Synthesis, Purification, and In-Silico Modeling of Second Generation

Anti-Epileptic CompoundsJoseph Schrader, Kyle Scully, Jahyun Koo, Rakesh Tiwari, Roberta King, David Worthen

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

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Di Girolamo, F., L. Campanella, et al. (2009). "Mass

spectrometric identification of hemoglobin

modifications induced by nitrosobenzene."

Ecotoxicol Environ Saf 72(5): 1601-8.

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.

INTRODUCTION

Ongoing Studies

Department of Biomedical and Pharmaceutical Sciences, College of Pharmacy

University of Rhode Island, Kingston, RI 02881

Conclusions

•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 Proposed Toxic Mechanism

DW 1JKC5

JKC5OH

JKC6

JKC6MO

JKC4

JKDA

Initial Metabolism Studies

0

0.1

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0.5

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Treatments (10 µM)

PXR Activity in HuH-7 Cell Line With Second Generation Compounds

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0.05

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0.2

0.25

0.3

DM

SO

DW

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DW

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DW

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MK

DW

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DW

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DW

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DW

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DW

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DW

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TCD

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Treatments (10 µM)

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.

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.

•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.

•Thiodianaline is an effective anticonvulsant In-vivo .

•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.

Separation and PurificationSynthesis

• 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.

•The reaction used to

produce compound

JKDA. Displayed on

either side of the

arrow are the varying

conditions and

reagents used for the

reaction.

Minutes

0 1 2 3 4 5 6 7 8 9 10

mA

U

0

500

1000

mA

U

0

500

1000

2.5

40

Multi-Chrom 1 (1: 212 nm, 4 nm)ACTYL

Retention Time

•Reaction mixtures are

separated using

Combiflash flash

chromatography,

based on the RF

determined by TLC.

•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 ESI-

MS analysis.

(also NMR, DSC, etc.)

Synthesis Rationale

•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 N-

hydroxy 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.

In-Silico Modeling

CA B

•Using Discovery Studio and

AutoDock 4; NMDA receptor

(1Y20) subunit zeta 1 was

modeled.

•The favorable binding

interactions of DW1 (A), JKDA

(B), and JKC5 (C) with NMDA

receptor were modeled and

residues within 5 angstroms

were identified.

•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 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.

Acknowledgments

References