Search

All-trans-retinoic acid (atRA), the major active metabolite of vitamin A, plays a role in many biological processes, including maintenance of epithelia, immunity, and fertility and regulation of apoptosis and cell differentiation. atRA is metabolized mainly by CYP26A1, but other P450 enzymes such as CYP2C8 and CYP3As also contribute to atRA 4-hydroxylation. Although the primary metabolite of atRA, 4-OH-RA, possesses a chiral center, the stereochemical course of atRA 4-hydroxylation has not been studied previously. (4S)- and (4R)-OH-RA enantiomers were synthesized and separated by chiral column HPLC. CYP26A1 was found to form predominantly (4S)-OH-RA. This stereoselectivity was rationalized via docking of atRA in the active site of a CYP26A1 homology model. The docked structure showed a well defined niche for atRA within the active site and a specific orientation of the β-ionone ring above the plane of the heme consistent with stereoselective abstraction of the hydrogen atom from the pro-(S)-position. In contrast to CYP26A1, CYP3A4 formed the 4-OH-RA enantiomers in a 1:1 ratio and CYP3A5 preferentially formed (4R)-OH-RA. Interestingly, CYP3A7 and CYP2C8 preferentially formed (4S)-OH-RA from atRA. Both (4S)- and (4R)-OH-RA were substrates of CYP26A1 but (4S)-OH-RA was cleared 3-fold faster than (4R)-OH-RA. In addition, 4-oxo-RA was formed from (4R)-OH-RA but not from (4S)-OH-RA by CYP26A1. Overall, these findings show that (4S)-OH-RA is preferred over (4R)-OH-RA by the enzymes regulating atRA homeostasis. The stereoselectivity observed in CYP26A1 function will aid in better understanding of the active site features of the enzyme and the disposition of biologically active retinoids.

Itraconazole (ITZ) is a mixture of four cis-stereoisomers that inhibit CYP3A4 potently and coordinate CYP3A4 heme via the triazole nitrogen. However, (2R,4S,2'R)-ITZ and (2R,4S,2'S)-ITZ also undergo stereoselective sequential metabolism by CYP3A4 at a site distant from the triazole ring to 3'-OH-ITZ, keto-ITZ, and N-desalkyl-ITZ. This stereoselective metabolism demonstrates specific interactions of ITZ within the CYP3A4 active site. To further investigate this process, the binding and metabolism of the four trans-ITZ stereoisomers by CYP3A4 were characterized. All four trans-ITZ stereoisomers were tight binding inhibitors of CYP3A4-mediated midazolam hydroxylation (IC(50) 16-26 nM), and each gave a type II spectrum upon binding to CYP3A4. However, instead of formation of 3'-OH-ITZ, they were oxidized at the dioxolane ring, leading to ring scission and formation of two new metabolites of ITZ. These two metabolites were also formed from the four cis-ITZ stereoisomers, although not as efficiently. The catalytic rates of dioxolane ring scission were similar to the dissociation rates of ITZ stereoisomers from CYP3A4, suggesting that the heme iron is reduced while the triazole moiety coordinates to it and no dissociation of ITZ is necessary before catalysis. The triazole containing metabolite [1-(2,4-dichlorophenyl)-2-(1H-1,2,4-triazol-1-yl)ethanone] also inhibited CYP3A4 (IC(50)15 μM) and showed type II binding with CYP3A4. The dioxolane ring scission appears to be clinically relevant because this metabolite was detected in urine samples from subjects that had been administered the mixture of cis-ITZ isomers. These data suggest that the dioxolane ring scission is a metabolic pathway for drugs that contain this moiety.

Three secondary amines desipramine (DES), (S)-fluoxetine [(S)-FLX], and N-desmethyldiltiazem (MA) undergo N-hydroxylation to the corresponding secondary hydroxylamines [N-hydroxydesipramine, (S)-N-hydroxyfluoxetine, and N-hydroxy-N-desmethyldiltiazem] by cytochromes P450 2C11, 2C19, and 3A4, respectively. The expected primary amine products, N-desmethyldesipramine, (S)-norfluoxetine, and N,N-didesmethyldiltiazem, are also observed. The formation of metabolic-intermediate (MI) complexes from these substrates and metabolites was examined. In each example, the initial rates of MI complex accumulation followed the order secondary hydroxylamine > secondary amine ≫ primary amine, suggesting that the primary amine metabolites do not contribute to formation of MI complexes from these secondary amines. Furthermore, the primary amine metabolites, which accumulate in incubations of the secondary amines, inhibit MI complex formation. Mass balance studies provided estimates of the product ratios of N-dealkylation to N-hydroxylation. The ratios were 2.9 (DES-CYP2C11), 3.6 [(S)-FLX-CYP2C19], and 0.8 (MA-CYP3A4), indicating that secondary hydroxylamines are significant metabolites of the P450-mediated metabolism of secondary alkyl amines. Parallel studies with N-methyl-d3-desipramine and CYP2C11 demonstrated significant isotopically sensitive switching from N-demethylation to N-hydroxylation. These findings demonstrate that the major pathway to MI complex formation from these secondary amines arises from N-hydroxylation rather than N-dealkylation and that the primary amines are significant competitive inhibitors of MI complex formation.

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.

A series of 6-O-ethers of 6 beta- and 6 alpha-naltrexol (6 and 7) were prepared to examine the effect of large aralkyl groups on affinity of the ligands for opioid receptors. The affinities of the 6 beta- and 6 alpha-O-ether with benzyl, biphenylmethyl, 1- and 2-naphthylmethyl, and 9-anthracylmethyl groups were determined. Preparation of the ligands was accomplished from suitably 3-O-protected derivatives of 6 and 7 by phase transfer catalyzed alkylation using aralkyl halides, followed by deprotection. Both 3-O-trityl and -benzyl protecting groups were used. In radioligand displacement assays, compounds from the 6 alpha-O ether series had higher affinity than the analogous diastereomers in the 6 beta-O series, with few exceptions. In the 6 alpha-O series, the benzyl ether (29) and the biphenylmethyl ether (30) had the highest affinity, similar to naltrexone. In the 6 beta-O series, the benzyl ether had the highest affinity. The larger aralkyl ethers had slightly less affinity. Large lipophilic 6 beta-O- and 6 alpha- O-aralkyl groups are readily accommodated in the drug-receptor interaction.

Female brownbanded cockroaches, Supella longipalpa, emit a sex pheromone that attracts males from a distance. This pheromone was isolated and identified as 5-(2,4-dimethylheptanyl)-3-methyl-2H-pyran-2-one (which we refer to as supellapyrone), and its structure was confirmed by synthesis. A racemic blend of the synthetic compound elicited behavioral and electrophysiological responses comparable to the natural pheromone across a range of doses. This compound is not only a very different type of cockroach pheromone but also makes up an additional class of natural products--namely, 3,5-dialkyl-substituted alpha-pyrones.

Asymmetric synthesis of the diastereomeric 7-(1-hydroxyethyl)-2-[1-hydroxy-2-(tert-butylamino)ethyl]benzofurans (2), the benzylic hydroxylation metabolites of bufuralol (1), is described, and the absolute configurations of these diastereomers are assigned. 1"-Oxobufuralol (3) was reduced with a complex of (2S)-(-)-2-amino-3-methyl-1,1-diphenylbutan-1-ol and borane, yielding 2, which had a 95:5 ratio of the possible 1"R and 1"S isomers as determined by HPLC. Separation of the resulting diastereomers was facilitated by derivatization with the enantiomers of 1-phenethyl isocyanate (PEIC). The absolute configurations 1'S,1"R and 1'R,1"R were assigned to the diastereomers formed in excess, 2c and 2b, on the basis of the known stereochemistry of reduction of closely related alkyl phenyl ketones to R alcohols by using this chiral borane reagent. The circular dichroism spectra of the four isomeric benzylic alcohols were in agreement with these assignments. In the presence of the rat liver microsomal fraction, benzylic hydroxylation of bufuralol was significantly product stereoselective favoring formation of diastereomers with the 1"R absolute stereochemistry at the new chiral center in products from (1'R)-1 by a ratio of 4.5:1 [(1'R,1"R)-2:(1'R,1"S)-2] and by nearly 8:1 [(1'S,1"R)-2:(1'S,1"S)-2] from (1'S)-1. (1'R)-Bufuralol was more rapidly hydroxylated than was (1'S)-1, by about 3-fold. In the presence of human liver microsomes, (1'R)-bufuralol was also more rapidly hydroxylated than was (1'S)-1, by ca. 2.5-fold. However, product stereoselectivity from the 1'R enantiomer was reversed from that observed in the rat liver microsomal oxidation, with more (1"S)-carbinol being formed than 1"R isomer by nearly 4-fold. From (1'S)-1, about equal amounts of the two possible hydroxybufuralol diastereomers were formed. The results from the human liver microsomal studies are consistent with observed enantioselectivity of hydroxylation of bufuralol in vivo in humans.

Benzomorphans with an electrophilic group in the nitrogen substituent were prepared as potentially irreversible ligands for the kappa-opioid receptor. These were synthesized from products of the reaction of normetazocine with the enantiomers of 5-(iodomethyl)-gamma-butyrolactone (11). alpha-Methylene gamma-lactones 5 and 7 and endocyclic alpha,beta-unsaturated gamma-lactones 8 and 9 were prepared from the corresponding saturated gamma-lactones 13 and 23 possessing the "active" (1R,5R,9R)-benzomorphan stereochemistry. Only gamma-lactones 8, 9, 13, and 23, lacking the exocyclic methylene group, retain significant affinities for opioid receptor binding sites when compared with the reference compounds (2"S)-3 and (2"R)-3. As observed with these references compounds, greater binding affinity is also seen with gamma-lactone diastereomers having the 2"S stereochemistry in the nitrogen substituent. Although the gamma-lactones do not bind irreversibly in opioid receptor preparations, they do show kappa-receptor selectivities comparable to those observed for the reference compounds.

2-(3,4-Dimethoxyphenyl)-2-isopropyl-5-[N-[4-(N-methyl-2-diazo- 3,3,3-trifluoropropionamido)phenethyl]methyl-amino]valeronitril e (3), a potential photoaffinity probe for Ca2+ channels related to verapamil (1), was prepared from N-methyl-4-nitrophenethylamine (7) and 2-(3,4-dimethoxyphenyl)-2-isopropyl-5-(methanesulfonoxy)valeron itrile (12). Compound 3 showed concentration-dependent negative inotropic effects in rat right myocardial ventricular strips, EC50 = (1.05 +/- 0.33) X 10(-7) M (mean +/- SD), being slightly less potent than gallopamil (2), EC50 = (2.18 +/- 0.66) X 10(-8) M. It displaced [3H]gallopamil in myocardial membranes, Ki = (3.76 +/- 1.55) X 10(-8) M, compared to 2, Ki = (1.55 +/- 0.16) X 10(-8) M. Photoactivation at 265 nm reduced the recoverable binding of [3H]gallopamil to 26% compared to no effect on 2. This agent may be a useful photoaffinity probe to aid in further characterization of Ca2+ channels.

Alpha- and beta-naltrexol derived esters 9 and 10 and ethers 11 and 12, each containing the alpha, beta-unsaturated ester functionality, were prepared as conformationally more flexible analogues of spiro-alpha-methylene-gamma-lactones 5 and 6. All were active in the opioid radioreceptor binding assay against [3H]bremazocine and more active against [3H]DAGO, indicating mu-subtype selectivity, but only ether 12 showed significant irreversible activity. We conclude that small structural changes, made in very closely related electrophilic opioids, lead to changes in receptor binding. All four compounds were long-acting antagonists to morphine in mice, with ester 10 being approximately equipotent with naltrexone.

Itraconazole (ITZ) has three chiral centers and is administered clinically as a mixture of four stereoisomers. This study evaluated stereoselectivity in ITZ metabolism. In vitro experiments were carried out using heterologously expressed CYP3A4. Only (2R,4S,2'R)-ITZ and (2R,4S,2'S)-ITZ were metabolized by CYP3A4 to hydroxy-ITZ, keto-ITZ, and N-desalkyl-ITZ. When (2S,4R,2'R)-ITZ or (2S,4R,2'S)-ITZ was incubated with CYP3A4, neither metabolites nor substrate depletion were detected. Despite these differences in metabolism, all four ITZ stereoisomers induced a type II binding spectrum with CYP3A4, characteristic of coordination of the triazole nitrogen to the heme iron (K(s) 2.2-10.6 nM). All four stereoisomers of ITZ inhibited the CYP3A4-catalyzed hydroxylation of midazolam with high affinity (IC(50) 3.7-14.8 nM). Stereochemical aspects of ITZ pharmacokinetics were evaluated in six healthy volunteers after single and multiple oral doses. In vivo, after a single dose, ITZ disposition was stereoselective, with a 3-fold difference in C(max) and a 9-fold difference in C(min) between the (2R,4S)-ITZ and the (2S,4R)-ITZ pairs of diastereomers, with the latter reaching higher concentrations. Secondary and tertiary ITZ metabolites (keto-ITZ and N-desalkyl-ITZ) detected in plasma were of the (2R,4S) stereochemistry. After multiple doses of ITZ, the difference in C(max) and C(min) decreased to 1.5- and 3.8-fold, respectively. The initial difference between the stereoisomeric pairs was most likely due to stereoselective metabolism by CYP3A4, including stereoselective first-pass metabolism as well as stereoselective elimination. However, stereoselective elimination was diminished after multiple dosing, presumably as a result of CYP3A4 autoinhibition. In conclusion, the metabolism of ITZ is highly stereoselective in vitro and in vivo.

N-Methyl-N-[(1S)-1-(4-isothiocyanatophenyl)-2-(1-pyrrolidinyl)ethyl-3,4- dichlorophenylacetamide (MITPD), is an isothiocyanate derivative of the kappa agonist ICI-199,441. In this study, interaction of MITPD with cloned mu, delta, and kappa opioid receptors was characterized. MITPD inhibited [3H]diprenorphine binding to kappa receptors with high affinity and with approximately 700- and approximately 870-fold selectivity over mu and delta receptors. Pretreatment with MITPD followed by extensive washing reduced kappa receptor binding with an IC50 value of 3.7 nM, but did not affect mu or delta binding ator = 0.1 microM. Preincubation with 1 microM MITPD abolished [3H]diprenorphine binding, while pretreatment with 1 microM ICI-199,441 increased Kd of [3H]diprenorphine binding with no change in Bmax. Thus, MITPD is a selective kappa irreversible ligand. The region of the kappa receptor that conferred selectivity for MITPD was determined by examining its binding to four mu/kappa chimeras. IC50 values of MITPD for inhibition of [3H]diprenorphine binding were determined to be 430 nM for Chimera III (kappa1-141/mu151-398), 1.8 nM for Chimera IV (mu1-150/kappa142-380), 40 nM for Chimera XI (mu1-268/kappa263-380) and 14 nM for Chimera XII (kappa1-262/mu269-398). Pretreatment with MITPD followed by extensive washing reduced binding to chimera IV with an IC50 value of 75 nM, but did not affect III, XI or XII binding (IC501 microM). Thus, the region from the third transmembrane helix to the C-terminus of the kappa receptor is important for the binding of MITPD.

The isothiocyanate group was attached to the 4'-, 5'-, 6'-, or 7'-position of naltrindole in an effort to determine the importance of the position of this electrophilic group on the selectivity for subtypes of delta opioid receptors. All of the ligands were delta-selective when tested against standard agonists in smooth muscle preparations. However, the rank-order delta antagonism of antinociception in mice did not parallel the in vitro pharmacologic data. The 5'-isothiocyanate 2 was the most potent and selective antagonist in vivo, causing a 52-fold increase of the ED50 for [D-Ser2,D-Leu5]enkephalin-Thr6 (DSLET) and no increase for [D-Pen2,D-Pen5]enkephalin (DPDPE). The effect of each of the ligands on the binding of [3H]DSLET and [3H]DPDPE to guinea pig brain membranes clearly differentiated between the binding sites that recognize these radioligands. These studies provide additional evidence for the presence of two subtypes of delta opioid receptors.

O6-Ether derivatives of 6 beta-naltrexol in which the ether substituent includes various electrophilic groups have been synthesized in an effort to examine structure-activity requirements at the 6 beta-substituent for receptor affinity and irreversibility of binding in opioid receptor preparations. A series of tethered 6 beta-ethers having terminal epoxides, alpha-methylene-gamma-lactones, and an isothiocyanate group were prepared. The stereochemistry of the alpha-methylene-gamma-lactones was established by convergent synthesis of their reduction products from epoxides of known absolute stereochemistry. In general, the tested compounds showed comparable affinity and selectivity for the receptor subtypes. All were found with high affinity for mu-sites. The terminal epoxide ether diastereomers 8 and 9 were not bound irreversibly in the assay for total opioid receptors. The alpha-methylene-gamma-lactone diastereomers 10 and 11, and their O14-acetyl precursors 20 and 21, respectively, varied in their irreversible effects, but where noted these effects were mu-site selective. Methacrylate ether 7 and isothiocyanate ether 12 were bound irreversibly at both mu- and delta-sites.

Isothiocyanate and alpha-methylene-gamma-lactone analogues of 6,14-endo-ethenotetrahydrothebaine and -oripavine were prepared with the electrophilic groups being located at C-19 in the C-7 alpha-side chain. Isothiocyanates were prepared in the N-Me and N-CPM (N-cyclopropylmethyl) series, both as the phenols and 3-O-methyl ethers from the diastereomeric amines formed from reductive amination of thevinone (2) and N-(cyclopropylmethyl)northevinone (13). Although addition of the organozinc reagent from methyl alpha-bromomethacrylate to 25 failed, addition to 3-O-protected aldehydes 27 and 35 produced, after subsequent deprotection, alpha-methylene-gamma-lactones 29 and 37, respectively. In the opioid receptor displacement assays against [3H]bremazocine as the radiolabeled ligand, the phenolic compounds were most potent with N-CPM isothiocyanates 20 and 21 showing IC50s of 0.32 and 0.76 nM, respectively, and N-CPM alpha-methylene-gamma-lactone 37 having an IC50 = 1.0 nM. Compound 37 showed irreversible effects in the binding assay which were mu-selective, as demonstrated by analogous experiments using [3H]DAGO, and naloxone was found to protect against the irreversible effects. This observation suggests that a receptor-bound nucleophile is located at a position where it can readily reach the alpha-methylene group of lactone 37.

Two 13C-labeled verapamil compounds were prepared. 2-(3,4-Dimethoxyphenyl)-2-isopropyl-5-[2–(3,4-dimethoxyphenyl-1–[13C]-ethyl)methylamino]-valeronitrile was synthesized from 2–(3,4-dimethoxyphenyl)-1–[13C]-ethylamine, which was prepared from 3,4-dimethoxybenzyl chloride by displacement with 13C-labeled KCN followed by diborane reduction. 2-(3,4-Di-[13C]-methoxyphenyl)-2-isopropyl-5–[2–(3,4-di–[13C]-methoxyphenethyl)methylamino]-valeronitrile containing 50% incorporation of 13C in the N-methyl group was prepared via demethylation of verapamil and subsequent re-methylation with 13C-labeled CH3I.

Enantiomers of metoprolol (1) containing six deuterium atoms in the isopropyl methyl groups [(2R-2], two deuterium atoms at C-2 and C-6 of the aromatic ring [(2S)-3], and two deuterium atoms at C-3 of the propanolamine side chain [(2S)-4] were prepared. Chiral 2,2-dimethyl-1,3-dioxolane-4-methanols [(4R)-5 and (4S)-5] were key synthons. Sources of deuterium were [2H6]-isopropylamine, 4-(2-methoxyethyl)-2,6-[2H2]-phenol (12), prepared by 2HCl/2H2O exchange, and (4S)-2,2-dimethyl-1,3-dioxolane-4-[2H2]-4-methanol (19), prepared by LiAl2H4 reduction of (4S)-methyl 2,2-dimethyl-1,3-dioxolane-4-carboxylate. Enantiomeric excess was greater than 94% for each of the prepared enantiomers, as determined independently by 1H NMR spectroscopy on diastereomeric derivatives and by chiral column HPLC.

Deuterated (-)-verapamil, (2S)-(-)-2-(2,5,6-2H3-3,4-dimethoxyphenyl)-2-isopropyl-5-[(2,5,6-2H3-3,4-dimethoxyphenethyl)methylamino]valeronitrile, and its analog (-)-2H5-gallopamil, (2S)-(-)-2-(2,6-2H2-3,4,5-trimethoxyphenyl)-2-isopropyl-5-[(2,5,6-2H3-3,4-dimethoxyphenethyl)methylamino]valeronitrile, were prepared. (-)-Gallopamil readily incorporated five atoms of deuterium into the available positions in the aromatic rings. (-)-2H6-Verapamil was prepared from ring trideuterated 3,4-dimethoxyphenylacetic acid and (2S)-(+)-triphenyl-methoxy-2-[(methanesulfonyl)oxy]propane. Three additional atoms of deuterium were incorporated into the aromatic ring of the N-methyl-3,4-dimethoxyphenethylamine moiety incorporated as the the short N-alkyl side chain. N-13C-Methylverapamil was prepared from N-13C-methyl-3,4-dimethoxyphenethylamine.

Preparation of analogs of 4-[N-(3-chlorophenyl) carbamoyloxy]-2-butynyltrimethylammonium chloride [1 (McN-A-343)], cis- and trans-4-[N-(4-chlorophenyl)carbamoyloxy]-2-butenyltrimethylammonium iodides (5 and 6), and the corresponding epoxides and cyclopropanes is reported. Pharmacological testing for ganglion-stimulating activity demonstrated that the trans olefin 6 and trans epoxide 8 have properties similar to 1, while the trans cyclopropane analog 10 was inactive. All cis compounds were inactive. The muscarinic ganglion-stimulating properties of the active compounds are interpreted in terms of similar fit at the receptor level by the alkyltrimethylammonium ion and the ether oxygen 5.7 A distant, as well as an electron-rich center midway between groups in the form of a double bond or unshared electron pairs. Comparison of smooth muscle and ganglion-stimulating properties of the compounds showed that trans epoxide 8 was the most selective for muscarinic ganglionic sites.

Reduction of 1 (verapamil) afforded amine 2, which was converted with thiophosgene to isothiocyanate 3, a chemoaffinity ligand for Ca2+ channels. Compound 3 showed concentration-dependent negative inotropic effects in rat right myocardial ventricular strips, EC50 = (4.56 +/- 3.40) X 10(-6) M (mean +/- SD), being slightly less potent than 4 (gallopamil), EC50 = (1.95 +/- 1.22) X 10(-6) M. It displaced [3H]gallopamil in rat myocardial membranes, IC50 = (3.42 +/- 2.51) X 10(-7) M, approximately equipotent with 1. It showed irreversible antagonism of [3H]gallopamil binding when preincubated at 10(-5) M; only 25% of [3H]gallopamil binding vs. control was observed. This agent may be a useful chemoaffinity ligand to aid in characterization of Ca2+ channels.

Deuteration of verapamil, 5-[(3,4-dimethoxyphenylethyl)methyl-amino]-2-(3,4-dimethoxyphenyl)-2-isopropyvaleronitrile, an important calcium channel antagonist was accomplished by direct exchange in 25% 2H2SO4 in 2H2O. Refluxing for 140 hr incorporated 4–6 atoms of deuterium, which were distributed into both aromatic rings, but primarily in the ring attached through position 5 of the valeronitrile moiety.

Synthesis and stereochemistry of various endo-2- and exo-2-substituted-7-oxabicyclo[2.2.1]-hept-5-enes and -heptanes are described. The nmr spectra of several derivatives are reported and discussed. Use is made of this data to allow determination of the stereochemical integrity of the system. Facile chromatographic separation methods were found for endo- and exo-2-sub-stituted-7-oxabicyclo[2.2.1 ]hept-5-enes and -heptanes.

The enantiomers of 2-[[[2-(2,6-dimethoxyphenoxy)ethyl]amino]methyl]-1,4-benzodioxane (4) were prepared from the chiral 2-[(tosyloxy)methyl]-1,4-benzodioxanes [(2S)- and (2R)-5]. The corresponding (2R)- and (2S)-2-(aminoethyl)-1,4-benzodioxanes [2R)- and (2S)-7] were prepared by a modified Gabriel synthesis and converted to the enantiomers of 4 by condensation with 2,6-dimethoxyphenoxyacetaldehyde (8) and reduction of the intermediate imine with NaBH4. The enantiomer (2S)-4 was 40--50 times as potent as the enantiomer (2R)-4 in antagonizing the alpha-adrenergic response of methoxamine-induced contraction of rabbit aortic strips, showing a pA2 = 9.0. This result is consistent with the previous observation that S enantiomers of 2-[(alkylamino)methyl]benzodioxanes are more potent antagonists at a alpha-adrenergic receptors than the R enantiomers.

Synthesis and opioid radioreceptor assay data on analogues closely related to 6-desoxy-6-spiro-alpha-methylene-gamma-lactone 5a, a compound with irreversible activity in this assay, are reported. Saturated lactones (7a,b), endocyclic alpha, beta-unsaturated gamma-lactones (8a,b and 9a), and 6 alpha,7 alpha-fused alpha-methylene-gamma-lactones (10a and 11a) were prepared. Related 6-desoxy-6-methylene 6 beta- and 6 alpha-oxides (12a,b and 13a) and glycidate esters 14a,b and 15a,b were also prepared with use of naltrexone (1a) and oxymorphone (1b) as starting material. Compounds in the N-cyclopropylmethyl (N-CPM) series were more potent than those in the N-Me series in displacing [3H]naltrexone in the opioid radioreceptor assay, usually by 2-16-fold in the absence of Na ion. The most potent N-CPM analogues were epoxides 12a and 13a and glycidate esters 14a and 15a, showing IC50's of 2-6 nM, similar to that of 5a. Of the N-Me analogues, 6 beta-oxide 12b was most active, with an IC50 of 8 nM in the absence of Na ion. For the N-CPM analogues, the Na ion ratios were generally less than 1, with two exceptions. The N-Me analogues showed expected larger Na ion effects of 7 or greater. None of the lactone analogues had irreversible effects when preincubated in the rat brain membrane preparation, even at 37 degrees C for 30 min, i.e., washing restored [3H]naltrexone binding to control levels. These results clearly show that the alpha-methylene-gamma-lactone moiety of 5a is required for irreversible effects, consistent with it serving as a conjugate addition acceptor of a nucleophilic group from a ligand at or near the receptor. The epoxides and glycidate esters also had no irreversible activity, indicating more electrophilic functional groups are needed and/or these electrophiles are not properly aligned to react with nucleophilic groups at or near the opioid receptor.

There is a strong association between aromatic hydrocarbon responsiveness and N-hydroxylation of phenacetin, acetanilide, and p-chloroacetanilide in the livers from progeny and the appropriate backcrosses and intercross involving B6 and D2 mice. Mutagenic activity in the Salmonella test system of several 2-acylaminofluorenes were compared using the S-9 liver fractions from B6 and D2 mice. Pretreatment of B6 mice with 3-MC increased the mutagenic activity of all the analogs in the S-9 liver fraction. No increase in mutagenic activity is found in S-9 liver fraction from D2 mice after 3-MC pretreatment, indicating that mutagenic activity correlates with aromatic hydrocarbon responsiveness and N-hydroxylase activity. ANF, an inhibitor of N-hydroxylation of 2-AAF in vitro, inhibits the mutagenic activity of these analogs indicating that N-hydroxylation is the first step in the activation of N-acylaminofluorenes into mutagens.

Oxprenolol (1), like related beta-adrenergic antagonists, undergoes oxidative N-dealkylation to form the corresponding 3-aryloxypropane-1,2-diol (2), 3-aryloxylactic acid (3), and 2-aryloxyacetic acid (4) metabolites. Compounds 3 and 4 were synthesized by conversion of 2-allyloxyphenol (5) to the aryloxyacetaldehyde 6 and subsequent elaboration to the desired acids. Both acids (3 and 4) and glycol 2 were confirmed as metabolites formed from 1 in vivo in the rat and in vitro in the rat liver 9000 X g supernatant fraction. Incubation of a pseudoracemate of 1, made up of equal molar amounts of (2S)-1-d0 and (2R)-1-d2, showed that 2 and 3 arise principally from (2S)-1 by S/R ratios of approximately 5:1 and 2:1, respectively. On the other hand, acetic acid derivative 4 arises about equally from both enantiomers of 1.

Reduction of 1 (verapamil) afforded amine 2, which was converted with thiophosgene to isothiocyanate 3, a chemoaffinity ligand for Ca2+ channels. Compound 3 showed concentration-dependent negative inotropic effects in rat right myocardial ventricular strips, EC50 = (4.56 +/- 3.40) X 10(-6) M (mean +/- SD), being slightly less potent than 4 (gallopamil), EC50 = (1.95 +/- 1.22) X 10(-6) M. It displaced [3H]gallopamil in rat myocardial membranes, IC50 = (3.42 +/- 2.51) X 10(-7) M, approximately equipotent with 1. It showed irreversible antagonism of [3H]gallopamil binding when preincubated at 10(-5) M; only 25% of [3H]gallopamil binding vs. control was observed. This agent may be a useful chemoaffinity ligand to aid in characterization of Ca2+ channels.

A new metabolic pathway of terminal hydroxylation (omega-hydroxylation) of the N-isopropyl group of propranolol (1) was established. Selected ion-monitoring GC-MS analysis, based on use of the synthesized mixture of diastereoisomers of 1-(1-hydroxy-2-propylamino)-3-(1-naphthoxy)-2-propanol (2) as a standard, established formation of both diastereoisomers of 2 as metabolites of 1. These diastereoisomers were formed in unequal amounts when 1, its hexadeuterated analogue 8 or heptadeuterated analogue 9, were incubated in the presence of the rat liver microsomal fraction. Authentic (2R,2"S)-2, obtained from the amide formed from (2S)-3-(1-naphthoxy)-2-hydroxypropionic acid [(2S)-5] and (2S)-alaninol by diborane reduction, facilitated examination of stereochemical aspects of this process. From incubations of the enantiomers of 1 and pseudoracemic propranolol [equimolar (2R)-propranolol-3,3-d2 and (2S)-propranolol-d0] in the presence of the rat liver microsomal fraction, we established that the diastereomeric products were formed in the order (2S,2"S)-2 approximately equal to (2S,2"R)-2 greater than (2R,2"R)-2 greater than (2R,2"S)-2. (2S)-1, which was metabolized to 2 to a greater extent than (2R)-1, showed no stereoselectivity, affording about equal amounts of (2S,2"S)-2 and (2S,2"R)-2. (2R)-1, which was metabolized to 2 to a lesser extent, afforded considerably more (2R,2"R)-2 than (2R,2"S)-2. omega-Hydroxylation was a minor metabolic pathway in the microsomal incubation. About 2000X less 2 than 1-amino-3-(1-naphthoxy)-2-propanol (3), the product of N-dealkylation of 1, was formed.

The optical isomers of alpha-adrenergic receptor antagonists prosympal (2), piperoxan (3), and dibozane (4) were prepared by methods establishing the absolute configuration of each. (2S)-3(2'-Hydroxyphenoxy)-1,2-propanediol ditosylate (10) was prepared from (2R)-3-tosyloxy-1,2-propanediol acetonide (6). Intramolecular displacement afforded (2S)-tosyloxymethylbenzodioxan [(2R)-11]. Reaction of (2R)-11 with the appropriate amine (diethylamine, piperidine, or piperazine) afforded the 2S isomers of 2, 3, and 12, respectively. Reaction of (2S)-12 with (2R)-11 afforded the SS isomer of 4. Reaction of (2S)-3-benzyloxy-1,2-propanediol ditosylate (14) with catechol (NaOMe) afforded (2R)-benzyloxymethylbenzodioxan (15). Subjecting 15 to hydrogenolysis, tosylation, and displacement with the appropriate amine afforded 2R isomers of 2, 3, and 12. Reaction of (2R)-12 with (2S)-11 afforded (RR)-4. Reaction of (2R)-12 with (2R)-11 afforded meso-4. The S isomers were more effective antagonists to the alpha-adrenergic response of methoxamine-induced contraction of rabbit aortic strips by twofold in 2 and 18-19-fold in 3 and 4. meso-4 was as effective as the SS isomer of 4. The results are interpreted in terms of a similar conformational distribution of aminoalkyl, oxygen, and aromatic functional groups of the (S)-benzodioxans and (R)-epinephrine.

The metabolic hydroxylation of 4'-deuteriooxprenolol [1-(isopropylamino)-3-[2'-(allyloxy)-4'-deuteriophenoxy]-2-propanol] prepared from the 4'-bromo compound was examined in the rat (in vivo). GC-MS analysis of the 4'-and 5'-hydroxyoxprenolol obtained showed 65% retention of deuterium in each of the metabolites. The results indicate that an arene oxide-NIH shift pathway is operative in these hydroxylation processes. The equal magnitude of deuterium retention is supportive of a 4',5'-arene oxide as a major contributor to their formation.

Naloxone (1a), naltrexone (1b), and oxymorphone (1c) were converted to the corresponding 6,6-diaziridines (4a-c), oximes (5a-c), and oxime O-methyl ethers (6a-c). The antagonist derivatives (R = CH2CH = CH2 and R = CH2-c-C3H7) were less active than the parent ketones in the tail-flick assay vs. morphine, by 2-10-fold, except for 6a, which was much less active. The agonist analogues (R = Me) were more active than morphine but less active than dihydromorphine in standard agonist assays. None were significantly longer in duration of action. Thus structural changes at the C-6 position to produce diaziridines, oximes, and oxime O-methyl ethers provide compounds retaining expected opioid activity.

The diastereomeric 6-desoxy-6-spiro-alpha-methylene-gamma-butyrolactone derivatives of naltrexone (4a and 5a) and of oxymorphone (4b and 5b) were prepared from their parent ketones. Diastereomers 4a and 4b were obtained from the 3,14-diacetate derivatives of naltrexone (6a) and oxymorphone (6b) by reaction with the Reformatsky reagent prepared from methyl alpha-(bromomethyl)acrylate. Deacetylation with methanol completed the synthesis. Diastereomers 5a and 5b were obtained from oxiranes 8a and 8b, respectively. The oxiranes were allowed to react with the sodium salt of ethyl acetoacetate, followed by methenation and deprotection to complete the synthesis of 5a and 5b, respectively. Compound 5a was the most potent agent tested in competition against [3H]naltrexone in the opioid radioreceptor assay. At a concentration of 5 nM this compound produced a 50% inhibition of binding. The majority of this inhibition (30%) was irreversible, i.e., it remained even after extensive washing of the membrane preparation in the presence and absence of Na+. Naloxone protected against this irreversible effect. The data suggest a receptor nucleophile, perhaps a sulfhydryl group, is located where it can add to the alpha, beta-unsaturated carbonyl system of 5a.