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use to maintain these levels. The impact of P450 2A6 activity on tobacco addiction is evidenced in
individuals who are slow metabolizers due to P450 2A6 polymorphism, and thus smoke less [37,39,40] .
[41]
Inhibition of P450 2A6, which is known to be well tolerated by humans , is emerging as the most
promising strategy for smoking cessation and treatment of tobacco-dependence. Development of P450 2A6-
specific inhibitors is currently pursued by many research groups [42-47] . Recent developments in this field
include the use of bioelectrochemical platforms that use the “molecular lego” approach [48,49] . In the study
reported by Castrignanò et al , genetically-fused P450 2A6 with Desulfovibrio vulgaris flavodoxin (FLD)
[50]
module is used for investigating the inhibitory effects of coumarins and nicotine. Such an approach could
improve and hasten the process of P450 enzyme inhibitor development. The development of mimicking
agents with structural similarities to nicotine through the modification of its pyridine ring substituents, by
our research group and the Cashman and Lazarus Research Groups [51-54] , has led to potent (with low
micromolar IC values) inhibitors of P450 2A6 [42,51,54] . For this study, we have identified two series of such
50
pyridine-based P450 2A6 inhibitors.

METHODS
Synthesis of 3-((prop-2-yn-1-yloxy)methyl)pyridine (6): 3-Hydroxymethylpyridine (1.0 eq) in
tetrahydrofuran (THF) was added dropwise to a cooled (0 C) suspension of sodium hydride (NaH, 95%, 2.1
o
eq) in dry THF. After 20 min, propargyl bromide (80% solution in toluene, 2.0 eq) was added slowly. The
reaction mixture was heated at 50 C overnight. It was then allowed to cool to room temperature before
o
careful quenching by the addition of water. The crude was extracted with ethyl acetate. The organic layer
was washed with brine and dried over anhydrous sodium sulfate (Na SO ). The solvent was removed under
4
2
vacuum. The residue was then purified by silica gel column chromatography using hexane: ethyl acetate
(1:3) as eluent to afford the desired product.

1
Compound 6: (96% yield; brown liquid) GC-MS showed > 99% purity. m/z: 146, 108, 92, 80, 65, 51. HNMR
(CDCl , 300 MHz) δ = 2.45 (t, J = 2.4 Hz, 1H), 3.42 (s, 2H), 4.51 (s, 2H), 7.32 (m, 1H), 7.62 (m, 1H), and 8.55
3
(m, 1H). C NMR (CDCl , 75 MHz) δ = 57.7, 69.1, 75.3, 79.3, 123.5, 133.0, 135.8, 149.5, and 149.6.
13
3
Syntheses of 4-(prop-2-yn-1-yloxy)pyridine (2), 2-(prop-2-yn-1-yloxy)pyridine (3) and 4-(3-(prop-2-yn-
1-yloxy)propyl)pyridine (5) were achieved using the same procedure.

Compound 2: (82% yield; white solid) GC-MS showed > 98% purity, m/z: 133.1, 104.1, 78.0, 52.0. HNMR
1
(CDCl , 300 MHz) δ = 2.63 (t, J = 2.56 Hz, 1H), 4.50 (d, J = 2.6 Hz, 2H), 6.35 (d, J = 7.3 Hz, 2H), and 7.42 (d,
3
13
J = 7.6 Hz, 2H). C NMR (CDCl , 75 MHz) δ = 45.5, 75.7, 77.3, 118.8, 139.6, and 179.1.
3
1
Compound 3: (76% yield; white solid) GC-MS showed > 99% purity, m/z: 133.1, 104.1, 78.0, 52.0. HNMR
(CDCl , 300 MHz) δ = 2.48 (t, J = 2.4 Hz, 1H), 4.72 (d, J = 2.3 Hz, 2H), 6.22 (t, J = 6.1 Hz, 1H), 6.54 (d, J = 5.2
3
13
Hz, 1H), 7.36 (d, J = 2.8 Hz, 1H), and 7.63 (d, J = 4.6 Hz, 1H). C NMR (CDCl , 75 MHz) δ = 37.5, 75.3, 77.3,
3
106.2, 120.1, 136.5, 139.9, and 161.6.
Compound 5: (67% yield; yellow liquid) GC-MS showed > 98% purity. m/z: 174, 158, 145, 118, 110, 93. 1
HNMR (CDCl , 300 MHz) δ = 2.0 (m, 2H), 2.42(t, J = 2.6 Hz, 1H), 2.89 (t, J = 7.4 Hz, 2H), 3.56 (t, J = 6.3 Hz,
3
2H), 4.14 (d, J = 2.3 Hz, 2H), 7.15 (m, 2H), 7.60 (m, 1H), and 8.52 (m, 1H). C NMR (CDCl , 75 MHz) δ =
13
3
30.1, 31.6, 58.2, 68.8, 74.6, 77.1, 124.1, 149.8, and 150.8.