MYPT1) phosphorylation after oral dosing.

Discovery of a Novel Class of d-Amino Acid Oxidase Inhibitors Using the Schrödinger Computational Platform.

d-Serine is a coagonist of the N-methyl d-aspartate (NMDA) receptor, a key excitatory neurotransmitter receptor. In the brain, d-serine is synthesized from its l-isomer by serine racemase and is metabolized by the D-amino acid oxidase (DAO, DAAO). Many studies have linked decreased d-serine concentration and/or increased DAO expression and enzyme activity to NMDA dysfunction and schizophrenia. Thus, it is feasible to employ DAO inhibitors for the treatment of schizophrenia and other indications. Powered by the Schrödinger computational modeling platform, we initiated a research program to identify novel DAO inhibitors with the best-in-class properties. The program execution leveraged an hDAO FEP+ model to prospectively predict compound potency. A new class of DAO inhibitors with desirable properties has been discovered from this endeavor. Our modeling technology on this program has not only enhanced the efficiency of structure-activity relationship development but also helped to identify a previously unexplored subpocket for further optimization.

Investigations of the reactivity, stability and biological activity of halido (NHC)gold(I) complexes.

The significance of the halido ligand (Cl-, Br-, I-) in halido[3-ethyl-4-phenyl-5-(2-methoxypyridin-5-yl)-1-propyl-1,3-dihydro-2H-imidazol-2-ylidene]gold(I) complexes (2-4) in terms of ligand exchange reactions, including the ligand scrambling to the bis[3-ethyl-4-phenyl-5-(2-methoxypyridin-5-yl)-1-propyl-1,3-dihydro-2H-imidazol-2-ylidene]gold(I) complex (5), was evaluated by HPLC in acetonitrile/water = 50:50 (v/v) mixtures. In the presence of 0.9% NaCl, the bromido (NHC)gold(I) complex 3 was immediately transformed into the chlorido (NHC)gold(I) complex 2. The iodido (NHC)gold(I) complex 4 converted under the same conditions during 0.5 h of incubation by 52.83% to 2 and by 8.77% to 5. This proportion remained nearly constant for 72 h. The halido (NHC)gold(I) complexes also reacted very rapidly with 1 eq. of model nucleophiles, e.g., iodide or selenocysteine (Sec). For instance, Sec transformed 3 in the proportion 73.03% to the (NHC)Au(I)Sec complex during 5 min of incubation. This high reactivity against this amino acid, present in the active site of the thioredoxin reductase (TrxR), correlates with the complete inhibition of the isolated TrxR enzyme at 1 µM. Interestingly, in cellular systems (A2780cis cells), even at a 5-fold higher concentration, no increased ROS levels were detected. The concentration required for ROS generation was about 20 µM. Superficially considered, the antiproliferative and antimetabolic activities of the halido (NHC)Au(I) complexes correlate with the reactivity of the Au(I)-X bond (2 < 3 < 4). However, it is very likely that degradation products formed during the incubation in cell culture medium participated in the biological activity. In particular, the high-cytotoxic [(NHC)2Au(I)]+ complex (5) distorts the results.

Internal and External Influences on Stability and Ligand Exchange Reactions in Bromido[3-ethyl-4-aryl-5-(2-methoxypyridin-5-yl)-1-propyl-1,3-dihydro-2H-imidazol-2-ylidene]gold(I) Complexes.

The ligand scrambling reaction of gold(I) complexes is a phenomenon occurring primarily in L-AuI-X (L = phosphine, N-heterocyclic carbene (NHC), and thiol; X = halide and thiol) complexes and has been observed among others for e.g., the bromido[3-ethyl-4-(4-methoxyphenyl)-5-(2-methoxypyridin-5-yl)-1-propyl-1,3-dihydro-2H-imidazol-2-ylidene]gold(I) complex (7a), which underwent ligand rearrangement in aqueous solutions. In this study, we investigated the influence of substituents on the 4-aryl ring of the related (NHC)AuIBr complexes (1a-9a) in terms of the conversion to the [(NHC)2AuI]+ (1b-9b) and [(NHC)2AuIIIBr2]+ (1c-9c) species. Furthermore, the influence of external factors such as solvent, temperature, concentration, and presence of halides (Cl-, Br-, and I-) or hydroxyl ions was studied to gain a deeper understanding of the ligand rearrangement reaction. The substituent on the 4-aryl ring has a marginal impact on the scrambling reaction. Out of the investigated organic solvents (dimethylformamide (DMF), dimethyl sulfoxide (DMSO), ethanol (EtOH), methanol (MeOH), and acetonitrile (ACN)), only ACN separates single complex molecules. In all other solvents, relatively stable ((NHC)AuIBr)2 dimers are present. The addition of water to ACN solutions forces the formation of such dimeric units, starting the transformation to [(NHC)2AuI]+ and [(NHC)2AuIIIBr2]+. The rate-determining step is the release of Br- from a T-shape intermediate because an excess of KBr terminates this reaction. Furthermore, it is obvious that only single molecules react with halides. The aurophilic interactions between two (NHC)AuIBr molecules are too strong in the presence of water and largely impeded reaction with halides. As a single molecule, the reaction with Cl- (e.g., in a 0.9% NaCl solution) is notable, while I- even leads to a fast and quantitative conversion to (NHC)AuII and finally to [(NHC)2AuI]+.

Synthesis, characterization and biological activity of bis[3-ethyl-4-aryl-5-(2-methoxypyridin-5-yl)-1-propyl-1,3-dihydro-2H-imidazol-2-ylidene]gold(i) complexes.

A series of bis[3-ethyl-4-aryl-5-(2-methoxypyridin-5-yl)-1-propyl-1,3-dihydro-2H-imidazol-2-ylidene]gold(i) complexes (2a-f) containing methyl, fluoro or methoxy substituents at various positions in the 4-aryl ring was synthesized and evaluated for their anti-cancer properties in A2780 (wild-type and Cisplatin-resistant) ovarian carcinoma as well as LAMA 84 (imatinib-sensitive and -resistant) and HL-60 leukemia cell lines. The bis-NHC gold(i) complexes were more active compared to their related mono-NHC gold(i) analogues and reduced proliferation and metabolic activity in a low micromolar range. With the exception of 2d (3-F), the compounds displayed higher potency than the established drugs Auranofin and Cisplatin. The lack of effects against non-cancerous lung fibroblast SV-80 cells indicated a high selectivity towards tumor cells. All tested complexes generated reactive oxygen species in A2780cis cells; however, the induction of apoptosis was very low. Furthermore, thioredoxin reductase is not the main target of these complexes, because its inhibition pattern did not correlate with their biological activity.

N-Heterocyclic Carbene Gold(I) Complexes: Mechanism of the Ligand Scrambling Reaction and Their Oxidation to Gold(III) in Aqueous Solutions.

N-Heterocyclic carbene (NHC) gold(I) complexes offer great prospects in medicinal chemistry as antiproliferative, anticancer, and antibacterial agents. However, further development requires a thorough understanding of their reaction behavior in aqueous media. Herein, we report the conversion of the bromido[3-ethyl-4-(4-methoxyphenyl)-5-(2-methoxypyridin-5-yl)-1-propylimidazol-2-ylidene]gold(I) ((NHC)AuIBr, 1) complex in acetonitrile/water mixtures to the bis[3-ethyl-4-(4-methoxyphenyl)-5-(2-methoxypyridin-5-yl)-1-propylimidazol-2-ylidene]gold(I) ([(NHC)2AuI]+, 7), which is subsequently oxidized to the dibromidobis[3-ethyl-4-(4-methoxyphenyl)-5-(2-methoxypyridin-5-yl)-1-propylimidazol-2-ylidene]gold(III) ([(NHC)2AuIIIBr2]+, 9). By combining experimental data from HPLC, NMR, and (LC-)/HR-MS with computational results from DFT calculations, we outline a detailed ligand scrambling reaction mechanism. The key step is the formation of the stacked ((NHC)AuIBr)2 dimer (2) that rearranges to the T-shaped intermediate Br(NHC)2AuI-AuIBr (3). The dissociation of Br- from 3 and recombination lead to (NHC)2AuI-AuIBr2 (5) followed by the separation into [(NHC)2AuI]+ (7) and [AuIBr2]- (8). [AuIBr2]- is not stable in an aqueous environment and degrades in an internal redox reaction to Au0 and Br2. The latter in turn oxidizes 7 to the gold(III) species 9. The reported ligand rearrangement of the (NHC)AuIBr complex differs from that found for related silver(I) analogous. A detailed understanding of this scrambling mechanism is of utmost importance for the interpretation of their biological activity and will help to further optimize them for biomedical and other applications.

Synthesis, characterization and biological activity of bromido[3-ethyl-4-aryl-5-(2-methoxypyridin-5-yl)-1-propyl-1,3-dihydro-2H-imidazol-2-ylidene]gold(i) complexes.

Bromido[3-ethyl-4-aryl-5-(2-methoxypyridin-5-yl)-1-propyl-1,3-dihydro-2H-imidazol-2-ylidene]gold(i) complexes (8a-h) with methoxy, methyl and fluorine substituents at different positions of the 4-aryl ring were synthesized and characterized. The relevance of the 2-methoxypyridin-5-yl residue and the substituents at the 4-aryl ring with regard to the activity against a series of cell lines was determined. Particularly against the Cisplatin-resistant ovarian cancer cell line A2780cis, the most active bromido[3-ethyl-4-(4-methoxyphenyl)-5-(2-methoxypyridin-5-yl)-1-propyl-1,3-dihydro-2H-imidazol-2-ylidene]gold(i) complex 8c was more active than Auranofin. It also inhibited thioredoxin reductase more effectively and induced high amounts of reactive oxygen species in A2780cis cells. Furthermore, its influence on non-cancerous SV 80 lung fibroblasts was lower than that of Auranofin. This fact, together with a high accumulation rate in tumor cells, determined on the example of MCF-7 cells, makes this complex an interesting candidate for further extensive studies.

Mucus permeating thiolated self-emulsifying drug delivery systems.

CONTEXT: Mucus represents a critical obstacle for self-emulsifying drug delivery systems (SEDDS) targeting the epithelial membrane site. OBJECTIVE: The aim of the study was the development of a novel SEDDS to overcome the mucus barrier. MATERIALS AND METHODS: Two novel conjugates N-dodecyl-4-mercaptobutanimidamide (thiobutylamidine-dodecylamine, TBA-D) and 2-mercapto-N-octylacetamide (thioglycolicacid-octylamine, TGA-O) were synthesized, incorporated into SEDDS and analyzed for stability, cytotoxicity and physico-chemical characteristics using dynamic light scattering. Mucus interaction studies were performed using in vitro assays based on multiple particle tracking, rotational silicone tubes and rheology. RESULTS AND DISCUSSION: TBA-D was synthesized using dodecylamine and iminothiolane as thiol precursor (yield=55 ± 5%). TGA-O was obtained via crosslinking of octylamine with SATA ((2,5-dioxopyrrolidin-1-yl) 2-acetylsulfanylacetate) (yield=70 ± 6%). The chemical structure of target compounds was confirmed via NMR analysis. The thiol-conjugates were incorporated in an amount of 3% (m/m) into SEDDS (Cremophor EL 30%, Capmul MCM 30%, Captex 355 30% and propylene glycol 10%), namely thiolated SEDDS leading to a droplet size around 50 nm and zeta potential close to 0 mV. Thiolated SEDDS with an effective diffusion coefficient 〈Deff〉 of up to 0.871 ± 0.122 cm(2) s(-1) × 10(-9) were obtained. Rotational silicone studies show increased permeation of the thiolated SEDDS A in comparison with unthiolated control. Rheological studies confirmed the mucolytic activity of the thiol-conjugates which differed only by 3% from DTT (dithiothreitol) serving as positive control. CONCLUSION: Low molecular weight thiol-conjugates were identified to improve the mucus permeation, leading to highly efficient mucus permeating SEDDS, which were superior to conventional SEDDS and might thus be a new carrier for lipophilic drug delivery.

Hydrosoluble and solvatochromic naphthalene diimides with NIR absorption.

Mimicking biochromophore anions containing phenolate moieties, eight conjugated naphthalene diimides (NDIs) have been synthesized in order to develop probes, displaying charge-transfer transitions affected by the nearby environment. NIR absorption of the resulting phenolates and their solvatochromic properties in both organic solvents and water are described.