7694 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24
Xie et al.
3,4-Dimethyl-4′-hydroxy-3′,4′-dihydroseselin (10). NaBH4 (11.4
g, 300 mmol) in water (50 mL) was dropped into a solution of 9
(27.2 g, 100 mmol) in MeOH (300 mL) at ice-bath temperature.
Adding a few of drops of 1 M aqueous NaOH kept the reaction in
a basic condition. The mixture was stirred at room temperature for
12 h until the reaction was complete (TLC, petroleum/EtOAc 4:1).
The pH was adjusted to 5-6 with aqueous 5% HCl, and the mixture
was extracted with EtOAc three times. The organic phase was
washed with 5% NaHCO3, water, and brine successively. After
drying over anhydrous Na2SO4, the solvent was removed in vacuo
to give crude 10 (25.5 g, 93% yield). The crude product could be
used in the next reaction without further purification. It was also
purified by a flash silica chromatography (eluant EtOAc/petroleum
with brine, dried (MgSO4), evaporated, and decolorized by passage
through a short silica gel column (cyclohexane/acetone/CH2Cl2 7:2:
1) to afford the pure desired nitrile 4 (5.0 g, 74%): white solid, mp
164-166 °C, 99.0% de (measured by HPLC), [R]D + 22.81° (c
1
1.01, CHCl3); H NMR δ 1.08-1.12 (ms, 18H, 6 × CH3), 1.46
and 1.49 (each s, 3H, 2′-CH3), 1.66, 1.91, 2.18, and 2.51 (each m,
2 × CH2), 2.52 (s, 3H, 4-CH3), 3.72 (s, 2H, 3-CH2), 5.40 (d, 1H,
J ) 4.8 Hz, 3′-H), 6.65 (d, 1H, J ) 4.8 Hz, 4′-H), 6.89 (d, 1H, J
) 8.8 Hz, 6-ArH), 7.62 (d, 1H, J ) 8.8 Hz, 5-ArH); 13C NMR δ
ppm 9.6 and 9.9 (2 × CH3), 15.7 (3-CH2), 16.3, 16.4, 16.6, and
16.7 (4 × CH3), 21.2 (2 × CH3) and 26.1 (4-CH3), 28.9, 29.0,
30.9, and 31.3 (4 × CH2), 54.6, 54.7, 54.9, and 55.1 (4 × C), 61.5
and 72.1 (2 × CH), 112.8 (CN), 90.7, 91.3, 106.3, 113.3, 116.3 (6
× C), 114.9 and 127.2 (2 × CH), 150.6, 152.2, 156.7, 159.2, 167.0,
167.4 (6 × C), 177.4 and 178.6 (CdO); IR cm-1 (KBr) 2972, 2935;
MS (ESI) m/z (%) 676.5 ([M + 1]+, 100), 693.5 (M + NH4+,48),
698.5 (M + Na+,75). Anal. (C37H41O11N·) C 66.08, H 6.25, N
1.64. Calculated: C 65.77, H 6.12, N 2.07.
1
ether 0-30%) to give a light-yellow solid: mp 166-167 °C; H
NMR δ 1.41 and 1.47 (each 3H, s, 2′-CH3), 2.13 (2H, d, J ) 5.2
Hz, 3′-CH2), 2.19 (3H, s, 3-CH3), 2.38 (3H, s, 4-CH3), 3.23 (1H,
s, 4′-OH), 5.26 (1H, m, 4′-H), 6.78 (1H, d, J ) 9.2 Hz, 6-H), 7.45
(1H, d, J ) 9.2 Hz, 5-H).
3,4-Dimethylseselin (11). Compound 10 (27.4 g, 100 mmol) in
anhydrous benzene (250 mL) in the presence of TsOH (2 g, catalytic
amount) was heated to reflux for 15 min with TLC monitoring
(petroleum ether/EtOAc 10:1). After the mixture was cooled to room
temperature, more EtOAc was added to the mixture, and the organic
phase was washed successively with 10% aqueous NaOH, water,
and brine and dried over anhydrous Na2SO4, After removing solvent
in vacuo, the residue was purified by a flash column (silica, eluant
0-10% EtOAc/petroleum ether) to give 25.0 g of 11: 98% yield,
colorless needle crystals from EtOH/H2O, mp 135-136 °C (lit.
Isonitrile 4b. A mixture of 13 (73 mg, 0.1 mmol), Bu4N+I-
(18.4 mg, 0.05 mmol), and sodium cyanide (11 mg, 0.22 mmol) in
10 mL of CH3CN was stirred at room temperature for 12 h. The
mixture was then poured into ice-water and extracted with EtOAc
three times. The combined organic phase was washed with brine
and dried over MgSO4. After removal of the solvent in vacuo, the
residue was separated by PTLC (eluant cyclohexane/acetone 2:1)
1
to give 4b (36 mg, 53% yield); H NMR δ 1.08-1.12 (ms, 18H,
6 × CH3), 1.46 and 1.50 (each s, 3H, 2′-CH3), 1.66, 1.91, 2.18,
and 2.51 (each m, 2 × CH2), 2.52 (s, 3H, 4-CH3), 5.27 (s, 2H,
3-CH2), 5.38 (d, 1H, J ) 4.8 Hz, 3′-H), 6.65 (d, 1H, J ) 4.8 Hz,
4′-H), 6.88 (d, 1H, J ) 8.8 Hz, 6-ArH), 7.63 (d, 1H, J ) 8.8 Hz,
5-ArH); 13C NMR δ ppm 9.55 and 9.63 (2 × CH3), 15.26, 16.26,
16.36, 16.60, 16.66, and 21.14 (6 × CH3) and 26.13 (4-CH3), 28.91,
28.96, 29.20, and 30.55 (4 × CH2), 54.20, 54.60, and 54.89, 55.04
(4 × C), 59.74 (3-CH2), 61.41 and 72.06 (2 × CH), 90.72 (NC),
91.07, 91.30, 106.06, 113.44, 116.39 (5 × C), 114.59 and 127.64
(2 × CH), 152.35, 152.70, 156.62, 159.35, 166.89, 167.25 (6 ×
C), 177.95, 178.01 and 178.51 (CdO); IR cm-1 (KBr) 2341, 2360
(NC); MS (ESI) m/z (%): 676.5 ([M + 1]+, 5), 649.5 (M - NC,10).
1
121-122 °C); H NMR δ 1.47 (6H, s, 2′-CH3 × 2), 2.18 (3H, s,
3-CH3), 2.35 (3H, s, 4-CH3), 5.71 (1H, d, J ) 9.6 Hz, 3′-H), 6.72
(1H, d, J ) 8.8 Hz, 6-H), 6.91 (1H, d, J ) 9.6 Hz, 4′-H), 7.35
(1H, d, J ) 8.8 Hz, 5-H).
3,4-Dimethyl-3′,4′-di-O-(S)-camphanoyl-(+)-cis-khellactone
(12). A mixture of K3Fe(CN)6 (20 g, 60 mmol), K2CO3 (10 g, 72.5
mmol), 2,5-diphenyl-4,6-bis(9-O-dihydroquinyl)pyrimidine [(DHQ)2-
PYR] (531 mg, 0.6 mmol), and K2OsO2(OH)4 (220 mg, 0.6 mmol)
was solubilized in 260 mL of t-BuOH/H2O (v/v, 1:1) at room
temperature. Then the solution was cooled to 0 °C, and methane-
sulfonamide (1.9 g, 20 mmol) added under stirring. When the
solution turned from light-yellow to orange, 11 (5.1 g, 20 mmol)
was added. The mixture was stirred at 0 °C for 24 h. Na2S2O5
(excess), water, and more CHCl3 were added. After being stirred
for 0.5 h at room temperature, the mixture was extracted with CHCl3
three times. The combined organic layer was dried over MgSO4,
then solvent was removed in vacuo to give 3,4-dimethyl-(+)-cis-
khellactone, which was acylated directly, without further purifica-
tion, with (S)-(-)-camphanic chloride (13.0 g, 60 mmol) in pyridine/
CH2Cl2 (100 mL with 3:5 v/v) for 24 h at room temperature
monitored by TLC (cyclohexane/EtOAc 7:3). The mixture was
diluted with EtOAc and washed successively with 10% aqueous
HCl, water, and brine. The organic phase was dried over anhydrous
MgSO4, filtered, and concentrated in vacuo. The residue was
separated by a flash column, eluting with a 0-50% gradiant of
EtOAc/hexane to give 5.0 g of pure 12 (77% yield), as calculated
from 11, with 99.0% stereoselectivity for (3′R,4′R)-isomer measured
by HPLC.
3-Bromomethyl-4-methyl-3′,4′-di-O-(S)-camphanoyl-(+)-cis-
khellactone (13). A mixture of 12 (19.5 g, 30 mmol) and
N-bromosuccinimide (NBS, 6.4 g, 36 mmol) in 50 mL of anhydrous
benzene was heated under reflux for 4 h until the reaction was
completed as monitored by TLC. The mixture was cooled at 4 °C
overnight, the solid filtered, and the solvent removed in vacuo to
give a 19.7 g of 13 (97% yield), which was sufficiently pure to use
in the next reaction without further purification.
3-Cyanomethyl-4-methyl-3′,4′-di-O-(S)-camphanoyl-(+)-cis-khel-
lactone (4). A solution of sodium cyanide (0.69 g, 14 mmol) in 40
mL of 95% EtOH was cooled in an ice bath. Compound 13 (7.29
g, 10 mmol) in 15 mL of DMF was added slowly to the above
solution over a 15-20 min period. After 2 h of stirring at room
temperature, the mixture was poured into ice-water and extracted
three times with EtOAc. The combined organic phase was washed
Detection of HIV-1 Replication As Measured by p24
Antigen Production. MT-2 and MT-4 cells, laboratory-adapted
HIV-1 strains, and primary HIV-1 isolates were obtained from the
NIH AIDS Research and Reference Reagent Program. The inhibi-
tory activity of compounds on HIV-1 infection was determined as
previously described.24 In brief, 1 × 104 MT-2 cells were infected
with an HIV-1 strain (100 TCID50) in 200 µL of RPMI 1640
medium containing 10% FBS in the presence or absence of a test
compound at graded concentrations overnight. Then the culture
supernatants were removed and fresh media containing no test
compounds were added. On the fourth day postinfection, an amount
of 100 µL of culture supernatants was collected from each well,
mixed with equal volumes of 5% Triton X-100, and assayed for
p24 antigen, which was quantitated by ELISA. Briefly, wells of
polystyrene plates (Immulon 1B, Dynex Technology, Chantilly, VA)
were coated with HIV immunoglobulin (HIVIG), which was
prepared from plasma of HIV-seropositive donors with high
neutralizingtitersagainstHIV-1IIIB,in0.085Mcarbonate-bicarbonate
buffer (pH 9.6) at 4 °C overnight, followed by washes with washing
buffer (0.01 M PBS containing 0.05% Tween-20) and blocking with
PBS containing 1% dry fat-free milk (Bio-Rad Inc., Hercules, CA).
Virus lysates were added to the wells and incubated at 37 °C for
1 h. After extensive washes, anti-p24 mAb (183-12H-5C), biotin
labeled antimouse IgG1 (Santa Cruz Biotechnology, Santa Cruz,
CA), streptavidin-labeled horseradish peroxidase (Zymed, S. San
Francisco, CA), and the substrate 3,3′,5,5′-tetramethylbenzidine
(Sigma Chemical Co., St. Louis, MO) were added sequentially.
Reactions were terminated by addition of 1 N H2SO4. Absorbance
at 450 nm was recorded in an ELISA reader (Ultra 386, TECAN,
Research Triangle Park, NC). Recombinant protein p24 purchased
from U.S. Biological (Swampscott, MA) was included for establish-
ing standard dose response curves. Each sample was tested in
triplicate. The percentage of inhibition of p24 production was