Ivan O. Edafiogho

Synthesis, neuronal activity and mechanisms of action of halogenated enaminones

Due to the excellent anticonvulsant activity of previously synthesized halogenated enaminones, more disubstituted analogs were synthesized and evaluated in vitro. The new enaminones either had no effect, depressed, or enhanced population spike (PS) amplitude in the rat hippocampus in a concentration-dependent manner. Structure-activity relationship (SAR) analysis indicated that compounds 21 and 25 (with dibromo substituents) were equipotent, and more potent than compound 2 (with dichloro substituents), with compound 25 being the most efficacious of all tested compounds. Both diiodo derivatives 30 and 31 tested produced no significant effect on PS. For PS depression, phenyl substitution on the cyclohexenone ring produced the most efficacious compound 25. PS depressing analogues also depressed evoked excitatory postsynaptic current (EPSC) and action potential firing frequency. Removal of phenyl or methyl group from position 6 on the cyclohexenone ring of enaminone esters produced compound 28 which exhibited pro-convulsant effects. There was no direct correlation between C log P values and anticonvulsant activity of the halogenated enaminones. The mechanisms of anticonvulsant activity were the indirect suppression of excitatory synaptic transmission by enhancing extracellular GABA, and the direct suppression of action potential firing of the neurons.

Evaluation of Anticonvulsant Actions of Dibromophenyl Enaminones Using In Vitro and In Vivo Seizure Models

Epilepsy and other seizure disorders are not adequately managed with currently available drugs. We recently synthesized a series of dibromophenyl enaminones and demonstrated that AK6 and E249 were equipotent to previous analogs but more efficacious in suppressing neuronal excitation. Here we examined the actions of these lead compounds on in vitro and in vivo seizure models. In vitro seizures were induced in the hippocampal slice chemically (zero Mg2+ buffer and picrotoxin) and electrically using patterned high frequency stimulation (HFS) of afferents. In vivo seizures were induced in rats using the 6 Hz and the maximal electroshock models. AK6 (10 µM) and E249 (10 µM) depressed the amplitude of population spikes recorded in area CA1 of the hippocampus by −50.5±4.3% and −40.1±3.1% respectively, with partial recovery after washout. In the zero Mg2+ model, AK6 (10 µM) depressed multiple population spiking (mPS) by −59.3±6.9% and spontaneous bursts (SBs) by −65.9±7.2% and in the picrotoxin-model by −43.3±7.2% and −50.0±8.3%, respectively. Likewise, E249 (10 µM) depressed the zero-Mg2+-induced mPS by −48.8±9.5% and SBs by −55.8±15.5%, and in the picrotoxin model by −37.1±5.5% and −56.5±11.4%, respectively. They both suppressed post-HFS induced afterdischarges and SBs. AK6 and E249 dose-dependently protected rats in maximal electroshock and 6 Hz models of in vivo seizures after 30 min pretreatment. Their level of protection in both models was similar to that obtained with phenytoin Finally, while AK6 had no effect on locomotion in rats, phenytoin significantly decreased locomotion. AK6 and E249, suppressed in vitro and in vivo seizures to a similar extent. Their in vivo activities are comparable with but not superior to phenytoin. The most efficacious, AK6 produced no locomotor suppression while phenytoin did. Thus, AK6 and E249 may be excellent candidates for further investigation as potential agents for the treatment of epilepsy syndromes with possibly less CNS side effects.

Novel Piperazino-Enaminones Suppress Pro-Inflammatory Cytokines and Inhibit Chemokine Receptor CCR2.

Pro-inflammatory mediators including TNF-alpha, IL-6, and nitric oxide are important for the regulation of the immune response when an infection is present, but when overproduced, it can be responsible for the development of tissue and organ injury seen in sepsis, as well as severe asthma, and autoimmune diseases such as Crohn’s disease and rheumatoid arthritis. Data from our lab to characterize the novel compound enaminone E121 have suggested that macrophages stimulated with lipopolysaccharide (LPS) release significantly decreased levels of TNF-alpha and IL-6 as measured by enzyme-linked immunosorbent assay as compared to the DMSO control group. Additionally, functional experiments in a mouse model of asthma have shown that E121 is efficacious in decreasing airway hyperresponsiveness. A new set of compounds synthesized in our lab (JODI) have an N-aryl piperazino motif incorporated on the aromatic side of the enaminone pharmacophore. It was hypothesized that this would enhance their immunosuppressive activity as anti-inflammatory agents by also acting as a chemokine receptor antagonist. Our studies suggest that JODI appears to suppress TNF-alpha and IL-6 in a dose-dependent manner. The JODI compounds were also more effective in reducing TNF-alpha after LPS stimulation when compared to dexamethasone. Lastly, studies using MCP-1 suggest that the JODI compounds, and not E121, are able to block CCR2 signaling as evidenced by decreased total ERK1/2. These studies indicate that E121 and its corresponding piperazino analogs could act as strong anti-inflammatory agents in asthma or other autoimmunities where efficacious therapeutic options are needed.

Antiallergic and Antiasthmatic Effects of a Novel Enhydrazinone Ester (CEE-1): Inhibition of Activation of Both Mast Cells and Eosinophils

Activation of mast cells and eosinophils is a fundamental process in the pathophysiology of allergic diseases. We have previously reported that the novel enhydrazinone ester CEE-1 (ethyl 4-phenylhydrazinocyclohex-3-en-2-oxo-6-phenyl-1-oate) possesses potent anti-inflammatory activity. We have now tested whether the compound also possesses antiallergic and antiasthmatic effects in vitro and in vivo. The compound significantly inhibited degranulation and leukotriene C4 (LTC4) release from activated human eosinophils, as well as IgE-dependent degranulation and LTC4 release from passively sensitized rat basophilic leukemia cells and bone marrow–derived mouse mast cells. In human eosinophils, the drug was more potent in inhibiting degranulation than LTC4 release {IC50 = 0.4 μM [confidence interval (CI): 0.1–0.9] versus 3.8 μM (CI: 0.9–8.3)}, whereas in mast cells the reverse was essentially the case. The drug did not affect stimulus-induced calcium transients in eosinophils but significantly inhibited early phosphorylation of extracellular signal-regulated kinases 1/2 and p38–mitogen-activated protein kinases (MAPK). In vivo, topical application of 4.5–15 mg/kg of the compound significantly inhibited allergen-induced passive cutaneous anaphylaxis in mice. Similarly, in the mouse asthma model, the intranasal administration of 6.5–12.5 mg/kg of the compound significantly inhibited bronchial inflammation and eosinophil accumulation in bronchial lavage fluid, as well as abolishing airway hyper-responsiveness to methacholine. These results show that CEE-1 inhibits the activation of both mast cells and eosinophils in vitro, probably by blocking MAPK-activation pathways, and that these effects are translated into antiallergic and antiasthmatic effects in vivo. The compound, therefore, has potential application in the treatment of asthma and other allergic diseases.

Current and novel anti-inflammatory drug targets for inhibition of cytokines and leucocyte recruitment in rheumatic diseases

Many studies of disease state mechanisms reveal that unbridled inflammation is to blame for many of the symptoms associated with autoimmune diseases such as Crohn’s and Rheumatoid Arthritis (RA). While therapies aimed at decreasing levels of pro‐inflammatory cytokines exist, some have failed clinically or have extensive adverse effects. The aim of this review is to discuss common drug targets for anti‐inflammatory therapies as well as explore potential mechanisms of action for new therapies. Various studies done on novel mechanisms targeting pro‐inflammatory cytokine release as well as leukocyte chemotaxis have been researched for discussion here. Both of these contribute to tissue injury and patient symptoms in inflammatory and autoimmune disease states.

Design, synthesis and biological evaluation of piperazino-enaminones as novel suppressants of pro-Inflammatory cytokines

Infection triggers the release of pro-inflammatory cytokines (TNF-alpha and IL-6). Over-production, however, cause tissue injury seen in severe asthma. The ability of enaminone E121 to reduce pro-inflammatory cytokines in our laboratory encouraged further examination of its structural scaffold. Piperazino-enaminones were designed by incorporating n-arylpiperazine motif into the aromatic enaminone. Four possible modifications were explored systematically. Synthesis was accomplished by amination of the corresponding methyl/ethyl 2,4-dioxo-6-(substituted)cyclohexane-carboxylate.. Sixteen novel compounds were synthesized. Biological activity was tested in J774 macrophages stimulated with lipopolysaccharides. The release of cytokines was measured via ELISA. Four compounds significantly suppressed TNF-alpha and IL-6 release in dose-dependent manner.