A general approach to calculating isotopic distributions for mass spectrometry.

Fundamental principles for obtaining mass spectral isotopic distributions are applied to a general computer program that can be used to calculate and present in tabular and graphic form the isotopic contributions for any molecular formula. A unique feature is the retention of the isotopic distribution, exact mass, and absolute abundance for all individual peaks at each mass. Special considerations have been made for the large number of isotopic combinations that occur for many higher mass compounds. The computer program accepts the input of a molecular formula followed by interactive input of a number of parameters that affect the final presentation of the theoretical distribution profile.

In vitro and in vivo metabolism of a potent and selective integrin alpha v beta 3 antagonist in rats, dogs, and monkeys.

Compound A (3-[2-oxo-3-[3-(5,6,7,8-tetrahydro-[1,8]naphthyrindin-2-yl)propyl]-imidazolidin-1-yl]-3(S)-(6-methoxy-pyridin-3-yl)-propionic acid), a potent and selective antagonist of integrin alpha(v)beta(3) receptor, is under development for treatment of osteoporosis. This study describes metabolism and excretion of A in vivo in rats, dogs, and monkeys, and metabolism of A in vitro in primary hepatocytes from rats, dogs, monkeys, and humans. In all three animal species studied, A was primarily excreted as unchanged drug and, to a lesser degree, as phase I and phase II metabolites. Major biotransformation pathways of A included glucuronidation/glucosylation on the carboxylic group to form acyl-linked glucuronides/glucosides; and oxidation on the tetrahydronaphthyridine moiety to generate a carbinolamine and its further metabolized products. Minor pathways involved O-demethylation and hydroxylations on the alkyl chain. Only in rats, a glutathione adduct of A was also observed, and its formation is proposed to be via an iminium intermediate on the tetrahydronaphthyridine ring. Similar metabolic pathways were observed in the incubates of hepatocytes from the corresponding animals as well as from humans. CYP 3A and 2D subfamilies were capable of metabolizing A to its oxidative products. Overall, these in vitro and in vivo findings should provide useful insight on possible biotransformation pathways of A in humans.

Improving metabolic stability of phosphodiesterase-4 inhibitors containing a substituted catechol: prevention of reactive intermediate formation and covalent binding.

A detailed study directed towards metabolic stability optimization of the alkoxy substituents on the catechol moiety of CDP-840 is reported. Replacement of the methoxy and cyclopentyloxy substituents by cyclobutyloxy and/or difluromethoxy groups resulted in the discovery of potent and selective PDE4 inhibitors where the formation of reactive metabolites that could covalently bind to microsomal protein was significantly reduced or eliminated.

Diversity in the oxidation of substrates by cytochrome P450 2D6: lack of an obligatory role of aspartate 301-substrate electrostatic bonding.

Cytochrome P450 (P450) 2D6 was first identified as the polymorphic human debrisoquine hydroxylase and subsequently shown to catalyze the oxidation of a variety of drugs containing a basic nitrogen. Residue Asp301 has been characterized as being involved in electrostatic interactions with substrates on the basis of homology modeling and site-directed mutagenesis experiments [Ellis, S. W., Hayhurst, G. P., Smith, G., Lightfoot, T., Wong, M. M. S., Simula, A. P., Ackland, M. J., Sternberg, M. J. E., Lennard, M. S., Tucker, G. T., and Wolf, C. R. (1995) J. Biol. Chem. 270, 29055-29058]. However, pharmacophore models based on the role of Asp301 in substrate binding are compromised by reports of catalytic activity toward substrates devoid of a basic nitrogen, which have generally been ignored. We characterized a high-affinity ligand for P450 2D6, also devoid of a basic nitrogen atom, spirosulfonamide [4-[3-(4-fluorophenyl)-2-oxo-1-oxaspiro[4.4]non-3-en-4-yl]benzenesulfonamide], with K(s) 1.6 microM. Spirosulfonamide is a substrate for P450 2D6 (k(cat) 6.5 min(-)(1) for the formation of a syn spiromethylene carbinol, K(m) 7 microM). Mutation of Asp301 to neutral residues (Asn, Ser, Gly) did not substantially affect the binding of spirosulfonamide (K(s) 2.5-3.5 microM). However, the hydroxylation of spirosulfonamide was attenuated in these mutants to the same extent (90%) as for the classic nitrogenous substrate bufuralol, and the effect of the D301N substitution was manifested on k(cat) but not K(m). Analogues of spirosulfonamide were also evaluated as ligands and substrates. Analogues in which the sulfonamide moiety was modified to an amide, thioamide, methyl sulfone, or hydrogen were ligands with K(s) values of 1.7-32 microM. All were substrates, and the methyl sulfone analogue was oxidized to the syn spiromethylene carbinol analogue of the major spirosulfonamide product. The D301N mutation produced varying changes in the oxidation patterns of the spirosulfonamide analogues. The peptidometic ritonavir and the steroids progesterone and testosterone had been reported to be substrates for P450 2D6, but the affinities (K(s)) were unknown; these were estimated to be 1.2, 1.5, and 15 microM, respectively (cf. 6 microM for the classic substrate bufuralol). The results are consistent with a role of Asp301 other than electrostatic interaction with a positively charged ligand. H-Bonding or electrostatic interactions probably enhance binding of some substrates, but our results show that it is not required for all substrates and explain why predictive models fail to recognize the proclivity for many substrates, especially those containing no basic nitrogen.

Discovery of a potent and selective COX-2 inhibitor in the alkoxy lactone series with optimized metabolic profile.

The COX-2 inhibitor DFP [5,5-dimethyl-3-(2-propoxy)-4-methanesulfonylphenyl)-2(5H)-furanone] was found to have a long half-life in humans. Analogues have been characterized in order to optimize pharmacokinetics. This has lead to the discovery of 5(S)-(5-ethyl-5-methyl-3-(2-propoxy)-4-methanesulfonylphenyl)-2(5H)-furanone analogue 11 a potent and selective COX-2 inhibitor which is metabolized to a greater extent than DFP upon incubation with rat and human hepatocytes, suggesting a shorter half-life in humans.