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where X is the fluorescence observed in presence of the test compound; A is the fluorescence observed in
the absence of an inhibitor (no inhibitor control); and B is the fluorescence observed for the positive
control. For graphing purposes, percent inhibition vs. anti-log[drug concentration] was plotted. A logistic
sigmoidal model was used to fit the data and obtain IC values using Graphpad Prism software.
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Docking studies
Docking studies were performed using methods previously published [55-57] . The coordinates of the reported
X-ray crystal structure of P450 2A6 bound to nicotine (4EJJ.pdb) were downloaded from the Protein Data
[58]
Bank website (http://www.rcsb.org/), and used for the docking studies. Heme was considered as part of the
receptor for docking purposes. Standard force fields were used for the compounds. MOE energy
minimization method was used using the MOE software platform from the ChemComp group.

RESULTS
Nicotine, the main addictive ingredient of tobacco products, is mainly (70%-80%) metabolized to cotinine
by P450 2A6 enzyme in the liver. Cotinine has a longer half-life but is much less active in inducing
dopamine release in smokers than nicotine . As nicotine is metabolized, maintenance of its blood plasma
[30]
levels compels the smokers to modify their smoking frequency to compensate. Tobacco usage has been
linked to several debilitating diseases, with lung cancer being the most common disease among smokers.
Inhibition of P450 2A6 can prove to be one of the most effective strategies for smoking cessation. Our
research group has been exploring several classes of compounds as possible P450 2A6 inhibitors. The use of
the pyridine molecule as a scaffold for P450 2A6 inhibitors has been explored by our research group and the
Cashman and Lazarus Research Groups [51-54] . The incorporation of 5-membered heterocyclic rings has been
pursued by both research groups. One of the distinctive features of the P450 2A6 inhibitors developed by
the Cashman and Lazarus Research Groups has been the use of a primary amino group as the main
functional group that binds to the heme-Fe of the enzyme. In contrast, our research group is focused on (1)
the incorporation of a triple bond that can lead to mechanism-based inhibition of the enzyme; and (2) the
incorporation of an imidazole ring that can mimic the pyrrolidine ring of nicotine, in which the -CH of the
5-membered ring faces the heme-Fe of the enzyme.

The syntheses of compounds 1 to 7 [Figure 1] were accomplished using the synthetic Schemes 1 and 2. The
binding mode of nicotine to the P450 enzyme in the X-ray crystal structure (4EJJ.pdb) indicates that
substituents at position 3 of the pyridine ring would be ideal. Nicotine-mimicking compounds containing
imidazole or methyl substituted imidazole side chains with varied sizes of 1-3 carbon alkyl spacers at
position 3 of the pyridine ring were synthesized using Scheme 1 (compounds 1, 4 and 7). Based on our
previous findings that acetylenic substituents can interact with the P450 active site amino acids and lead to
mechanism-based inhibition, triple bond in the form of propargyl ether was incorporated in a second series
of compounds at positions 2, 3 or 4 of the pyridine ring as an ether linkage (compounds 3, 6 and 2,
respectively). Positions 2 and 4 were used to investigate whether substituted compounds at those positions
would show a difference in their inhibition activities compared to the 3-position substituted compound. A
longer spacer in between the propargyl ether and the 4-position of the pyridine ring was introduced to
investigate whether a greater flexibility in the positioning of the key functional groups (pyridine ring and
the alkyne moiety) in the binding site would be achieved. The Vivid CYP450 Screening Kit (Life
Technologies, catalog #PV6140) was used for the P450 2A6 inhibition assays. An initial high-throughput
screening was performed at a 10 µM concentration followed by a dose-response curve determination.
Compounds 1, 4 and 6, all three containing a substituent on position 3 of the pyridine ring, were found to
have single digit micromolar IC values [Figure 2, Table 1]. Compounds 5 and 7 were found to have
50
moderate inhibition activity, with compound 7, a 3-position substituted compound, showing better