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3209
and (S)-7 displayed optical rotations of opposite signs
and equal absolute values.
chromatography failed to separate the remaining start-
ing material from the product, only showing a mixture
of 11 (70%) and 9 (30%) in the proton NMR. The mix-
ture was used in the next reaction without further puri-
fication. The pure product 120 was obtained from the
Raney nickel desulfurization15 and purification by col-
umn chromatography (Scheme 3). The final transforma-
tion involved the hydrolysis12,16 of the dithiane 10 with
excess methyl iodide and sodium bicarbonate in acetoni-
trile and water at 40 °C to yield the ketone product 320
(Scheme 4).
(R)-7 was converted to 2-iodomethyl-1,4-dioxane 89,10
with imidazole, triphenylphosphine, and iodine in a
mixed solvent of toluene and tetrahydrofuran at ambi-
ent temperature.11 Displacement of iodide from interme-
diate 89,10 with the lithium anion of 1,3-dithiane
afforded the dithioacetal 9, which was deprotonated
with n-butyllithium and reacted with the iodide 89,10
in a mixed solvent of THF and HMPA to afford the
penultimate intermediate 10.12–14 Finally, the desired
product 220 was obtained through the desulfurization15
reaction with Raney nickel in refluxing ethanol
(Scheme 2).
Biological assays were focused on testing for inhibition
of Sindbis virus production and for cytotoxicity in unin-
fected BHK cells. Two assays were employed to look for
antiviral activity: virus production using SIN-IRES-Luc
and in vitro core-like particle assembly. SIN-IRES-Luc
is Sindbis virus with a firefly luciferase gene inserted at
the 30 end of the genome, which is expressed off of an
internal ribosomal entry site (IRES). The infectivity of
the virus could be assayed directly as a measure of the
luciferase amounts produced in infected cells over a per-
iod of time.
The first and second alkylation intermediates 9 and 10
were useful to provide additional target compounds 1
and 3 (Schemes 3 and 4). Deprotonation of intermediate
9 with n-butyllithium in THF–HMPA, followed by reac-
tion of the anion with (2-iodoethyl)benzene, provided
the product 11. There was no Rf value difference be-
tween the product 11 and starting material 9 during
TLC with an EtOAc–hexane solvent system. Column
A cell viability assay was performed initially to plot a
cytotoxicity curve for the compounds. A standard
XTT-based colorimetric assay was employed, which in-
volved treating BHK cells with various concentrations
of the compounds and then using an XTT-conjugated
substrate that formed an orange precipitate on reaction
with mitochondrial dehydrogenases to determine cell
viability. Spectrophotometric readings were taken at
450 nm and the intensity of the color was proportional
to the viability of the cells. The cytotoxic concentrations
for each of the compounds were established by compar-
ing the spectrophotometric readings for the treated and
untreated cells. In the assay for the inhibition of Sindbis
virus production, BHK cells were grown to confluency
in 96-well plates. Renilla luciferase plasmid (Promega
Corp.) was transfected into these cells. Expression of
Renilla luciferase was used as an internal control to en-
sure that cellular transcription and translation were not
affected by addition of the compounds. Four hours post-
infection, cells were infected with SIN-IRES-Luc virus
at a multiplicity of infection (MOI) of less than 1. Media
containing the compounds at concentrations less than
cytotoxic concentrations were added onto the infected
cells, and the cells were further incubated at 37 °C for
12 h. Cell extracts were taken and luciferase assays were
performed on the extracts using the LmaxII 96-well
plate luminometer (Molecular Devices).17,18 The Renilla
luciferase amounts were found to be equivalent in all the
cells, untreated as well as treated with the compounds,
thus indicating that the compounds had no detectable
effect on the cellular transcriptional and translational
machinery.
Scheme 2. Reagents and conditions: (a) imidazole, PPh3, I2, toluene-
THF, rt, 3 h, 93%; (b) 1,3-dithiane, n-BuLi, THF–HMPA 9:1, ꢁ70 °C
to rt, 98%; (c) n-BuLi, 8, THF-HMPA 9:1, ꢁ70 °C to rt, 76%; (d)
Raney Ni, absolute ethanol, reflux, 5 h, 44%.
Scheme 3. Reagents and conditions: (a) n-BuLi, (2-iodoethyl)benzene,
THF–HMPA 9:1, ꢁ70 °C to rt, 70%; (b) Raney Ni, absolute ethanol,
reflux, 15 h, 71%.
As judged by a reduction in firefly luciferase activity,
compound 2 inhibited virus production with an EC50
of 40 lM, which was not cytotoxic in uninfected BHK
cells (Fig. 2). The concentration of 2 for 50% growth
inhibition in uninfected BHK cells is greater than
1 mM (Table 1).
Scheme 4. Reagents and conditions: (a) MeI, NaHCO3, acetonitrile–
water 1:1, 40 °C, 12 h, 80%.