Anti-AIDS agents. 42. Synthesis and anti-HIV activity of disubstituted (3′R,4′R)-3′,4′-di-O-(S)-camphanoyl-(+)-cis- khellactone analogues

Article abstract of DOI:10.1021/jm000070g

A series of disubstituted 3′,4′-di-O-(S)-camphanoyl-(+)-cis-khellactone (DCK) analogues (1-10) were synthesized and evaluated for inhibition of HIV-1 replication in H9 lymphocytes. 5-Methoxy-4-methyl DCK (8) was the most promising compound with an EC₅₀ value of 7.21 × 10^-6 μM and a therapeutic index of >2.08 × 10,⁷ which were much better than those of lead compound DCK in the same assay. Another six disubstituted DCK analogues (1-5 and 7) were more potent than AZT but less active than DCK. Conformational analysis suggested that resonance of the coumarin system is an essential structural feature for potent anti-HIV activity. Steric compression of C(4) and C(5) substituents of the coumarin moiety can reduce the overall planarity and thus resonance of the coumarin nucleus, resulting in a decrease or lack of anti-HIV activity.

Full text of DOI:10.1021/jm000070g

664
J . Med. Chem. 2001, 44, 664-671
Articles
An ti-AIDS Agen ts. 42. Syn th esis a n d An ti-HIV Activity of Disu bstitu ted
(3′R,4′R)-3′,4′-Di-O-(S)-ca m p h a n oyl-(+)-cis-k h ella cton e An a logu es
Lan Xie,† Yasuo Takeuchi,† L. Mark Cosentino,‡ Andrew T. McPhail,# and Kuo-Hsiung Lee*,†
Natural Products Laboratory, School of Pharmacy, University of North Carolina, Chapel Hill, North Carolina 27599,
BBI-Biotech Research Laboratories, Inc., Gaithersburg, Maryland 20877, and Department of Chemistry,
Paul M. Gross Chemical Laboratory, Duke University, Durham, North Carolina 27708
Received February 18, 2000
A series of disubstituted 3′,4′-di-O-(S)-camphanoyl-(+)-cis-khellactone (DCK) analogues (1-
10) were synthesized and evaluated for inhibition of HIV-1 replication in H9 lymphocytes.
5-Methoxy-4-methyl DCK (8) was the most promising compound with an EC50 value of 7.21 ×
10-6 µM and a therapeutic index of >2.08 × 10,7 which were much better than those of lead
compound DCK in the same assay. Another six disubstituted DCK analogues (1-5 and 7) were
more potent than AZT but less active than DCK. Conformational analysis suggested that
resonance of the coumarin system is an essential structural feature for potent anti-HIV activity.
Steric compression of C(4) and C(5) substituents of the coumarin moiety can reduce the overall
planarity and thus resonance of the coumarin nucleus, resulting in a decrease or lack of anti-
HIV activity.
In tr od u ction
designed, synthesized, and evaluated in an in vitro anti-
HIV assay.3 Among them, 3-methyl DCK, 4-methyl
DCK, 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 × 10-5 to 2.39 × 10-7 µM and
2.15 × 106 to 3.97 × 108, respectively. Thus, these first
structure-activity relationship (SAR) studies indicated
that alkyl/O-alkyl substituents at the 3-, 4-, and 5-posi-
tions on the coumarin nucleus are favorable for en-
hanced anti-HIV activity and decreased toxicity of DCK
and a methyl group is preferred to other substituents.
Even though the mechanism of action of DCK and its
analogues is still unknown, current mechanistic studies
indicate that they do not inhibit RT, integrase, or
protease in the HIV life cycle but are strongly syner-
gistic with approved drugs such as AZT. Therefore, DCK
and its analogues might inhibit HIV-1 replication by a
novel mechanism. These exciting results prompted us
to further modify DCK in order to ascertain the phar-
macophore(s) for this compound type as potent anti-HIV
agents. In this paper, we report the synthesis and the
anti-HIV bioassay results of 10 disubstituted DCK
analogues (1-10).
Since the early 1980s, acquired immune-deficiency
syndrome (AIDS) has spread rapidly across the world
and has become a serious global threat to human health
and life. Millions of people have been killed by the AIDS
epidemic, and the total number of people living with the
virus has risen to more than 33.4 million worldwide.
As of now, the FDA has approved 14 anti-HIV agents
as drugs for clinical use, including 6 nucleoside reverse
transcriptase inhibitors (NRTIs), 3 nonnucleoside re-
verse transcriptase inhibitors (NNRTIs), and 5 protease
inhibitors (PIs). All these drugs are currently used alone
or as a part of a combination regimen to treat HIV
infection/AIDS disease. These available drugs have
made a marked impact on suppression of HIV infection
but still have limited or transient clinical benefits in
HIV-infected individuals due to rapid development of
HIV resistance, side effects, and/or toxicity. Drug re-
sistance is a large problem for all anti-HIV drugs,
because HIV has the potential to constantly mutate and
eventually is no longer sensitive to chemotherapy. Thus,
HIV continues its life cycle as before drug treatment
and damages the immune system. Consequently, these
limitations still necessitate development of novel anti-
HIV agents with new mechanism(s) of action.
Ch em istr y
In our previous studies, (3′R,4′R)-3′,4′-di-O-(S)-cam-
phanoyl-(+)-cis-khellactone (DCK) was discovered as a
potent anti-HIV agent with an EC50 value of 2.56 × 10-4
µM and a therapeutic index (TI) of 1.37 × 105.2 Subse-
quently, 24 (3′R,4′R)-(+)-cis-khellactone derivatives were
On the basis of previous results with monomethylated
DCK analogues, we first wanted to explore the effects
of two methyl substituents on anti-HIV activity. Ac-
cordingly, 3,4-dimethyl (1), 4,5-dimethyl (2), 3,5-di-
methyl (3), and 4,6-dimethyl (4) DCK analogues were
synthesized. After reviewing their bioassay data, other
3,4-disubstituted (5-7) and 4,5-disubstituted (8-10)
DCK analogues were investigated. Each of these de-
signed target compounds, except 7, contains at least one
* To whom correspondence should be addressed. Tel: 919-962-0066.
Fax: 919-966-3893. E-mail: khlee@email.unc.edu.
† University of North Carolina.
‡ BBI-Biotech Research Laboratories, Inc.
#
Duke University.
10.1021/jm000070g CCC: $20.00 © 2001 American Chemical Society
Published on Web 01/12/2001

Anti-AIDS Agents
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 5 665
pared using a base-catalyzed Kostanecki condensation8
between 2,4-dihydroxyacetophenone and phenylacetyl
chloride with anhydrous potassium carbonate in ac-
etone.
These nine disubstituted 7-hydroxycoumarin inter-
mediates 11a -i were then converted to the correspond-
ing seselin derivatives 16a-i by alkylation with 3-chloro-
3-methyl-1-butyne in dimethylformamide (DMF) at 70-
80 °C in the presence of potassium iodide and potassium
carbonate followed by cyclization in N,N-diethylaniline
at reflux temperature (Scheme 2). Finally, asymmetric
dihydroxylation9 followed by acylation gave disubsti-
tuted DCK analogues 1-9, respectively.
The asymmetric dihydroxylation (AD) afforded most
target compounds with >76% diastereomeric excess
(d.e.). However, under the same conditions, 4-isopropyl-
5-methyl DCK (9) was obtained in a 1:1 diastereomeric
1
mixture, as per H NMR. The isomers were separated
by crystallization from ethanol. Compound 9 was soluble
in ethanol, while the (3′S,4′S)-isomer crystallized. Both
diastereomers were obtained in >95% d.e.
F igu r e 1. DCK, 4-methyl DCK, and disubstituted DCK
A different synthetic route was explored for the
synthesis of 10 and 8 as shown in Scheme 3. Friedel-
Crafts acylation of 1,3,5-trihydroxybenzene with 3,3-
dimethylacrylic acid10 in the presence of boron trifluo-
ride diethyl etherate gave 5,7-dihydroxy-2,2-dimethyl-
4-chromanone (17). The 7-phenolic group in 17 was
selectively benzylated using benzyl bromide (1:1 molar
ratio) in acetone in the presence of K2CO3 to afford
7-benzyloxy-2,2-dimethyl-5-hydroxy-4-chromanone (18).
Next, the 4-carbonyl group in 18 was reduced readily
to a methylene with NaBH4 in refluxing THF solution
under basic conditions. Then the lactone ring was
formed by a Pechmann reaction to afford 5-benzyloxy-
4-methyl-3′,4′-dihydroseselin (20). Satisfactory yields
were obtained using boron trifluoride diethyl etherate,
rather than sulfuric acid, as a catalyst. Compound 20
was dehydrogenated with DDQ in dioxane at reflux to
afford 5-benzyloxy-4-methylseselin (22) in 70% yield.11
Compound 22 was then converted to 5-benzyloxy-4-
methyl DCK (10) by asymmetric dihydroxylation and
acylation in the same manner as described above. Using
the same synthetic route, 5-methoxy-4-methyl DCK (8)
was also synthesized successfully.
analogues.
Sch em e 1. Syntheses of Disubstituted
7-Hydroxycoumarins 11a -d ,f-i
Bioa ssa y Resu lts
Disubstituted DCK analogues 1-10 were screened for
inhibition of HIV replication in H9 lymphocytes, and
their bioassay data are shown in Table 1. 5-Methoxy-
4-methyl DCK (8) was the most promising compound.
It exhibited extremely potent inhibitory activity against
HIV-1 replication with an EC50 value of 7.21 × 10-6 µM
and a TI of >2.08 × 107, which were much better than
those of DCK and AZT and similar to those of 4-methyl
DCK (EC50 1.83 × 10-6 µM, TI >6.89 × 107) in the same
assay. Dimethyl DCK analogues 1-4 were more potent
than AZT but less active than DCK, although the
previously prepared monomethyl DCK analogues were
more active than DCK.3 3-Chloro-4-methyl DCK (5) and
3,4-cyclohexano DCK (7) also showed potent anti-HIV
activity, comparable to those of the dimethyl DCK
compounds. These results indicated that anti-HIV activ-
ity could be maintained when two methyl or other
aliphatic substituent(s) were placed on the DCK cou-
methyl group on the coumarin nucleus (Figure 1) in
order to maintain potent anti-HIV activity.
As shown in Scheme 1, disubstituted 7-hydroxycou-
marins 11a ,b,d ,g-i were synthesized from different 1,3-
dihydroxybenzene derivatives using a Pechmann reac-
tion with various â-keto esters in the presence of sulfuric
acid at room temperature.4 2,4-Dihydroxy-6-methylbenz-
5
aldehyde (14), prepared by reacting DMF/POCl3 with
3,5-dihydroxytoluene (12), underwent a Wittig reaction
followed by an intramolecular cyclization to afford 3,5-
dimethyl-7-hydroxycoumarin (11c).6,7 3-Chloro-4-meth-
yl-7-hydroxycoumarin (11e) was commercially available.
7-Hydroxy-4-methyl-3-phenylcoumarin (11f) was pre-

666 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 5
Xie et al.
Sch em e 2. Syntheses of Disubstituted DCK Analogues 1-9a
a
(i) 3-Chloro-3-methyl-1-butyne, KI, K2CO3 in DMF or acetone, 70-80 °C; (ii) N,N-diethylaniline, reflux; (iii) K2Os2(OH)4, K2CO3,
K3Fe(CN)6, (DHQ)2-PYR in t-BuOH/H2O (v/v ) 1:1), ice bath; (iv) (S)-(-)-camphanoyl chloride, pyridine in CH2Cl2.
Sch em e 3. Syntheses of 4,5-Disubstituted DCK
Analogues 10 and 8a
a
(i) K2OsO2(OH)4, K2CO3, K3Fe(CN)6, (DHQ)2-PYR in t-BuOH/
H2O (v/v ) 1:1), ice bath; (ii) (S)-(-)-camphanic chloride, pyridine
in CH2Cl2.
F igu r e 2. Torsional angles of 4-methyl DCK, 2, 8, and 9. The
Ta ble 1. Anti-HIV Activity of Disubstituted DCK Analogues
torsion angle A-B-C-D is defined as positive if, when viewed
along the B-C bond, atom A must be rotated clockwise to
eclipse atom D.
1-10a in H9 Lymphocytesb
compd
IC50 (µM)
EC50 (µM)
TI
1
2
3
4
>154
>154
>154
3.75
1.92 × 10-3
4.19 × 10-3
9.10 × 10-3
4.69 × 10-3
2.01 × 10-3
>8.02 × 104
>3.68 × 104
>1.69 × 104
1.25 × 103
5.17 × 104
>3.20
anti-HIV activity and were more active than DCK,
dimethyl DCK analogues 1-4 all showed lower anti-
HIV activity than DCK in H9 lymphocytes. However,
5-methoxy-4-methyl DCK (8) was more active than
DCK, AZT, and 4,5-dimethyl DCK (2) and was compa-
rable to 4-methyl DCK. In contrast, 4-isopropyl-5-
methyl DCK (9) was completely inactive in the same
assay. These results prompted us to consider whether
steric compression between adjacent substituents could
alter the molecular three-dimensional orientation,
thereby directly affecting the molecular affinity with
target receptor/enzyme and resulting in different anti-
HIV activity.
5
104
6
7
8
9
>140
>148
>150
>147
135
43.7
2.12 × 10-3
>6.98 × 104
7.21 × 10-6
>2.08 × 107
no suppression
10
DCK
AZT
1.54
87.7
35
1875
2.56 × 10-4
1.37 × 105
4.50 × 10-2
4.17 × 104
a
All data presented are averages of at least two separate
b
experiments. Assay in H9 lymphocytes was performed by BBI-
Biotech Research Laboratories, Inc., Gaithersburg, MD.
marin nucleus. However, 4-methyl-3-phenyl DCK (6)
and 5-benzyloxy-4-methyl DCK (10) suppressed HIV-1
replication only slightly, suggesting that an aromatic
substituent on the coumarin nucleus is not favorable
for anti-HIV activity. Furthermore, 4-isopropyl-5-methyl
DCK (9) did not suppress HIV-1 replication in this
assay.
To obtain a better understanding of the SARs of
disubstituted DCK analogues, we performed conforma-
tional searches for 4-methyl DCK, 2, 8, and 9 using
Sybyl software. The resulting stable three-dimensional
structures and torsional angles (Figure 2) suggested
that the steric compression between two bulky substit-
uents at the 4- and 5-positions caused deformation of
the coumarin system. The 5-methyl groups in the
structures of 9 and 2 were forced out of the resonance
plane with a torsional angle [C(4)-C(4a)-C(5)-C(Me-
5)] of 42.9° in 9 and 29.5° in 2. Comparatively, the
Discu ssion
Although monomethyl DCK analogues (3-, 4-, and
5-methyl DCK) previously exhibited extremely potent

Anti-AIDS Agents
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 5 667
marin system. With 2 and 9, the corresponding absorp-
tion bands shifted to 298 and 300 nm, respectively. This
blue shift of the absorption band indicates a loss of
coumarin resonance because of steric compression be-
tween the two substitutents at the 4- and 5-positions.
In contrast, 8 possessed an absorption band at 318 nm,
very similar to that found in 4-methyl DCK.
The foregoing studies indicated that the presence of
substituents at both the 4- and 5-positions results in
overcrowding, which is relieved in part by out-of-plane
displacements thereby disturbing the overall planarity
of the coumarin system. Accordingly, these steric effects
and consequent skeletal deformation might be respon-
sible for the lack of anti-HIV activity in 9 and obvious
decrease in 2. On the other hand, with 5-methoxy-4-
methyl DCK (8), the unfavorable steric interaction of
the methoxy group with the 4-methyl group is mini-
mized by (i) rotation of the C(5)-O bond and (ii) the
C(4)-CH3 bond. Therefore, the coumarin moiety in
compound 8 maintains maximum resonance and is
significantly more active than DCK as an anti-HIV
agent.
In summary, these studies indicated the following
conclusions: (1) A planar coumarin system is probably
an essential structural feature for potent anti-HIV
activity. (2) Steric compression of C(4) and C(5) sub-
stituents of the coumarin moiety can reduce the overall
planarity and thus resonance of the coumarin system,
resulting in decreased or completely lost anti-HIV
activity. (3) Methyl or other aliphatic substitutions on
the coumarin nucleus are favorable for anti-HIV activ-
ity, whereas aromatic substituents are not.
F igu r e 3. X-ray structure of 4-methyl DCK. ORTEP diagram
(40% probability ellipsoids) showing the crystallographic atom-
numbering scheme and solid-state conformation; small filled
circles represent hydrogen atoms.
Exp er im en ta l Section
Ch em istr y. Melting points were measured with a Fisher
J ohns melting apparatus without correction. 1H NMR spectra
were measured on a Bruker AC-300 MHz spectrometer using
TMS as internal standard. The solvent used was CDCl3 unless
indicated. UV spectra were measured on a Shimadzu UV-
2101PC, UV-vis scanning spectrophotometer using EtOH as
solvent. Elemental analyses were performed by Atlantic Mi-
crolab, Inc., Norcross, GA. All target compounds were analyzed
for C, H and gave values within (0.4% of the theoretical
values. Optical rotations were measured with a J asco Dip-1000
digital polarimeter at 25 °C at the sodium D-line. The diaste-
reoisomeric excess percentages were determined from intensity
1
of protons at the 3′-position in the H NMR spectra. TLC was
performed on a precoated silica gel GF plate purchased from
Analtech, Inc. Silica gel (200-400 mesh) from Aldrich, Inc.,
was used for column chromatography. All other chemicals were
obtained from Aldrich, Inc.
Gen er a l P r oced u r e for Syn th esizin g Disu bstitu ted
7-Hyd r oxycou m a r in s 11. To a mixture of an appropriate
phenol and a â-keto ester in a 1:1.5 molar ratio was slowly
added excess H2SO4 (98%) with stirring at 0 °C within 1 h.
Then, the mixture was stirred at room temperature until the
reaction was complete as monitored by TLC. The mixture was
poured into ice-water and allowed to stand overnight. The
precipitated solid was filtered, washed with water until
neutral, and dried in vacuo to afford the product.
F igu r e 4. UV spectra of 4-methyl DCK, 2, 8, and 9.
corresponding torsional angles in 8 and 4-methyl DCK
were 3.3° and 2.0°, respectively. Other torsional angles
in 2 and 9 also show obvious deviations from planarity,
unlike those in 4-methyl DCK and 8, which are close to
0° or 180°. Furthermore, the solid-state conformation
and torsion angle data for 4-methyl DCK from an X-ray
diffraction study (see Figure 3) were similar to the
computational data.
The UV spectral data of 4-methyl DCK, 2, 8, and 9
(Figure 4) supported their calculated conformations. The
longest wavelength absorption band for 4-methyl DCK
was at 320 nm, indicating full resonance in the cou-
3,4-Dim eth yl-7-h ydr oxycou m ar in (11a): 87% yield (start-
ing with 8 mmol of 1,3-dihydroxybenzene and 12 mmol of ethyl
2-methylacetoacetate for 16 h); colorless crystals from EtOH;
1
mp 220 °C dec; H NMR (CD3OD) δ 2.11 (3H, s, CH3-3), 2.38
(3H, s, CH3-4), 6.65 (1H, d, J ) 2.4 Hz, H-8), 6.77 (1H, dd, J
) 2.4 and 8.8 Hz, H-6), 7.57 (1H, d, J ) 8.8 Hz, H-5).
4,5-Dim eth yl-7-h ydr oxycou m ar in (11b): 77% yield (start-
ing with 4 mmol of 3,5-dihydroxytoluene and 6 mmol of ethyl
acetoacetate for 4 h); yellow solid; mp 235 °C dec; 1H NMR

668 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 5
Xie et al.
(CD3OD) δ 2.31 (3H, s, CH3-5), 2.60 (3H, s, CH3-4), 5.98 (1H,
s, H-3), 6.54 (1H, s, H-8), 6.59 (1H, s, H-6).
2.0 and 8.8 Hz, H-5), 6.28 (1H, J ) 2.0 Hz, H-3), 6.84 (1H, d,
J ) 8.8 Hz, H-6).
Gen er a l P r oced u r e for Syn th esizin g Disu bstitu ted
Seselin s 16. A mixture of disubstituted 7-hydroxycoumarin
11 (5 mmol), K2CO3 (12.5 mmol), KI (5 mmol), and 3-chloro-
3-methyl-1-butyne (1-2 mL, excess) in DMF (10 mL) was
heated to 70-80 °C with stirring until the reaction was
complete as monitored by TLC. Solid K2CO3 was filtered. The
filtrate was concentrated in vacuo. The residue, without
purification, was directly heated to reflux in 10 mL of N,N-
diethylaniline for 4-6 h. The reaction mixture was cooled to
room temperature, diluted with EtOAc and washed with 10%
aq HCl, water and brine. The organic layer was separated and
solvent was removed in vacuo. The residue was purified by
column chromatography or TLC with an eluant of hexane:
EtOAc ) 7:3 to afford the substituted seselin 16.
7-Hydr oxy-4,6-dim eth ylcou m ar in (11d): 92% yield (start-
ing with 4 mmol of 2,4-dihydroxytoluene 15 and 6 mmol of
ethyl acetoacetate for 24 h); yellow solid; mp 268 °C dec; H
NMR (CD3OD) δ 2.39 (3H, s, CH3-6), 2.57 (3H, s, CH3-4), 6.21
(1H, s, H-3), 6.58 (1H, s, H-8), 7.61 (1H, s, H-5).
7-Hyd r oxy-3,4-tetr a h yd r oben zocou m a r in (11g): 98%
yield (starting with 8 mmol of 1,3-dihydroxybenzene and 12
mmol of ethyl 2-cyclohexanonecarboxylate for 24 h); yellow
solid; mp 225-7 °C; 1H NMR (DMSO) δ 1.73 (4H, m, 2 × CH2),
2.39 (2H, t, J ) 5.7 Hz, CH2-4), 2.73 (2H, t, J ) 5.7 Hz, CH2-
3), 6.69 (1H, d, J ) 2.4 Hz, H-8), 6.77 (1H, dd, J ) 2.4 and 8.8
Hz, H-6), 7.54 (1H, d, J ) 8.8 Hz, H-5), 10.35 (1H, br, OH-7).
1
7-Hyd r oxy-5-m eth oxy-4-m eth ylcou m a r in (11h ): 72%
yield (starting with 4 mmol of 5-methoxyresorcinol and 6 mmol
3,4-Dim eth ylseselin (16a ): yield 38% (starting with 950
mg of 11a ); mp 121-2 °C; 1H NMR δ 1.48 (6H, s, 2 × CH3-2′),
2.20 (3H, s, CH3-3), 2.36 (3H, s, CH3-4), 5.72 (1H, d, J ) 10.2
Hz, H-3′), 6.74 (1H, d, J ) 8.8 Hz, H-6), 6.94 (1H, d, J ) 10.2
Hz, H-4′), 7.36 (1H, d, J ) 8.8 Hz, H-5).
1
of ethyl acetoacetate for 4 h); yellow solid; mp 223-6 °C; H
NMR δ 2.46 (3H, s, CH3-4), 3.77 (3H, s, CH3O-5), 5.93 (1H, s,
H-3), 6.31 (1H, d, J ) 2.4 Hz, H-8), 6.42 (1H, d, J ) 2.4 Hz,
H-6).
7-Hyd r oxy-4-isop r op yl-5-m eth ylcou m a r in (11i): 33%
yield (starting with 4 mmol of 3,5-dihydroxytoluene and 6
mmol of ethyl isobutyryl acetate for 16 h); white solid; mp
4,5-Dim eth ylseselin (16b): yield 38% (starting with 440
1
mg of 11b); mp 95-6 °C; H NMR δ 1.49 (6H, s, 2 × CH3-2′),
2.35 (3H, s, CH3-4), 2.60 (3H, s, CH3-5), 5.64 (1H, d, J ) 10.2
Hz, H-3′), 6.04 (1H, s, H-3), 6.48 (1H, d, J ) 10.2 Hz, H-4′),
6.69 (1H, s, H-6).
1
173-4 °C; H NMR δ 1.22 (6H, d, J ) 6.9 Hz, CH(CH3)2-4),
2.30 (3H, s, CH3-5), 4.11 (1H, m, J ) 6.9 Hz, CH(CH3)2-4), 4.59
(br, OH-7), 6.10 (1H, s, H-3), 6.55 (1H, s, H-8), 6.63 (1H, H-6).
3,5-Dim eth ylseselin (16c): yield 40% (starting with 250
mg of 11c); mp 184-7 °C; 1H NMR δ 1.46 (6H, s, 2 × CH3-2′),
2.20 (3H, s, CH3-3), 2.45 (3H, s, CH3-5), 5.66 (1H, d, J ) 10.2
Hz, H-3′), 6.58 (1H, s, H-6), 6.88 (1H, d, J ) 10.2 Hz, H-4′),
7.62 (1H, s, H-4).
4,6-Dim eth ylseselin (16d ): yield 43% (starting with 700
mg of 11d ); mp 153-5 °C; 1H NMR δ 1.46 (6H, s, 2 × CH3-2′),
2.22 (3H, s, CH3-6), 2.36 (3H, s, CH3-4), 5.70 (1H, d, J ) 10.2
Hz, H-3′), 6.09 (1H, s, H-3), 6.90 (1H, d, J ) 10.2 Hz, H-4′),
7.19 (1H, s, H-5).
3-Ch lor o-4-m eth ylseselin (16e): yield 26% (starting with
378 mg of 11e); mp 154-5 °C; 1H NMR δ 1.55 (6H, s, 2 × CH3-
2′), 2.51 (3H, s, CH3-4), 5.72 (1H, d, J ) 10.2 Hz, H-3′), 6.76
(1H, d, J ) 8.8 Hz, H-6), 6.86 (1H, d, J ) 10.2 Hz, H-4′), 7.35
(1H, d, J ) 8.8 Hz, H-5).
4-Meth yl-3-p h en ylseselin (16f): yield 38% (starting with
504 mg of 11f); light yellow plate; mp 97-9 °C; 1H NMR δ
1.50 (6H, s, 2 × CH3-2′), 2.26 (3H, s, CH3-4), 5.75 (1H, d, J )
10.2 Hz, H-3′), 6.78 (1H, J ) 8.8 Hz, H-6), 7.00 (1H, d, J )
10.2 Hz, H-4′), 7.30 (1H, J ) 8.8 Hz, H-5), 7.45 (5H, m, ArH-
3).
7-Hyd r oxy-3,5-d im eth ylcou m a r in (11c). To a solution of
14 (273 mg, 1.8 mmol) in 5 mL of anhydrous DMF under N2
was added Ph3PdCCH3COOMe (2.0 mmol). The mixture was
stirred and heated to reflux for 6 h. After cooling to room
temperature, the mixture was diluted with EtOAc and ex-
tracted three times with 10% aq KOH. The combined water
layer was acidified with 10% aq HCl to pH 3. Then, the
precipitated solid was filtered and solution was extracted again
with EtOAc three times. The organic phase was washed with
water until neutral, then dried over Na2SO4. Finally, removal
of the solvent furnished 277 mg of pure 11c: 81% yield; mp
1
147 °C dec; H NMR δ 2.12 (3H, s, CH3-3), 2.39 (3H, s, CH3-
5), 6.57 (2H, s, H-6 & H-8), 7.61 (1H, s, H-4).
7-Hyd r oxy-4-m eth yl-3-p h en ylcou m a r in (11f). A mixture
of 2,4-dihydroxyacetophenone (1.52 g, 10 mmol) and phenyl-
acetyl chloride (4,4 mL, 30 mmol) in acetone in the presence
of K2CO3 was heated to reflux for 30 h. K2CO3 was filtered
and solvent was removed. The residue was crystallized from
EtOH to produce needle crystals of 1.2 g of 11f: 48% yield;
1
mp 225-8 °C dec; H NMR (CD3Cl + CD3OD) δ 2.23 (3H, s,
CH3-4), 6.78 (1H, d, J ) 2.4 Hz, H-8), 6.80 (1H, dd, J ) 2.4
and 8.8 Hz, H-6), 7.23-7.41 (5H, m, ArH), 7.50 (1H, d, J )
8.8 Hz, H-5).
3,4-Tetr a h yd r oben zoseselin (16g): yield 33% (starting
1
with 648 mg of 11g); mp 152-4 °C; H NMR δ 1.49 (6H, s, 2
× CH3-2′), 1.82 (4H, m, 2 × CH2), 2.57 (2H, m, CH2-4), 2.73
(2H, m, CH2-3), 5.72 (1H, d, J ) 10.2 Hz, H-3′), 6.73 (1H, J )
8.8 Hz, H-6), 6.94 (1H, d, J ) 10.2 Hz, H-4′), 7.32 (1H, J ) 8.8
Hz, H-5).
2,4-Dih yd r oxy-6-m eth ylben za ld eh yd e (14). 1 mL of
phosphorus oxychloride (POCl3) was added dropwise into 5 mL
of DMF below 10 °C with rapid stirring over 0.5 h. 3,5-
Dihydroxytoluene (12) (1.10 g, 8.88 mmol) in 5 mL of DMF
was added slowly keeping the temperature below 10 °C. The
mixture was warmed to room temperature and stirred for 1
h. Ice and 10% aq NaOH were then added successively until
the pH was 9-10 and solid appeared. The mixture was heated
to boiling for 10 min, then adjusted to pH 3 with 10% aq HCl
after cooling to room temperature. The solid product was
collected, washed with water until neutral, and dried to give
5-Meth oxy-4-m eth ylseselin (16h ): yield 40% (starting
1
with 500 mg of 11h ); mp 174-5 °C; H NMR δ 1.45 (6H, s, 2
× CH3-2′), 2.53 (3H, s, CH3-4), 3.87 (3H, s, CH3O-5), 5.58 (1H,
d, J ) 10.2 Hz, H-3′), 5.94 (1H, s, H-3), 6.26 (1H, s, H-6), 6.84
(1H, d, J ) 10.2 Hz, H-4′).
4-Isop r op yl-5-m eth ylseselin (16i): yield 22% (starting
with 218 mg of 11i); mp 65-8 °C; 1H NMR δ 1.27 (6H, d, J )
6.9 Hz, CH(CH3)2-4), 1.50 (6H, s, 2 × CH3-2′), 2.35 (3H, s, CH3-
5), 4.00 (1H, m, J ) 6.9 Hz, CH(CH3)2-4), 5.65 (1H, d, J )
10.2 Hz, H-3′), 6.22 (1H, s, H-3), 6.50 (1H, d, J ) 10.2 Hz,
H-4′), 6.72 (1H, s, H-6).
1
14: 1.25 g, 93% yield; mp 152-4 °C; H NMR δ 2.55 (3H, s,
CH3), 6.20 and 6.22 (each 1H, s, ArH), 10.20 (1H, CHO), 5.50
and 12.40 (OH-4 and OH-2, disappeared in D2O).
2,4-Dih yd r oxytolu en e (15). To a solution of 2,4-dihy-
droxybenzaldehyde (690 mg, 5 mmol) and sodium cyanoboro-
hydride (1.0 g) in 30 mL of THF was added methyl orange as
an indictor, giving the solution a yellow color; aq HCl solution
(15 mL, 1 N in water) was slowly added to the reaction system,
keeping the solution orange. The mixture was stirred for 3 h
at room temperature. Water was added, and the mixture was
extracted with Et2O three times. After removal of solvent,
product 15 (611 mg) was obtained: 98.6% yield; mp 105-6
Gen er a l P r oced u r e of Asym m etr ic Dih yd r oxyla tion
a n d Acyla tion for Syn th esizin g Su bstitu ted (3′R,4′R)-Di-
O-(-)-ca m p h a n oyl-(+)-cis-k h ella cton es. A mixture of K3-
Fe(CN)6 (150 mg, 0.75 mmol), K2CO3 (105 mg, 0.75 mmol), 2,5-
diphenyl-4,6-bis(9-O-dihydroquinyl)pyrimidine [(DHQ)2-PYR]
(4.4 mg, 0.005 mmol), and K2OsO2(OH)4 (1.8 mg, 0.005 mmol)
was solubilized in 5 mL of t-BuOH/H2O (v/v, 1:1) at room
temperature. Then, the solution was cooled to 0 °C and
methanesulfonamide (0.25 mmol) added under stirring. When
the solution turned from a light yellow to an orange color, the
1
°C; H NMR (CD3OD) δ 2.06 (3H, s, CH3), 6.20 (1H, dd, J )

Anti-AIDS Agents
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 5 669
substituted seselin compound (0.25 mmol) was added. The
mixture was stirred at 0 °C for 2-4 days. Na2S2O5 (excess),
water, and CHCl3 were added. After stirring 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. The residue was separated by TLC to
obtain the pure substituted (+)-cis-khellactone. However, the
substituted (+)-cis-khellactone could be directly acylated,
without further purification, with (S)-(-)-camphanic chloride
(excess) in Py/CH2Cl2 for 1-2 days at room temperature. The
mixture was diluted with EtOAc and washed with 10% aq HCl,
water and brine, successively. The organic phase was dried
over anhydrous MgSO4, filtered, and concentrated. The residue
was separated by TLC (eluant: hexane/EtOAc ) 7:3) and
afforded the appropriately substituted 3′,4′-di-O-(S)-campha-
noyl-(+)-cis-khellactone derivative.
2.24, and 2.52 (each 2H, m, CH2 in camphanoyl group), 1.80
(4H, m, 2 × CH2), 2.54 (2H, m, CH2), 2.72 (2H, m, CH2), 5.39
(1H, d, J ) 4.8 Hz, H-3′), 6.64 (1H, d, J ) 4.8 Hz, H-4′), 6.82
(1H, d, J ) 8.8 Hz, H-6), and 7.50 (1H, d, J ) 8.8 Hz, H-5);
75% d.e.; [R]D +9.00° (c 2.29, CHCl3). Anal. (C38H44O11‚1/2H2O)
C, H.
(3′R,4′R)-3′,4′-Di-O-(S)-ca m p h a n oyl-5-m eth oxy-4-m eth -
yl-(+)-cis-k h ella cton e (8): yield 44% (starting with 125 mg
of 16h ); mp 144-6 °C; UV λmax nm (ꢀ) 318 (7215), 257 (5225),
1
248 (5135), 215 (13440); H NMR δ 0.98-1.14 (15H, ms, 5 ×
CH3), 1.44, 1.57 and 2.14 (each 3H, s, CH3), 1.69, 1.97, 2.17,
and 2.52 (each 2H, m, CH2 in camphanoyl group), 2.64 (3H, s,
CH3-4), 3.89 (3H, s, CH3O-5), 5.36 (1H, d, J ) 4.8 Hz, H-3′),
5.95 (1H, s, H-3), 6.27 (1H, s, H-6), 6.58 (1H, d, J ) 4.8 Hz,
H-4′); 90% d.e.; [R]D -9.62° (c 0.52, CHCl3). Anal. (C36H42O12
‚
1/2H2O) C, H.
(3′R,4′R)-3,4-Dim et h yl-3′,4′-d i-O-(S)-ca m p h a n oyl-(+)-
cis-k h ella cton e (1): yield 64% (starting with 256 mg of 16a );
white solid; mp 118-20 °C; 1H NMR δ 0.93-1.12 (15H, ms, 5
× CH3), 1.27, 1.49, and 1.55 (each 3H, s, CH3), 1.73, 1.92, 2.20,
and 2.48 (each 2H, m, CH2 in camphanoyl group), 2.13 (3H, s,
CH3-3), 2.38 (3H, s, CH3-4), 5.40 (1H, d, J ) 4.8 Hz, H-3′),
6.66 (1H, d, J ) 4.8 Hz, H-4′), 6.63 (1H, d, J ) 8.8 Hz, H-6),
and 7.54 (1H, d, J ) 8.8 Hz, H-5); 88% d.e.; [R]D +6.14° (c 2.15,
CHCl3). Anal. (C36H42O11‚2H2O) C, H.
(3′R,4′R)-3′,4′-Di-O-(S)-cam ph an oyl-4-isopr opyl-5-m eth -
yl-(+)-cis-k h ella cton e (9): yield 36% (starting with 110 mg
of 16i); white solid; mp 218-20 °C; UV λmax nm (ꢀ) 300 (6392),
208 (14875); 1H NMR δ 0.96-1.15 and 1.27-1.30 (18H, ms, 6
× CH3), 1.50 and 1.57 (each 3H, s, CH3), 1.69, 1.94, 2.19, and
2.49 (each 2H, m, CH2 in camphanoyl group), 1.12 and 1.13
(each 3H, d, J ) 6.6 Hz, CH(CH3)2-4), 2.24 (3H, s, CH3-5), 3.12
(1H, m, J ) 6.6 Hz, CH(CH3)2-4), 5.39 (1H, d, J ) 4.8 Hz, H-3′),
6.28 (1H, s, H-3), 6.40 (1H, d, J ) 4.8 Hz, H-4′), 6.86 (1H, s,
(3′R,4′R)-4,5-Dim et h yl-3′,4′-d i-O-(S)-ca m p h a n oyl-(+)-
cis-k h ella cton e (2): yield 70% (starting with 256 mg of 16b);
white solid; mp 137-8 °C; UV λmax nm (ꢀ) 298 (6141), 215
H-6); 95% d.e.; [R]D -54.74° (c 0.38, CHCl3). Anal. (C38H46O11
)
C, H.
5,7-Dih yd r oxy-2,2-d im eth yl-4-ch r om a n on e (17). To a
mixture of 1,3,5-benzenetriol dihydrate (6.48 g, 40 mmol) and
3,3-dimethylacrylic acid (4.80 g, 48 mmol) was added 20 mL
of BF3‚Et2O at room temperature, and the mixture was heated
to 70 °C for 2.5 h, when the solution color changed to orange
from light yellow. After cooling to room temperature, the
reaction mixture was poured into ice-water and 10% aq KOH
added to pH 10. The solution was washed with EtOAc three
times. Then the aqueous solution was acidified by 10% aq HCl
and stirred for 10 min. The resulting precipitate was collected
by filtration, and washed with water until neutral. After
drying, 6.90 g of 17 was obtained: 83% yield; mp 189-90 °C;
1H NMR δ 1.46 (6H, s, 2 × CH3-2), 2.70 (2H, s, CH2-3), 5.37
(1H, br, OH-7), 5.88 and 5.94 (each 1H, d, J ) 2.1 Hz, ArH-6
and ArH-8), 12.04 (1H, s, OH-5, chelating with carbonyl at
C-4).
1
(12782); H NMR δ 0.96-1.15 (18H, ms, 6 × CH3), 1.50 and
1.56 (each 3H, s, CH3), 1.73, 1.93, 2.22, and 2.46 (each 2H, m,
CH2 in camphanoyl group), 2.24 (3H, s, CH3-4), 2.58 (3H, s,
CH3-5), 5.38 (1H, d, J ) 4.8 Hz, H-3′), 6.12 (1H, s, H-3), 6.41
(1H, d, J ) 4.8 Hz, H-4′), 6.83 (1H, s, H-6); 72% d.e.; [R]D
-70.77° (c 0.38, CHCl3). Anal. (C36H42O11‚H2O) C, H.
(3′R,4′R)-3,5-Dim et h yl-3′,4′-d i-O-(S)-ca m p h a n oyl-(+)-
cis-k h ella cton e (3): yield 82% (starting with 70 mg of 16c);
white solid; mp 98-100 °C; 1H NMR δ 0.93-1.12 (15H, ms, 5
× CH3), 1.27, 1.42, and 1.46 (each 3H, s, CH3), 1.69, 1.89, 2.20,
and 2.50 (each 2H, m, CH2 in camphanoyl group), 2.16 (3H, s,
CH3-3), 2.46 (3H, s, CH3-5), 5.36 (1H, d, J ) 4.8 Hz, H-3′),
6.61 (1H, d, J ) 4.8 Hz, H-4′), 6.65 (1H, s, H-6), and 7.61 (1H,
s, H-4); 80% d.e.; [R]D -4.47° (c 0.76, CHCl3). Anal. (C36H42O11
‚
1/2H2O) C, H.
(3′R,4′R)-4,6-Dim et h yl-3′,4′-d i-O-(S)-ca m p h a n oyl-(+)-
cis-k h ella cton e (4): yield 68% (starting with 400 mg of 16d );
white solid; mp 250 °C dec; 1H NMR δ 0.99-1.11 (18H, ms, 6
× CH3), 1.47, and 1.49 (each 3H, s, CH3), 1.65, 1.92, 2.20, and
2.45 (each 2H, m, CH2 in camphanoyl group), 2.25 (3H, s, CH3-
6), 2.98 (3H, s, CH3-4), 5.39 (1H, d, J ) 4.8 Hz, H-3′), 6.09
(1H, s, H-3), 6.65 (1H, d, J ) 4.8 Hz, H-4′), and 7.37 (1H, s,
7-Ben zyloxy-2,2-dim eth yl-5-h ydr oxy-4-ch r om an on e (18).
A mixture of compound 17 (624 mg, 3 mmol), benzyl bromide
(0.37 mL, 3 mmol), and K2CO3 (828 mg, 6 mmol) was refluxed
in acetone (20 mL) for 2 h. The solid was filtered, solvent was
removed, and the residue was recrystallized from 90% EtOH
to give white needle crystals of 18: 644 mg, 72%; mp 131-2
°C; 1H NMR δ 1.47 (6H, s, 2 × CH3-2), 2.70 (2H, s, CH2-3),
5.07 (2H, s, CH2Ph-7), 6.02 and 6.10 (each 1H, d, J ) 2.1 Hz,
ArH-6 and ArH-8), 7.39 (5H, m, PhH-7), 12.02 (1H, s, OH-5,
chelating with carbonyl at C-4).
2,2-Dim eth yl-5-h ydr oxy-7-m eth oxy-4-ch r om an on e (19).
A mixture of 17 (5.20 g, 25.0 mmol), K2CO3 (16.91 g, 50.0
mmol), and MeI (2.34 mL, 35 mmol) in acetone (50 mL) was
refluxed for 1 h. The reaction mixture was filtered and solvent
was removed in vacuo. The residue was extracted with hexane
several times. The hexane was removed to give 19: 4.11 g,
74%; mp 71-3 °C; 1H NMR δ 1.47 (6H, s, 2 × CH3-2), 2.70
(2H, s, CH2-3), 3.82 (3H, s, CH3O), 5.95 and 6.02 (each 1H, d,
J ) 2.1 Hz, ArH-6 and ArH-8), 12.03 (1H, s, OH-5, chelating
with carbonyl at C-4).
5-Ben zyloxy-4-m eth yl-3′,4′-d ih yd r oseselin (20). Com-
pound 18 (298 mg, 1 mmol) in THF (15 mL) was added
dropwise to NaBH4 (140 mg, 5 mmol) in THF (5 mL), then 1
mL of 10% aq KOH was added to completely solubilize the
NaBH4. The mixture was refluxed for 24 h. After cooling to
room temperature, the reaction mixture was poured into ice-
water, acidified with 10% aq HCl to neutral, and extracted
with Et2O three times. The organic phase was washed with
brine, and dried over anhydrous Na2SO4. After removal of the
solvent, crude product was dissolved in anhydrous CH2Cl2 (8
mL), and ethyl acetoacetate (0.8 mL, 0.67 mmol) and BF3‚Et2O
H-5); 86% d.e.; [R]D -2.00° (c 0.50, CHCl3). Anal. (C36H42O11
‚
H2O) C, H.
(3′R,4′R)-3-Ch lor o-3′,4′-d i-O-(S)-ca m p h a n oyl-4-m eth yl-
(+)-cis-k h ella cton e (5): yield 31% (starting with 69 mg of
16e); white solid; mp 203-4 °C; MS m/z (%) 670 (M+, 10), 672
(M + 2, 3); 1H NMR δ 0.99-1.13 (15H, ms, 5 × CH3), 1.28,
1.46, and 1.49 (each 3H, s, CH3), 1.72, 1.94, 2.23, and 2.52 (each
2H, m, CH2 in camphanoyl group), 2.64 (3H, s, CH3-4), 5.38
(1H, d, J ) 4.8 Hz, H-3′), 6.60 (1H, d, J ) 4.8 Hz, H-4′), 6.85
(1H, d, J ) 8.8 Hz, H-6), and 7.56 (1H, d, J ) 8.8 Hz, H-5);
76% d.e.; [R]D -343.2° (c 0.50, CHCl3). Anal. (C35H39O11Cl).
(3′R,4′R)-3′,4′-Di-O-(S)-ca m p h a n oyl-4-m eth yl-3-p h en yl-
(+)-cis-k h ella cton e (6): yield 61% (starting with 140 mg of
1
16f); white solid; mp 157-9 °C; H NMR δ 0.96-1.15 (15H,
ms, 5 × CH3), 1.27, 1.47, and 1.52 (each 3H, s, CH3), 1.71, 1.92,
2.20, and 2.52 (each 2H, m, CH2 in camphanoyl group), 2.29
(3H, s, CH3-4), 5.44 (1H, d, J ) 4.8 Hz, H-3′), 6.70 (1H, d, J )
4.8 Hz, H-4′), 6.88 (1H, d, J ) 8.8 Hz, H-6), 7.44 (5H, m, ArH-
3) and 7.62 (1H, d, J ) 8.8 Hz, H-5); 87% d.e.; [R]D +20.08° (c
2.58, CHCl3). Anal. (C41H44O11‚1/2H2O) C, H.
(3′R,4′R)-3′,4′-Di-O-(S)-cam ph an oyl-3,4-tetr ah ydr oben zo-
(+)-cis-k h ella cton e (7): yield 37% (starting with 141 mg of
1
16g); white solid; mp 160-3 °C; H NMR δ 0.96-1.12 (15H,
ms, 5 × CH3), 1.29, 1.44, and 1.48 (each 3H, s, CH3), 1.70, 1.92,

670 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 5
Xie et al.
were added under N2 protection with stirring at room tem-
perature. After 24 h, the reaction was complete as monitored
by TLC. The mixture was poured into ice-water and extracted
with CH2Cl2 three times. The organic phase was washed with
water, brine, and dried over anhydrous Na2SO4. The solvent
was removed and the residue was purified by TLC to give 20:
supernatants. Toxicity was determined by performing cell
counts on a Coulter counter on the mock-infected H9 lympho-
cytes, which had either received culture medium (no toxicity)
or test sample or AZT.
X-r a y Cr ysta l Str u ctu r e An a lysis of 4-Meth yl-(3′R,4′R)-
3′,4′-d i-O-(S)-ca m p h a n oyl-(+)-cis-k h ella cton e.
Crystal
1
193 mg, 55%; mp 137-9 °C; H NMR δ 1.37 (6H, s, 2 × CH3-
data: C35H40O11, mol wt ) 636.70, hexagonal, space group P65-
(C63) No. 170, a ) b ) 13.748(1) Å, c ) 29.990(4) Å, R ) 90.0(-
2′), 1.84 (2H, t, J ) 6.6 Hz, CH2-3′), 2.49 (3H, s, CH3-4), 2.83
(2H, t, J ) 6.6 Hz, CH2-4′), 5.05 (2H, s, OCH2-7), 5.94 (1H, s,
H-3), 6.33 (1H, s, H-6), 7.39 (5H, m, ArH-7).
)°, â ) 90.0(-)°, γ ) 120.0(-)°, V ) 4909(1) Å3, Z ) 6, Dcalcd
)
,
1.292 g cm-3, µ (Cu KR radiation, λ ) 1.5418 Å) ) 7.6 cm-1
crystal dimensions ) 0.40 × 0.40 × 0.60 mm.
5-Meth oxy-4-m eth yl-3′,4′-d ih yd r oseselin (21). To a mix-
ture of 19 (4.44 g, 20.0 mmol) in 10% aq KOH (100 mL) and
toluene (50 mL) was added NaBH4 (3.78 g, 20.0 mmol) and
the solution refluxed for 4 h. The subsequent procedure was
the same as in the preparation of 20. 21: 2.62 g, 48%; mp
174-5 °C; 1H NMR δ 1.38 (6H, s, 2 × CH3-2′), 1.85 (2H, t, J )
6.6 Hz, CH2-3′), 2.55 (3H, s, CH3-4), 2.83 (2H, t, J ) 6.6 Hz,
CH2-4′), 3.83 (3H, s, OCH3), 5.94 (1H, s, H-3), 6.23 (1H, s, H-6).
5-Ben zyloxy-4-m eth ylseselin (22). A solution of 20 (100
mg, 0.30 mmol) in 1,4-dioxane (12 mL) was added to a solution
of DDQ (160 mg, 0.7 mmol) in anhydrous 1,4-dioxane (12 mL)
over 1 h under a nitrogen atmosphere at room temperature.
The darkened reaction mixture was then heated to reflux for
24 h and solvent removed in vacuo. The residue was dissolved
in CH2Cl2 and passed through a short column of Florisil (60-
100 mesh), then washed with CH2Cl2 to obtain 22: 70 mg, 67%;
mp 153-4 °C; 1H NMR δ 1.47 (6H, s, 2 × CH3-2′), 2.47 (3H, s,
CH3-4), 5.09 (2H, s, OCH2-7), 5.58 (1H, d, J ) 10 Hz, H-3′),
5.93 (1H, s, H-3), 6.36 (1H, s, H-6), 6.85 (1H, d, J ) 10 Hz,
H-4′), 7.42 (5H, m, ArH-7).
All measurements were made at 298 K on an Enraf-Nonius
CAD-4 diffractometer (Cu KR radiation, graphite monochro-
mator). Laue symmetry and systematic absences (00l when l
* 6n) indicated that the space group was P61 or P65; the latter
alternative was shown during the course of the analysis to be
the correct choice (vide infra). Refined unit cell parameters
were calculated from the diffractometer setting angles for 25
reflections (35° < θ < 40°) widely separated in reciprocal space.
Intensity data (3421 nonequivalent +h,+k,-l reflections),
recorded by means of θ-2θ scans (scan width (0.80 + 0.14 tan
θ)°; θmax ) 75°), yielded 2897 reflections with I > 2.0σ(I) for
use in the structure analysis and parameter refinement. The
intensities of four reference reflections, monitored every 2 h
during data collection, showed no significant variation (<1%
overall). The data were corrected for the usual Lorentz and
polarization effects.
The crystal structure was solved by direct methods. Ap-
proximate coordinates for all non-hydrogen atoms were ob-
tained from an E-map. Of the two alternative space goup
choices, P65 gave the enantiomer consistent with the known
absolute stereochemistry of the camphanoyl moiety. Positional
and temperature factor parameters (first isotropic and then
anisotropic) were adjusted by means of several rounds of full-
matrix least-squares calculations during which Σw∆2 [w )
1/σ2|Fo|, ∆ ) (|Fo| - |Fc|)] was minimized. Hydrogen atom
positional and isotropic thermal parameteres were also refined
during the later least-squares iterations and an extinction
correction (g) was included as a variable. The parameter
5-Meth oxy-4-m eth ylseselin (16h ). The procedure was
identical to that used for the preparation of 22: yield 48%
(starting with 4.45 g of 21); mp 174-5 °C.
(3′R,4′R)-5-Ben zyloxy-3′,4′-di-O-(S)-cam ph an oyl-4-m eth -
yl-(+)-cis-k h ella cton e (10). The procedure was the same as
for general preparation of other DCK analogues: yield 56%
1
(starting with 76 mg of 22); mp 111-3 °C; H NMR δ 0.98-
1.16 (15H, ms, 5 × CH3), 1.27, 1.44 and 1.50 (each 3H, s, CH3),
1.71, 1.94, 2.17, and 2.48 (each 2H, m, CH2 in camphanoyl
group), 2.47 (3H, s, CH3-4), 5.11 (2H, s, CH2O-5), 5.36 (1H, d,
J ) 4.8 Hz, H-3′), 5.94 (1H, s, H-3), 6.36 (1H, s, H-6), 6.59
(1H, d, J ) 4.8 Hz, H-4′), 7.42 (5H, m, ArH-5); 78% d.e.; [R]D
-18.13° (c 0.75, CHCl3). Anal. (C42H46O12‚1/2H2O) C, H.
refinement converged (maximum shift esd ) 0.03) at R ) Σ-
2
(||Fo| - |Fc||/|Fo|) ) 0.030, Rw ) [Σw(|Fo| - |Fc|)2/Σw|Fo| ]1/2
)
)
0.040, GOF ) [Σw(|Fo| - |Fc|)2/(Nobservations - Nparameters)]1/2
1.27. A final difference Fourier synthesis contained no unusual
features [∆F (e/Å3) ) 0.14 (maximum), -0.12 (minimum)].
HIV Gr ow th In h ibition Assa y in H9 Lym p h ocytes. The
T cell line, H9, was maintained in continuous culture with
complete medium (RPMI 1640 with 10% fetal calf serum (FCS)
supplemented with L-glutamine) at 5% CO2 and 37 °C. 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 concen-
trations routinely used for screening: 100, 20, 4 and 0.8 µg/
mL, but for active agents additional dilutions were prepared
for subsequent testing so that an accurate EC50 value could
be achieved. 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 was used for toxicity determina-
tions (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 aliquot
of H9 cells. The other aliquot of H9 cells only received culture
medium and then was incubated under identical conditions
as the HIV-infected H9 cells. After a 4-h incubation at 37 °C
and 5% CO2, both cell populations were washed 3 times with
fresh medium and 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 37 °C and 5% CO2 for 4 days. Cell-free superna-
tants were collected on day 4 for use in our in-house p24
antigen ELISA assay. p24 antigen is a core protein of HIV and
therefore is an indirect measure of virus present in the
Crystallographic calculations were performed by use of the
Enraf-Nonius Structure Determination Package (SDP 3.0). For
all structure factor calculations, neutral atom scattering
factors and their anomalous scattering corrections were taken
from International Tables.12
Ack n ow led gm en t. The authors thank the National
Cancer Institute, National Institutes of Health, for
performing the in vitro anti-HIV assay in the CEM-SS
cell line. This investigation was supported by Grant AI-
33066 from the National Institute of Allergies and
Infectious Diseases awarded to K.-H. Lee.
Su p p or tin g In for m a tion Ava ila ble: Tables of fractional
atomic coordinates and temperature factor parameters, bond
lengths, bond angles, and torsion angles for 4-methyl-(3′R,4′R)-
3′,4′-di-O-(S)-camphanoyl-(+)-cis-khellactone (4-methyl DCK).
This material is available free of charge via the Internet at
http://pubs.acs.org.
Refer en ces
(1) Yang, Z. Y.; Xia, Y.; Xia, P.; Brossi, A.; Cosentino, L. M.; Lee, K.
H. Anti-AIDS 41. Synthesis and anti-HIV activity of 3′,4′-di-O-
(-)-camphanoyl-(+)-cis-khelloactone (DCK) lactam analogues,
Bioorg. Med. Chem. Lett. 2000, 10, 1003-1005.
(2) Huang, L.; Kashiwada, Y.; Cosentino, L. M.; Fan, S., Chen, C.
H.; McPhail, A. T.; Fujioka, T.; Mihashi, K.; Lee, K. H. Anti-
AIDS agents 15. Synthesis and anti-HIV activity of dihydrose-
selins and related analogues. J . Med. Chem. 1994, 37, 3947-
3955.

Anti-AIDS Agents
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 5 671
(3) Xie, L.; Takeuchi, Y.; Cosentino, M. L.; Lee, K. H. Anti-AIDS
agents 37. Synthesis and structure-activity relationships of
monosubstituted (+)-cis-khellactone derivatives as potent anti-
HIV agents. J . Med. Chem. 1999, 42, 2662-2672.
(4) Fall, Y.; Santana, L.; Teijeira, M.; Uriarte, E. A convenient
synthesis of benzofuran-3-acetic acids. Heterocycles 1995, 41,
647-650.
(5) Smith, G. F. Indoles Part I. The formylation of indole and some
reactions of 3-formylindole. J . Chem. Soc. 1954, 3842-3846.
(6) Harayama, T.; Nakatsuka, K.; Nishioka, H.; Murakami, K.;
Ohmori, Y.; Takeuchi, Y.; Ishii, H.; Kenmotsu, K. Convenient
synthesis of a simple coumarin from salicyaldehyde and Wittig
reagent (III): Synthesis of nitrocoumarins. Heterocycles 1994,
38, 2729-2738.
(8) Neelakantan, S.; Raman, P. V.; Tinabaye, A. A new and
convenient synthesis of 4-methyl-3-phenyl-coumarins and 3-phe-
nylcoumarins. Indian J . Chem. 1982, 21B, 256-257.
(9) Xie, L.; Crimmins, M. T.; Lee, K. H. Asymmetric synthesis of
3′,4′-di-O-(S)-camphanoyl-(+)-cis-khellactone (DCK), a potent
anti-HIV agent. Tetrahedron Lett. 1995, 36, 4529-4532.
(10) Vijayalakshmi, C. S.; Subramanian, M. Rajendra Prasad, K. J .
Synthesis of 1-aryl-3-methyl-but-2-en-1-ones intermediates for
the synthesis of chromanone derivatives. Indian J . Chem. 1990,
29B, 661-663.
(11) Schuda, P. F.; Price, W. A. Total synthesis of isoflavones:
jamaicin, calopogonium isoflavone-B, pseudibaptigenin, and
maxima substance-B. Friedel-Crafts acylation reactions with
acid-sensitive substrates. J . Org. Chem. 1987, 52, 1972-1979.
(12) Ibers, J . A., Hamilton, W. C., Eds. International Tables for X-ray
Crystallography; Kynoch: Birmingham, U.K., 1974; Vol. IV.
(7) Harayama, T.; Katsano, K.; Nishioka, H.; Fujii, M.; Nishita, Y.;
Ishii, H.; Kaneko, Y. A convenient synthesis of a simple coumarin
from salicyaldehyde and Wittig reagent (I): A synthesis of
methoxy- and hydroxycoumarins. Heterocycles 1994, 39, 613-
622.
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