Synthesis, Purification, and In-Silico Modeling of Second Generation Anti-Epileptic Compounds Joseph Schrader, Kyle Scully, Jahyun Koo, Rakesh Tiwari, Roberta King, David Worthen Department of Biomedical and Pharmaceutical Sciences, College of Pharmacy University of Rhode Island, Kingston, RI 02881
INTRODUCTION •Thiodianiline and related compounds have profound in vivo anti-epileptic effects. However, thiodianline and many of its structural analogs are known to be carcinogenic. •In-silico modeling of thiodianiline has shown low energy binding affinity for the NMDA receptor in the brain. •Autodock 4, a computer program designed to predict how small molecules (e.g. substrates or drug candidates) bind to receptors with known 3D strutures. •The N-methyl D-aspartate (NMDA) receptor, important in excitatory neurotransmission, is ubiquitously present in the brain, and vital for normal CNS function, making it a prime target for epilepsy drugs.
JK Series Thiodianaline Derivatives JKC5
•Thiodianaline is an effective anticonvulsant In-vivo . •Using Discovery Studio and AutoDock 4; NMDA receptor (1Y20) subunit zeta 1 was modeled.
•Using AutoDock, a 56-56-56 size grid box was used to direct the ligands to the binding pocket area. •The exact location of the grid box of each dockings were 47.939, -12.274, and 9.094. •The grid box covers all top three binding pockets of 1Y20 NMDA receptors •150 dockings were run for each ligand and binding energy was determined.
•The favorable binding interactions of DW1 (A), JKDA (B), and JKC5 (C) with NMDA receptor were modeled and residues within 5 angstroms were identified.
Proposed Toxic Mechanism
•Based on the proposed toxic mechanism, metabolism directly contributes to the toxicity of thiodianaline. •Screening of rationally designed thiodianaline derivatives in silico indicates that the JK Series likely bind NMDA receptor. •Second generation derivatives are readily sythesized.
Ongoing Studies •Large scale synthesis producing the JK Series for testing in multiple testing models.
•NMDA receptor screening in xenopus oocyte model in collaboration with the Kovoor lab. •Invertebrate neuro-activity screening in Hydra in collaboration with the Kass-Simon lab.
•Anti-Convulsant Screening Program in collaboration with the NIH.
0.4 0.3 0.2 0.1 0
Treatments (10 µM)
0.2 0.15 0.1 0.05 0
Thin Layer Chromatography
Example of thin layer chromatography (TLC), used to separate mixtures of synthesized products. The mobile phase, 1:1 cyclohexane:ethyl acetate, was optimized based on chromatographic separation. The products were isolated using preparative TLC, where the products were applied in a line on the plate, separated using the same mobile phase, and then scraped off. These were then extracted into methanol and then evaporated. TLC served as a guide for flash chromatography.
Treatments (10 µM)
Separation and Purification
•The reaction used to produce compound JKDA. Displayed on either side of the arrow are the varying conditions and reagents used for the reaction.
• The reaction used to produce compound JKC5, with stirring at room temperature under a fume hood. Reactants and reagents are displayed on either side of the reaction arrow.
Multi-Chrom 1 (1: 212 nm, 4 nm) ACTYL
AhR Activity in HuH-7 Cell Line With Second Generation Compounds
HuH-7 Cells transfected with DNA response elements for AhR (left) and PXR expression plasmids (right) treated with test compounds, including second generation compounds, and positive controls (Rifamp and TCDD). *Denotes significance compared to control (DMSO) when p ≤ 0.05, n = 8.
DMSO DW 1 DW 9 DW 11 MK DW 2 DW 10 DW 3 DW 5 DW 6 DW 7 TCDD
PXR Activity in HuH-7 Cell Line With Second Generation Compounds
Relative Luciferase Activity
Relative Luciferase Acitivity
Initial Metabolism Studies
•It is necessary to “protect” the nitrogen atoms from being oxidized to the N-hydroxy metabolite. •Using di-bromo-alkanes the NH₂ groups could sequentially displace both of the bromines, first inter-molecularly and secondly intra-molecularly, resulting in N-cycloalkyls, with the size depending on the length of the carbon chain from the original di-bromo alkane molecule. •The inductive effects of the ring, as well as the formation of a tertiary amine, should prevent Nhydroxy metabolite formation. •If the NH₂ was substituted with an acetyl group, the oxygen’s inductive effect would likely render the nitrogen non-basic. The relative binding of these compounds might indicate whether or not a protonateable nitrogen is required for the molecule to bind to the receptor site. • In-silico modeling suggested that the di-acetyl would bind without a protonateable nitrogen. •The JK Series of molecules are currently undergoing further investigation.
Bluhm, R. E., A. Adedoyin, et al. (1999). "Development of dapsone toxicity in patients with inflammatory dermatoses: activity of acetylation and hydroxylation of dapsone as risk factors." Clin Pharmacol Ther 65(6): 598-605. Di Girolamo, F., L. Campanella, et al. (2009). "Mass spectrometric identification of hemoglobin modifications induced by nitrosobenzene." Ecotoxicol Environ Saf 72(5): 1601-8.
•Reaction mixtures are separated using Combiflash flash chromatography, based on the RF determined by TLC.
Worthen, D. R., A. K. Bence, et al. (2009). "In vivo evaluation of diaminodiphenyls: anticonvulsant agents with minimal acute neurotoxicity." Bioorg Med Chem Lett 19(17): 5012-5.
•In order to assess separation and purity, fractions are examined by HPLC.
•In order to confirm that the collected fraction is the theoretical product samples are examined by direct injection ESIMS analysis. (also NMR, DSC, etc.)
The authors gratefully acknowledge technical instrumentation and poster printing support from the RI-INBRE Centralized Research Core Facility supported by Grant # P20RR16457-10 from NCRR, NIH, with special thanks to Mr. Nathan Nous. Financial support for this project was generously provided in part by an Undergraduate Research Grant from the URI Division of Research and Economic Development to JK and JS.