Dan Fishlock

Photo-control of nitric oxide synthase activity using a caged isoform specific inhibitor.

Nitric oxide (NO) plays a critical role in a number of physiological processes and is produced in mammalian cells by nitric oxide synthase (NOS) isozymes. Because of the diverse functions of NO, pharmaceutical interventions which seek to abrogate adverse effects of excess NOS activity must not interfere with the normal regulation of NO levels in the body. A method has been developed for the control of NOS enzyme activity using the localized photochemical release of a caged isoform-specific NOS inhibitor. The caged form of an iNOS inhibitor has been synthesized and tested for photosensitivity and potency. UV and multiphoton uncaging were verified using a hemoglobin-based assay. IC(50) values were determined for the inhibitor (70+/-11 nM), the caged inhibitor (1098+/-172 nM), the UV uncaged inhibitor (67+/-26 nM) and the multiphoton uncaged inhibitor (73+/-11 nM). UV irradiation of the caged inhibitor resulted in a 86% reduction in iNOS activity after 5 min. Multiphoton uncaging had an apparent first order time constant of 0.007+/-0.001 min(-1). A therapeutic range exists, with molar excess of inhibitor to enzyme from 3- to 7-fold, over which the full dynamic range of the inhibition can be exploited.

Synthetic studies on Et-743. Assembly of the pentacyclic core and a formal total synthesis.

A formal total synthesis of the potent anticancer agent Et-743 is described. The tetrahydroisoquinoline core is stereoselectively constructed using a novel radical cyclization of a glyoxalimine. Further elaboration of this core rapidly accessed the pentacyclic core of Et-743, but a mixture of regiosisomers was obtained in the key Pictet-Spengler ring closure. A known advanced intermediate in the synthesis of Et-743 was intercepted, constituting a formal synthesis of the molecule.

Synthesis of N-protected N-methyl serine and threonine

Abstract Two efficient and convenient syntheses of N -Cbz and N -Fmoc N -methyl serine and threonine are described. The amino acid side-chain alcohol can be protected as a TBDMS ether in very good yield or left free, followed by the formation and subsequent reduction of the corresponding oxazolidinone.

Probing Persistent Intramolecular C−H···X (X = O, S, Br, Cl, and F) Bonding in Solution Using Benzyl Meldrum’s Acid Derivatives

Persistent intramolecular interactions between acidic C-H hydrogens and a variety of acceptors (X) (X = O, S, Br, Cl, and F) in solution were probed by (1)H NMR experiments, using 5-benzyl Meldrums acid derivatives. To bring about formation of intramolecular C-H...X bonding, ortho-substituted benzyl Meldrums acids were designed, for which hydrogen bonding occurred through a six-membered ring. Introduction of substituents on the aromatic moiety and in the tether allowed variation of electronic and steric factors. The superior acidity of Meldrums acids impacted the ability of the C-H hydrogen to engage in nonclassical C-H...X bonds and drove the conformational properties of benzyl Meldrums acid, in combination with steric factors, namely A(1,3)-allylic strain. Further understanding of intramolecular C-H...X bonds was gained by characterization of benzyl Meldrums acids in the solid state, by X-ray analysis, and by the conformation correlated to the observations made in solution.

Synthesis and evaluation of trans 3,4-cyclopropyl l-arginine analogues as isoform selective inhibitors of nitric oxide synthase

Four optically pure conformationally restricted L-arginine analogues syn- 1 and anti- 2 trans-3,4-cyclopropyl L-arginine, and syn- 3 and anti-trans-3,4-cyclopropyl N-(1-iminoethyl) L-ornithine 4 were synthesized. These compounds were tested as potential inhibitors against the three isoforms of nitric oxide synthase (NOS). Compound 1 was determined to be a poor substrate of NOS, while compound 2 was determined to be a poor mixed type inhibitor and did not exhibit any isoform selectivity. Syn- 3 and anti-trans-3,4-cyclopropyl N-(1-iminoethyl) L-ornithine 4 were found to be competitive inhibitors of NOS. These compounds were time dependent inhibitors of inducible NOS (iNOS), but not of neuronal NOS (nNOS) or endothelial NOS (eNOS). Compound 3 was 10- to 100-fold more potent an inhibitor than 4, exhibited a 5-fold increase in nNOS/iNOS and eNOS/iNOS selectivity over 4, and displayed tight binding characteristics against iNOS. These results indicate that the relative configuration of the cyclopropyl ring in the L-arginine analogues significantly affects their inhibitory potential and NOS isoform selectivity.