Ureidosulfocoumarin Derivatives As Selective and Potent Carbonic Anhydrase IX and XII Inhibitors.

Owing to severe allergic reactions (anaphylaxis) and resistance exhibited by sulfonamide-based carbonic anhydrase (CA) inhibitors, non-classical or non-sulfonamide CA inhibitors are gaining increased attention by medicinal chemists. In this context, we report the design and synthesis of 30 new non-sulfonamide sulfocoumarin derivatives as CA inhibitors. They were investigated against hCA I and II (cytosolic isozymes) as well as hCA IX and XII (transmembrane, tumor-associated enzymes). All compounds showed prominent selectivity for the tumor-associated isoenzymes hCA IX and XII over the cytosolic isoenzymes hCA I and II. Among all synthesized compounds, 1-(2,2-dioxidobenzo[e][1,2]oxathiin-6-yl)-3-(o-tolyl)urea(5 j)and1-(3-fluorophenyl)-3-(8-methoxy-2,2-dioxidobenzo[e][1,2]oxathiin-6-yl)urea(5 q)were found to be more potent and to have better inhibition constant values against hCA IX than the standard acetazolamide (AAZ), with Ki values of 23.6 and 23.3 nM, respectively. All other compounds were found to be active under Ki =920 nM against hCA IX and XII.This study provides a new perspective for the future development of non-sulfonamide derivatives as selective CA inhibitors.

Synthesis and Evaluation of Antimicrobial and Cytotoxic Activity of Oxathiine-Fused Quinone-Thioglucoside Conjugates of Substituted 1,4-Naphthoquinones.

A series of new tetracyclic oxathiine-fused quinone-thioglycoside conjugates based on biologically active 1,4-naphthoquinones and 1-mercapto derivatives of per-O-acetyl d-glucose, d-galactose, d-xylose, and l-arabinose have been synthesized, characterized, and evaluated for their cytotoxic and antimicrobial activities. Six tetracyclic conjugates bearing a hydroxyl group in naphthoquinone core showed high cytotoxic activity with EC50 values in the range of 0.3 to 0.9 µM for various types of cancer and normal cells and no hemolytic activity up to 25 µM. The antimicrobial activity of conjugates was screened against Gram-positive bacteria (Staphylococcus aureus, Bacillus cereus), Gram-negative bacteria (Pseudomonas aeruginosa and Escherichia coli), and fungus Candida albicans by the agar diffusion method. The most effective juglone conjugates with d-xylose or l-arabinose moiety and hydroxyl group at C-7 position of naphthoquinone core at concentration 10 µg/well showed antimicrobial activity comparable with antibiotics vancomicin and gentamicin against Gram-positive bacteria strains. In liquid media, juglone-arabinosidic tetracycles showed highest activity with MIC 6.25 µM. Thus, a positive effect of heterocyclization with mercaptosugars on cytotoxic and antimicrobial activity for group of 1,4-naphthoquinones was shown.

Receptor and circuit mechanisms underlying differential procognitive actions of psychostimulants.

Psychostimulants, including methylphenidate (MPH), improve cognitive processes dependent on the prefrontal cortex (PFC) and extended frontostriatal circuitry. In both humans and animals, systemic MPH improves certain cognitive processes, such as working memory, in a narrow inverted-U-shaped manner. In contrast, other processes, including attention-related, are improved over a broader/right-shifted dose range. The current studies sought to elucidate the potential circuit and receptor mechanisms underlying the divergent dose-dependent procognitive effects of psychostimulants. We first observed that, as with working memory, although sustained attention testing was highly dependent on multiple frontostriatal regions, only MPH infusion into the dorsomedial PFC improved task performance. Importantly, the dose-response curve for this action was right-shifted relative to working memory, as seen with systemic administration. Additional studies examined the receptor mechanisms within the PFC associated with the procognitive actions of MPH across working memory and sustained attention tasks. We observed that PFC α2 and D1 receptors contributed to the beneficial effects of MPH across both cognitive tasks. However, α1 receptors only contributed to MPH-induced improvement in sustained attention. Moreover, activation of PFC α1 receptors was sufficient to improve sustained attention. This latter action contrasts with the impairing actions of PFC α1 receptors reported previously for working memory. These results provide further evidence for a prominent role of the PFC in the procognitive actions of MPH and demonstrate the divergent dose sensitivity across cognitive processes aligns with the differential involvement of PFC α1 receptors.

Prelimbic α1-adrenoceptors are involved in the regulation of depressive-like behaviors in the hemiparkinsonian rats.

At present, it is not clear whether α1-adrenoceptors in the prelimbic cortex (PrL) are involved in Parkinson's disease-related depression. Here we examined effects of PrL α1-adrenoceptors on depressive-like behaviors in rats with unilateral 6-hydroxydopamine lesions of the medial forebrain bundle. The lesion induced depressive-like responses as measured by the sucrose preference and forced swim tests compared to sham-operated rats. Intra-PrL injection of α1-adrenoceptor agonist phenylephrine induced or increased the expression of depressive-like behaviors in sham-operated and the lesioned rats. Further, intra-PrL injection of α1-adrenoceptor antagonist benoxathian produced antidepressant effects in two groups of rats. Intra-PrL injection of phenylephrine increased the mean firing rate of PrL pyramidal neurons in both sham-operated and the lesioned rats, while benoxathian decreased the mean firing rate of the neurons. Compared to sham-operated rats, the duration of phenylephrine and benoxathian action on the firing rate of the pyramidal neurons was shortened in the lesioned rats. Neurochemical results showed that intra-PrL injection of phenylephrine or benoxathian increased or decreased dopamine and noradrenaline and serotonin levels in the medial prefrontal cortex, ventral hippocampus and habenula in sham-operated and the lesioned rats, respectively. Altogether, these results suggest that activation and blockade of α1-adrenoceptors in the PrL change the firing activity of the pyramidal neurons, and then increase or decrease levels of three monoamines in the limbic and limbic-related brain regions, which are involved in the regulation of depressive-like behaviors. Additionally, the results also suggest that the dopaminergic lesion leads to hypofunctionality of α1-adrenoceptors on pyramidal neurons of the PrL.

GABA-A receptor activity in the noradrenergic locus coeruleus drives trigeminal neuropathic pain in the rat; contribution of NAα1 receptors in the medial prefrontal cortex.

Trigeminal neuropathic pain is described as constant excruciating facial pain. The study goal was to investigate the role of nucleus locus coeruleus (LC) in a model of chronic orofacial neuropathic pain (CCI-ION). The study examines LC's relationship to both the medullary dorsal horn receiving trigeminal nerve sensory innervation and the medial prefrontal cortex (mPFC). LC is a major source of CNS noradrenaline (NA) and a primary nucleus involved in pain modulation. Although descending inhibition of acute pain by LC is well established, contribution of the LC to facilitation of chronic neuropathic pain is also reported. In the present study, a rat orofacial pain model of trigeminal neuropathy was induced by chronic constrictive injury of the infraorbital nerve (CCI-ION). Orofacial neuropathic pain was indicated by development of whisker pad mechanical hypersensitivity. Hypersensitivity was alleviated by selective elimination of NA neurons, including LC (A6 cell group), with the neurotoxin anti-dopamine-ß-hydroxylase saporin (anti-DßH-saporin) microinjected either intracerebroventricularly (i.c.v.) or into trigeminal spinal nucleus caudalis (spVc). The GABAA receptor antagonist, bicuculline, administered directly into LC (week 8) inhibited hypersensitivity. This indicates a valence shift in which increased GABAA signaling ongoing in LC after trigeminal nerve injury paradoxically produces excitatory facilitation of the chronic pain state. Microinjection of NAα1 receptor antagonist, benoxathian, into mPFC attenuated whisker pad hypersensitivity, while NAα2 receptor antagonist, idazoxan, was ineffective. Thus, GABAA-mediated activation of NA neurons during CCI-ION can facilitate hypersensitivity through NAα1 receptors in the mPFC. These data indicate LC is a chronic pain generator.

The α1 adrenoceptors in ventrolateral orbital cortex contribute to the expression of morphine-induced behavioral sensitization in rats.

The aim of the present study was to investigate the effect of microinjection of benoxathian, selective α1 adrenoceptor antagonist, into the ventrolateral orbital cortex (VLO) on morphine-induced behavioral sensitization and its underlying molecular mechanism in rats. A single morphine treatment protocol was used in establishing the behavioral sensitization model. The effect of bilateral intra-VLO benoxathian injection on locomotor activity was examined and the protein expression levels of α1 adrenoceptors and activation of extracellular signal-regulated kinase (ERK) in the VLO were detected after locomotor test. The results showed that a single injection of morphine could induce behavioral sensitization by a low challenge dosage of morphine after a 7-days drug free period. Benoxathian significantly suppressed the expression but not the development of morphine-induced behavioral sensitization. Morphine treatment significantly elicited ERK phosphorylation and downregulated the expression level of α1 adrenoceptors in the VLO. In addition, intra-VLO benoxathian injection enhanced the expression levels of α1 adrenoceptors and phosphorylated ERK. These results suggest that α1 adrenoceptors in the VLO are involved in regulating the expression of morphine-induced behavioral sensitization. The effect of decreased locomotor activity by blocking α1 adrenoceptors might be associated with activation of ERK in the VLO.

New synthesis method for sultone derivatives: synthesis, crystal structure and biological evaluation of S-CA.

There has been no remarkable progress in the synthesis of sultones in recent years. To facilitate more detailed studies of this functional group, we found a new method to synthesize the sulfonic acid lactone derivatives and finish its ring-closing reaction. A new sultone derivative, (E)-ethyl 4-oxo-6-styryl-3,4-dihydro-1,2-oxathiine-5-carboxylate 2,2-dioxide (S-CA), was synthesized and structurally identified by 1H-NMR, 13C-NMR, HMQC and X-ray single crystal diffraction analysis. The new rapid synthesis extended the method of ring-closing reaction of sulfonic acid lactone derivatives. The angiogenesis activities of S-CA were evaluated by the chick chorioallantoic membrane (CAM) model. It could selectively suppress small angiogenesis in CAM, without influencing either middle and large angiogenesis. In addition, anticancer efficacy of S-CA was evaluated in vivo using a murine sarcoma S180 model. Reduction of the tumor weight and tumor HE staining regions demonstrated that S-CA (10 mg/kg, intraperitoneal injection) had potent inhibition effects and a 44.71% inhibitory rate in S180 mice. Moreover, an acute toxicity test showed that the LD50 value of S-CA via intraperitoneal injection was 25.624 mg/kg.

6-Substituted 1,2-benzoxathiine-2,2-dioxides are isoform-selective inhibitors of human carbonic anhydrases IX, XII and VA.

A series of 6-substituted 2-benzoxathiine-2,2-dioxides were synthesized starting from 2,5-dihydroxybenzaldehyde, and then screened in vitro for their inhibition properties against five human carbonic anhydrase (hCA, EC 4.2.1.1) isoforms. All the compounds showed excellent selectivity against the mitochondrial (hCA VA) and the tumor associated (hCA IX and XII) enzymes.

Impaired adrenergic-mediated plasticity of prefrontal cortical glutamate synapses in rats with developmental disruption of the ventral hippocampus.

Neonatal ventral hippocampus (nVH) lesion in rats is a useful model to study developmental origins of adult cognitive deficits and certain features of schizophrenia. nVH lesion-induced reorganization of excitatory and inhibitory neurotransmissions within prefrontal cortical (PFC) circuits is widely believed to be responsible for many of the behavioral abnormalities in these animals. Here we provide evidence that development of an aberrant medial PFC (mPFC) α-1 adrenergic receptor (α-1AR) function following neonatal lesion markedly affects glutamatergic synaptic plasticity within PFC microcircuits and contributes to PFC-related behavior abnormalities. Using whole-cell patch-clamp recording, we report that norepinephrine-induced α-1AR-dependent long-term depression (LTD) in a subset of cortico-cortical glutamatergic inputs is strikingly diminished in mPFC slices from nVH-lesioned rats. The LTD impairment occurs in conjunction with completely blunted α-1AR signaling through extracellular signal-regulated kinase 1/2. These α-1AR abnormalities have functional significance in a mPFC-related function, that is, extinction of conditioned fear memory. Post-pubertal animals with nVH lesion show significant resistance to extinction of fear by repeated presentations of the conditioned tone stimulus. mPFC infusion of an α-1AR antagonist (benoxathian) or LTD blocking peptide (Tat-GluR23Y) impaired fear extinction in sham controls, but had no significant effect in the lesioned animals. The data suggest that impaired α-1 adrenergic regulation of cortical glutamatergic synaptic plasticity may be an important mechanism in cognitive dysfunctions reported in neurodevelopmental psychiatric disorders.

Chronic effects of antidepressants on serotonin release in rat raphe slice cultures: high potency of milnacipran in the augmentation of serotonin release.

Most clinically-used antidepressants acutely increase monoamine levels in synaptic clefts, while their therapeutic effects often require several weeks of administration. Slow neuroadaptive changes in serotonergic neurons are considered to underlie this delayed onset of beneficial actions. Recently, we reported that sustained exposure of rat organotypic raphe slice cultures containing abundant serotonergic neurons to selective serotonin (5-HT) reuptake inhibitors (citalopram, fluoxetine and paroxetine) caused the augmentation of exocytotic serotonin release. However, the ability of other classes of antidepressants to evoke a similar outcome has not been clarified. In this study, we investigated the sustained actions of two tricyclic antidepressants (imipramine and desipramine), one tetracyclic antidepressant (mianserin), three 5-HT and noradrenaline reuptake inhibitors (milnacipran, duloxetine and venlafaxine) and one noradrenergic and specific serotonergic antidepressant (mirtazapine) on serotonin release in the slice cultures. For seven of nine antidepressants, sustained exposure to the agents at concentrations of 0.1-100 µ m augmented the level of increase in extracellular serotonin. The rank order of their potency was as follows: milnacipran>duloxetine>citalopram>venlafaxine>imipramine>fluoxetine>desipramine. Neither mirtazapine nor mianserin caused any augmentation. The highest augmentation by sustained exposure to milnacipran was partially attenuated by an α 1-adrenoceptor antagonist, benoxathian, while the duloxetine-, venlafaxine- and citalopram-mediated increases were not affected. These results suggest that inhibition of the 5-HT transporter is required for the enhancement of serotonin release. Furthermore, the potent augmentation by milnacipran is apparently due to the accompanied activation of the α 1-adrenoceptor.

Benzosultines as sulfur dioxide (SO2) donors.

In order to understand precise biological roles of sulfur dioxide (SO(2)), reliable SO(2) donors, compounds that produce SO(2) under physiological conditions, are necessary. The design and development of 1-phenyl-benzosultine as an efficient SO(2) donor is reported. This compound undergoes cycloreversion to generate SO(2) upon dissolution in aqueous buffer at 37 °C with a yield of 89% and a half-life of 39 min and shows SO(2)-like biological activity in a DNA cleavage assay.

In vitro bioactivation of a selective estrogen receptor modulator (2S,3R)-(+)-3-(3-hydroxyphenyl)-2-[4-(2-pyrrolidin-1-ylethoxy)phenyl]-2,3-dihydro-1,4-benzoxathiin-6-ol (I) in liver microsomes: formation of adenine adducts.

As part of our efforts to develop safer selective estrogen receptor modulators (SERMs), compound I {(2S,3R)-(+)-3-(3-hydroxyphenyl)-2-[4-(2-pyrrolidin-1-ylethoxy)-phenyl]-2,3-dihydro-1,4-benzoxathiin-6-ol} was previously identified as a lead for further development. Subsequent studies showed that compound I is genotoxic in both in vitro Chinese hamster ovary (CHO) cells and in vivo mouse studies. To better understand the possible mechanisms for the observed genetoxicity effects, in vitro incubations of I with liver microsomes of human, monkey, and mouse in the presence of adenine were performed, which led to the detection of five adenine adducts. The formation of these adducts was NADPH-dependent, suggesting the involvement of oxidative bioactivation catalyzed by cytochrome P450 enzymes. The mechanism for the formation of the major adenine adduct (A1) involves the formation of a reactive ring-opened para-quinone intermediate. The formation of four other adenine adducts may involve the formation of a reactive epoxide or ortho-quinone intermediate. Furthermore, incubations of compound I with human hepatocytes showed dose-dependent DNA damages in Comet assays. All of the above suggest that some reactive metabolites of compound I, formed through bioactivation mechanisms, have a potential to interact with DNA molecules in vitro and in vivo. This may be one of the causes of the genotoxicity observed preclinically both in vitro and in vivo. This case study demonstrated an approach using in vitro DNA trapping assays for assessing the genotoxicity potential of drug candidates.

Inhibition of glycogen synthase kinase-3 attenuates psychotomimetic effects of ketamine.

N-methyl-D-aspartate (NMDA) glutamate receptors mediate fast neurotransmission and regulate synaptic plasticity in the brain. Disruption of NMDA receptor-mediated signaling by noncompetitive antagonists, such as PCP or ketamine, evokes psychotomimetic behaviors, although the cellular mechanisms by which hypofunctional NMDA receptor signaling drives behavioral pathology are still unclear. Activation of glycogen synthase kinase-3 (GSK-3) has been implicated in the cellular neurotoxicity of NMDA receptor antagonists. Accordingly, in the present study we examined the ability of GSK-3 inhibitors, SB216763 and 1-azakenpaullone, to reverse the behavioral aberrations induced by ketamine. Male NMRI mice received intracerebroventricular (i.c.v.) injection of the GSK-3 inhibitors, SB216763 and 1-azakenpaullone, 5 min prior to ketamine administration. Locomotor activity, rotarod performance, prepulse inhibition, novel object recognition, and duration of loss of righting reflex were monitored. GSK-3 inhibitors attenuated ketamine-induced locomotor hyperactivity, motor incoordination, sensorimotor impairment, and cognitive deficits, but did not affect ketamine anesthesia. These data support an important role of GSK-3 in the expression of behavioral aberrations associated with NMDA receptor hypofunction, and suggest that GSK-3 inhibitors may ameliorate certain behavioral and cognitive dysfunctions in patients with schizophrenia.

Investigation of the diastereomerism of dihydrobenzoxathiin SERMs for ER alpha by molecular modeling.

Molecular dynamics simulations were performed to investigate the distinct uterine activity of ten dihydrobenzoxathiin diastereomers against human estrogen receptor (ER) α. These diastereomers share similar binding mode to ER α ligand binding domain (LBD). Dihydrobenzoxathiin diastereomers with full antagonistic activity form more stable hydrogen bonds with Glu353 and His524 of ER α LBD than corresponding diastereomers. The molecular mechanics based generalized born surface area (MM-GBSA) analysis revealed that van der Waals interactions are predominant to the binding of dihydrobenzoxathiin diastereomers to ER α LBD. The per-residue free energy decomposition analysis revealed that the uterine activity difference is contributed mainly by electrostatic interactions. Our study provides mechanistic insights into the difference of uterine activity for dihydrobenzoxathiin diastereomers.

α1-Adrenergic drugs modulate differentiation and cell death of human erythroleukemia cells through non adrenergic mechanism.

Preliminary data showed that α1-adrenergic antagonists induce apoptosis and a switch towards megakaryocytic differentiation in human erythroleukemia cells. To test the hypothesis whether survival and differentiation of erythroleukemia cells are under control of α1-adrenergic signalling, we examined α1-adrenoceptor expression of erythroleukemia cells and compared the in vitro effects of α-adrenergic antagonists with those of agonists. We discovered that α1-adrenergic agonists suppress both erythroid differentiation and growth of erythroleukemia cells concomitant with lipofuscin accumulation, autophagy and necrotic cell death. α1-adrenergic agonists also inhibit the in vitro growth of physiologic hematopoietic progenitors obtained from umbilical cord blood with high selectivity for the erythroid lineage. Interestingly, the observed effects could not be related to α1-adrenoceptors, even though agonists and antagonists displayed opposing effects regarding cellular growth and differentiation of erythroleukemia cells. Our data suggest that the effects of α1-adrenergic drugs are related to a non-adrenoceptor binding site, controlling the fate of erythroid progenitor cells towards differentiation and cell death. Since the observed effects are not mediated through adrenoceptors, the physiologic relevance of our data remains unclear, so far. Nevertheless, the identification of the still unknown binding site(s) might disclose new insights into regulation of erythroid differentiation and cell death.

Interhemispheric regulation of the medial prefrontal cortical glutamate stress response in rats.

While stressors are known to increase medial prefrontal cortex (PFC) glutamate (GLU) levels, the mechanism(s) subserving this response remain to be elucidated. We used microdialysis and local drug applications to investigate, in male Long-Evans rats, whether the PFC GLU stress response might reflect increased interhemispheric communication by callosal projection neurons. We report here that tail-pinch stress (20 min) elicited comparable increases in GLU in the left and right PFC that were sodium and calcium dependent and insensitive to local glial cystine-GLU exchanger blockade. Unilateral ibotenate-induced PFC lesions abolished the GLU stress response in the opposite hemisphere, as did contralateral mGlu(2/3) receptor activation. Local dopamine (DA) D(1) receptor blockade in the left PFC potently enhanced the right PFC GLU stress response, whereas the same treatment applied to the right PFC had a much weaker effect on the left PFC GLU response. Finally, the PFC GLU stress response was attenuated and potentiated, respectively, following alpha(1)-adrenoreceptor blockade and GABA(B) receptor activation in the opposite hemisphere. These findings indicate that the PFC GLU stress response reflects, at least in part, activation of callosal neurons located in the opposite hemisphere and that stress-induced activation of these neurons is regulated by GLU-, DA-, norepinephrine-, and GABA-sensitive mechanisms. In the case of DA, this control is asymmetrical, with a marked regulatory bias of the left PFC DA input over the right PFC GLU stress response. Together, these findings suggest that callosal neurons and their afferentation play an important role in the hemispheric specialization of PFC-mediated responses to stressors.

Dihydrobenzo[1,4]oxathiine: A multi-potent pharmacophoric heterocyclic nucleus.

2,3-dihydrobenzo[b][1,4]oxathiine represents a valuable pharmacophoric heterocyclic nucleus known since very long time. Initially, together with some patents reporting the use of these compounds as herbicides or lipogenesis inhibitors, several papers reported their ability as melatonin, histamine and serotonin receptor ligands, alpha-adrenoreceptor blockers as well as non-glycoside sweeteners. This wide range of biological activities has been recently further improved by studies stating their activity as antimycotics, multi-defense antioxidants and estrogen receptor ligands. The last insights regarding the preparation, the biological activity and the structure activity relationship (SAR) of derivatives containing the dihydrobenzoxathiine skeleton will be discussed in this review.

Synthesis, structure-activity relationships, and biological profiles of a dihydrobenzoxathiin class of histamine H(3) receptor inverse agonists.

A series of novel dihydrobenzoxathiin derivatives was synthesized and evaluated as potent human histamine H(3) receptor inverse agonists. After systematic modification of lead 1a, the potent and selective histamine H(3) inverse agonist 1-(3-{4-[(2S,3S)-8-methoxy-3-methyl-4,4-dioxido-2,3-dihydro-1,4-benzoxathiin-2-yl]phenoxy}propyl)pyrrolidine (5k) was identified. Compound 5k showed good pharmacokinetic profiles and brain penetrability in laboratory animals. After 3mg/kg oral administration of 5k, significant elevation of brain histamine levels was observed in rats where the brain H(3) receptor was fully occupied.

The alpha1-adrenergic receptor antagonists, benoxathian and prazosin, induce apoptosis and a switch towards megakaryocytic differentiation in human erythroleukemia cells.

The erythroleukemia cell lines K562 and human erythroleukemia (HEL) are established models to study erythroid and megakaryocytic differentiation in vitro. In this study, we show that the alpha1-adrenergic antagonists, benoxathian and prazosin, inhibit the proliferation and induce apoptosis in K562 and HEL cells. Furthermore, both tested substances induced the expression of the megakaryocytic marker CD41a, whereas the expression of the erythroid marker glycophorin-a was decreased or unchanged. Even though the expression of differentiation markers was similar after benoxathian and prazosin treatment in both cell lines, endomitosis of erythroleukemia cells was observed only after prazosin treatment. So far, benoxathian and prazosin are the first described extracellular ligands, which cause megakaryocytic differentiation in K562 and HEL cells. In summary, these results indicate a possible role of alpha1-adrenergic receptor signaling in the regulation of erythroid and megakaryocytic differentiation, even though the receptor dependence of the observed effects needs further investigation.