1-Phenyl-3-(2-thiazolyl)-2-thiourea inhibits melanogenesis via a dual-action mechanism.

1-Phenyl-3-(2-thiazolyl)-2-thiourea (PTTU) is a well-characterized dopamine ß-hydroxylase inhibitor that prevents 6-hydroxydopamine-induced degenerative neuronal disease. However, the effect of PTTU on melanogenesis has not been reported. In this study, we examined the effect of PTTU on melanogenesis and studied its mechanism of action. We found that PTTU decreased melanin biosynthesis in a dose-dependent manner in normal human epidermal melanocytes (NHEMs). PTTU also inhibited tyrosinase catalytic activity in NHEMs. Moreover, PTTU treatment led to reduced protein levels of tyrosinase in NHEMs, while the protein levels of tyrosinase-related protein-1, tyrosinase-related protein-2, and microphthalmia-associated transcription factor were not affected. However, PTTU treatment did not affect the mRNA expression of tyrosinase. We found that PTTU-accelerated tyrosinase degradation via the ubiquitin-dependent proteasome pathway. In summary, we found that PTTU decreased melanin biosynthesis by decreasing the enzymatic activity and stability of tyrosinase. Our results indicate that PTTU could be used as a depigmentation agent for hyperpigmentation disorder.

Structure-based design, parallel synthesis, structure-activity relationship, and molecular modeling studies of thiocarbamates, new potent non-nucleoside HIV-1 reverse transcriptase inhibitor isosteres of phenethylthiazolylthiourea derivatives.

In this paper we describe our structure-based ligand design, synthetic strategy, and structure-activity relationship (SAR) studies that led to the identification of thiocarbamates (TCs), a novel class of non-nucleoside reverse transcriptase inhibitors (NNRTIs), isosteres of phenethylthiazolylthiourea (PETT) derivatives. Assuming as a lead compound O-[2-(phthalimido)ethyl]phenylthiocarbamate 12, one of the precursors of the previously described acylthiocarbamates (Ranise, A.; et al. J. Med. Chem. 2003, 46, 768-781), two targeted solution-phase TC libraries were prepared by parallel synthesis. The lead optimization strategy led to para-substituted TCs 31, 33, 34, 39, 40, 41, 44, 45, and 50, which were active against wild-type HIV-1 in MT-4-based assays at nanomolar concentrations (EC50 range: 0.04-0.01 microM). The most potent congener 50 (EC50 = 0.01 microM) bears a methyl group at position 4 of the phthalimide moiety and a nitro group at the para position of the N-phenyl ring. Most of the TCs showed good selectivity indices, since no cytotoxic effect was detected at concentrations as high as 100 microM. TCs 31, 37, 39, 40, and 44 significantly reduced the multiplication of the Y181C mutant, but they were inactive against K103R and K103N + Y181C mutants. Nevertheless, the fold increase in resistance of 41 was not greater than that of efavirenz against the K103R mutant in enzyme assays. The docking model predictions were consistent with in vitro biological assays of the anti-HIV-1 activity of the TCs and related compounds synthesized.

Phenyl thiazolyl urea and carbamate derivatives as new inhibitors of bacterial cell-wall biosynthesis.

Over 50 phenyl thiazolyl urea and carbamate derivatives were synthesized for evaluation as new inhibitors of bacterial cell-wall biosynthesis. Many of them demonstrated good activity against MurA and MurB and gram-positive bacteria including MRSA, VRE and PRSP. 3,4-Difluorophenyl 5-cyanothiazolylurea (3p) with clog P of 2.64 demonstrated antibacterial activity against both gram-positive and gram-negative bacteria.

Role of catecholamines in the courtship behavior of male ring doves.

The role of catecholamines in the expression of male courtship behavior in ring doves was examined using central administration of pharmacological agents. Males treated with 6-hydroxydopamine or U-14,624, which depleted norepinephrine (NE) levels in the preoptic-hypothalamic area, showed increased levels of bow-coo and nest-coo displays. Conversely, males treated with tyramine or desipramine, which elevated NE levels in the preoptic-hypothalamic area, showed decreased levels of bow-coo and nest-coo displays. Drug-induced changes in dopamine levels were not consistent with any changes in behavior. This suggests that in the male ring dove NE in the preoptic-hypothalamic area is important in the expression of courtship displays.

Norepinephrine does not contribute to methamphetamine-induced changes in hippocampal serotonergic system.

The purpose of this study was to determine whether norepinephrine (NE) mediated the reduction in the activity of tryptophan hydroxylase (TPH) in the hippocampus and other serotonergic changes, induced by a single or multiple administrations of methamphetamine. The NE in the hippocampus was depleted by injecting rats with DSP4 intraperitoneally, 10 days prior to administration of methamphetamine. A single administration of methamphetamine (15 mg/kg) reduced the activity of TPH to 40% of control after 3 hr. A 90 to 98% reduction in the concentration of NE in the hippocampus, failed to alter this methamphetamine-induced response. The reduction in serotonin (5-HT) in the hippocampus induced by methamphetamine, was not altered by the treatment with DSP4. These observations were confirmed by a lack of effect on methamphetamine-induced changes in 5-HT and TPH after inhibition of the synthesis of NE with U-14,624. The pretreatment with DSP4 also failed to block the decline in activity of TPH in the hippocampus or concentrations of 5-HT measured 18 hr after the last of 4 doses of methamphetamine (15 mg/kg, s.c.). The results presented in this study indicate that NE is not involved in the response of the serotonergic system to methamphetamine.

Some catecholamine inhibitors do not cause accumulation of nuclear estrogen receptors in rat hypothalamus and anterior pituitary gland.

We have recently shown that the dopamine-beta-hydroxylase inhibitor, U-14,624, decreases the concentration of cytosol estrogen receptors in the mediobasal hypothalamus (MBH) and anterior pituitary gland (AP) in ovariectomized rats, but that it also causes cell nuclear accumulation of estrogen receptors. We tried to determine if this is the mechanism by which other catecholaminergic inhibitors decrease the concentration of cytosol estrogen receptors in either the MBH or AP. The previously reported decrease in the concentration of cytosol estrogen receptors in AP by the tyrosine hydroxylase inhibitor alpha-methyl-p-tyrosine was confirmed. Also, the decrease in the concentration of cytosol estrogen receptors in MBH after treatment with the dopamine-beta-hydroxylase inhibitors, diethyldithiocarbamate and FLA 63 was demonstrated. In no case was an increase in the concentration of nuclear estrogen receptor accumulation detected after treatment with the drugs. Results of assays of norepinephrine and dopamine levels in MBH after the various treatments suggest that, at the dosage used, U-14,624 has a greater effect on norepinephrine and dopamine levels that the other dopamine-beta-hydroxylase inhibitors. The results of these experiments suggest that inhibitors of dopamine-beta-hydroxylase and tyrosine hydroxylase cause decreases in the concentration of cytosol estrogen receptors in either the MBH or AP that are not referable to increased cell nuclear accumulation of estrogen receptors.

Cell nuclear accumulation of estrogen receptors in rat brain and pituitary gland after treatment with a dopamine-beta-hydroxylase inhibitor.

In a recent experiment, it was found that the dopamine-beta-hydroxylase inhibitor, U-14,624, decreases the concentration of cytosol progestin receptors in guinea pig hypothalamus and causes an increase in the concentration of nuclear progestin receptors. In this series of experiments, the possibility that similar effects would be seen in the rat estrogen receptor system in mediobasal hypothalamus and pituitary was tested. U-14,624 caused a time-dependent decrease in the concentration of cytosol estrogen receptors and increase in the concentration of nuclear estrogen receptors in both mediobasal hypothalamus and anterior pituitary gland in ovariectomized rats, both in the absence and presence of low levels of estradiol, as well as in ovariectomized-adrenalectomized rats. The nuclear estrogen receptors that accumulate after U-14,624 injection do not require incubation at 25 degrees C to be assayed, suggesting that they are not occupied by an estradiol-like ligand. The nuclear estrogen receptors that accumulate after U-14,624 treatment are high affinity, with an apparent dissociation constant of approximately 0.1 nM. U-14,624 does not compete with (3H)estradiol, in vitro, suggesting that it does not directly interact with estrogen receptors. These results suggest that under some conditions, inhibition of dopamine-beta-hydroxylase causes a modification in unoccupied estrogen receptors so that they develop a higher affinity for cell nuclear components.

Noradrenergic inhibitors cause accumulation of nuclear progestin receptors in guinea pig hypothalamus.

A series of experiments was performed to study the possible behavioral relevance of the apparent regulation of hypothalamic cytosol progestin receptors by noradrenergic transmission in female guinea pigs. In the first experiment, ovariectomized guinea pigs were injected with estradiol benzoate followed 36 h later by the dopamine-beta-hydroxylase inhibitor, U-14,624 or vehicle. Twelve h later, they were injected with progesterone and tested hourly for sexual behavior. Six h after the progesterone injection, a time at which inhibition of sexual behavior by the U-14,624 was confirmed, they were killed, and cytosol and nuclear progestin receptors were assayed in the hypothalamus and cerebral cortex. No difference was seen in the concentration of progestin receptors after drug treatment in these animals that also received a progesterone injection. In subsequent experiments, it was found that the U-14,624-inhibition of progesterone-facilitated sexual behavior is not accompanied by an inhibition of nuclear progestin receptor accumulation. Furthermore, it was found that the decreased cytosol progestin receptor level caused by U-14,624 prior to progesterone injection was accompanied by an increase in the concentration of nuclear progestin receptors. The increase in nuclear progestin receptors was also seen after treatment with the alpha-adrenergic antagonist, prazosin, U-14,624 does not compete with [3H]R 5020 for binding to the progestin receptor, suggesting that it does not directly cause translocation of progestin receptors. The results of these experiments suggest that the decrease in the concentration of cytosol progestin receptors caused by noradrenergic inhibitors is not due entirely to an interference with the formation of cytosol progestin receptors. Rather, it seems that these drugs, in some way, also cause the accumulation of progestin receptors in cell nuclei. Furthermore, they suggest that the mechanism by which these drugs inhibit sexual behavior may not be by interference with the progestin receptor system.

Effect of dopamine receptor blockade or norepinephrine synthesis inhibition on acute, ovariectomy-induced increases in pulsatile luteinizing hormone release in the rat.

The initial aim of the present studies was to examine the influence of blockade of dopamine (DA) receptors with pimozide or inhibition of norepinephrine (NE) synthesis with U-14,624 on acute, ovariectomy (OVX)-induced changes in pulsatile LH release. Either treatment instituted at the time of OVX suppressed or inhibited the rapid increase in LH pulse amplitude and frequency normally occurring within 24 hr following ovarian removal on diestrus 1. While administration of pimozide at either 24 hr or 48 hr following OVX suppressed pulsatile LH release by selectively reducing LH pulse frequency, by 8 days following OVX pimozide failed to exert any effect on LH pulse frequency and therefore on pulsatile LH secretion. To determine if there was a transient critical period following OVX of at least 2 days but less than 8 when endogenous DA was excitatory to pulsatile LH release, piribedil (a DA receptor agonist) was given 24 hr following OVX. Rather than increase LH secretion, piribedil markedly suppressed pulsatile LH release indicating that DA does not stimulate LH secretion in acutely ovariectomized rats. These experiments indicate that (1) NE is involved in stimulating the acute, OVX-induced increase that occurs in pulsatile LH release; (2) DA receptor blockade by pimozide has a differential effect on pulsatile LH secretion which depends on the time following OVX when the compound is administered; (3) this differential effect cannot be explained by a transient critical period of a few days duration following OVX during which DA is excitatory to pulsatile LH release.

Brain monoaminergic control of male reproductive behavior. III. Norepinephrine and the post-ejaculatory refractory period.

The present study was performed to examine the role of brain norepinephrine in the control of copulation and the post-ejaculatory refractory period in the male rat. Disruption of central noradrenergic systems was achieved by (1) selective electrolytic lesion of noradrenergic cell bodies in the locus coeruleus or (2) administration of specific inhibitors of norepinephrine synthesis, sodium diethyldithiocarbamate (DDC) or 1-phenyl-3-(2-thiazolyl)-2 thiourea (U-14, 624). Electrolytic lesions of the locus coeruleus produced a significant increase in the duration of the post-ejaculatory refractory period and its concomitant 22-kHz ultrasonic vocalization. Administration of norepinephrine synthesis inhibitors significantly increased both mount and intromission latencies and caused a dramatic increase in the length of the post-ejaculatory refractory period. These findings support the hypothesis that norepinephrine-containing neural pathways are involved in the control of sexual arousal and suggest that a functional noradrenergic system is essential to the integrity of normal masculine copulatory behavior.

Studies on memory: the cerebral spread of an engram in mice as affected by inhibitors of dopamine beta-hydroxylase.

Bitemporal injections of puromycin that primarily affect the hippocampal-entorhinal areas consistently cause amnesia of maze-learning in mice for 3 days after training but become consistently ineffective if given 6 or more days after training. At these later times, additional puromycin injection sites covering widespread areas of the forebrain are necessary to induce amnesia. These observations are interpreted to indicate that the locus of the engram has become more widespread within the 6-day period. Treatment with inhibitors of dopamine beta-hydroxylase for 3 days following training, retarded the spread of memory from a matter of days to a period of weeks. Repeated treatment with the inhibitors restricted engram spread for about 3 months; again spread was evident about a month after the last treatment. These observations imply that the mechanisms responsible for engram spread are capable of surviving for extraordinarily long periods of time.

Actometric effects of intravenous cocaine in rats.

Intravenous infusions of cocaine, in dosages which have been reported to maintain self-administration behavior, were administered to cannulated rats. Ten identical infusions were administered at 6 min intervals within a session. The activity occuriring in the initial minute following infusions was compared to that produced by saline infusions. Dosages of 200, 400, 800 and 1200 microgram/kg significantly increased activity. However, this effect was not maintained throughout the session. The tenth infusion no longer increased activity as compared to the initial infusion. Therefore these data would not support the hypothesis that cocaine-induced activity was responsible for maintaining cocaine self-administration behavior in this species. Pretreatment with agents which disrupt the synthesis of dopamine and/or norepinephrine failed to antagonize this initial increase in activity. These data would suggest that the activity effect of cocaine is not dependent on newly synthesized pools of the catecholamines.

Noradrenergic role in the self-administration of ethanol.

Involvement of noradrenergic and/or dopaminergic processes of the brain in self-administration behavior toward ethanol was assessed in rats allowed to lever-press for 25 mg/kg intragastric doses on a CRF schedule. Initial access to infusions of saline for establishing an operant baseline was followed by one 10-hr session on acquisition contingencies for ethanol and then one extinction session on saline. Prior to a reacquisition session, rats were treated with either (a) saline, (b) alpha-methyl-p-tyrosine (AMT; 225 mg/kg), (c) 1-phenyl-3-(2-thiazolyl)-2-thiourea (U-14,624; 600 mg/kg or 300 mg/kg), or (d) haloperidol (3.5 mg/kg). Only the saline-pretreated control group and the haloperidol-treated rats reacquired lever-press behavior. Groups treated in like fashion, but pressing for a sweet milk reinforcer, all showed reacquisition. Thus, the effects of AMT and U-14,624 are attributed to an inteference with the reinforcing effect of ethanol infusions. Brain levels of norepinephrine were depleted by both compounds, dopamine was depleted only by AMT, and serotonin was elevated by 600 mg/kg of U-14,624 but unaffected by 300 mg/kg. These results suggest that a cerebral noradrenergic system plays an important role in the reinforcing effect of ethanol without an involvement of dopaminergic systems.