Ocular pharmacology of bicyclic hexahydroaporphines.

This paper explores the ocular hypotensive actions of bicyclic analogs of hexahydroaporphine (HHA), specifically nor-HHA, in an attempt to shed light on the mechanism(s) by which they lower intraocular pressure (IOP). Studies involving the measurement of IOP and aqueous humor production were conducted in ocular normotensive albino rabbits, while those involving smooth muscle contractility utilized isolated bovine iris. The ability of nor-HHA to produce a sustained drop in IOP is linked to both a functioning adrenergic nervous system and the availability of the products of cyclooxygenase metabolism. Although aqueous flow is not impacted by the bicyclic structures, the significant enhancement of outflow facility points to a probable mechanism of IOP-lowering action. Nor-HHA had no direct contractile or relaxant action on bovine irides, but does cause a concentration-dependent inhibition of carbachol-evoked contractions. This inhibition was reversed by inhibitors of phospholipase A(2) and cyclooxygenase, but not by inhibitors of lipoxygenase, again indicating a role for prostaglandins in the ocular pharmacological action of bicyclic HHAs. Pretreatment with a nitric oxide (NO) scavenger also reversed the ability of nor-HHA to inhibit carbachol-induced contractions, implying a role for NO in the postjunctional actions of HHAs.

Assessment of the in vivo and in vitro opioid activity of bridged hexahydroaporphine and isoquinoline molecules.

Four novel racemic bridged hexahydroaporphine (1 and 2) and isoquinoline (3 and 4) analogues have been synthesized in an attempt to generate bicyclic derivatives of the morphinan ring system. The opioid activity of these analogues has been assessed through membrane-binding studies, in vitro studies in isolated guinea pig ileum and mouse vas deferens, and in vivo studies utilizing the mouse hot plate technique. The bridged isoquinoline precursor molecules were inactive as antinociceptives. Both the racemic phenolic hexahydroaporphine 1 and its 10-methoxy congener 2 demonstrated dose-dependent, albeit weak, antinociceptive activity when administered icv, but they induced lethal convulsions when given subcutaneously. The antinociception elicited by 1 appeared to show very weak opioid character while that caused by 2 was totally nonopioid.

An assessment of potential chemoprotectant activity against ricin toxicity by mechanism based glycosidase inhibitors in macrophage J744A.1 cell cultures.

The abilities of potential chemoprotectants to inhibit cytotoxicity of ricin have been determined in vitro, using the macrophage cell line J744A.1. Six compounds were tested: alpha- and beta-galactopyranosylamine; N-bromoacetyl-alpha-D-galactopyranosylamine; N-bromoacetyl-beta-D-galactopyranosylamine; N-bromoacetylglucopyranosylamine; and N-bromoacetylmannopyranosylamine. Of the six compounds which were tested, only N-bromoacetyl-alpha-D-galactopyranosylamine and N-bromoacetyl-beta-D-galactopyranosylamine exhibited significant activity against ricin toxicity, as indicated by the release of lactate dehydrogenase (LDH) and aspartate aminotransferase (AST). The alpha-isomer provided greater protection against ricin toxicity and also exhibited less inherent cytotoxicity in the absence of ricin, as compared to the beta-isomer. Neither the alpha- and beta-galactopyranosylamines nor the glucose and mannose analogs were promising as potential chemoprotectants.

Synthesis of a secondary N-desmethyl and a tertiary N-cyclopropylmethyl bridged hexahydroaporphine as precursors to bicyclic opioid ligands.

In an attempt to generate a bicyclic 5,8-ethano derivative of N-methylmorphinan, an isomeric bicyclic hexahydroaporphine 2 was synthesized. The phenolic analogue of 2 has demonstrated affinity for mu opioid receptors in vitro and, along with 2, provided weak, primarily nonopioid analgesic action when injected intracerebroventricularly in mice. It was of interest to assess the potential opioid antagonist action of bicyclic hexahydroaporphine analogues containing cyclopropylmethyl and allyl nitrogen substituents. As the first steps in the generation of these potential opioid antagonists, the secondary bicyclic hexahydroaporphine 3 and its N-cyclopropylmethyl congener 4 were synthesized. N-Demethylation of 2 was initially attempted via the von Braun reaction, but acid-catalyzed hydrolysis of the crude N-cyano intermediate resulted in product decomposition. A successful approach to 3 involved the hydrolysis of the N-formyl precursor 1 in ethanolic potassium hydroxide. Direct alkylation of the secondary amine 3 utilizing cyclopropylmethyl bromide and sodium bicarbonate successfully generated the alkylated derivative 4. Both products were purified in hydrochloride salt form and characterized by standard analytical and spectroscopic methods. The free base form of 3 was highly sensitive to photooxidation. Opioids are known to oxidize to 10-keto structures, and secondary amines can oxidize to hydroxylamines. Infrared analysis of the decomposition product indicated the presence of both hydroxy and carbonyl groups which were absent in the spectrum of the salt. Structures of potential oxidation products are proposed.

Bicyclic derivatives of hexahydroaporphine: novel agents that lower intraocular pressure.

We have synthesized a number of novel bicyclic hexahydroaporphines containing a phenethylamine moiety for preliminary study as intraocular pressure lowering agents. The target molecules were synthesized in a twelve step process. The final products were secondary or tertiary amines containing either an aromatic methoxy or phenolic substituent. These molecules, in hydrochloride salt form, were assayed in doses ranging from 0.1-1.5%. All products and vehicle were administered topically to one eye of normotensive rabbits and intraocular pressure was measured in both eyes for up to six hours. Four of the five compounds examined produced a significant and, in some cases, prolonged, ocular hypotensive response. Secondary and N-methylated tertiary amines were equally effective, as were compounds containing either the 10-methoxy group of free phenol. Studies are currently in progress to optimize potency and identify functional and molecular mechanisms of action.

Assessment of the intraocular pressure-lowering activity of bicyclic derivatives of 1-substituted benzyloctahydroisoquinoline.

In our study of IOP-lowering agents, we have synthesized several bicyclic analogs of 1-benzyloctahydroisoquinoline. The target molecules were synthesized in an eleven-step process. Structures were proved through spectrometry, elemental analysis and, in selected cases, high resolution mass spectrometry. The final products were secondary or tertiary amines containing a 1-benzyl moiety substituted at the p-position with a methoxy, methyl or chloro group. All target molecules were analyzed in 1% solution in distilled water in normotensive rabbits. After topical administration, IOP was monitored in both eyes for up to seven hours. The 1-p-methoxybenzyl molecule 2 was the most active, and caused a maximal IOP drop of 8.8 +/- 1.9 (n = 7) mm Hg in the ipsilateral eye at 4 hours post-administration, with only partial recovery at seven hours. All other compounds tested either showed very weak activity (3-6) or were inactive (1). All compounds produced a contralateral effect, and 5 induced rebound ocular hypertension. We conclude that selected tertiary bicyclic 1-p-methoxybenzyl-octahydroisoquinolines, particularly N-methylated structures, exhibit a significant IOP-lowering effect in normotensive rabbits.

Potential chemoprotectant activity of mechanism-based glycosidase inhibitors against ricin toxicity in Chinese hamster ovary and macrophage J774A.1 cell cultures.

The abilities of the triacetylated galacto- and gluco-derivatives of 2-deoxy-2-fluoro-D-pyranosyl fluoride as well as alpha- and beta-N-bromoacetyl-D-galactopyranosylamine to inhibit the cytotoxicity of ricin in vitro in macrophage J774A.1 and Chinese hamster ovary (CHO) cell lines were determined. Leakage of lactate dehydrogenase (LDH) and aspartate aminotransferase (AST) from the cells into the culture media were used as indicators of ricin cytotoxicity. The potential chemoprotectants were used in concentrations ranging from 10(-8) to 10(-4) g ml-1. Of the four potential mechanism-based, site-specific glycosidase inhibitors that were tested, 3,4,6-tri-O-acetyl-2-deoxy-2-fluoro- beta-D-glucopyranosyl fluoride exhibited the greatest chemoprotectant activity. The ricin-induced LDH release was inhibited in a concentration-dependent manner by this compound, with the LDH leakage returning to control values in the presence of the highest concentration of this chemoprotectant in both cell cultures when given 4 h prior to ricin. This compound exhibited a small but significant inhibition of AST release from both cell cultures when given simultaneously with ricin. 3,4,6-Tri-O-acetyl-2-deoxy-2-fluoro-beta-D-galactopyranosyl fluoride exhibited a small but significant chemoprotective effect only at the highest concentration in both cell cultures when given simultaneously with ricin. Both the alpha- and beta-isomers of N-bromoacetyl-D-galactopyranosylamine exhibited activity against ricin toxicity in the CHO cell line, with the beta-isomer exhibiting greatest activity. The beta-isomer exhibited greater cytotoxicity in the absence of ricin, as demonstrated by the release of both enzymes from the cultured CHO cells. Further studies will be required to assess the utility of these compounds as chemoprotectants against ricin toxicity in vivo.

Evaluation of potential chemoprotectants against microcystin-LR hepatotoxicity in mice.

Microcystin-LR (MCLR) is a potent cyclic heptapeptide hepatotoxin produced by the blue-green algae, Microcystis aeruginosa. Toxic blooms of this cyanobacteria have been reported throughout the temperate world. In spite of the potential economic loss and health hazard posed by this toxin, few studies on the development of an antidote have been conducted. Thus, a number of biologically active compounds were tested in mice for effectiveness in preventing the toxicity of a lethal dose of MCLR (100 micrograms kg-1). Efficacy was evaluated based upon the percentage of surviving mice, time to death and serum lactate dehydrogenase activity 45 min after treatment with the toxin. The biologically active compounds were separated into groups based upon proposed mechanisms of action. Enzyme induction by phenobarbital but not by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) resulted in partial protection against toxicity. Calcium channel blockers, free-radical scavengers and water-soluble antioxidants produced little protection against toxicity. The membrane-active antioxidants vitamin E and silymarin, as well as glutathione and the monoethyl ester of glutathione, produced significant protection from lethality. Rifampin and cyclosporin-A, both immunosuppressive and membrane-active agents, which also block the bile acid uptake system of hepatocytes, produced complete protection from the toxicity of MCLR. Thus, lipophilic antioxidants provide partial protection against MCLR toxicity while cyclosporin-A and rifampin are highly effective and potentially useful antidotes. The toxicity of MCLR may depend upon stimulation of the immune system and may be mediated by membrane alterations.

Pharmacological properties of novel bicyclic isoquinoline analogs in isolated guinea pig atria, trachea and in human platelets: relationship to trimetoquinol.

1. Antiplatelet and beta-adrenoceptor activities of a set of secondary and tertiary N-methyl substituted amine analogs of trimetoquinol (TMQ, I and II, respectively) and 5,8-ethano-l-(p-methoxybenzyl)-1,2,3,4,5,6,7,8-octahydroisoquin oline (bicyclic isoquinoline compounds III and IV, respectively) were examined. 2. Compounds III and IV induced relaxations of guinea pig trachea which were blocked by propranolol whereas neither compound acted as an agonist nor antagonist of beta-adrenoceptors (chronotropy) in guinea pig atria. TMQ analogs (I and II) were agonists in both beta-adrenoceptor systems. 3. When tested in human platelets, compounds III and IV, like the TMQ analogs, blocked several inducers of the prostaglandin-dependent and -independent pathways, and the alpha 2-adrenoceptor-mediated pathway of platelet activation. 4. The bicyclic isoquinoline analogs (III and IV) possessed more selective beta 2-adrenoceptor stimulatory activity and equal or greater inhibitory activity against inducers of the prostaglandin-independent pathways of platelet function than the corresponding TMQ analogs (I and II). 5. These chemically novel lipophilic bicyclic compounds provide a new lead to the development of agents useful for the treatment of asthma and thrombotic disorders.