Alexis Perry

First C−H Activation Route to Oxindoles using Copper Catalysis

The preparation of 3,3-disubstituted oxindoles by a formal C-H, Ar-H coupling of anilides is described. Highly efficient conditions have been identified using catalytic (5 mol %) Cu(OAc)(2).H(2)O with atmospheric oxygen as the reoxidant; no additional base is required, and the reaction can be run in toluene or mesitylene. Optimization studies are reported together with a scope and limitation investigation based on variation of the anilide precursors. The application of this methodology to prepare a key intermediate for the total synthesis of the anticancer, analgesic oxindole alkaloid Horsfiline is also described.

AP39, a novel mitochondria-targeted hydrogen sulfide donor, stimulates cellular bioenergetics, exerts cytoprotective effects and protects against the loss of mitochondrial DNA integrity in oxidatively stressed endothelial cells in vitro

The purpose of the current study was to investigate the effect of the recently synthesized mitochondrially-targeted H2S donor, AP39 [(10-oxo-10-(4-(3-thioxo-3H-1,2-dithiol-5yl)phenoxy)decyl) triphenylphosphonium bromide], on bioenergetics, viability, and mitochondrial DNA integrity in bEnd.3 murine microvascular endothelial cells in vitro, under normal conditions, and during oxidative stress. Intracellular H2S was assessed by the fluorescent dye 7-azido-4-methylcoumarin. For the measurement of bioenergetic function, the XF24 Extracellular Flux Analyzer was used. Cell viability was estimated by the combination of the MTT and LDH methods. Oxidative protein modifications were measured by the Oxyblot method. Reactive oxygen species production was monitored by the MitoSOX method. Mitochondrial and nuclear DNA integrity were assayed by the Long Amplicon PCR method. Oxidative stress was induced by addition of glucose oxidase. Addition of AP39 (30-300 nM) to bEnd.3 cells increased intracellular H2S levels, with a preferential response in the mitochondrial regions. AP39 exerted a concentration-dependent effect on mitochondrial activity, which consisted of a stimulation of mitochondrial electron transport and cellular bioenergetic function at lower concentrations (30-100 nM) and an inhibitory effect at the higher concentration of 300 nM. Under oxidative stress conditions induced by glucose oxidase, an increase in oxidative protein modification and an enhancement in MitoSOX oxidation was noted, coupled with an inhibition of cellular bioenergetic function and a reduction in cell viability. AP39 pretreatment attenuated these responses. Glucose oxidase induced a preferential damage to the mitochondrial DNA; AP39 (100 nM) pretreatment protected against it. In conclusion, the current paper documents antioxidant and cytoprotective effects of AP39 under oxidative stress conditions, including a protection against oxidative mitochondrial DNA damage.

Involvement of GSK-3 in Regulation of Murine Embryonic Stem Cell Self-Renewal Revealed by a Series of Bisindolylmaleimides

The ability to propagate embryonic stem cells (ESCs) while maintaining their pluripotency is critical if their potential use in regenerative medicine is to be realized. The mechanisms controlling ESC self-renewal are under intense investigation, and glycogen synthase kinase 3 (GSK-3) has been implicated in regulating both self-renewal and differentiation. To clarify its role in ESCs we have used chemical genetics. We synthesized a series of bisindolylmaleimides, a subset of which inhibit GSK-3 in murine ESCs and robustly enhance self-renewal in the presence of leukemia inhibitory factor (LIF) and serum, but not in the absence of LIF. Importantly, these molecules appear selective for GSK-3 and do not perturb other signaling pathways regulating self-renewal. Our study clarifies the functional importance of GSK-3 in regulation of ESC self-renewal and provides tools for investigating its role further.

Regulation of Heart Function by Endogenous Gaseous Mediators—Crosstalk Between Nitric Oxide and Hydrogen Sulfide

Both nitric oxide (NO) and hydrogen sulfide (H(2)S) are two important gaseous mediators regulating heart function. The present study examined the interaction between these two biological gases and its role in the heart. We found that l-arginine, a substrate of NO synthase, decreased the amplitudes of myocyte contraction and electrically induced calcium transients. Sodium hydrogen sulfide (an H(2)S donor), which alone had minor effect, reversed the negative inotropic effects of l-arginine. The effect of l-arginine + sodium hydrogen sulfide was abolished by three thiols (l-cysteine, N-acetyl-cysteine, and glutathione), suggesting that the effect of H(2)S + NO is thiol sensitive. The stimulatory effect on heart contractility was also induced by GYY4137, a slow-releasing H(2)S donor, when used together with sodium nitroprusside, an NO-releasing donor. More importantly, enzymatic generation of H(2)S from recombinant cystathionine-γ-lyase protein also interacted with endogenous NO generated from l-arginine to stimulate heart contraction. In summary, our data suggest that endogenous NO may interact with H(2)S to produce a new biological mediator that produces positive inotropic effect. The crosstalk between H(2)S and NO also suggests an intriguing potential for the endogenous formation of a thiol-sensitive molecule, which may be of physiological significance in the heart.

The synthesis and functional evaluation of a mitochondria-targeted hydrogen sulfide donor, (10-oxo-10-(4-(3-thioxo-3H-1,2-dithiol-5-yl)phenoxy)decyl) triphenylphosphonium bromide (AP39)

Synthesis and bioavailability of the endogenous gasomediator hydrogen sulfide (H2S) is perturbed in many disease states, including those involving mitochondrial dysfunction. There is intense interest in developing pharmacological agents to generate H2S. We have synthesised a novel H2S donor molecule coupled to a mitochondria-targeting moiety (triphenylphosphonium; TPP+) and compared the effectiveness of the compound against a standard non-TPP+ containing H2S donor (GYY4137) in the inhibition of oxidative stress-induced endothelial cell death. Our study suggests mitochondria-targeted H2S donors are useful pharmacological tools to study the mitochondrial physiology of H2S in health and disease.

A Direct CH/ArH Coupling Approach to Oxindoles, Thio‐oxindoles, 3,4‐Dihydro‐1 H‐quinolin‐2‐ones, and 1,2,3,4‐Tetrahydroquinolines

A copper(II)-catalysed approach to oxindoles, thio-oxindoles, 3,4-dihydro-1H-quinolin-2-ones, and 1,2,3,4-tetrahydroquinolines via formal C-H, Ar-H coupling is described. In a new variant, copper(II) 2-ethylhexanoate has been identified as an inexpensive and efficient catalyst for this transformation, which utilises atmospheric oxygen as the re-oxidant.

Effects of AP39, a novel triphenylphosphonium derivatised anethole dithiolethione hydrogen sulfide donor, on rat haemodynamic parameters and chloride and calcium Cav3 and RyR2 channels.

H2S donor molecules have the potential to be viable therapeutic agents. The aim of this current study was (i) to investigate the effects of a novel triphenylphosphonium derivatised dithiolethione (AP39), in the presence and absence of reduced nitric oxide bioavailability and (ii) to determine the effects of AP39 on myocardial membrane channels; CaV3, RyR2 and Cl(-). Normotensive, L-NAME- or phenylephrine-treated rats were administered Na2S, AP39 or control compounds (AP219 and ADT-OH) (0.25-1 µmol kg(-1)i.v.) and haemodynamic parameters measured. The involvement of membrane channels T-type Ca(2+) channels CaV3.1, CaV3.2 and CaV3.3 as well as Ca(2+) ryanodine (RyR2) and Cl(-) single channels derived from rat heart sarcoplasmic reticulum were also investigated. In anaesthetised Wistar rats, AP39 (0.25-1 µmol kg(-1) i.v) transiently decreased blood pressure, heart rate and pulse wave velocity, whereas AP219 and ADT-OH and Na2S had no significant effect. In L-NAME treated rats, AP39 significantly lowered systolic blood pressure for a prolonged period, decreased heart rate and arterial stiffness. In electrophysiological studies, AP39 significantly inhibited Ca(2+) current through all three CaV3 channels. AP39 decreased RyR2 channels activity and increased conductance and mean open time of Cl(-) channels. This study suggests that AP39 may offer a novel therapeutic opportunity in conditions whereby (•)NO and H2S bioavailability are deficient such as hypertension, and that CaV3, RyR2 and Cl(-) cardiac membrane channels might be involved in its biological actions.

The novel mitochondria-targeted hydrogen sulfide (H2S) donors AP123 and AP39 protect against hyperglycemic injury in microvascular endothelial cells in vitro

Graphical abstract

Investigating the generation of hydrogen sulfide from the phosphonamidodithioate slow-release donor GYY4137

A combination of NMR spectroscopy, mass spectrometry and chemical synthesis was used to elucidate the two-step hydrolytic decomposition pathway of the slow-release hydrogen sulfide (H2S) donor GYY4137 and the key decomposition product was also prepared by an independent synthetic route. The (dichloromethane-free) sodium salt of the phosphonamidodithioate GYY4137 was also produced as a pharmaceutically more acceptable salt. In contrast with GYY4137 and its sodium salt, the decomposition product did not generate H2S or exert cytoprotective or anti-inflammatory effects in oxidatively stressed human Jurkat T-cells and LPS-treated murine RAW264.7 macrophages. The decomposition product represents a useful control compound for determining the biological and pharmacological effects of H2S generated from GYY4137.

Semi-Rigid Bis-Phosphane Ligands for Metallamacrocycle Formation

Efficient syntheses are described for the new bridging bis-phosphanes DPPN (4) and DPEN (5) built around the restricted rigidity of a 2,7-dialkoxynaphthalene backbone. These show marked preference for bridging pairs of metal atoms (PtII, Mo0) to form metallamacrocycles or oligomers. Single-crystal X-ray structure determinations of four dimeric complexes with PtII and Mo0 are reported. The dimeric platinum complexes can be obtained as the kinetically favoured trans,trans products, which isomerise to the cis,cis forms in the presence of free ligand. cis,cis-[Pt2Cl4(DPEN)2] (12) shows marked hydroformylation catalytic activity.