Development of an in vitro primary screen for skin depigmentation and antimelanoma agents.

An in vitro cell culture assay was developed to identify inhibitors of melanogenesis and agents which produce cytostatic or cytotoxic effects specifically in melanocytes. A total of 50 compounds related to tyrosine, dihydroxyphenylalanine, and hydroquinone (HQ) were tested in vitro in order to determine their effects upon a murine melanocyte cell line, Mel-Ab, that produces copious amounts of melanin in culture. The agents that demonstrated an inhibition of growth or pigment production by 50% (IC50) at < 100 micrograms/ml were considered active. The cytotoxicity of melanocyte-active compounds were also tested in vitro on a control nonmelanocyte cell line (HT 1080), using a simple crystal violet staining method to quantitate adherent cell number after treatment. The cell culture assay was validated with known potent melanocyte cytotoxic agents, including HQ and 4-S-cysteaminylphenol (4-S-CAP). Although most cytotoxic chemicals were nonspecific in this primary screen (i.e. killing both Mel-Ab and HT-1080 cells), several of the compounds tested exhibited high melanocyte-specific cytotoxicity, similar to HQ and 4-S-CAP. Potentially these compounds may be useful as either antimelanoma or skin depigmentation agents. All of the compounds identified as active in this primary screen were cytotoxic or cytostatic to melanocytes, except for the methyl ester of gentisic acid, which uniquely inhibited the de novo synthesis of melanin without cytotoxicity.

Synthesis and biological activity of spirocyclic benzopyran imidazolone potassium channel openers.

A series of novel spirocyclic benzopyran imidazolones were synthesized as rigid analogues of cromakalim. These compounds cause a dose-dependent membrane hyperpolarization of A10 rat aorta cells. This hyperpolarization was blocked by pretreatment with glyburide, indicating that the spirocyclic benzopyran imidazolones were acting by increasing the open probability of ATP-sensitive potassium channels in A10 cells. Representative compounds also showed potent in vivo activity as hypotensive agents in normotensive rats. Many of the compounds described are much more potent than cromakalim both in vitro and in vivo, with one of the most potent compounds being 2,3-dihydro-2,2-dimethyl-6-nitro-2'-(propylamino)spiro[4H-1-benzopyran- 4,4'-[4H]imidazol]-5'(1'H)-one (5r). It is concluded that the N1' nitrogen of the imidazolone is an effective substitute for the carbonyl oxygen of cromakalim. The rigid spirocyclic ring fusion holds this nitrogen in an optimum orientation relative to the benzopyran ring.

Limitation of myocardial injury with the potassium channel opener cromakalim and the nonvasoactive analog U-89,232: vascular vs. cardiac actions in vitro and in vivo.

Cromakalim has been shown to have anti-ischemic properties, but it also produces profound hypotension upon systemic administration. We hypothesized that U-89,232, a cromakalim analog, would reduce infarct size in an ischemia-reperfusion injury model without hemodynamic alteration. Twenty-four anesthetized, open chest New Zealand White rabbits were instrumented for occlusion of a marginal branch of the left coronary artery. All animals were subjected to coronary artery occlusion (30 min) and reperfusion (2 hr). Study animals received either cromakalim (20 micrograms/kg, i.v.) or U-89,232 (20 micrograms/kg, i.v.), which was given as a pretreatment 30 min before occlusion. Control animals (n = 10) received vehicle (10% dimethyl sulfoxide). At termination of the experiment, the necrotic area and the area at risk were determined with tetrazolium and India ink staining, and infarct size was calculated using planimetry. Treatment with cromakalim produced profound hypotension (greater than 30% decrease in mean arterial pressure), whereas U-89,232 had no such hemodynamic effect. With comparable areas at risk, infarct size (as a percent of risk area) in the control animals was 46.8 +/- 3.4%. Treatment with cromakalim or U-89,232 reduced infarct size to 33.1 +/- 4.4 and 24.4 +/- 4.0%, respectively (P < .05, both compared to control). In vitro studies demonstrate that although both of these compounds shorten the duration of the cardiac action potential, only cromakalim is active in vascular smooth muscle. We conclude that U-89,232 exhibits myoprotection without hypotension, and that its mechanism of action is most likely due to ability to affect cardiac electrophysiology.

Synthesis and evaluation of a miniature organic model of chymotrypsin.

An artificial chymotrypsin, with all the features of the real chymotrypsin, namely a binding site (from cyclodextrin) attached to a catalytic site containing an imidazolyl group, a carboxylate group and a hydroxyl group, has been synthesized. This artificial chymotrypsin has a molecular weight of only 1,365 while the real enzyme has a molecular weight of 24,800. However, from preliminary measurements, both the real and artificial enzymes have approximately the same catalytic activity (both rate and binding constants).