Anti-AIDS agents. Part 56: Synthesis and anti-HIV activity of 7-thia-di-O-(-)-camphanoyl-(+)-cis-khellactone (7-thia-DCK) analogs

Article abstract of DOI:10.1016/j.bmc.2004.09.038

Two thia-DCK analogs (3a,b) were synthesized and evaluated for inhibition of HIV-1 replication in H9 lymphocytes. Compound 3a showed potent anti-HIV activity with an EC₅₀ value of 0.14 μM and a therapeutic index of 1110. However, the corresponding 6-tert-butyl-substituted compound (3b) showed no suppression. The bioassay results indicated that thia-DCK analogs merit attention as potential HIV-1 inhibitors.

Full text of DOI:10.1016/j.bmc.2004.09.038

Bioorganic & Medicinal Chemistry 12 (2004) 6383–6387
Anti-AIDS agents. Part 56: Synthesis and anti-HIV activity of
7-thia-di-O-(ꢀ)-camphanoyl-(+)-cis-khellactone
(7-thia-DCK) analogsI
Ying Chen,a Qian Zhang,a Beina Zhang,a Peng Xia,a,* Yi Xia,b Zheng-Yu Yang,b
Nicole Kilgore,c Carl Wild,c Susan L. Morris-Natschkeb and Kuo-Hsiung Leeb,*
aDepartment of Medicinal Chemistry, School of Pharmacy, Fudan University, Shanghai 200032, China
bNatural Products Laboratory, School of Pharmacy, University of North Carolina, Chapel Hill, NC 27599, USA
cPanacos Pharmaceuticals, Inc., 209 Perry Parkway, Gaithersburg, MD 20877, USA
Received 2 July 2004; accepted 20 September 2004
Abstract—Two thia-DCK analogs (3a,b) were synthesized and evaluated for inhibition of HIV-1 replication in H9 lymphocytes.
Compound 3a showed potent anti-HIV activity with an EC50 value of 0.14lM and a therapeutic index of 1110. However, the cor-
responding 6-tert-butyl-substituted compound (3b) showed no suppression. The bioassay results indicated that thia-DCK analogs
merit attention as potential HIV-1 inhibitors.
ꢀ 2004 Elsevier Ltd. All rights reserved.
5
4
1. Introduction
6
3
7 O
O
O
Acquired immunodeficiency syndrome (AIDS), which is
caused by human immunodeficiency virus (HIV) infec-
tion, has become a serious global threat to human health
and life. The US FDA has now approved 19 anti-HIV
drugs for clinical use. However, these drugs cannot com-
pletely eliminate HIV in the human body and also show
side effects and toxicity and are subject to drug resist-
ance by mutated virus. Thus, it is necessary to further
explore novel anti-HIV agents with new mechanisms
of action.
2
1
10
8
O
O
9
O
O
O
O
O
OOCCH2CH(CH3)2
O
O
OAc
O
O
2
1
Figure 1. Structures of 1 and 2.
numerous DCK derivatives were synthesized and evalu-
ated for activity in an in vitro anti-HIV assay. Among
them, 3-methyl, 4-methyl, and 5-methyl DCK were
much more potent than DCK and AZT in the same
assay with EC50 and TI values ranging from 5.25 · 10ꢀ5
to 2.39 · 10ꢀ7 lM and 2.15 · 106 to 3.97 · 108, respec-
tively. A preliminary mechanistic study showed that
DCK and its analogs did not inhibit HIV-RT, integrase,
or protease and might inhibit HIV-1 replication by a
novel mechanism. Currently, their mechanism of action
is still under investigation.
Suksdorfin (1, Fig. 1), isolated from the fruit of
Lomatium suksdorfii, is a khellactone with interesting
biological properties, especially anti-HIV activity.2
Modification of 1 has already provided 30,40-di-O-(ꢀ)-
camphanoyl-(+)-cis-khellactone (DCK) (2, Fig. 1),
which showed extremely potent inhibitory activity
against HIV-1 replication in H9 lymphocytic cells with
an EC50 value of 2.56 · 10ꢀ4 lM and a therapeutic index
(TI) of 1.37 · 105.3 In further structural research,
q For Part 55, see Ref. 1.
Based on the concept of bioisosterism, we designed a
new series of analogs by replacing the oxygen atom in
ring C (pyran ring) of DCK with a sulfur atom. Such
*
Corresponding authors. Tel.: +86 21 5427563 (P.X.); tel.: +1 919 926
0066 (K.-H.L.); e-mail addresses: pxia@shmu.edu.cn; khlee@unc.edu
0968-0896/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.09.038

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Y. Chen et al. / Bioorg. Med. Chem. 12 (2004) 6383–6387
R
S
R
O
R
O
C
S
C
ii
iii
iv
O
O
O
O
HO
O
O
N
N
5a R=H
6a R=H
4a R=H
i
5b R=t-Bu
6b R=t-Bu
4b R=t-Bu
R
R
S
R
v
vi
vii
S
O
O
O
O
HS
O
O
9a R=H
7a R=H
8a R=H
9b R=t-Bu
7b R=t-Bu
8b R=t-Bu
R
S
R
S
viii
O
O
O
O
OR1
OR1
OH
O
OH
10a R=H
10b R=t-Bu
3a R=H
R1=
O
O
3b R=t-Bu
Scheme 1. Reagents and conditions: (i) t-BuOH, H3PO4/P2O5; (ii) dimethylthiocarbamoyl chloride, DMAP/CH2Cl2; (iii) heating; (iv) (1) NaOCH3/
MeOH, (2) HCl; (v) 3-bromo-1-propyne, K2CO3, KI, acetone/N2; (vi) N,N-diethylaniline, reflux; (vii) (1) K2OsO2(OH)4, K3Fe(CN)6, (DHQ)2–
PHAL, K2CO3, (2) Na2SO3; (viii) camphanic chloride, DMAP/CH2Cl2.
Table 1. Anti-HIV activity of compounds 3a and 3b in acutely infected
H9 lymphocytes
novel 7-thia-DCK analogs would provide more insight
into the SAR of this molecular scaffold. In this paper,
we report the synthesis and anti-HIV bioassay results
Compound
IC50 (lM)a
EC50 (lM)b
TIc
of two initial 7-thia-4-methyl-8-nonsubstituted DCK
analogs. Further structural modification and biological
screening are ongoing.
3a
3b
DCKe
AZT
155d
126d
35
0.141
1110
No suppression
0.000256
0.036
No suppression
136,719
51,972
1871
a Concentration that inhibits uninfected H9 cell growth by 50%.
b Concentration that inhibits viral replication by 50%.
2. Chemistry
c TI = therapeutic index IC50/EC50
.
d Maximum IC50 value possible for this assay due to DMSO, which is
used to solubilize the agents tested.
As shown in Scheme 1, the reaction of 7-hydroxy-4-
methyl-coumarin (4a) with tert-butanol in the presence
of H3PO4 and P2O5 at 80ꢁC gave 7-hydroxy-4-methyl-
6-tert-butyl-coumarin (4b). Compounds 5a and 5b,
which were obtained by reacting intermediates 4a and
4b with dimethylthiocarbamoyl chloride, underwent
rearrangement upon heating followed by hydrolysis in
the presence of NaOCH3/CH3OH and acidification with
HCl to afford the key intermediate 7-mercapto-couma-
rins (7a,b).4,5 The 6-tert-butyl- and 6-nonsubstituted-7-
mercapto-coumarins were then converted to the corre-
sponding seselin analogs (9a,b) by alkylation with 3-bro-
mo-1-propyne in the presence of potassium iodide and
potassium carbonate in acetone at room temperature,
followed by cyclization in N,N-dimethylaniline at reflux
temperature. Finally, asymmetric dihydroxylation6 of
(9a,b) followed by acylation with camphanic chloride
gave the desired 7-thia-DCK analogs (3a,b).
e The data for DCK were cited in Ref. 7.
1110. In the same assay, the EC50/TI values of AZT
and DCK were 0.036lM/51,972 and 0.000256lM/
136,719, respectively. These bioassay results indicated
that thia-DCK analogs could be potential anti-HIV
agents. Interestingly, compound 3b with a tert-butyl
group at the 6-position showed no anti-HIV-1 activity.
This result suggested that the 6-position might be crucial
for potency against HIV replication and sensitive to
modification. A bulky group at this position might not
be accommodated at the target receptor. The bioactivity
comparison between 3a and DCK implies that two
methyl groups at the 8-position might be favorable for
anti-HIV activity. In order to gain more information
on this thia-DCK analog class, further modification is
under investigation.
3. Results and discussion
4. Experimental
Compounds 3a and3b were screened for anti-HIV activ-
ity in H9 lymphocytes, and their bioassay data are
shown in Table 1. The 8-nonsubstituted analog 3a
exhibited potent inhibitory activity against HIV-1 repli-
cation with an EC50 value of 0.14lM and TI value of
Melting points were measured with the capillary
tube method without correction. 1H NMR spectra
were determined on Bruker-AM-300, DPX300 and

Y. Chen et al. / Bioorg. Med. Chem. 12 (2004) 6383–6387
6385
DRX-400MHz spectrometers using TMS as internal
standard. The solvent used was CDCl3 unless otherwise
indicated. Optical rotations were measured with a Jasco
P-1020 digital polarimeter at 17.4ꢁC at the sodium
D-line. MS and HRMS were measured on HP5989A
and Concept 1H mass spectrometers.
was then acidified with dry HCl. After being diluted
with 100mL CH2Cl2 and washed with 40mL H2O, the
aqueous layer was extracted with CH2Cl2 three times.
The organic phase was dried over Na2SO4. Finally, re-
moval of the solvent furnished a light yellow solid
(870mg, yield 59.2%), mp 176–180ꢁC. MS (m/z, %):
1
248 (M+, 52.27), 233 (M+ꢀ15, 100); H NMR: d 1.51
4.1. 4-Methyl-6-(tert-butyl)-7-hydroxy-2H-chromen-2-
one (4b)
(9H, s, t-Bu), 2.41 (3H, s, –CH3), 3.87 (1H, s, –SH),
6.20 (1H, s, 3-H), 7.18 (1H, s, 8-H), 7.53 (1H, s, 5-H).
tert-Butanol (10g, 0.134mol) was added to a mixture of
7-hydroxy-4-methyl-2H-chromen-2-one (3.0g, 17.0mmol),
H3PO4 (200mL), and P2O5 (15g) at 60–70ꢁC. After
stirring for 1h, additional P2O5 (15g) and tert-butanol
(15g) were added. This addition was repeated every
2h, and the reaction was continued for an additional
11h at the same temperature. The reaction mixture
was then cooled to rt, poured into 1500mL ice water,
and filtered to afford crude product, which was
purified by column chromatography on silica H with
an eluent of petroleum–EtOAc = 3:1 to provide pure
4b (1.45g, yield 36.7%), mp 243–244ꢁC; MS (m/z, %):
4.5. 4-Methyl-6-(tert-butyl)-7-(prop-2-ynylthio)-2H-chro-
men-2-one (8b)
Under nitrogen, a mixture of compound 7b (500mg,
2.0mmol), K2CO3 (5.57g, 40.3mmol), KI (50mg,
0.30mmol),
and
3-bromo-1-propyne
(956mg,
8.37mmol) in 100mL acetone was stirred for 15min at
rt. Water (50mL) was added to the reaction mixture,
which was then extracted with EtOAc four times. The
organic phase was dried over Na2SO4 and filtered. After
removing the solvent in vacuo, the residue was purified
by column chromatography on silica H (eluent: petro-
leum–EtOAc = 8:1) to give 8b (330mg, yield 57.2%),
mp 158–160ꢁC. MS (m/z, %): 286 (M+, 3.45), 271
1
232 (M+, 22.08), 217 (M+ꢀ15, 100); H NMR: d 1.45
(9H, s, t-Bu), 2.45 (3H, s, 4-CH3), 6.14 (1H, s, 3-H),
7.32 (1H, s, 8-H), 7.46 (1H, s, 5-H), 7.55–7.59 (1H, br,
OH).
1
(M+ꢀ15, 77.14); H NMR: d 1.46 (9H, s, t-Bu), 2.20
(1H, t, J = 1.62Hz, 30-H), 2.35 (3H, s, 4-CH3), 3.66
(2H, d, J = 1.62Hz, 10-H), 6.17 (1H, s, 3-H), 7.37 (1H,
s, 8-H), 7.49 (1H, s, 5-H).
4.2. 4-Methyl-6-(tert-butyl)-7-[(N,N-dimethyl)thiocarb-
amoyloxy]-2H-chromen-2-one (5b)
4.6. 4-Methyl-6-(tert-butyl)-8H-thiopyrano[6,5-h]-2H-chro-
men-2-one (9b)
Under nitrogen, dimethylthiocarbamoyl chloride (3.2g,
26mmol) and DMAP (3.16g, 26mmol) were added to
a solution of compound 4b (1.0g, 4.3mmol) in CH2Cl2
(60mL). After 96h, the solvent was removed in vacuo,
and the residue was poured into ice water and filtered.
Compound 5b was obtained as white solid (1.35g, yield
98.2%, mp 234–238ꢁC), which was used in next reaction
without purification. An analytical sample was purified
by column chromatography on silica H (eluent: petro-
leum–EtOAc = 6:1), mp 236–237ꢁC. MS (m/z, %): 319
Under nitrogen, compound 8b (200mg, 0.7mmol) was
heated to reflux in N,N-diethylaniline for 75min. The
solvent was removed in vacuo. The residue was purified
by column chromatography on silica H (eluent: petro-
leum–EtOAc = 30:1) to afford 9b as a yellow solid
(158mg, yield 79%), mp 142–144ꢁC. MS (m/z, %): 286
1
(M+, 57.26), 271 (M+ꢀ15, 100); H NMR: d 1.48 (9H,
s, t-Bu), 2.35 (3H, s, –CH3), 3.27 (2H, dd, J1 = 1.2Hz,
J2 = 5.3Hz, 8-H), 6.10 (1H, m, J1 = 5.3Hz,
J2 = 10.4Hz, 9-H), 6.15 (1H, s, 3-H), 7.15 (1H, d,
J = 10.1Hz, 10-H), 7.39 (1H, s, 5-H).
1
(M+ꢀ1, 17.27), 88 (100); H NMR: d 1.41 (9H, s, t-
Bu), 2.44 (3H, d, J = 0.92Hz, 4-CH3), 3.43 (3H, s, N–
CH3), 3.50 (3H, s, N–CH3), 6.25 (1H, d, J = 0.97Hz,
3-H), 7.04 (1H, s, 8-H), 7.58 (1H, s, 5-H).
4.7. (9R,10R)-9,10-Dihydroxy-4-methyl-6-(tert-butyl)-
8H,9H,10H-thiopyrano[6,5-h]-2H-chromen-2-one (10b)
4.3. 4-Methyl-6-(tert-butyl)-7-[(N,N-dimethyl)carbamo-
ylmercapto]-2H-chromen-2-one (6b)
A mixture of K3Fe(CN)6 (0.92g, 2.8mmol), K2CO3
(0.38g, 2.8mmol), K2OsO2(OH)4 (8mg, 0.02mmol),
and [(DHQ)2–PHAL] (20mg, 0.03mmol) was dissolved
in 48mL of t-BuOH/H2O (v/v, 1:1) at rt. The solution
was cooled to 0ꢁC, compound 9b was added, and the
reaction continued for 6h at 0–5ꢁC. Then, Na2SO3
(684mg, 0.54mmol) was added. After stirring for 2h
at rt, the mixture was extracted with CHCl3 five times
and EtOAc four times. The combined organic layers
were dried over Na2SO4, and then solvent was removed.
The residue was purified by column chromatography on
silica H (eluent: petroleum–EtOAc = 2:1) to afford 10b
as a white solid (126mg, yield 71.3%), mp 227–228ꢁC.
Compound 5b (1.40g) was heated to 230–250ꢁC with
stirring for 3h. The crude product (6b) (1.4g) was ob-
tained in quantitative yield. An analytical sample was
obtained by recrystallization from EtOAc, mp 186–
1
187ꢁC. MS (m/z, %): 318 (M+ꢀ1, 6.71), 88 (100); H
NMR: d 1.52 (9H, s, t-Bu), 2.44 (3H, d, J = 1.27Hz,
4-CH3), 3.06 (6H, br, N–(CH3)2), 6.29 (1H, d,
J = 1.23Hz, 3-H), 7.47 (1H, s, 8-H), 7.64 (1H, s, 5-H).
4.4. 4-Methyl-6-(tert-butyl)-7-mercapto-2H-chromen-2-
one (7b)
1
Compound 6b (1.89g, 5.92mmol) was hydrolyzed in the
presence of NaOCH3/CH3OH (5mL, 25% w/v) in 80mL
of MeOH under nitrogen for 36h. The reaction mixture
MS (m/z, %): 320 (M+, 57.12), 276 (M+ꢀ45, 100); H
NMR: d 1.53 (9H, s, t-Bu), 2.42 (3H, s, 4-CH3), 2.85
(1H, dd, J1 = 3.6Hz, J2 = 11.9Hz, 8-H), 3.18 (1H, d,

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Y. Chen et al. / Bioorg. Med. Chem. 12 (2004) 6383–6387
J = 9.7Hz, 9-OH), 3.33 (1H, t, J = 11.9Hz, 8-H), 3.77
(1H, s, 10-OH), 4.13 (1H, m, J1 = 3.7Hz, J2 = 9.7Hz,
9-H), 5.40 (1H, t, J = 3.5Hz, 10-H), 6.21 (1H, s, 3-H),
7.53 (1H, s, 5-H); 1H NMR (CDCl3+D2O): d 1.53
(9H, s, t-Bu), 2.42 (3H, s, 4-CH3), 2.84 (1H, dd,
J1 = 3.6Hz, J2 = 11.9Hz, 8-H), 3.31 (1H, t,
J = 11.9Hz, 8-H), 4.11 (1H, m, J = 11.9Hz, 9-H), 5.36
(1H, d, J = 3.7Hz, 10-H), 6.21 (1H, s, 3-H), 7.53 (1H,
s, 5-H); HRMS: C17H20O4S calcd 320.10823. Anal.
320.10750.
4.12. 4-Methyl-7-prop-2-ynylthio-2H-chromen-2-one (8a)
Under nitrogen, a mixture of compound 7a (2.60g,
13.3mmol), K2CO3 (28.0g, 203mmol), KI (337mg,
2.03mmol), and 3-bromo-1-propyne (6mg, 47mmol) in
100mL acetone was stirred for 45min at rt. Water
(100mL) was added, and the mixture extracted four
times with CHCl3. The organic phase was dried over
Na2SO4 and filtered. After removing the solvent, the res-
idue was purified by column chromatography on silica
H (eluent: petroleum–EtOAc = 15:1) to obtain pure
compound 8a (2.77g), yield 89.13%, mp 127–128ꢁC.
4.8. (9R,10R)-9,10-Dicamphanoyloxy-4-methyl-6-(tert-
butyl)-8H,9H,10H-thiopyrano[6,5-h]-2H-chromen-2-one
(3b)
1
MS (m/z, %): 229 (M+ꢀ1, 100), 230 (M+, 69.62); H
NMR: d 2.29 (1H, s, 30-H), 2.37 (3H, s, 4-CH3), 3.72
(2H, s, 10-H), 6.72 (1H, s, 3-H), 7.26–7.36 (2H, m,
6-H, 8-H), 7.54 (1H, d, J = 8.33Hz, 5-H); IR (cmꢀ1):
2119 (alkyne).
Compound 10b (140mg, 0.44mmol) was acylated with
(S)-(ꢀ)-camphanic chloride (379mg, 1.75mmol) in
40mL CH2Cl2 in the presence of DMAP (320mg,
2.62mmol) for 1h at rt. After removing the solvent,
the target product 3b (200mg) was obtained by column
4.13. 4-Methyl-8H-thiopyrano[6,5-h]-2H-chromen-2-one
(9a)
chromatography on silica
H (eluent: petroleum–
EtOAc = 2:1), yield 94.1%, mp 256–258ꢁC. MS (m/z,
%): 680 (M+, 23.53), 482 (M+ꢀcamphanoyl group,
20.13), 285 (M+ꢀ2 · camphanoyl group, 100); 1H
NMR: d 0.77–2.58 (24H, m, H in camphanoyl group),
1.55 (9H, s, t-Bu), 2.40(3H, s, 4-CH3), 2.84 (1H, m, 8-
H), 3.52 (1H, m, 8-H), 5.89 (1H, m, 9-H), 6.18 (1H, s,
3-H), 6.87 (1H, m, 10-H), 7.59 (1H, s, 5-H); [a]D
ꢀ37.39 (c 0.0023, CDCl3), HRMS: C33H26O10S calcd
680.26552. Anal. 680.26686.
Under nitrogen, compound 8a (512mg, 2.22mmol) was
heated to reflux in N,N-diethylaniline for 3h. The sol-
vent was removed in vacuo and the residue was purified
by column chromatography on silica H (eluent: petro-
leum–EtOAc = 8:1) to afford compound 9a (397mg,
yield 77.6%), mp 164–167ꢁC. MS (m/z, %): 230 (M+,
100), 299 (M+ꢀ1, 99); 1H NMR: d 2.33 (3H, s, 4-
CH3), 3.42 (2H, dd, J1 = 1.5Hz, J2 = 5.2Hz, 8-H),
5.997 (1H, m, J1 = 5.2Hz, J2 = 10.4Hz, 9-H), 6.15
(1H, s, 3-H), 7.07 (1H, d, J = 10.4Hz, 10-H), 7.07 (1H,
d, J = 8.3Hz, 6-H), 7.25 (1H, d, J = 8.3Hz, 5-H).
4.9. 7-N,N-Dimethylthiocarbamoyloxy-4-methyl-2H-chro-
men-2-one (5a)
4.14. (9R,10R)-9,10-Dihydroxy-4-methyl-8H,9H,10H-
thiopyrano[6,5-h]-2H-chromen-2-one (10a)
7-Hydroxy-4-methyl-2H-chromen-2-one (1.76g, 10mmol)
and DMAP (2.44g, 20mmol) were solubilized in 15mL
DMF, and reacted with dimethylthiocarbamoyl chloride
(2.47g, 20mmol) for 3h at rt. The mixture was poured
into 400mL ice water and filtered to afford crude prod-
uct 5a (2.27g, yield 86.3%), mp 213–215ꢁC (lit.4 mp
208–209ꢁC). Recrystallization from acetone afforded a
pure sample, mp 215–217ꢁC.
A mixture of K3Fe(CN)6 (4.29g, 13.1mmol), K2CO3
(1.79g, 13.1mol), K2OsO2(OH)4 (38mg, 0.10mmol),
and [(DHQ)2–PHAL] (98 mg, 0.12mmol) was solubi-
lized in 174mL of t-BuOH/H2O (v/v, 1:1) at rt. The solu-
tion was cooled to 0ꢁC, compound 9a (600mg,
2.6mmol) was added, and reaction was continued for
32h at 0–5ꢁC. Then, Na2SO3 (3.20g, 25.6mmol) was
added. After stirring for 2h at rt, the mixture was ex-
tracted with CHCl3 five times and EtOAc nine times.
The combined organic layers were dried over Na2SO4,
and then solvent was removed. The residue was purified
by column chromatography on silica H (eluent: petro-
leum–EtOAc = 2:1) to afford dihydroxy compound 10a
(353mg, yield 51.3%), mp 182–185ꢁC. MS (m/z, %):
264 (M+, 63.63), 220 (M+ꢀ44, 100), 192 (7-SH-heterocy-
4.10. 7-N,N-Dimethylcarbamoylmercapto-4-methyl-2H-
chromen-2-one (6a)
Compound 5a (1.50g, 5.70mmol) was heated to 225–
230ꢁC with stirring for 1h. The crude product (6a)
was recrystallized from acetone to give light yellow crys-
tals (1.31g, yield 87.3%), mp 160–162ꢁC [lit.4 mp 159–
160ꢁC].
1
cle core, 69.47); H NMR (DMSO-d6): d 2.35 (3H, s, 4-
4.11. 4-Methyl-7-mercapto-2H-chromen-2-one (7a)
CH3), 2.69 (1H, dd, J1 = 2.8Hz, J2 = 11.5Hz, 8-H), 3.27
(1H, t, J = 11.6Hz, 8-H), 3.82 (9-H), 5.03 (1H, br,
10-H), 5.40 (2H, m, 2 · OH), 6.26 (1H, s, 3-H), 7.07
(1H, d, J = 8.5Hz, 6-H), 7.53 (1H, d, J = 8.6Hz, 5-H);
1H NMR (DMSO-d6 + D2O): d 2.31 (3H, s, 4-CH3),
2.68 (1H, dd, J1 = 2.9Hz, J2 = 11.6Hz, 8-H), 3.24 (1H,
t, J = 11.8Hz, 8-H), 3.83 (1H, m, J1 = 2.8Hz,
J2 = 8.8Hz, 9-H), 5.01 (1H, s, 10-H), 6.20 (1H, s, 3-H),
7.05 (1H, d, J = 8.6Hz, 6-H), 7.50 (1H, d, J = 8.6Hz,
5-H); HRMS C13H12O4S calcd 264.04563. Anal.
264.04125.
Under nitrogen, compound 6a (5.0g, 19mmol) was
hydrolyzed in the presence of NaOCH3/MeOH (30mL,
25% w/v) in 400mL MeOH for 19h at rt, then acidified
with dry HCl. After being diluted with 100mL H2O, the
reaction mixture was extracted with CH2Cl2 three times.
The organic phase was dried over Na2SO4. Finally,
removal of the solvent furnished 7a as a light yellow
solid (2.7g, yield 74.0%), mp 137–138ꢁC [lit.4 mp 138–
139ꢁC].

Y. Chen et al. / Bioorg. Med. Chem. 12 (2004) 6383–6387
6387
4.15. (9R,10R)-9,10-Dicamphanoyloxy-4-methyl-8H,9H,
10H-thiopyrano[6,5-h]-2H-chromen-2-one (3a)
then added to the appropriate wells of a 24-well plate
containing the various concentrations of the test drug
or culture medium (positive infected control/negative
drug control). In addition, AZT was also assayed during
each experiment as a positive drug control. The plates
were incubated at 37ꢁC and 5% CO2 for 4days. Cell-free
supernatants were collected on day 4 for use in-house
p24 antigen ELLSA assay. P24 antigen is protein of
HIV and therefore is an indirect measure of virus pre-
sent in the supernatants. Toxicity was determined by
performing cell counts on a Coulter counter on the
mock-infected H9 lymphocytes, which had either re-
ceived culture medium (no toxicity) or test sample or
AZT.
Compound 10a (200mg, 0.76mmol) was acylated with
(S)-(ꢀ)-camphanic chloride (656mg, 3.02mmol) in
60mL CH2Cl2 in presence of DMAP (5.54g, 4.54mmol)
for 1h at rt. After removing the solvent, the target prod-
uct 3a (450mg) was obtained by column chromatogra-
phy on silica H (eluent: petroleum–EtOAc = 2:1), yield
95.2%, mp 177–179ꢁC. MS (m/z, %): 624 (M+, 11.68),
229 (M+ꢀ2 · camphanoyl group, 100); 1H NMR: d
0.77–2.61 (24H, m, H in camphanoyl group), 2.40 (3H,
s, 4-CH3), 2.81 (1H, m, 8-H), 3.55 (1H, m, 8-H), 5.56
(1H, m, 9-H), 6.19 (1H, s, 3-H), 6.87 (1H, m, 10-H),
7.09 (1H, d, J = 8.48Hz, 6-H), 7.48 (1H, d,
J = 8.33Hz, 5-H); [a]D ꢀ53.40 (c 0.002, CDCl3), HRMS:
C33H26O10S calcd 624.20292. Anal. 624.20460.
Acknowledgements
Supported by National Natural Science Foundation of
China, Grant No 20272010 awarded to P. Xia and in
part by Grant AI-33066 from the National Institute of
Allergies and Infectious Diseases awarded to K. H. Lee.
5. HIV growth inhibition assay in H9 lymphocytes
The T cell line, H9, was maintained in continuous cul-
ture with complete medium (RPMI 1640 with 10% fetal
serum (FCS) supplemented with L-glutamine) at 5%
CO2 and 37ꢁC. Aliquots of this cell line were used in
experiments only when in log phase of growth. Test
samples were first dissolved in dimethyl sulfoxide
(DMSO). The following were the final drug concentra-
tions routinely used for screening: 100, 20, 4, and
0.8lg/mL. As the test samples were being prepared, an
aliquot of the T cell line, H9, was infected with HIV-1
(IIIB isolate) while another aliquot was mock-infected
with complete medium. The mock-infected aliquot was
used for toxicity determinations (IC50). The stock virus
used for these studies typically had a TCID50 value of
104 infectious units/mL. The appropriate amount of
virus for a multiplicity of infection (moi) between 0.1
and 0.01 infectious units/cell was added to the first ali-
quot of H9 cells. The other aliquot of H9 cells received
only culture medium and then was incubated under
identical conditions as the HIV-infected H9 cells. After
a 4h incubation at 37ꢁC and 5% CO2, both cell popula-
tions were washed three times with fresh medium and
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