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Page 4 of 12 Goyal et al. J Cancer Metastasis Treat 2021;7:18 https://dx.doi.org/10.20517/2394-4722.2020.143

Synthesis of 3-((1H-imidazol-1-yl)methyl)pyridine (4): The solution of 3-chloromethylpyridine
hydrochloride salt (1.0 eq) and imidazole (10.0 eq) in dimethylformamide (DMF) (10 mL) was heated at 100
o C until all the salt starting material was consumed (~3 h). The light brown solution was cooled down to
room temperature, and the DMF was removed under vacuum. The residue was dissolved in
dichloromethane (DCM), and washed with water. The DCM layer was dried over anhydrous Na SO . The
4
2
solvent was evaporated under vacuum to afford the desired product.
Compound 4: (14% yield; brown solid) GC-MS showed > 99% purity. m/z: 159, 132, 92. 65. HNMR
1
(CDCl , 300 MHz) δ = 5.10 (s, 2H), 6.88 (s, 1H), 7.00 (s, 1H), 7.31 (m, 2H), 7.65 (s, 1H), 8.46 (m, 1H), and
3
8.56 (m, 1H). C NMR (CDCl , 75 MHz) δ = 48.3, 119.3, 123.9, 130.0, 135.0, 137.3, 148.7, and 149.7.
13
3
Syntheses of 3-(3-(2-methyl-1H-imidazol-1-yl))propyl]pyridine (1) and 3-[(2-methyl-1H-imidazol-1-
yl)methyl] pyridine (7) were achieved using the same procedure.

Compound 1: (86% yield; yellow oil) GC-MS showed > 99% purity. m/z: 201, 186, 118, 96, 55. HNMR
1
(CDCl , 300 MHz) δ = 2.05 (m, 2H), 2.32 (s, 3H), 2.62 (t, J = 7.7 Hz, 2H), 3.85 (t, J = 7.4 Hz, 2H), 6.80 (d, J =
3
1.0 Hz, 1H), 6.90 (d, J = 1.1 Hz, 1H), 7.22 (m, 1H), 7.45 (m, 1H), and 8.45 (m, 2H). C NMR (CDCl , 75
13
3
MHz) δ = 12.6, 29.3, 31.3, 44.7, 118.7, 120.9, 123.1, 126.6, 135.3, 135.6, 143.8, 147.3, and 149.3.
Compound 7: (15% yield; brown oil) GC-MS showed > 98% purity. m/z: 173, 146, 92, 65. HNMR (CDCl ,
1
3
300 MHz) δ = 2.35 (s, 3H), 5.0 (s, 2H), 6.84 (s, 1H), 6.97 (s, 1H), 7.31 (m, 2H), 8.46 (m, 1H), and 8.56 (m,
1H). C NMR (CDCl , 75 MHz) δ = 13.1, 47.4, 119.8, 123.9, 127.8, 132.1, 132.4, 144.8, 148.4, and 149.6.
13
3
P450 2A6 inhibition assay
Cytochrome P450 2A6 (Cyp2A6) activity was determined using the Vivid CYP450 Screening Kit (Life
Technologies, catalog #PV6140) according to the manufacturer’s instructions. Briefly, a master pre-mix
containing baculosomes and regeneration system was prepared using 0.5× Vivid reaction buffer II. The test
compounds were dissolved in dimethyl sulfoxide (DMSO) at a concentration of 100 mM. From the stock
solutions, each compound was serially diluted in 0.5× Vivid reaction buffer II to make working stocks of 100
µM, 50 µM, 25 µM and so on up to 10 dilutions. It is essential to dilute the DMSO at least 1000-fold when
making the working dilutions to prevent its interference with the enzyme activity. An initial high-
throughput screening was performed at 10 µM concentration for each compound. For the dose-response
curve determination, in a 96-well plate, 40 µL of the diluted solutions of each test compound were added to
each well, followed by 50 µl of the master pre-mix, before incubation for 10 min at room temperature. A 10×
mixture of Vivid substrate (reconstituted with acetonitrile) and NADP was then prepared. At the end of the
+
incubation period, 10 µL of this solution was added to each well to start the reaction. After 2 hours of
incubation in the dark at room temperature, the plate was read at 415 nm on a plate reader (Synergy H1,
Biotek). For a positive inhibition control, tranylcypromine (Sigma-Aldrich, cat. #P8511) was used at a final
concentration of 100 µM. For a negative (no inhibitor) control, a 1:1000 dilution of pure DMSO in 0.5×
Vivid reaction buffer was used. Each concentration in the dose-response curve was set up in triplicates, and
each data point was the average of triplicate wells. The % inhibition was calculated using the following
equation.