Synthesis of SMT022357 enantiomers and in vivo evaluation in a Duchenne muscular dystrophy mouse model.

Following on from ezutromid, the first-in-class benzoxazole utrophin modulator that progressed to Phase 2 clinical trials for the treatment of Duchenne muscular dystrophy, a new chemotype was designed to optimise its physicochemical and ADME profile. Herein we report the synthesis of SMT022357, a second generation utrophin modulator preclinical candidate, and an asymmetric synthesis of its constituent enantiomers. The pharmacological properties of both enantiomers were evaluated in vitro and in vivo. No significant difference in the activity or efficacy was observed between the two enantiomers; activity was found to be comparable to the racemic mixture.

2-Arylbenzo[d]oxazole Phosphinate Esters as Second-Generation Modulators of Utrophin for the Treatment of Duchenne Muscular Dystrophy.

Utrophin modulation is a promising therapeutic strategy for Duchenne muscular dystrophy (DMD), which should be applicable to all patient populations. Following on from ezutromid, the first-generation utrophin modulator, we describe the development of a second generation of utrophin modulators, based on the bioisosteric replacement of the sulfone group with a phosphinate ester and substitution of the metabolically labile naphthalene with a haloaryl substituent. The improved physicochemical and absorption, distribution, metabolism, and excretion (ADME) properties, further reflected in the enhanced pharmacokinetic profile of the most advanced compounds, 30 and 27, led to significantly better in vivo exposure compared to ezutromid and alleviation of the dystrophic phenotype in mdx mice. While 30 was found to have dose-limiting hepatotoxicity, 27 and its enantiomers exhibited limited off-target effects, resulting in a safe profile and highlighting their potential utility as next-generation utrophin modulators suitable for progression toward a future DMD therapy.

Isolation, Structural Identification, Synthesis, and Pharmacological Profiling of 1,2-trans-Dihydro-1,2-diol Metabolites of the Utrophin Modulator Ezutromid.

5-(Ethylsulfonyl)-2-(naphthalen-2-yl)benzo[d]oxazole (ezutromid, 1) is a first-in-class utrophin modulator that has been evaluated in a phase 2 clinical study for the treatment of Duchenne muscular dystrophy (DMD). Ezutromid was found to undergo hepatic oxidation of its 2-naphthyl substituent to produce two regioisomeric 1,2-dihydronaphthalene-1,2-diols, DHD1 and DHD3, as the major metabolites after oral administration in humans and rodents. In many patients, plasma levels of the DHD metabolites were found to exceed those of ezutromid. Herein, we describe the structural elucidation of the main metabolites of ezutromid, the regio- and relative stereochemical assignments of DHD1 and DHD3, their de novo chemical synthesis, and their production in systems in vitro. We further elucidate the likely metabolic pathway and CYP isoforms responsible for DHD1 and DHD3 production and characterize their physicochemical, ADME, and pharmacological properties and their preliminary toxicological profiles.

L-cysteine stimulates hydrogen sulfide synthesis in myocardium associated with attenuation of ischemia-reperfusion injury.

Hydrogen sulfide (H( 2)S) is a biological mediator produced by enzyme-regulated pathways from L-cysteine, which is a substrate for cystathionine-gamma-lyase (CSE). In myocardium, endogenously and exogenously administered H(2)S has been shown to protect against ischemia-reperfusion injury. We hypothesized that L-cysteine exerts its protective action through stimulation of H(2)S production. Rat isolated hearts were Langendorff-perfused and underwent 35-minute regional ischemia and 120-minute reperfusion. L-cysteine perfusion from 10 minutes before ischemia until 10 minutes after reperfusion limited infarct size in a concentration-dependent manner, maximal at 1 mmol/L (control 36.4% +/- 2.4% vs L-cysteine 24.3% +/- 3.4%, P < .05). This protective action was attenuated by the CSE inhibitor, DL-propargylglycine (PAG) 1 mmol/L (31.4 +/- 5.9%, not significant vs control) but administration of the CSE cofactor pyridoxal-5'-phosphate (PLP) 50 mumol/L did not enhance the effect of L-cysteine. Ten minutes normoxic perfusion with L-cysteine 1 mmol/L caused a 3-fold increase in myocardial H(2)S concentration (0.64 +/- 0.16 vs 2.01 +/- 0.07 mumol/g protein, P < .01), an effect that was significantly attenuated by PAG (1.17 +/- 0.15 mumol/g protein). These data provide evidence that exogenous L-cysteine administration limits ischemia-reperfusion injury through a mechanism that appears to be at least partially dependent on H(2)S synthesis.

Regulation of cardiovascular cell function by hydrogen sulfide (H(2)S).

Since the discovery of endogenously-produced hydrogen sulfide (H(2)S) in various tissues, there has been an explosion of interest in H(2)S as a biological mediator alongside other gaseous mediators, nitric oxide and carbon monoxide. The identification of enzyme-regulated H(2)S synthetic pathways in the cardiovascular system has led to a number of studies examining specific regulatory actions of H(2)S. We review evidence showing that endogenously-generated and exogenously-administered H(2)S exerts a wide range of actions in vascular and myocardial cells including vasodilator/vasoconstrictor effects via modification of the smooth muscle tone, induction of apoptosis and anti-proliferative responses in the smooth muscle cells, angiogenic actions, effects relevant to inflammation and shock, and cytoprotection in models of myocardial ischemia-reperfusion injury. Several molecular mechanisms of action of H(2)S have been described. These include interactions of H(2)S with NO, redox regulation of multiple signaling proteins and regulation of K(ATP) channel opening. The gaps in our current understanding of precise mechanisms, the absence of selective pharmacological tools and the limited availability of H(2)S measurement techniques for living tissues, leave many questions about physiological and pathophysiological roles of H(2)S unanswered at present. Nevertheless, this area of investigation is advancing rapidly. We believe H(2)S holds promise as an endogenous mediator controlling a wide range of cardiovascular cell functions and integrated responses under both physiological and pathological conditions and may be amenable to therapeutic manipulation.