(M1130-05-26) Hit to Lead Optimization Studies of Meta Substituted Enaminone Derivatives as Potential Anti-Seizure Agents for Generalized and Drug-Resistant Epilepsy

Purpose: Epilepsy is one of the most common neurological disorders, with 3 million people in the United States and 65 million people worldwide living with this disorder. In America, 1 in 26 individuals will eventually develop epilepsy in their life. According to several reports, there’s an ongoing need for research strategies that aim to develop novel anti-seizure drugs (ASD) that act on known and unknown molecular targets, while improving upon the incidence of unwanted side effects, with ASD use. Our lab engages in early drug design and development of novel anti-seizure analogs as potential pharmacotherapeutics for epilepsy. Enaminones are enamines of β-dicarbonyl compounds and their usefulness in medicinal chemistry as building blocks for novel drug candidates, has been vastly studied for decades. Research efforts in our lab are focused on elucidating the antiseizure properties of benzamide enaminones, which have proven to be an excellent pharmacophore for antiseizure active analogs.

Methods: Electrophysiological techniques were performed to conduct whole-cell patch clamp experiments. Differentiated mouse neuroblastoma x Rat neuron hybrid (ND7/23 cells), were treated with 50 μM of mono and disubstituted N-fluorinated benzamide enaminones (in the μM range).¹ To validate the molecular target, recently, our drug discovery program established a pentylenetetrazol (PTZ) seizure model as previously described by Mussulini etal, utilizing adult zebrafish.² The test zebrafish were immersed in varying concentrations of PTZ (2.5 mM - 7.5mM). Behavioral activity was monitored with video recording software (Noldus Ethovison XT7, video tracking software) and compared to the control group (without PTZ exposure). The following endpoints were determined for the experiment 1) distanced traveled (cm) 2) average velocity cm/s 3) time spent in upper and lower zones of the tank 4) number of erratic movements.

Results: Preliminary target identification studies, led to the discovery of a potent T-VGCC blocker, lead compound IAA65 (di-meta trifluoromethylated enaminone). IAA65 is selective for the Cav3.2 and Cav3.3 Ca⁺² channel subunits. In vitro analysis of IAA65 revealed this analog altered the steady state inactivation of the Cav3.2 T-type Ca⁺² channel subunit, which resulted in an increase in the inactivation recovery of the channels.³ In the Cav3.3 subunit, IAA65 altered the gating mechanism of the T-type Ca⁺² subunit, as there was a decrease in the activation and inactivation time constant of the channels. These results indicate inhibition of the voltage-gated T-type calcium channel occurs via different mechanisms depending on the subunit. Figure 4 shows the preliminary results of the PTZ anti-seizure model. The average velocity was significantly increased in the 7.5 mM PTZ group when compared to control. The control fish spent equal time in both halves of the tank, whereas there was concentration-dependent effect seen in the PTZ-treated groups. Fish immersed into 5 mM PTZ solution spent more time in the upper half of the tank compared to control (n=10; p= 0.0282) while those immersed in 7.5 mM PTZ solution spent less time in the upper half of the tank (n=6; p=0.0002). This response could also be explained by the lack of mobility following the loss of posture. Representative traces generated by Ethovision XT7 software generally supported a normal rhythmic swimming pattern in the control and 2.5 mM PTZ groups. However, a trend of seizure-like behavior, such as erratic movement, was typically noted in zebrafish treated with 5 mM PTZ (n=6; p=0.1058).

Conclusion: Based on the small molecule IAA65, our drug discovery program next steps are: 1) determine the pharmacological efficacy of meta-substituted enaminones inhibitory action of T-VGCC, 2) use a new preclinical in vivo approach (for our research) to assess the effects of N-aromatic enaminones against PTZ induced seizures in adult zebrafish, and 3) to conduct hit to lead optimization studies to discover new lead(s) small molecules. The outcome of this project will provide detailed insight into the key structural and electronic requirements needed for aromatic enaminones to target neuronal T-VGCC and to have anti-seizure pharmacological effects in adult zebrafish.

References: (1) Amaye, I. J.; Heinbockel, T.; Woods, J.; Wang, Z.; Martin-Caraballo, M.; Jackson-Ayotunde, P. 6 Hz Active Anticonvulsant Fluorinated N-Benzamide Enaminones and Their Inhibitory Neuronal Activity. International Journal of Environmental Research and Public Health 2018, 15 (8), 1784.
(2) Mussulini, B. H. M.; Leite, C. E.; Zenki, K. C.; Moro, L.; Baggio, S.; Rico, E. P.; Rosemberg, D. B.; Dias, R. D.; Souza, T. M.; Calcagnotto, M. E.; et al. Seizures induced by pentylenetetrazole in the adult zebrafish: a detailed behavioral characterization. PloS one 2013, 8 (1), e54515-e54515. DOI: 10.1371/journal.pone.0054515 PubMed.
(3) Amaye, I. J.; Jackson-Ayotunde, P. L.; Martin-Caraballo, M. Evaluation of potential anticonvulsant fluorinated N-benzamide enaminones as T-type Ca2+ channel blockers. Bioorganic & Medicinal Chemistry 2022, 116766.

Acknowledgements: We gratefully acknowledge Title III University of Maryland Eastern Shore – School of Pharmacy and Health professions for financial support. Dr. Seth Friese, Professor, Salisbury University, Salisbury, MD, for his assistance with NMR spectrometry analysis.


Behavioral responses to vehicle and PTZ (2.5 – 7.5 mM) in adult zebrafish in the 20-minute trial.