Importance of amine pKa and distribution coefficient in the metabolism of fluorinated propranolol derivatives. Preparation, identification of metabolite regioisomers, and metabolism by CYP2D6.

A series of 1"-mono-, di-, and trifluorinated analogs of propranolol and related steric congeners was prepared, and their metabolism was examined in recombinant-expressed CYP2D6. The structural changes in this series of compounds, principally added fluorines and methyl groups in the 1"-position of the N-isopropyl group, provided compounds that varied in pK(a) by more than 5 log units and also varied in lipophilicity and in steric size. Products of both aromatic hydroxylation and N-dealkylation were observed in the metabolic experiments. The regiochemistry of aromatic hydroxylation at the 4'- and 5'-positions was assigned based on high-pressure liquid chromatography, fluorescence, and mass spectral characteristics of the products and standards. Correlations of the metabolic kinetic parameters K(m) and catalytic efficiency (k(cat)/K(m)) with substituent parameters of the added groups showed that increased basicity (higher pK(a) values) was associated with increased enzyme affinity (low K(m) values) and increased catalytic efficiency. More basic methyl-substituted compounds showed higher affinities for CYP2D6 than the structurally analogous less basic fluorinated congeners, indicating the decrease in affinity of the fluorinated compounds was not due to the size of the N-alkyl substituent. Correlations with log D reflected the degree of ionization and showed that the less lipophilic substrates (more basic compounds) had higher affinity for CYP2D6. These results are consistent with the proposal in the literature that ion pairing of the protonated amine of the substrate with Asp301 in the active site of CYP2D6 is very important to substrate affinity.

Importance of amine pKa and distribution coefficient in the metabolism of fluorinated propranolol analogs: metabolism by CYP1A2.

A series of 1"-mono-, di-, and trifluorinated analogs of propranolol and related steric congeners was prepared, and their metabolism was examined with recombinant-expressed CYP1A2. The structural changes in this series of compounds, principally added fluorines and methyl groups in the 1"-position of the N-isopropyl group, provided compounds that varied in pK(a) by more than 5 log units, in log D by 3 log units, and in size of the added substituents. N-Dealkylation and aromatic hydroxylation (formation of the 4'- and 5'-regioisomers) were catalyzed by CYP1A2. Correlations of the metabolic kinetic parameters K(m) and catalytic efficiency (k(cat)/K(m)) with physicochemical properties pK(a) and log D showed that increased lipophilicity (higher log D values) was associated with increased affinity (lower K(m)) and increased catalytic efficiency for CYP1A2. Comparison of log K(m) and log k(cat)/K(m) with pK(a) showed that the less basic analogs had higher affinities and increased catalytic efficiencies. The changes associated with pK(a) reflect increased lipid partitioning of substrate (increased log D) caused by an increase in the proportion of nonionized substrate. Increased steric bulk in the N-substituent alone did not decrease substrate affinity for CYP1A2 but did increase the amount of aromatic hydroxylation versus N-dealkylation. Removal of the hydroxyl group from the propanolamine side chain of propranolol resulted in a similar change in regioselectivity of metabolism.

Carbinolamines, imines, and oxazolidines from fluorinated propranolol analogs. (19)F NMR and mass spectral characterization and evidence for formation as intermediates in cytochrome P450-catalyzed N-dealkylation.

Formation of carbinolamine, imine, and oxazolidines from the reactions of desisopropylpropranolol (5), its O-methyl ether (10), and 3-(1-naphthoxy)propylamine (11) with trifluoroacetone and trifluoroacetaldehyde methyl hemiacetal was investigated by (19)F NMR and tandem mass spectrometry. Products from the metabolism of the related secondary amine substrates trifluoropropranolol (7), its O-methyl ether (23), and its N-trifluoroethyl-O-methyl ether analog (24) in the presence of rat liver microsomes and CYP1A2 were examined to determine whether these species were formed. The (19)F NMR experiments showed the presence of carbinolamine and imine species from these primary amines and fluorinated carbonyl compounds in solution. Mass spectral experiments under atmospheric pressure chemical ionization and electrospray ionization-ion trap conditions showed formation of imine metabolites (and/or oxazolidine from 7) as well as products of N-dealkylation and aromatic hydroxylation when the secondary amine substrates were incubated with rat liver microsomes or CYP1A2. In spite of mass spectral evidence for these imines as metabolites, we were unable to detect the carbinolamines under the conditions used in these studies. Their presence is inferred from the results of the (19)F NMR experiments.

Mass spectral fragmentation pathways of propranolol related beta-fluorinated amines studied by electrospray and electron impact ionization.

Propranolol, its 1"-mono-, di-, and trifluorinated analogs, and other related compounds were analyzed under electrospray ionization ion trap collision-induced dissociation (ESI-CID) and electron impact (EI) conditions. Interesting trends were observed in the fragment ions formed in both cases. Under ESI conditions, the abundances of product ions easily explained by protonation on the amine nitrogen decreased relative to the abundances of those formed from the ether-protonated species as the number of fluorines increased from zero to three. Under EI conditions, the distribution of fragment ions was shifted away from those arising from a nitrogen-centered cation radical and toward those arising from an ether oxygen-centered cation radical. The changes observed in apparent molecular sites of protonation and of ion radical formation in the mass spectra are consistent with the electron-withdrawing effects of the sequentially added fluorines. These effects are correlated with changes in solution phase pK( a)'s of the fluorinated amines.

Fragmentation pathways of selectively labeled uropranolol using electrospray ionization on an ion trap mass spectrometer and comparison with ions formed by electron impact.

Propranolol, deuterium- and 18O-labeled propranolol and related compounds were analyzed using an ion trap mass spectrometer equipped with a modified Finnigan API electrospray interface. Sequential product ion (MSn) experiments were used to elucidate fragmentation pathways for these compounds. The observed ions were compared to those observed under electron impact (EI) conditions. The electrospray ionization (ESI) ion trap spectra, as well as the EI spectra, afford useful information to allow assignments of most product ions, many of which retain portions of the aliphatic three-carbon side chain.

(E)- and (Z)-7-arylidenenaltrexones: synthesis and opioid receptor radioligand displacement assays.

The E-isomer of 7-benzylidenenaltrexone (BNTX, la) was reported by Portoghese as a highly selective delta-opioid antagonist. The corresponding Z-isomer 1b was not readily available through direct aldol condensation of naltrexone (6) with benzaldehyde. Using the photochemical methods employed by Lewis to isomerize cinnamamides, we have obtained Z-isomer 1b in good yield from E-isomer 1a. A series of (E)- and (Z)-7-arylidenenaltrexone derivatives was prepared to study the effect of larger arylidene groups on opioid receptor affinity in this series. By aldol condensation of naltrexone (6) with benzaldehyde, 1-naphthaldehyde, 2-naphthaldehyde, 4-phenylbenzaldehyde, and 9-anthracaldehyde, the (E)-arylidenes were readily obtained. Photochemical isomerization afforded the corresponding Z-isomers. These compounds were evaluated via opioid receptor radioligand displacement assays. In these assays, the Z-isomers generally had higher affinity and were more delta-selective than the corresponding E-isomers. The (Z)-7-(1-naphthylidene)naltrexone (3b) showed the greatest selectivity (delta:mu ratio of 15) and highest affinity delta-binding (Ki = 0.7 nM). PM3 semiempirical geometry optimizations suggest a significant role for the orientation of the arylidene substituent in the binding affinity and delta-receptor selectivity. This work demonstrates that larger groups may be incorporated into the arylidene portion of the molecule with opioid receptor affinity being retained.