Anti-AIDS agents 85. Design, synthesis, and evaluation of 1R,2R-dicamphanoyl-3,3-dimethyldihydropyrano-[2,3-c]xanthen-7(1H)-one (DCX) derivatives as novel anti-HIV agents

Article abstract of DOI:10.1016/j.ejmech.2011.10.025

In this study, 1R,2R-dicamphanoyl-3,3-dimethydihydropyrano[2,3-c]xanthen- 7(1H)-one (DCX) derivatives were designed and synthesized as novel anti-HIV agents against both wild-type and non-nucleoside reverse transcriptase (RT) inhibitor-resistant HIV-1 (RTMDR-1) strains. Twenty-four DCX analogs (6-29) were synthesized and evaluated against the non-drug-resistant HIV-1 NL4-3 strain, and selected analogs were also screened for their ability to inhibit the RTMDR-1 strain. Compared with the control 2-ethyl-3′,4′-di-O-(-)- camphanoyl-2′,2′-dimethyldihydropyrano[2,3-f]chromone (2-EDCP, 2), one of the best anti-HIV coumarin derivatives in our prior study, three DCX compounds (7, 12, and 22) showed better activity against both HIV strains with an EC₅₀ range of 0.062-0.081 μM, and five additional compounds (8, 11, 16, 18, and 21) exhibited comparable anti-HIV potency. Six DCX analogs (7, 11-12, 18, and 21-22) also showed enhanced selectivity index (SI) values in comparison to the control. Structure-activity relationship (SAR) information suggested that the extended conjugated system of the pyranoxanthone skeleton facilitates the interaction of the small DCX molecule within the viral binding pocket, consequently leading to enhanced anti-HIV activity and selectivity. Compared to DCP compounds, DCX analogs are a more promising new class of anti-HIV agents.

Full text of DOI:10.1016/j.ejmech.2011.10.025

European Journal of Medicinal Chemistry 47 (2012) 86e96
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Anti-AIDS agents 85. Design, synthesis, and evaluation of 1R,2R-dicamphanoyl-
3,3-dimethyldihydropyrano-[2,3-c]xanthen-7(1H)-one (DCX) derivatives as novel
anti-HIV agents
,
b
b
b
a
Ting Zhou a, Qian Shi a , Chin-Ho Chen , Li Huang , Phong Ho , Susan L. Morris-Natschke ,
**
*
,c,
Kuo-Hsiung Lee a
a Natural Products Research Laboratories, UNC Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, NC 27599-7568, United States
b Duke University Medical Center, Box 2926, SORF, Durham, NC 27710, United States
c Chinese Medicine Research and Development Center, China Medical University and Hospital, Taichung, Taiwan
a r t i c l e i n f o
a b s t r a c t
Article history:
In this study, 1R,2R-dicamphanoyl-3,3-dimethydihydropyrano[2,3-c]xanthen-7(1H)-one (DCX) deriva-
tives were designed and synthesized as novel anti-HIV agents against both wild-type and non-nucleoside
reverse transcriptase (RT) inhibitor-resistant HIV-1 (RTMDR-1) strains. Twenty-four DCX analogs (6e29)
were synthesized and evaluated against the non-drug-resistant HIV-1 NL4-3 strain, and selected analogs
were also screened for their ability to inhibit the RTMDR-1 strain. Compared with the control 2-ethyl-
30,40-di-O-(ꢀ)-camphanoyl-20,20-dimethyldihydropyrano[2,3-f]chromone (2-EDCP, 2), one of the best
anti-HIV coumarin derivatives in our prior study, three DCX compounds (7, 12, and 22) showed better
Received 23 June 2011
Received in revised form
11 October 2011
Accepted 12 October 2011
Available online 20 October 2011
Keywords:
activity against both HIV strains with an EC50 range of 0.062e0.081 mM, and five additional compounds
1R,2R-dicamphanoyl-3,3-
dimethydihydropyrano[2,3-c]xanthen-
7(1H)-one (DCX)
Anti-HIV activity
Structureeactivity relationship (SAR)
(8, 11, 16, 18, and 21) exhibited comparable anti-HIV potency. Six DCX analogs (7, 11e12, 18, and 21e22)
also showed enhanced selectivity index (SI) values in comparison to the control. Structureeactivity
relationship (SAR) information suggested that the extended conjugated system of the pyranoxanthone
skeleton facilitates the interaction of the small DCX molecule within the viral binding pocket, conse-
quently leading to enhanced anti-HIV activity and selectivity. Compared to DCP compounds, DCX analogs
are a more promising new class of anti-HIV agents.
Ó 2011 Elsevier Masson SAS. All rights reserved.
1. Introduction
reported DCP analogs, 2-ethyl DCP (2-EDCP, 2) and 2,5-dimethyl
DCP (3) exhibited the best anti-HIV activity against both wild-
AIDS has been epidemic for over 30 years, but still no cure has
been identified. Although over 30 formulations are now approved
by the US FDA to treat AIDS, drug resistance problems have
dramatically reduced the efficacy of current anti-HIV agents.
Therefore, research to find new anti-HIV agents with either higher
potency or novel mechanism has attracted great attention to
overcome this problem.
type and drug-resistant strains with remarkable EC50 values of
0.07 and 0.11 M for 2 and 0.036 and 0.49 M for 3 (Fig. 1) [1,2].
m
m
More specifically, preliminary mechanism of action-related studies
indicated that a DCK analog (4-methyl DCK, 5) inhibited the activity
of HIV-RT through inhibition of DNA-dependent DNA polymerase
activity [3], in contrast to currently available non-nucleoside
reverse transcriptase inhibitors (NNRTIs) that block HIV-RT by
inhibiting RNA-dependent DNA polymerization [4,5]. The mecha-
nistic and structural uniqueness of DCK and DCP analogs opened
a new avenue for us to discover more potent, more effective novel
anti-HIV drugs for AIDS therapy.
As previously described, structureeactivity relationship (SAR)
study and pharmacophore analysis based on DCP suggested that
the planar ring system is an important pharmacophore to maintain
anti-HIV activity against both wild-type and multi-drug resistant
HIV strains [6]. We speculated that adding an aromatic ring onto
the 4-pyrone ring of DCPs could produce a more rigid planar
system, and that the extended conjugation might sustain or even
In our prior studies, 30R,40R-di-O-(ꢀ)-camphanoyl-(þ)-cis-
khellactones (DCKs) and their positional isomers, 4H-chrom-4-one
derivatives (DCPs), showed high potency against wild-type HIV-1
replication (Fig. 1). DCP analogs also showed promising anti-HIV
potency against drug-resistant HIV strains. Among previously
* Corresponding author. Tel.: þ1 919 843 6325.
** Corresponding author. Natural Products Research Laboratories, UNC Eshelman
School of Pharmacy, University of North Carolina, Chapel Hill, North Carolina
27599-7568, United States. Tel.: þ1 919 962 0066; fax: þ1 919 966 3893.
E-mail addresses: qshi1@email.unc.edu (Q. Shi), khlee@unc.edu (K.-H. Lee).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.10.025

T. Zhou et al. / European Journal of Medicinal Chemistry 47 (2012) 86e96
87
and selectivity index (SI, CC50 O EC50) (Fig. 2, compounds 8, 9, and
19e29). In addition to its inductive electron withdrawing effect,
a cyano group could further expand the conjugation of the xan-
thenone ring, and perhaps result in potent anti-HIV activity. A
hydroxy group could not only increase polarity, but also could be
derivatized to obtain water soluble salts.
3. Chemistry
The general synthetic route to obtain pyrano-xanthones is
shown in Scheme 1. Hydroxylated xanthones (32bei, 32keq) were
produced through a cyclization reaction between phloroglucinol
(30) and appropriate substituted salicylic acids (31bej, 31leq), in
the presence of Eaton’s reagent (phosphorus pentoxide solution in
methanesulfonic acid) as a catalytic condensation agent [7]. A slight
excess of phloroglucinol was used to complete the desired reaction
and avoid the formation of side products (hydroxychromeno[3,2-b]
xanthenediones). The desired products (32bei, 32keq) were
obtained as brown solids after precipitation in ice-water, and
were collected by filtration. They were used in the next reaction
step without further purification. 6-Hydroxy-3,3-dimethylpyrano
[2,3-c]xanthen-7(3H)-ones (33bei, 33keq and 34a) were synthe-
sized from the xanthones (32, 32a is commercially available)
in anhydrous pyridine by microwave-assisted alkylation and
cyclization at 220 ꢁC for 4 h [8]. Methylation of the resulting 6-
hydroxy-3,3-dimethylpyrano[2,3-c]xanthen-7(3H)-ones (33) with
methyl iodide and potassium carbonate yielded 6-methoxy-3,3-
dimethylpyranoxanthones (34beq) [9].
DCX analogs 6, 7, and 10e24 were synthesized from 34 by
following the established literature procedures [5] for Sharpless
asymmetric dihydroxylation [10,11], which gave intermediate diols
(35), followed by esterification [11,12] with excess (S)-camphanoyl
chloride (Scheme 2).
Reaction of 7 with NH2OH$HCl in anhydrous pyridine afforded
DCX oxime analog 8. Demethylation of 7 with 48% hydrobromic acid
solution yielded 6-hydroxy-DCX (9) in 63% yield [9] (Scheme 3).
Brominated DCXs (25e27) were synthesized by reaction of 7
and 12 with N-bromosuccinimide (NBS) under appropriate condi-
tions. Bromination of 7 and 12 at the 5-position was accomplished
in dichloromethane under microwave conditions to generate 25
and 26, respectively [13], while benzylic bromination of 12 with
NBS in anhydrous carbon tetrachloride in the presence of m-
chloroperoxybenzoic acid (mCPBA) as a radical initiator led to 27
(Scheme 4) [6].
Fig. 1. Structures of DCP (1e3) and DCK (4e5) analogs.
increase the
pep stacking interaction between the ligand and the
target protein.
Therefore, in order to further understand the interaction
between the compounds’ planar ring and the binding pocket, we
designed
a series of tri-aryl conjugated compounds, with
a xanthen-9-one moiety replacing the chromone ring in the DCP
series (Fig. 2). We postulated that this modification could enhance
the interaction between the drug molecules and the target proteins,
and therefore, improve the anti-HIV activity and selectivity profiles.
2. Design
In previous studies, introducing appropriate alkyl or O-alkyl
groups on the planar ring system dramatically improved the anti-
HIV activity. For example, 2-EDCP (2) and 4-methyl DCK (5)
exhibited four- and five-fold better anti-HIV activity than the
parent DCP (1) and DCK (4), respectively. Thus, in our current study,
we first designed DCX analogs with a series of small alkyl and O-
alkyl groups substituted on the C ring (R8eR11) and A ring (R6) to
evaluate the effects of alkyl substitutions (Fig. 2, compounds 6, 7,
and 10e18). We then introduced other functional moieties,
such as halogen, cyano, and hydroxy groups, with different physi-
cochemical properties, as well as prepared a DCX oxime analog, to
explore how these functionalities affect the anti-HIV activity (EC50
)
Fig. 2. Structures of novel DCX analogs.

88
T. Zhou et al. / European Journal of Medicinal Chemistry 47 (2012) 86e96
Scheme 1. Synthesis of compounds 34aeq. Reagents and conditions: (i) Eaton’s reagent, reflux; (ii) 4,4-dimethoxy-2-methyl-2-butanol, pyridine, microwave, 220 ꢁC/4 h; (iii) MeI,
K2CO3, acetone, reflux.
Scheme 5 illustrates the synthetic pathway to 6-methoxy-10-
cyano-DCX (28) and 6-methoxy-9-cyano-DCX (29). Compounds
34o and 34p were treated with zinc cyanide in the presence of
tetrakis(triphenylphosphine)palladium(0) [Pd(PPh3)4] as a catalyst
to afford 36 and 37 [14]. After dihydroxylation and esterification
following the existing procedures mentioned above [6e8],
compounds 28 and 29 were obtained.
The structures of 6, 7, and 9 differ only by the substituent at C-6;
however, this slight structural differentiation resulted in significant
deviation in anti-HIV activity. Analog 7, with a methoxy group at C-
6, had an EC50 value of 0.063 mM, and was five-fold more potent
than the unsubstituted 6. Analog 7 was also 1.5-fold more potent
than 2, the positive control, and had a two-fold higher SI value,
suggesting a better selectivity between HIV inhibition and cytotoxic
activity. In contrast, compound 9, a 6-hydroxy DCX analog, did not
show notable activity or selectivity (an average SI value less than 4
was observed). One possible explanation for the lack of anti-HIV
potency is that intramolecular H-bonding (HB) between the OH
at C-6 and the carbonyl oxygen at C-7 might disrupt HB interaction
between this oxygen and the target protein. However, 19, with
4. Results and discussion
All synthesized DCX analogs were screened against the wild-
type HIV-1 NL4-3 strain in a single cycle infection assay using
TZM-bl cells. The results are shown in Table 1.
Scheme 2. Synthesis of compounds 6, 7, 10e24. Reagents and conditions. (i) K3Fe(CN)6, (DHQ)2PYR, K2OsO2(OH)4, K2CO3, methanesulfonamide, t-butanol/H2O, 0 ꢁC; (ii) (S)-
camphanoyl chloride, DMAP, CH2Cl2, rt.

T. Zhou et al. / European Journal of Medicinal Chemistry 47 (2012) 86e96
89
activity (smaller CC50 value was observed), which consequently led
to lower SI values.
In our previous study, the 2- and 3-positions of the chromone
system in DCP analogs, displayed equivalent characteristics when
substituted with the same functionalities [6]. Therefore, in this
study, we compared the introduction of methyl and methoxy
substituents at the 9- or 10-position of DCX compounds (analogs to
the 2- and 3-position, respectively, in DCP compounds). 9- (12) and
10-Methyl- (11) DCX analogs showed similar EC50 values (0.065 vs
0.095
while 9-methoxy-DCX (16) was three-fold more potent than 10-
methoxy-DCX (15) (EC50 0.12 and 0.362 M, respectively). Rela-
mM, respectively), with 12 being slightly more potent than 11,
m
tive to 7, methylation at C-9- or C-10 did not affect anti-HIV
potency, but methoxylation at these positions decreased the
potency.
The effect of substitution at C-8 on anti-HIV activity was also
studied, and the rank order of potency was 8-F (22) > 8-CH3
(13) > 8-OH (19) > 8-OCH3 (17). 8-Fluoro-substiuted DCX (22)
exhibited the greatest anti-HIV activity and selectivity, with an EC50
Scheme 3. Synthesis of compounds 8 and 9. Reagents and conditions: (i) NH2OH$HCl,
pyridine, reflux; (ii) HBr/CH3COOH, reflux.
value of 0.062
mM and SI value greater than 224, which were
a hydroxy group at C-8 rather than C-6, exhibited some anti-HIV
equivalent to the values for 7. Thus, the F atom with a similar size to
H led to similar activity. None or a small substituent, such as H or F,
is required at C-8 to maintain or enhance anti-HIV activity. Simi-
larly, 9-fluoro-DCX (21) also showed good anti-HIV potency and
selectivity.
However, bromination of the A- or C-ring in DCX led to reduced
anti-HIV activity. Except for 10-Br-DCX (24), which exhibited only
weak anti-HIV efficacy (EC50 1.47 mM), all other compounds (23,
activity (EC50 0.33
mM) and selectivity (SI 24.5), indicative of
unequal HB effects between the carbonyl oxygen and the two
similar adjacent OH groups (6-OH versus 8-OH). An unequal
molecular environment or point-by-point interactions of the
substituents on the drug molecule with the target protein could, in
turn, weaken the intramolecular interaction of the 8-OH. Overall,
the data suggested that introducing a hydrophobic, rather than
hydrophilic, substituent at C-6 is a good strategy to maintain or
enhance anti-HIV activity and selectivity, and consequently, all
other target compounds have a 6-OMe group.
The DCX oxime analog, 8, exhibited somewhat decreased anti-
HIV activity against wild-type HIV strain in comparison with its
oxo counterpart 7, indicating that the carbonyl group at C-7 likely
interacts with the binding pocket as a HB acceptor, while the NOH
group in the oxime analog attenuates such interaction.
25e27) lacked significant anti-HIV potency.
Introducing a cyano group at C-10 or C-9 resulted in compounds
28 and 29, respectively. Both compounds exhibited considerable
anti-HIV activity, with EC50 values of 0.20 and 0.29 mM.
Eight new DCX analogs, which showed significant anti-HIV
activity against the wild-type HIV strain, were selected and
screened against the drug-resistant RTMDR1 HIV strain. The data
are listed in Table 2. Interestingly, all selected DCX analogs (6, 7,
11e13, 16, 21, 22) also showed activity against the drug-resistant
strain. Based on both EC50 and SI values, compound 22 was the
most active compound followed by 7, 12, and 21. The activity profile
from the drug-resistant HIV strain was consistent with that from
the wild-type HIV strain.
In conclusion, the bioassay data generated from this study
demonstrated that the new DCX compounds are potent and
promising anti-HIV agents, with some analogs being more active
than the lead DCP compound 2. The following SAR conclusions
were derived from this study.
Compounds 7, 10, 14, and 20 have different substituents at C-11
(R11). The unsubstituted 7 (R11 ¼ H) exhibited higher potency than
20 (R11 ¼ F), which was more potent than 10 (R11 ¼ CH3) (EC50
0.063, 0.23 and 1.52
mM, respectively). 11-Methoxy-DCX (14)
showed no detectable anti-HIV activity or selectivity against wild-
type HIV-1 NL4-3 strain. The R11 substituent may influence the
orientation of the 1-camphanoyl group, as was previously shown to
be significant to maintain high anti-HIV potency in the DCP series
[2]. Introducing R11 substitutions also led to increased cytotoxic
1. The planar ring extension from a two-ring conjoined system
(pyranochromone in DCP) to a three-ring conjoined system
(pyranoxanthone in DCX) retained or even increased the anti-
HIV activity against both wild-type and drug-resistant strains,
suggesting that a conjugated planar ring system may be
essential to interact with the target protein through a pep ring
stacking interaction. The larger DCX molecule with enlarged
conjugating planer system perhaps, would interact more effi-
ciently with viral amino acid residues.
2. Compared to DCPs, selected DCXs, such as 7 and 22, exhibited
increased selectivity (SI values), suggesting DCX analogs target
the virus-infected cells efficiently and are less cytotoxic to
normal cells.
3. The positioning of the substituents in the molecule dramati-
cally affected the anti-HIV activity against both viral strains.
Adding a methyl group at C-11 generated an almost inactive
compound (10), while adding the same group at C-9 or C-10 led
to two potent anti-HIV compounds (12 and 11, respectively). A
Scheme 4. Synthesis of compounds 25e27. Reagents and conditions: (i) NBS, CH2Cl2,
microwave 100 ꢁC/2 h; (ii) mCPBA, NBS, CCl4, reflux.

90
T. Zhou et al. / European Journal of Medicinal Chemistry 47 (2012) 86e96
Scheme 5. Synthesis of compounds 28 and 29. Reagents and conditions: (i) Zn(CN)2, tetrakis (triphenylphosphine)palladium(0), DMF, 160 ꢁC; (ii) K3Fe(CN)6, K2CO3, (DHQ)2PYR,
K2OsO2(OH)4, methanesulfonamide, t-butanol/H2O, 0 ꢁC; (iii) (S)-camphanoyl chloride, DMAP, CH2Cl2, rt.
methoxy group was essential at C-6 for enhanced anti-HIV
potency, and a second methoxy group was more efficacious
at C-9 (16) than at C-10 (15), C-8 (17), or C-11 (14). Although the
6-hydroxy-DCX analog (9) did not exhibit anti-HIV activity, the
6-methoxy-8-hydroxy-DCX compound (19) was relatively
active with an EC50 value of 0.3 mM.
4. The properties of the substitutions at the positions also played
an important role. As mentioned above, a free OH group at C-6
resulted in an inactive compound (9), while changing the OH to
OCH3 at this position led to one of the most active compounds
(7). Generally, methyl substituted compounds showed more
potent anti-HIV activity than the corresponding methoxy
substituted compounds. A cyano group at C-9 or C-10 was well
tolerated. An oxime at C-7 lowered the anti-HIV activity, likely
by reducing the HB accepting capacity relative to a 7-carbonyl
group.
Table 1
Anti-HIV activity of DCX analogs (6e29) against HIV-1 NL4-3 strain.a
SId
c
Compound
R5
R6
R8
R9
R10
R11
X
CC50b m
M
EC50
mM
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Br
Br
H
H
H
H
H
H
H
H
H
H
H
CH3
H
H
H
OCH3
CH3
OH
H
H
F
H
H
H
H
H
H
H
H
CH3
H
H
H
OCH3
H
H
H
H
F
H
H
H
H
H
H
H
CH3
H
H
H
OCH3
H
H
OCH3
H
H
H
H
Br
H
H
H
H
H
H
CH3
H
H
H
OCH3
H
H
H
H
H
F
H
H
H
O
O
NOH
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
>29.8
>14.3
>13.9
N/A
4.32
>14.0
11.4
11.6
N/A
>13.7
10.8
11.6
>26.8
8.1
0.308
0.063
0.12
N/A
1.52
0.095
0.065
0.15
N/A
0.362
0.12
1.70
0.14
0.33
0.23
0.10
0.062
N/A
>96.8
>227.0
>118.2
ee
OCH3
OCH3
OH
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
2.84
>147.4
175.4
77.3
e
>37.8
88.8
6.83
>191.5
24.5
33.9
260
>224.2
e
7.8
26.0
>13.9
N/A
9.23
N/A
Br
H
CH3
CH2Br
H
H
H
H
H
H
H
1.47
N/A
N/A
4.53
0.20
0.29
0.089
6.83
e
H
H
H
H
H
H
CN
H
N/A
e
>25.2
9.08
>13.8
12.1
>5.55
45.4
>47.8
136.0
H
CN
2-EDCP (2)
a
All data presented in this table were averaged from at least three independent experiments.
Cytotoxic activity was determined using a Promega CytoTox-GloÔ assay kit.
This assay was performed in TZM-bl cell infected with HIV-1 NL4-3 strain.
Selectivity index ¼ CC50 O EC50.
b
c
d
e
No selective anti-HIV activity (CC50 O EC50 < 4).

T. Zhou et al. / European Journal of Medicinal Chemistry 47 (2012) 86e96
91
Table 2
d, J ¼ 8.4 Hz, H-5), 7.52 (1H, d, J ¼ 8.4 Hz, H-6), 6.41 (1H, s, H-4), 6.22
Anti-HIV activity of DCX analogs against drug-resistant RTMDR1 HIV strain.a
(1H, s, H-2), 2.44 (3H, s, CH3-7).
SIc
b
Compound
CC50
(
m
M)
EC50
(
m
M)
6.1.1.3. 1,3,8-Trihydroxy-9H-xanthen-9-one (32k). 1H NMR
d 11.86
6
7
>29.8
>14.3
>14.0
11.4
0.546
0.074
0.363
0.081
0.37
>54.6
>193.2
>38.6
140.7
31.4
(1H, s, OH-1), 11.80 (1H, s, OH-8), 7.68 (1H, t, J ¼ 8.4, 8.4 Hz, H-6),
7.01 (1H, d, J ¼ 8.4 Hz, H-5), 6.80 (1H, d, J ¼ 8.4 Hz, H-7), 6.39 (1H, d,
J ¼ 2.1 Hz, H-4), 6.23 (1H, d, J ¼ 2.1 Hz, H-2).
11
12
13
16
21
22
2
11.6
10.8
0.42
25.7
6.1.2. Synthesis of 6-hydroxy-3,3-dimethylpyrano[2,3-c]-7(3H)-
ones (33)
26.0
>13.9
12.1
0.16
0.065
0.11
162.5
>213.8
110
A mixture of starting compound (32cei, 32keq) (1 equiv), 4,4-
dimethoxy-2-methyl-2-butanol (1.5e2 equiv) and anhydrous
pyridine (2e3 mL) was heated at 220 ꢁC for 4 h under high-
absorption microwave conditions. The reaction mixture was
cooled to rt, diluted with EtOAc, and washed with aqueous HCl
(10%), and brine. The organic layer was separated, and solvent was
removed in vacuo. Crude products 33b, 33d and 33e were used
without further purification in the methoxylation reaction, while
the remaining compounds were purified by column chromatog-
raphy with hexane:EtOAc ¼ 97:3.
a
All data presented in this table were averaged from at least three independent
experiments.
b
c
This assay was performed in TZM-bl cell infected with HIVRTMDR1 strain.
Selectivity index ¼ CC50 O EC50
.
5. Conclusion
Our study indentified a new entity, DCX, and a series of DCX
analogs, as potent anti-HIV agents. Most of the synthesized DCX
analogs were active against both wild-type and drug-resistant HIV
strains. Compared to the control 2-EDCP (2), three compounds (7,12,
and 22) showed better activity against both HIV strains, and another
five compounds (8,11,16,18, and 21) exhibited comparable anti-HIV
potency against the wild-type HIV strain. The compounds that
exhibited high anti-HIV potency also had high SI values. Six analogs
(7,11,12,18, 21, 22) showed enhanced SI values in comparison to the
control. We also established an anti-HIV SAR study based on both
wild-type and drug-resistant HIV strains. Further modification is
currently ongoing to further improve the anti-HIV potency and
pharmacological profile of this compound type.
6.1.2.1. 6-Hydroxy-3,3,11-trimethylpyrano[2,3-c]xanthen-7(3H)-one
(33c). 43.6% yield (starting from 1.0 g of crude 32c); 1H NMR
d
13.22 (1H, s, OH-6), 8.09 (1H, d, J ¼ 7.6 Hz, H-8), 7.54 (1H, d,
J ¼ 7.2 Hz, H-10), 7.25 (1H, t, J ¼ 7.6, 7.2 Hz, H-9), 6.83 (1H, d,
J ¼ 9.6 Hz, H-1), 6.27 (1H, s, H-5), 5.63 (1H, d, J ¼ 9.6 Hz, H-2), 2.58
(3H, s, CH3-11), 1.49, 1.49 (each 3H, s, CH3-3,3).
6.1.2.2. 6-Hydroxy-3,3,8-trimethylpyrano[2,3-c]xanthen-7(3H)-one
(33f). 30.0% Yield (starting from 1.0 g of crude 32f); 1H NMR
d 13.32
(1H, s, OH-6), 7.53 (1H, t, J ¼ 8.0, 8.0 Hz, H-10), 7.30 (1H, d,
J ¼ 8.0 Hz, H-9), 7.11 (1H, d, J ¼ 8.0 Hz, H-11), 6.82 (1H, d, J ¼ 9.6 Hz,
H-1), 6.24 (1H, s, H-5), 5.50 (1H, d, J ¼ 9.6 Hz, H-2), 2.90 (3H, s, CH3-
8), 1.48, 1.48 (each 3H, s, CH3-3,3).
6. Experimental section
6.1. Chemistry
6.1.2.3. 6-Hydroxy-3,3-dimethyl-11-methoxy-pyrano[2,3-c]xanthen-
The proton nuclear magnetic resonance (1H NMR) spectra were
measured on Varian Inova 400 MHz and 300 MHz Varian Gemini
2000 spectrometers using TMS as internal standard. The carbon
nuclear magnetic resonance (13C NMR) spectra were measured on
Varian Inova 400 MHz Varian Gemini 2000 spectrometer using
TMS as internal standard. The solvent used was CDCl3 unless
indicated. Microwave reactions were performed with a Biotage
initiator EXP US. Mass spectra were measured on Shimadzu LCMS-
2010 (ESI-MS). Biotage Flash and Isco Companion systems were
used for medium-pressure column chromatography. Commercial
chemicals were obtained from Aldrich.
7(3H)-one (33g). 28.0% Yield (starting from 1.5 g of crude 32g); 1H
NMR
d
12.93 (1H, s, OH-6), 7.81 (1H, d, J ¼ 8.4 Hz, H-8), 7.29 (1H, d,
J ¼ 8.0 Hz, H-10), 7.24 (1H, t, J ¼ 8.4, 8.0 Hz, H-9), 6.92 (1H, d,
J ¼ 10.0 Hz, H-1), 6.28 (1H, s, H-5), 5.62 (1H, d, J ¼ 10.0 Hz, H-2), 4.02
(3H, s, OCH3-11), 1.48, 1.48 (each 3H, s, CH3-3,3).
6.1.2.4. 6-Hydroxy-3,3-dimethyl-10-methoxy-pyrano[2,3-c]xanthen-
7(3H)-one (33h). 12.8% Yield (starting from 1.0 g of crude 32h); 1H
NMR
d
13.07 (1H, s, OH-6), 8.11 (1H, d, J ¼ 9.3 Hz, H-8), 6.92 (1H, d,
J ¼ 9.3 Hz, H-9), 6.82 (1H, s, H-11), 6.81 (1H, d, J ¼ 9.9 Hz, H-1), 6.24
(1H, s, H-5), 5.61 (1H, d, J ¼ 9.9 Hz, H-2), 3.93 (3H, s, OCH3-10), 1.48,
1.48 (each 3H, s, CH3-3,3).
6.1.1. Synthesis of substituted 1,3-dihydroxy-9H-xanthen-9-ones(32)
To a mixture of commercially available phloroglucinol (1.2 equiv)
and an appropriate substituted salicylic acid (1 equiv), 20 mL of
Eaton’s reagent (P2O5eCH3SO3H) was added slowly. The mixture
was stirred for 3 h at 80 ꢁC, cooled to rt, and poured onto ice. After
vigorous stirring at rt for 2 h, a brown solid was precipitated. The
solid was collected by filtration, washed with water (pH w 6), and
dried at 60 ꢁC. In most instances, this intermediate 1,3-dihydroxy-
9H-xanthen-9-one was used without further purification.
6.1.2.5. 6-Hydroxy-3,3-dimethyl-9-methoxy-pyrano[2,3-c]xanthen-
7(3H)-one (33i). 42.0% Yield (starting from 2.0 g of crude 32i); 1H
NMR
d
12.98 (1H, s, OH-6), 7.61 (1H, d, J ¼ 3.0 Hz, H-8), 7.41 (1H, d,
J ¼ 9.3 Hz, H-11), 7.32 (1H, dd, J ¼ 9.3, 3.0 Hz, H-10), 6.84 (1H, d,
J ¼ 9.9 Hz, H-1), 6.27 (1H, s, H-5), 5.61 (1H, d, J ¼ 9.9 Hz, H-2), 3.91
(3H, s, OCH3-9), 1.49, 1.49 (each 3H, s, CH3-3,3).
6.1.2.6. 6,8-Dihydroxy-3,3-dimethylpyrano[2,3-c]xanthen-7(3H)-one
(33k). 28.0% Yield (starting from 1.0 g of crude 32k); 1H NMR
6.1.1.1. 1,3-Dihydroxy-9H-xanthen-9-one (32b). 1H NMR
d
8.15 (1H,
d
12.07, 11.95 (each 1H, s, OH-6,8), 7.56 (1H, t, J ¼ 8.1, 8.7 Hz, H-10),
d, J ¼ 8.1 Hz, H-8), 7.85 (1H, t, J ¼ 8.1, 6.9 Hz, H-6), 7.61 (1H, d,
J ¼ 8.1 Hz, H-5), 7.49 (1H, t, J ¼ 8.1, 6.9 Hz, H-7), 6.41 (1H, s, H-4),
6.23 (1H, s, H-2).
6.90 (1H, d, J ¼ 8.1 Hz, H-9), 6.81 (1H, d, J ¼ 10.2 Hz, H-1), 6.79 (1H,
d, J ¼ 8.7 Hz, H-11), 6.26 (1H, s, H-5), 5.63 (1H, d, J ¼ 10.2 Hz, H-2),
1.49, 1.49 (each 3H, s, CH3-3,3).
6.1.1.2. 1,3-Dihydroxy-7-methyl-9H-xanthen-9-one (32e). 1H NMR
6.1.2.7. 6-Hydroxy-11-fluoro-3,3-dimethylpyrano[2,3-c]xanthen-
7(3H)-one (33l). 30.0% Yield (starting from 1.0 g of crude 32l); 1H
d
12.88 (1H, s, OH-1), 11.03 (1H, s, OH-3), 7.92 (1H, s, H-8), 7.68 (1H,

92
T. Zhou et al. / European Journal of Medicinal Chemistry 47 (2012) 86e96
NMR
d
12.77 (1H, s, OH-6), 8.01 (1H, d, J ¼ 8.4 Hz, H-8), 7.51 (1H, t,
6.1.3.3. 6-Methoxy-3,3,10-trimethylpyrano[2,3-c]xanthen-7(3H)-one
J ¼ 8.4, 9.6 Hz, H-9), 7.29 (1H, d, J ¼ 9.6 Hz, H-10), 6.86 (1H, d,
J ¼ 10.4 Hz, H-1), 6.30 (1H, s, H-5), 5.63 (1H, d, J ¼ 10.4 Hz, H-2),
1.50, 1.50 (each 3H, s, CH3-3,3).
(34d). 100% Yield (starting from 200 mg of 33d); ESIþ (m/z, %), 323
(Mþ þ 1, 100); 1H NMR
8.17 (1H, d, J ¼ 8.1 Hz, H-8), 7.20 (1H, s, H-
d
11), 7.14 (1H, d, J ¼ 8.1 Hz, H-9), 6.89 (1H, d, J ¼ 9.9 Hz, H-1), 6.30
(1H, s, H-5), 5.62 (1H, d, J ¼ 9.9 Hz, H-2), 3.97 (3H, s, OCH3-6), 2.48
(3H, s, CH3-10), 1.50, 1.50 (each 3H, s, CH3-3,3).
6.1.2.8. 6-Hydroxy-9-fluoro-3,3-dimethylpyrano[2,3-c]xanthen-7(3H)-
one (33m). 20.0% Yield (starting from 1.0 g of crude 32m); 1H NMR
d
12.77 (1H, s, OH-6), 7.90 (1H, dd, J ¼ 8.0, 2.8 Hz, H-10), 7.43e7.46
6.1.3.4. 6-Methoxy-3,3,9-trimethylpyrano[2,3-c]xanthen-7(3H)-one
(2H, m, H-8,11), 6.83 (1H, d, J ¼ 10.0 Hz, H-1), 6.29 (1H, s, H-5), 5.63
(1H, d, J ¼ 10.0 Hz, H-2), 1.49, 1.49 (each 3H, s, CH3-3,3).
(34e). 86% Yield (starting from 399 mg of 33e); ESIþ (m/z, %), 323
(Mþ þ 1, 100); 1H NMR
8.06 (1H, s, H-8), 7.45 (1H, d, J ¼ 8.4 Hz, H-
d
11), 7.31 (1H, d, J ¼ 8.4 Hz, H-10), 6.90 (1H, d, J ¼ 9.9 Hz, H-1), 6.30
(1H, s, H-5), 5.62 (1H, d, J ¼ 9.9 Hz, H-2), 3.98 (3H, s, OCH3-6), 2.45
(3H, s, CH3-9), 1.50, 1.50 (each 3H, s, CH3-3,3).
6.1.2.9. 6-Hydroxy-8-fluoro-3,3-dimethylpyrano[2,3-c]xanthen-7(3H)-
one (33n). 46.0% Yield (starting from 1.0 g of crude 32n); 1H NMR
d
12.94 (1H, s, OH-6), 7.64 (1H, t, J ¼ 8.1, 8.4 Hz, H-10), 7.24 (1H, d,
J ¼ 8.4 Hz, H-11), 7.04 (1H, d, J ¼ 8.1 Hz, H-9), 6.78 (1H, d, J ¼ 10.2 Hz,
H-1), 6.25 (1H, s, H-5), 5.63 (1H, d, J ¼ 10.2 Hz, H-2), 1.49, 1.49 (each
3H, s, CH3-3,3).
6.1.3.5. 6-Methoxy-3,3,8-trimethylpyrano[2,3-c]xanthen-7(3H)-one
(34f). 84% Yield (starting from 700 mg of 33f); 1H NMR
d 7.45 (1H, t,
J ¼ 8.0, 7.6 Hz, H-10), 7.24 (1H, d, J ¼ 8.0 Hz, H-9), 7.06 (1H, d,
J ¼ 7.6 Hz, H-11), 6.87 (1H, d, J ¼ 10.0 Hz, H-1), 6.28 (1H, s, H-5), 5.60
(1H, d, J ¼ 10.0 Hz, H-2), 3.98 (3H, s, OCH3-6), 2.90 (3H, s, CH3-8),
1.50, 1.50 (each 3H, s, CH3-3,3).
6.1.2.10. 6-Hydroxy-10-bromo-3,3-dimethylpyrano[2,3-c]xanthen-
7(3H)-one (33o). 50.0% Yield (starting from 1.8 g of crude 32o);
1H NMR
d
12.79 (1H, s, OH-6), 8.10 (1H, d, J ¼ 8.4 Hz, H-8), 7.67
(1H, s, H-11), 7.50 (1H, d, J ¼ 8.4 Hz, H-9), 6.79 (1H, d, J ¼ 10.0 Hz,
H-1), 6.28 (1H, s, H-5), 5.64 (1H, d, J ¼ 10.0 Hz, H-2), 1.49, 1.49
(each 3H, s, CH3-3,3).
6.1.3.6. 6,11-Dimethoxy-3,3-dimethylpyrano[2,3-c]xanthen-7(3H)-one
(34g). 40% Yield (starting from 110 mg of 33g); ESIþ (m/z, %), 339
(Mþ þ 1, 100); 1H NMR
d
7.87 (1H, d, J ¼ 8.0 Hz, H-8), 7.24 (1H, t,
J ¼ 8.0, 8.0 Hz, H-9), 7,15 (1H, d, J ¼ 8.0 Hz, H-10), 6.97 (1H, d,
J ¼ 10.0 Hz, H-1), 6.32 (1H, s, H-5), 5.63 (1H, d, J ¼ 10.0 Hz, H-2),
4.01, 3.98 (each 3H, s, OCH3-6,11), 1.51, 1.51 (each 3H, s, CH3-3,3).
6.1.2.11. 6-Hydroxy-9-bromo-3,3-dimethylpyrano[2,3-c]xanthen-
7(3H)-one (33p). 20.0% Yield (starting from 1.0 g of crude 32p);
1H NMR
d
12.72 (1H, s, OH-6), 8.24 (1H, d, J ¼ 2.4 Hz, H-8), 7.78
(1H, dd, J ¼ 8.8, 2.4 Hz, H-10), 7.35 (1H, d, J ¼ 8.8 Hz, H-11), 6.80
(1H, d, J ¼ 6.4 Hz, H-1), 6.27 (1H, s, H-5), 5.63 (1H, d, J ¼ 6.4 Hz, H-
2), 1.49, 1.49 (each 3H, s, CH3-3,3).
6.1.3.7. 6,10-Dimethoxy-3,3-dimethylpyrano[2,3-c]xanthen-7(3H)-one
(34h). 95% Yield (starting from 110 mg of 33h); 1H NMR
d 8.17 (1H, d,
J ¼ 8.1 Hz, H-8), 7.20 (1H, s, H-11), 7.15 (1H, d, J ¼ 8.1 Hz, H-9), 6.89
(1H, d, J ¼ 9.9 Hz, H-1), 6.30 (1H, s, H-5), 5.62 (1H, d, J ¼ 9.9 Hz, H-2),
3.97 (3H, s, OCH3-6), 2.48 (3H, s, OCH3-10), 1.50. 1.50 (each 3H, s, CH3-
3,3).
6.1.2.12. 6-Hydroxy-10-methoxy-3,3,8-trimethylpyrano[2,3-c]xanthen-
7(3H)-one (33q). 30.0% Yield (starting from 1.0 g of crude 32q); 1H
NMR
d
13.44 (1H, s, OH-6), 6.76 (1H, d, J ¼ 10.0 Hz, H-1), 6.67 (1H, s,
H-9), 6.62 (1H, s, H-11), 6.18 (1H, s, H-5), 5.56 (1H, d, J ¼ 10.0 Hz, H-2),
3.87 (3H, s, OCH3-10), 2.81 (3H, s, CH3-8), 1.45,1.45 (each 3H, s, CH3-
3,3).
6.1.3.8. 6,9-Dimethoxy-3,3-dimethylpyrano[2,3-c]xanthen-7(3H)-one
(34i). 72% Yield (starting from 400 mg of 33i); ESIþ (m/z, %), 377
(Mþ þ K, 100); 1H NMR
d
7.66 (1H, d, J ¼ 3.0 Hz, H-8), 7.32 (1H, d,
J ¼ 9.0 Hz, H-11), 7.20 (1H, dd, J ¼ 9.0, 3.0 Hz, H-10), 6.86 (1H, d,
J ¼ 9.9 Hz, H-1), 6.27 (1H, s, H-5), 5.59 (1H, d, J ¼ 9.9 Hz, H-2), 3.95
(3H, s, OCH3-6), 3.87 (3H, s, OCH3-9), 1.47, 1.47 (each 3H, s, CH3-3,3).
6.1.3. Synthesis of 6-methoxy-3,3-dimethylpyrano[2,3-c]xanthen-
7(3H)-ones (34beq)
A mixture of compound (33bei, keq) (1 equiv) and K2CO3
(3 equiv) in anhydrous acetone (20 mL) was heated to reflux
temperature for 3 h, then allowed to cool to rt. Methyl iodide
(2e3 equiv) was added at rt and the reaction was kept stirring
overnight, monitored by TLC. At completion, the reaction mixture
was filtered, and the filtrate was evaporated to dryness. The crude
products 34j and 34n were carried directly to the dihydroxylation
reaction without purification. The remaining crude products were
purified by column chromatography using EtOAc and hexane to
give compounds 34bei, kem and oeq.
6.1.3.9. 6,8-Dimethoxy-3,3-dimethyl-pyrano[2,3-c]xanthen-7(3H)-
one (34j). 80 mg crude product was obtained from 100 mg of 33k.
6.1.3.10. 6-Methoxy-3,3-dimethyl-8-hydroxy-pyrano[2,3-c]xanthen-
7(3H)-one (34k). 95% Yield (starting from 100 mg of 33k); 1H NMR
d
13.21 (1H, s, OH-8), 7.49 (1H, t, J ¼ 8.4, 8.0 Hz, H-10), 6.85 (1H, d,
J ¼ 9.6 Hz, H-1), 6.83 (1H, d, J ¼ 8.4 Hz, H-9), 6.74 (1H, d, J ¼ 8.0 Hz,
H-11), 6.31 (1H, s, H-5), 5.63 (1H, d, J ¼ 9.6 Hz, H-2), 4.03 (3H, s,
OCH3-6), 1.51, 1.51 (each 3H, s, CH3-3,3).
6.1.3.1. 6-Methoxy-3,3-dimethylpyrano[2,3-c]xanthen-7(3H)-one (34b)
95% Yield (starting from 150 mg of 33b); ESIþ (m/z, %), 309 (Mþ,
6.1.3.11. 6-Methoxy-3,3-dimethyl-11-fluoro-pyrano[2,3-c]xanthen-
7(3H)-one (34l). 40% Yield (starting from 303 mg of 33l); 1H NMR
100); 1H NMR
d
8.29 (1H, d, J ¼ 8.1 Hz, H-8), 7.62 (1H, t, J ¼ 7.2,
d
8.04 (1H, d, J ¼ 8.4 Hz, H-8), 7.41 (1H, t, J ¼ 8.4, 9.6 Hz, H-9), 7.24
7.8 Hz, H-10), 7.39 (1H, d, J ¼ 7.8 Hz, H-11), 7.32 (1H, t, J ¼ 7.2, 8.1 Hz,
H-9), 6.89 (1H, d, J ¼ 9.9 Hz, H-1), 6.31 (1H, s, H-5), 5.62 (1H, d,
J ¼ 9.9 Hz, H-2), 3.98 (3H, s, OCH3-6),1.50,1.50 (each 3H, s, CH3-3,3).
(1H, d, J ¼ 9.6 Hz, H-10), 6.93 (1H, d, J ¼ 10.0 Hz, H-1), 6.33 (1H, s,
H-5), 5.64 (1H, d, J ¼ 10.0 Hz, H-2), 3.98 (3H, s, OCH3-6), 1.51, 1.51
(each 3H, s, CH3-3,3).
6.1.3.2. 6-Methoxy-3,3,11-trimethylpyrano[2,3-c]xanthen-7(3H)-one
6.1.3.12. 6-Methoxy-3,3-dimethyl-9-fluoro-pyrano[2,3-c]xanthen-
7(3H)-one (34m). 61% Yield (starting from 188 mg of 33m); 1H
(34c). 78% Yield (starting from 300 mg of 33c); ESIþ (m/z, %), 323
(Mþ þ 1, 100); 1H NMR
d
8.14 (1H, d, J ¼ 7.6 Hz, H-8), 7.48 (1H, d,
NMR
d
7.92 (1H, d, J ¼ 8.4 Hz, H-10), 7.27e7.41 (2H, m, H-8,11),
J ¼ 7.6 Hz, H-10), 7.22 (1H, t, J ¼ 7.6, 7.6 Hz, H-9), 6.88 (1H, d,
J ¼ 9.6 Hz, H-1), 6.31 (1H, s, H-5), 5.62 (1H, d, J ¼ 9.6 Hz, H-2), 3.98
(3H, s, OCH3-6), 2.54 (3H, s, CH3-11), 1.51, 1.51 (each 3H, s, CH3-3,3).
6.87 (1H, d, J ¼ 10.4 Hz, H-1), 6.31 (1H, s, H-5), 5.62 (1H, d,
J ¼ 10.4 Hz, H-2), 3.98 (3H, s, OCH3-6), 1.51, 1.51 (each 3H, s, CH3-
3,3).

T. Zhou et al. / European Journal of Medicinal Chemistry 47 (2012) 86e96
93
6.1.3.13. 6-Methoxy-10-bromo-3,3-dimethylpyrano[2,3-c]xanthen-
4.5 Hz, H-1), 3.90 (3H, s, OCH3-6), 3.87 (1H, t, J ¼ 4.5, 4.5 Hz, H-2),
3.84 (1H, d, J ¼ 4.5 Hz, OH-1), 3.47 (1H, d, J ¼ 4.5 Hz, OH-2), 2.42
(3H, s, CH3-9), 1.55, 1.48 (each 3H, s, CH3-3,3).
7(3H)-one (34o). 95% Yield (starting from 295 mg of 33o); 1H NMR
d
8.13 (1H, d, J ¼ 8.4 Hz, H-8), 7.60 (1H, s, H-11), 7.44 (1H, d,
J ¼ 8.4 Hz, H-9), 6.83 (1H, d, J ¼ 10.0 Hz, H-1), 6.31 (1H, s, H-5), 5.63
(1H, d, J ¼ 10.0 Hz, H-2), 3.97 (3H, s, OCH3-6), 1.50, 1.50 (each 3H, s,
CH3-3,3).
6.1.4.5. (1R,2R)-1,2-Dihydroxy-6-methoxy-3,3,8-trimethylpyrano
[2,3-c]xanthen-7(3H)-one (35f). 40% Yield (starting from 180 mg of
34f); ESIþ (m/z, %), 357 (Mþ þ 1, 100); 1H NMR
7.42 (1H, t, J ¼ 8.4,
d
6.1.3.14. 6-Methoxy-9-bromo-3,3-dimethylpyrano[2,3-c]xanthen-
7.6 Hz, H-10), 7.20 (1H, d, J ¼ 8.4 Hz, H-9), 7.07 (1H, d, J ¼ 7.6 Hz, H-
11), 6.26 (1H, s, H-5), 5.22 (1H, d, J ¼ 4.8 Hz, H-1), 3.95 (3H, s, OCH3-
6), 3.87 (1H, d, J ¼ 4.8 Hz, H-2), 3.32, 3.22 (each 1H, s, OH-1,2), 2.05
(3H, s, CH3-8), 1.50, 1.46 (each 3H, s, CH3-3,3).
7(3H)-one (34p). 72% Yield (starting from 70 mg of 33p); ESIþ
(m/z, %), 387 (Mþ, 100); 1H NMR
d
8.36 (1H, d, J ¼ 2.4 Hz, H-8),
7.69 (1H, dd, J ¼ 8.8, 2.4 Hz, H-10), 7.28 (1H, d, J ¼ 8.8 Hz, H-11),
6.84 (1H, d, J ¼ 10.0 Hz, H-1), 6.30 (1H, s, H-5), 5.61 (1H, d,
J ¼ 10.0 Hz, H-2), 3.96 (3H, s, OCH3-6), 1.48, 1.48 (each 3H, s, CH3-
3,3).
6.1.4.6. (1R,2R)-1,2-Dihydroxy-6,11-dimethoxy-3,3-dimethylpyrano
[2,3-c]xanthen-7(3H)-one (35g). Starting from 35 mg of crude 34g;
ESIþ (m/z, %), 339 (Mþ þ 1, 100); ESIþ (m/z, %), 373 (Mþ þ 1, 100).
6.1.3.15. 6,10-Dimethoxy-3,3,8-trimethylpyrano[2,3-c]xanthen-7(3H)-
one (34q). 54% Yield (starting from 295 mg of 33q); 1H NMR
d
6.85
6.1.4.7. (1R,2R)-6,10-Methoxy-3,3-dimethylpyrano[2,3-c]xanthen-
(1H, d, J ¼ 10.0 Hz, H-1), 6.67 (1H, s, H-9), 6.63 (1H, s, H-11), 6.28 (1H,
s, H-5), 5.59 (1H, d, J ¼ 10.0 Hz, H-2), 3.96 (3H, s, OCH3-6), 3.88 (3H, s,
OCH3-10), 2.86 (3H, s, CH3-8), 1.49, 1.49 (each 3H, s, CH3-3,3).
7(3H)-one (35h). 30% Yield (starting from 110 mg of 34h); ESIþ
(m/z, %), 373 (Mþ þ 1, 100); 1H NMR
d
8.18 (1H, d, J ¼ 8.7 Hz, H-8),
6.90 (1H, d, J ¼ 8.7 Hz, H-9), 6.71 (1H, s, H-11), 6.26 (1H, s, H-5), 5.20
(1H, br, H-1), 3.92 (3H, s, OCH3-6), 3.90 (3H, s, OCH3-10), 3.50 (1H,
br, H-2), 3.30 (2H, br, OH-1,2), 1.55, 1.48 (each 3H, s, CH3-3,3).
6.1.4. General procedure for the preparation of 35aeq and 38e39
A mixture of K3Fe(CN)6 (3 equiv), K2CO3 (3 equiv), (DHQ)2-PYR
(2% equiv), and K2OsO2(OH)4 (2% equiv) was dissolved in t-BuOH/
H2O (v/v, 1:1) at rt. The solution was cooled to 0 ꢁC and meth-
anesulfonamide (1 equiv) was added with stirring. After 20 min,
substituted pyranochromone (34aeq, and 36e37) was added. The
mixture was stirred at 0 ꢁC for 1e2 days, monitored by TLC. At
completion, Na2S2O5 (excess), water and CH2Cl2 were added, and
stirring was continued for 1 h at rt. The mixture was extracted with
CH2Cl2 three times, and the combined organic layer was dried over
MgSO4. The solvent was removed under reduced pressure, and the
residue was purified by column chromatography with
hexanes:EtOAc ¼ 3:7 to afford the pure substituted (þ)-cis-30,40-
dihydroxypyranoxanthenones (35aeb, def, hen, peq). Crude
compounds 35c, 35g, 35o, and 38e39 were carried to the next step
without purification.
6.1.4.8. (1R,2R)-1,2-Dihydroxy-6,9-dimethoxy-3,3-dimethylpyrano
[2,3-c]xanthen-7(3H)-one (35i). 40% Yield (starting from 200 mg of
34i); ESIþ (m/z, %), 373 (Mþ þ 1, 100); 1H NMR
d 7.65 (1H, s, H-8),
7.31 (1H, d, J ¼ 9.0 Hz, H-11), 7.22 (1H, d, J ¼ 9.0 Hz, H-10), 6.27 (1H,
s, H-5), 5.23 (1H, br, H-1), 3.94 (3H, s, OCH3-6), 3.90 (3H, s, OCH3-9),
3.35 (1H, br, H-2), 3.23, 3,23 (each 1H, s, OH-1,2), 1.51, 1.46 (each 3H,
s, CH3-3,3).
6.1.4.9. (1R,2R)-1,2-Dihydroxy-6,8-dimethoxy-3,3-dimethylpyrano
[2,3-c]xanthen-7(3H)-one (35j). 50% Yield (starting from 60 mg of
34j); ESIþ (m/z, %), 373 (Mþ þ 1, 100); 1H NMR
7.51 (1H, t, J ¼ 8.7,
d
8.4 Hz, H-10), 6.96 (1H, d, J ¼ 8.7 Hz, H-11), 6.79 (1H, d, J ¼ 8.4 Hz,
H-9), 6.26 (1H, s, H-5), 5.22 (1H, br, H-1), 3.97, 3.91 (each 3H, s,
OCH3-6,8), 3.19 (1H, br, H-2), 1.49, 1.45 (each 3H, s, CH3-3,3).
6.1.4.1. (1R,2R)-1,2-Dihydroxy-3,3-dimethylpyrano[2,3-c]xanthen-
6.1.4.10. (1R,2R)-1,2-Dihydroxy-6-methoxy-8-hydroxy-3,3-dimethyl-
7(3H)-one (35a). 22% Yield (starting from 70 mg of 34a); ESIþ
pyrano[2,3-c]xanthen-7(3H)-one (35k). 45% Yield (starting from
(m/z, %), 313 (Mþ þ 1, 100); 1H NMR
d
8.35 (1H, d, J ¼ 7.8 Hz, H-8),
50 mg of 34k); ESIþ (m/z, %), 357 (Mþ þ 1,100); 1H NMR
d 13.14 (1H,
8.20 (1H, d, J ¼ 9.3 Hz, H-6), 7.71 (1H, t, J ¼ 8.1, 8.1 Hz, H-10), 7.49
(1H, d, J ¼ 8.1 Hz, H-11), 7.41 (1H, t, J ¼ 8.1, 7.8 Hz, H-9), 6.89 (1H,
d, J ¼ 9.3 Hz, H-5), 5.35 (1H, d, J ¼ 5.4 Hz, H-1), 3.93 (1H, br, H-2),
3.34, 3.10 (each 1H, br, OH-1,2), 1.52, 1.46 (each 3H, s, CH3-3,3).
s, OH-8), 7.52 (1H, t, J ¼ 8.4, 8.4 Hz, H-10), 6.96 (1H, d, J ¼ 8.4 Hz, H-
9), 6.79 (1H, d, J ¼ 8.4 Hz, H-11), 6.32 (1H, s, H-5), 5.23 (1H, d,
J ¼ 4.8 Hz, H-1), 3.99 (3H, s, OCH3-6), 3.89 (1H, d, J ¼ 4.8 Hz, H-2),
3.11, 3.10 (each 1H, s, OH-1,2), 1.50, 1.47 (each 3H, s, CH3-3,3).
6.1.4.2. (1R,2R)-1,2-Dihydroxy-6-methoxy-3,3-dimethylpyrano[2,3-c]-
6.1.4.11. (1R,2R)-1,2-Dihydroxy-6-methoxy-11-fluoro-3,3-dimethyl-
pyrano[2,3-c]xanthen-7(3H)-one (35l). 50% Yield (starting from
xanthen-7(3H)-one (35b). 75% Yield (starting from 188 mg of 34b);
ESIþ (m/z, %), 343 (Mþ, 100); 1H NMR
d
8.30 (1H, d, J ¼ 7.8 Hz, H-8),
107 mg of 34l); ESIþ (m/z, %), 361 (Mþ þ 1, 100); 1H NMR
d 8.06 (1H,
7.63 (1H, t, J ¼ 6.9, 8.1 Hz, H-10), 7.38 (1H, d, J ¼ 8.1 Hz, H-11), 7.36
(1H, t, J ¼ 7.8, 6.9 Hz, H-9), 6.30 (1H, s, H-5), 5.25 (1H, br, H-1), 3.95
(3H, s, OCH3-6), 3.89 (1H, br, H-2), 3.37, 3.24 (each 1H, br, OH-1,2),
1.52, 1.48 (each 3H, s, CH3-3,3).
d, J ¼ 8.0 Hz, H-8), 7.44 (1H, t, J ¼ 8.0, 9.6 Hz, H-9), 7.30 (1H, d,
J ¼ 9.6 Hz, H-10), 6.34 (1H, s, H-5), 5.29 (1H, d, J ¼ 4.4 Hz, H-1), 3.98
(3H, s, OCH3-6), 3.91 (1H, s, OH-1), 3.39 (1H, d, J ¼ 4.4 Hz, H-2), 3.21
(1H, s, OH-2), 1.51, 1.46 (each 3H, s, CH3-3,3).
6.1.4.3. (1R,2R)-1,2-Dihydroxy-6-methoxy-3,3,10-trimethylpyrano
6.1.4.12. (1R,2R)-1,2-Dihydroxy-6-methoxy-9-fluoro-3,3-dimethyl-
pyrano[2,3-c]xanthen-7(3H)-one (35m). 50% Yield (starting from
[2,3-c]xanthen-7(3H)-one (35d). 45% Yield (starting from 200 mg of
34d); 1H NMR
d
8.15 (1H, d, J ¼ 8.4 Hz, H-8), 7.14e7.16 (2H, br, H-
110 mg of 34m); ESIþ (m/z, %), 361 (Mþ þ 1, 100); 1H NMR
d 7.92
9,11), 6.27 (1H, s, H-5), 5.23 (1H, br, H-1), 3.94 (3H, s, OCH3-6), 3.88
(1H, s, H-2), 3.55 (H, d, J ¼ 3.6 Hz, OH-1), 3.30 (1H, d, J ¼ 6.9 Hz, OH-
2), 2.46 (3H, s, CH3-10), 1.55, 1.49 (each 3H, s, CH3-3,3).
(1H, d, J ¼ 8.4 Hz, H-10), 7.56 (1H, m, H-8), 7.39 (1H, m, H-11), 6.34
(1H, d, J ¼ 4.0 Hz, H-1), 6.33 (1H, s, H-5), 5.24 (1H, d, J ¼ 4.0 Hz, H-2),
3.97 (2H, s, OCH3-6), 3.60, 3.40 (each 1H, s, OH-1,2), 1.49, 1.47 (each
3H, s, CH3-3,3).
6.1.4.4. (1R,2R)-1,2-Dihydroxy-6-methoxy-3,3,9-trimethylpyrano
[2,3-c]xanthen-7(3H)-one (35e). 59% Yield (starting from 250 mg
6.1.4.13. (1R,2R)-1,2-Dihydroxy-6-methoxy-8-fluoro-3,3-dimethyl-
pyrano[2,3-c]xanthen-7(3H)-one (35n). 60% Yield (starting from
of 34e); 1H NMR
d
7.97 (1H, s, H-8), 7.34 (1H, d, J ¼ 8.4 Hz, H-10),
7.19 (1H, d, J ¼ 8.4 Hz, H-11), 6.20 (1H, s, H-5), 5.20 (1H, t, J ¼ 4.5,
50 mg of 34n); ESIþ (m/z, %), 361 (Mþ þ 1, 100); 1H NMR
d 7.56 (1H,

94
T. Zhou et al. / European Journal of Medicinal Chemistry 47 (2012) 86e96
d, J ¼ 9.0 Hz, H-11), 7.21 (1H, d, J ¼ 9.0 Hz, H-9), 6.98 (1H, t, J ¼ 9.0,
9.0 Hz, H-10), 6.28 (1H, s, H-5), 5.2 (1H, br, H-1), 3.93 (3H, s, OCH3-
6), 3.90 (1H, br, H-2), 3.12, 3.11 (each 1H, s, OH-1,2), 1.50, 1.57 (each
3H, s, CH3-3,3).
H-8), 7.72 (1H, t, J ¼ 7.5, 8.1 Hz, H-10), 7.46 (1H, t, J ¼ 7.8, 7.5 Hz, H-9),
7.39 (1H, d, J ¼ 8.1 Hz, H-11), 6.98 (1H, d, J ¼ 4.5 Hz, H-1), 6.46 (1H, s,
H-5), 5.53 (1H, d, J ¼ 4.5 Hz, H-2), 4.12 (3H, s, OCH3-6), 2.60, 2.33, 2.10,
1.80 (each 2H, m, camphanoyl-CH2), 1.69, 1.61 (each 3H, s, CH3-3,3),
1.25, 1.25, 1.15, 1.12, 1.02, 1.01 (each 3H, s, camphanoyl-CH3);
6.1.4.14. (1R,2R)-1,2-Dihydroxy-6-methoxy-9-bromo-3,3-dimethyl-
[
a
]
ꢀ40.9ꢁ (c ¼ 0.0069, CH3Cl); HRMS for (Mþ þ Na): calcd m/z
D
pyrano[2,3-c]xanthen-7(3H)-one (35p). 45% Yield (starting from
725.2574, found: 725.2603; 13C NMR:
d 9.78, 9.92, 16.74, 16.85, 16.93,
50 mg of 34p); ESIþ (m/z, %), 421 (Mþ, 100); 1H NMR
d
8.34 (1H, d,
17.01, 21.53, 27.12, 29.20, 29.18, 31.00, 31.54, 54.59, 54.94, 55.15, 55.20,
61.57, 72.86, 72.82, 90.95, 91.49, 96.01, 96.06, 98.66, 107.82, 117.24,
123.20, 124.76, 127.06, 134.20, 154.47, 157.60, 159.07, 163.39, 167.17,
167.65, 175.13, 178.11, 178.18.
J ¼ 1.6 Hz, H-8), 7.69 (1H, dd, J ¼ 8.8, 1.6 Hz, H-10), 7.25 (1H, d,
J ¼ 8.8 Hz, H-11), 6.27 (1H, s, H-5), 5.20 (1H, d, J ¼ 4.4 Hz, H-1),
3.92 (3H, s, OCH3-6), 3.86 (1H, d, J ¼ 4.4 Hz, H-2), 3.20, 3.12 (each
1H, s, OH-1,2), 1.49, 1.45 (each 3H, s, CH3-3,3).
6.1.7.3. 1R,2R-(ꢀ)-Dicamphanoyl-3,3,11-trimethyl-6-methoxy-1,2-
dihydropyrano[2,3-c]xanthen-7(1H)-one (10). 85% Yield (starting
6.1.4.15.
pyrano[2,3-c]xanthen-7(3H)-one (35q). 50% Yield (starting from
50 mg of 34q); 1H NMR
6.67 (1H, s, H-9), 6.67 (1H, s, H-11), 6.28
(1H, s, H-5), 5.23 (1H, d, J ¼ 4.4 Hz, H-1), 4.10 (1H, d, J ¼ 4.4 Hz, H-
2), 3.93 (3H, s, OCH3-6), 3.89 (3H, s, OCH3-10), 3.13, 3.11 (each 1H,
s, OH-1,2), 2.87 (3H, s, CH3-8), 1.49, 1.46 (each 3H, s, CH3-3,3).
(1R,2R)-1,2-Dihydroxy-6,10-dimethoxy-3,3,8-trimethyl-
from 41 mg of 35c); white solid; mp 174e175 ꢁC; 1H NMR
d 8.12
d
(1H, d, J ¼ 7.6 Hz, H-8), 7.36 (1H, d, J ¼ 7.2 Hz, H-10), 7.24 (1H, t,
J ¼ 7.6, 7.2 Hz, H-9), 6.87 (1H, d, J ¼ 4.4 Hz, H-1), 6.34 (1H, s, H-5),
5.44 (1H, d, J ¼ 4.4 Hz, H-2), 4.01 (3H, s, OCH3-6), 2.45, 2.25, 1.90,
1.67 (each 2H, m, camphanoyl-CH2), 2.39 (3H, s, CH3-11), 1.60, 1.49
(each 3H, s, CH3-3,3), 1.14, 1.14, 1.03, 1.00, 0.93, 0.89 (each 3H, s,
6.1.5. 6-Methoxy-10-cyano-3,3-dimethylpyrano[2,3-c]xanthen-
7(3H)-one (36)
camphanoyl-CH3); [
a
]
D
ꢀ40.9ꢁ (c ¼ 0.0069, CH3Cl); [
a
]
ꢀ42.5ꢁ
D
(c ¼ 0.0051, CH3Cl); HRMS for (Mþ þ Na): calcd m/z 739.2731,
A mixture of 34o (10 mg, 0.025 mmoL), zinc cyanide (3.5 mg,
0.03 mmoL) and Pd(PPh3)4 (12 mg, 0.011 mmoL) was dissolved in
anhydrous DMF (0.5 mL) and heated to 130 ꢁC for 16 h, monitored
by TLC. At completion, the reaction mixture was filtered through
Celite, and the crude product was purified by PTLC with
EtOAc:hexane ¼ 1:3 to afford pure 36 as yellow solid. 90% yield;
found: 739.2736.
6.1.7.4. 1R,2R-(ꢀ)-Dicamphanoyl-3,3,10-trimethyl-6-methoxy-1,2-
dihydropyrano[2,3-c]xanthen-7(1H)-one (11). 80% Yield (starting
from 50 mg of 35d); white solid; mp 174e175 ꢁC; 1H NMR
d 8.15
(1H, d, J ¼ 8.1 Hz, H-8), 7.13 (1H, d, J ¼ 8.1 Hz, H-9), 7.07 (1H, s, H-11),
6.87 (1H, d, J ¼ 4.8 Hz, H-1), 6.32 (1H, s, H-5), 5.39 (1H, d, J ¼ 4.8 Hz,
H-2), 3.99 (3H, s, OCH3-6), 2.41 (3H, s, CH3-10), 2.45, 2.20, 1.95, 1.80
(each 2H, m, camphanoyl-CH2), 1.56, 1.49 (each 3H, s, CH3-3,3), 1.14,
MS-ESIþ (m/z, %) 334 (Mþ þ 1, 100); 1H NMR
d 8.35 (1H, d,
J ¼ 8.0 Hz, H-8), 7.72 (1H, s, H-11), 7.55 (1H, d, J ¼ 8.0 Hz, H-9), 6.82
(1H, d, J ¼ 10.0 Hz, H-1), 6.32 (1H, s, H-5), 5.64 (1H, d, J ¼ 10.0 Hz, H-
2), 3.96 (3H, s, OCH3-6), 1.57, 1.49 (each 3H, s, CH3-2,2).
1.13, 1.04, 1.00, 0.90, 0.87 (each 3H, s, camphanoyl-CH3); [
a
]
D ꢀ37.2ꢁ
(c ¼ 0.0018, CH3Cl); HRMS for (Mþ þ Na): calcd m/z 739.2731,
6.1.6. 6-Methoxy-9-cyano-3,3-dimethylpyrano[2,3-c]xanthen-7(3H)-
one (37)
found: 739.2724.
The procedure was identical to that used for the preparation of
36. The crude 37 was carried to the next step without purification.
MS-ESIþ (m/z, %) 334 (Mþ þ 1, 100).
6.1.7.5. 1R,2R-(ꢀ)-Dicamphanoyl-3,3,9-trimethyl-6-methoxy-1,2-
dihydropyrano[2,3-c]xanthen-7(1H)-one (12). 77% Yield (starting
from 100 mg of 35e); light yellow solid; mp 179e180 ꢁC; 1H NMR
d
8.05 (1H, s, H-8), 7.41 (1H, d, J ¼ 8.7 Hz, H-11), 7.18 (1H, d,
6.1.7. General procedure for the preparation of 6e7, 10e24, 18 and
28e29
J ¼ 8.7 Hz, H-10), 6.86 (1H, d, J ¼ 4.5 Hz, H-1), 6.32 (1H, s, H-5),
5.40 (1H, d, J ¼ 4.5 Hz, H-2), 4.00 (3H, s, OCH3-6), 2.44 (3H, s, CH3-
9), 2.45, 2.20, 1.90, 1.70 (each 2H, m, camphanoyl-CH2), 1.57, 1.49
(each 3H, s, CH3-3,3), 1.14, 1.13, 1.03, 1.00, 0.90, 0.88 (each 3H, s,
The substituted 1R,2R-dihydroxypyranoxanthone(35aeq, 38e39)
(1 equiv), (S)-(ꢀ)-camphanoyl chloride (3 equiv), and DMAP (4 equiv)
were stirred in CH2Cl2 for 1e2 h at rt, monitored by TLC. At comple-
tion, the mixture was diluted with CH2Cl2 and washed separately
with water and brine. The solvent was then removed under
reduced pressure and the residue was purified by PTLC with
hexanes:EtOAc ¼ 3:2 to afford the appropriatelysubstituted 1R,2R-di-
O-(ꢀ)-camphanoyl-3,3-dimethyldihydroprano[2,3-c]xanthone (6e7,
10e24, and 28e29).
camphanoyl-CH3); [
a
]
ꢀ34.6ꢁ (c ¼ 0.0028, CH3Cl); HRMS for
D
(Mþ þ Na): calcd m/z 739.2731, found: 739.2724.
6.1.7.6. 1R,2R-(ꢀ)-Dicamphanoyl-3,3,8-trimethyl-6-methoxy-1,2-
dihydropyrano[2,3-c]xanthen-7(1H)-one (13). 75% Yield (starting
from 70 mg of 35f); white solid; mp 240e242 ꢁC; 1H NMR
d 7.39
(1H, t, J ¼ 8.4, 7.6 Hz, H-10), 7.09 (1H, d, J ¼ 8.4 Hz, H-9), 7.06 (1H,
d, J ¼ 7.6 Hz, H-11), 6.82 (1H, d, J ¼ 4.8 Hz, H-1), 6.29 (1H, s, H-5),
5.37 (1H, d, J ¼ 4.8 Hz, H-2), 3.98 (3H, s, OCH3-6), 2,86 (3H, s, CH3-
8), 2.46, 2.18, 1.90, 1.65 (each 2H, m, camphanoyl CH2), 1.56, 1.55
(each 3H, s, CH3-3,3), 1.11, 1.10, 1.01, 1.00, 0.88, 0.86 (each 3H, s,
6.1.7.1. 1R,2R-(ꢀ)-Dicamphanoyl-3,3-dimethyl-1,2-dihydropyrano
[2,3-c]xanthen-7(1H)-one (6). 85% Yield (starting from 15 mg of
35a); white solid; mp 146e148 ꢁC; 1H NMR
d
8.33 (1H, d, J ¼ 7.8 Hz,
H-8), 8.31 (1H, d, J ¼ 9.0 Hz, H-6), 7.67 (1H, t, J ¼ 7.2, 8.4 Hz, H-10),
7.39 (1H, t, J ¼ 7.2, 7.8 Hz, H-9), 7.35 (1H, d, J ¼ 8.4 Hz, H-11), 6.94
(1H, d, J ¼ 9.0 Hz, H-5), 6.93 (1H, d, J ¼ 4.5 Hz, H-1), 5.43 (1H, d,
J ¼ 4.5 Hz, H-2), 2.52, 2.322 1.90, 1.75 (each 2H, m, camphanoyl-
CH2), 1.57, 1.50 (each 3H, s, CH3-3,3), 1.14, 1.12, 1.03, 1.00, 0.93, 0.89
camphanoyl CH3); [
a
]
ꢀ29.3ꢁ (c ¼ 0.003, CH3Cl); HRMS for
D
(Mþ þ Na): calcd m/z 739.2731, found: 739.2713.
6.1.7.7. 1R,2R-(ꢀ)-Dicamphanoyl-3,3-dimethyl-6,11-dimethoxy-1,2-
dihydropyrano[2,3-c]xanthen-7(1H)-one (14). 53% Yield (starting
(each 3H, s, camphanoyl-CH3); [
a
]
D
ꢀ22.7ꢁ (c ¼ 0.0015, CH3Cl);
from 25 mg of 35g); white solid; mp 179e180 ꢁC; 1H NMR
d 7.82
HRMS for (Mþ þ Na): calcd m/z 695.2469, found: 695.2485.
(1H, d, J ¼ 8.0 Hz, H-8), 7.25 (1H, t, J ¼ 8.0, 8.0 Hz, H-9), 7.09 (1H, d,
J ¼ 8.0 Hz, H-10), 6.82 (1H, d, J ¼ 4.4 Hz, H-1), 6.33 (1H, s, H-5), 5.44
(1H, d, J ¼ 4.4 Hz, H-2), 4.00, 3.91 (each 3H, s, OCH3-6,11), 2.44, 2.27,
1.95, 1,70 (each 2H, m, camphanoyl-CH2), 1.57, 1.47 (each 3H, s, CH3-
3,3), 1.13, 1.13, 1.03, 1.01, 0.91, 0.87 (each 3H, s, camphanoyl-CH3);
6.1.7.2. 1R,2R-(ꢀ)-Dicamphanoyl-3,3-dimethyl-6-methoxy-1,2-dihydro-
pyrano[2,3-c]xanthen-7(1H)-one (7). 88% Yield (starting from 52 mg
of 35b); white solid; mp 134e135 ꢁC; 1H NMR
d
8.40 (1H, d, J ¼ 7.8 Hz,

T. Zhou et al. / European Journal of Medicinal Chemistry 47 (2012) 86e96
95
[
a
]
ꢀ40.8ꢁ (c ¼ 0.0013, CH3Cl); HRMS for (Mþ þ Na): calcd m/z
1.06, 1.04, 1.00, 0.99 (each 3H, s, camphanoyl-CH3); [
a
]
ꢀ41.2ꢁ
D
D
755.2680, found: 755.2660.
(c ¼ 0.0052, CHCl3); HRMS for (Mþ þ Na): calcd m/z 743.2480, found:
743.2483.
6.1.7.8. 1R,2R-(ꢀ)-Dicamphanoyl-3,3-dimethyl-6,10-dimethoxy-1,2-
dihydropyrano[2,3-c]xanthen-7(1H)-one (15). 43% Yield (starting
6.1.7.14. 1R,2R-(ꢀ)-Dicamphanoyl-3,3-trimethyl-6-methoxy-9-fluoro-
1,2-dihydropyrano[2,3-c]xanthen-7(1H)-one (21). 48% Yield (starting
from 10 mg of 35h); white solid; mp 177e178 ꢁC; 1H NMR
d 8.12
(1H, d, J ¼ 8.7 Hz, H-8), 6.84 (1H, dd, J ¼ 8.7, 2.4 Hz, H-9), 6.79 (1H, d,
J ¼ 4.5 Hz, H-1), 6.59 (1H, d, J ¼ 2.4 Hz, H-11), 6.26 (1H, s, H-5), 5.33
(1H, d, J ¼ 4.5 Hz, H-2), 3.92 (3H, s, OCH3-6), 3.77 (3H, s, OCH3-10),
2.40, 2.15, 1.95, 1.65 (each 2H, m, camphanoyl-CH2), 1.50, 1.42 (each
3H, s, CH3-3,3), 1.07, 1.06, 0.97, 0.93, 0.85, 0.81 (each 3H, s, cam-
from 45 mg of 35m); white solid; mp 204e205 ꢁC; 1H NMR
d 7.92
(1H, d, J ¼ 8.0 Hz, H-10), 7.28e7.36 (2H, m, H-9,11), 6.85 (1H, d,
J ¼ 4.4 Hz, H-1), 6.35 (1H, s, H-5), 5.40 (1H, d, J ¼ 4.4 Hz, H-2), 4.00
(3H, s, OCH3-6), 2.49, 2.19, 1.90, 1.68 (each 2H, m, camphanoyl-CH2),
1.56, 1.49 (each 3H, s, CH3-3,3), 1.14, 1.13, 1.04, 1.00, 0.90, 0.90 (each
phanoyl-CH3); [
calcd m/z 733.2860, found: 733.2874.
a
]
D ꢀ16.2ꢁ (c ¼ 0.0052, CH3Cl); HRMS for (Mþ þ 1):
3H, s, camphanoyl-CH3); [
a]
D ꢀ23.6ꢁ (c ¼ 0.0025, CHCl3); HRMS for
(Mþ þ Na): calcd m/z 743.2480, found: 743.2469.
6.1.7.9. 1R,2R-(ꢀ)-Dicamphanoyl-3,3-dimethyl-6,9-dimethoxy-1,2-
dihydropyrano[2,3-c]xanthen-7(1H)-one (16). 60% Yield (starting
from 20 mg of 35i); white solid; mp 168e170 ꢁC; ESIþ (m/z, %), 732
6.1.7.15. 1R,2R-(ꢀ)-Dicamphanoyl-3,3-trimethyl-6-methoxy-8-fluoro-
1,2-dihydropyrano[2,3-c]xanthen-7(1H)-one (22). 80% Yield (starting
from 5 mg of 35n); white solid; mp 148e150 ꢁC; 1H NMR
d 7.44 (1H, t,
(Mþ, 100); 1H NMR
d
7.66 (1H, s, H-8), 7.21, 7.21 (each 1H, d,
J ¼ 8.7, 8.4 Hz, H-10), 7.01 (1H, d, J ¼ 8.4 Hz, H-11), 6.92 (1H, d,
J ¼ 8.7 Hz, H-9), 6.76 (1H, d, J ¼ 4.5 Hz, H-1), 6.26 (1H, s, H-5), 5.32 (1H,
d, J ¼ 4.5 Hz, H-2), 3.91 (3H, s, OCH3-6), 2.40, 2.12, 1.91, 1.67 (each 2H,
m, camphanoyl-CH2), 1.51, 1.42 (each 3H, s, CH3-3,3), 1.07, 1.05, 0.97,
J ¼ 9.2 Hz, H-10,11), 6.85 (1H, d, J ¼ 4.8 Hz, H-1), 6.32 (1H, s, H-5),
5.40 (1H, d, J ¼ 4.8 Hz, H-2), 4.00 (3H, s, OCH3-6), 3.88 (3H, s, OCH3-
9), 2.5, 2.20, 1.90, 1.70 (each 2H, m, camphanoyl-CH2), 1.57, 1.49
(each 3H, s, CH3-3,3), 1.14, 1.13, 1.03, 1.00, 0.90, 0.88 (each 3H, s,
0.93, 0.83, 0.83 (each 3H, s, camphanoyl-CH3); [
a
]D ꢀ17.1ꢁ (c ¼ 0.0035,
camphanoyl-CH3); [
a
]
D
ꢀ35.3ꢁ (c ¼ 0.0024, CHCl3); HRMS for
CHCl3), HRMS for (Mþ þ Na): calcd m/z 743.2480, found: 743.2476.
(Mþ þ 1): calcd m/z 733.2860, found: 733.2864.
6.1.7.16. 1R,2R-(ꢀ)-Dicamphanoyl-3,3-dimethyl-6-methoxy-10-bromo-
1,2-dihydropyrano[2,3-c]xanthen-7(1H)-one (23). 52% Yield (starting
6.1.7.10. 1R,2R-(ꢀ)-Dicamphanoyl-3,3-dimethyl-6,8-dimethoxy-1,2-
dihydropyrano[2,3-c]xanthen-7(1H)-one (17). 52% Yield (starting
from 35 mg of 35o); white solid; mp 159e160 ꢁC; 1H NMR
d 8.11 (1H,
from 10 mg of 35j); white solid; mp 155e156 ꢁC; 1H NMR
d
7.44 (1H,
d, J ¼ 8.4 Hz, H-8), 7.48 (1H, d, J ¼ 1.6 Hz, H-11), 7.44 (1H, dd,
J ¼ 8.4 Hz, H-9), 6.80 (1H, d, J ¼ 4.8 Hz, H-1), 6.32 (1H, s, H-5), 5.46
(1H, d, J ¼ 4.8 Hz, H-2), 3.97 (3H, s, OCH3-6), 2.45, 2.08, 1.90, 1.70 (each
2H, m, camphanoyl CH2),1.61,1.61 (each 3H, s, CH3-3,3), 1.46, 1.14, 1.12,
t, J ¼ 8.4, 8.4 Hz, H-10), 6.82 (1H, d, J ¼ 8.4 Hz, H-11), 6.80 (1H, d,
J ¼ 4.4 Hz, H-1), 6.75 (1H, d, J ¼ 8.4 Hz, H-9), 6.27 (1H, s, H-5), 5.36
(1H, d, J ¼ 4.4 Hz, H-2), 3.94, 3.93 (each 3H, s, OCH3-6.8), 2.50, 2.20,
1.90, 1.70 (each 2H, camphanoyl-CH2), 1.54, 1.46 (each 3H, s, CH3-
3,3), 1.13, 1.11, 1.03, 1.00, 0.88, 0.85 (each 3H, s, camphanoyl-CH3);
1.05, 1.04, 1.02 (each 3H, s, camphanoyl CH3); [
a]
D ꢀ25.3ꢁ (c ¼ 0.0035,
CHCl3); HRMS for (Mþ þ Na): calcd m/z 803.1679, found: 803.1691.
[
a
]
ꢀ131.7ꢁ (c ¼ 0.003, CHCl3); HRMS for (Mþ þ Na): calcd m/z
D
755.2680, found: 755.2671.
6.1.7.17. 1R,2R-(ꢀ)-Dicamphanoyl-3,3-dimethyl-9-bromo-6-methoxy-
1,2-dihydropyrano[2,3-c]xanthen-7(1H)-one (24). 50% Yield (starting
6.1.7.11. 1R,2R-(ꢀ)-Dicamphanoyl-3,3,8-trimethyl-6,10-dimethoxy-
1,2-dihydropyrano[2,3-c]xanthen-7(1H)-one (18). 52% Yield (start-
from 10 mg of 35p); white solid; mp 159e160 ꢁC; 1H NMR
d 8.35 (1H,
d, J ¼ 2.8 Hz, H-8), 7.67 (1H, dd, J ¼ 8.8, 2.8 Hz, H-10), 7.20 (1H, d,
J ¼ 8.8 Hz, H-11), 6.81 (1H, d, J ¼ 4.4 Hz, H-1), 6.32 (1H, s, H-5), 5.47
(1H, d, J ¼ 4.4 Hz, H-2), 3.98 (3H, s, OCH3-6), 2.40, 2,10, 1.90, 1.60
(each 2H, m, camphanoyl-CH2), 1.56, 1.46 (each 3H, s, CH3-3,3), 1.14,
ing from 10 mg of 35q); white solid; mp 183e184 ꢁC; 1H NMR
d 6.85
(1H, d, J ¼ 4.8 Hz, H-1), 6.64 (1H, d, J ¼ 2.4 Hz, H-9), 6.53 (1H, d,
J ¼ 2.4 Hz, H-11), 6.30 (1H, s, H-5), 5.39 (1H, d, J ¼ 4.8 Hz, H-2), 3.99
(3H, s, OCH3-6), 3.81 (3H, s, OCH3-10), 2.84 (3H, s, CH3-8), 2.45, 2.20,
1.90, 1.70 (each 2H, m, camphanoyl CH2), 1.56, 1.48 (each 3H, s, CH3-
3,3), 1.13, 1.13, 1.04, 1.00, 0.91, 0.88 (each 2H, s, camphanoyl CH3);
1.12, 1.03, 1.02, 1.01, 0.77 (each 3H, s, camphanoyl-CH3); [
a]
D ꢀ13.0ꢁ
(c ¼ 0.004, CHCl3); HRMS for (Mþ þ Na): calcd m/z 803.1679, found:
803.1684.
[
a
]
ꢀ23.6ꢁ (c ¼ 0.0012, CHCl3); HRMS for (Mþ þ Na): calcd m/z
D
769.2836, found: 769.2835.
6.1.7.18. 1R,2R-(ꢀ)-Dicamphanoyl-3,3-dimethyl-6-methoxy-10-cyano-
1,2-dihydropyrano[2,3-c]xanthen-7(1H)-one (28). 80% Yield (starting
6.1.7.12. 1R,2R-(ꢀ)-Dicamphanoyl-3,3-dimethyl-6-methoxy-8-hydroxy-
1,2-dihydropyrano[2,3-c]xanthen-7(1H)-one (19). 30% Yield (starting
from 36 mg of 36); white solid; mp 164e165 ꢁC; 1H NMR
d 8.36 (1H,
d, J ¼ 8.0 Hz, H-8), 7.60 (1H, d, J ¼ 8.0 Hz, H-9), 7.55 (1H, s, H-11), 6.78
(1H, d, J ¼ 4.4 Hz, H-1), 6.35 (1H, s, H-5), 5.39 (1H, d, J ¼ 4.4 Hz, H-2),
3.99 (3H, s, OCH3-6), 2.50, 2.20, 1.90, 1.70 (each 2H, m, camphanoyl-
CH2), 1.56, 1.47 (each 3H, s, CH3-3,3), 1.12, 1.11, 1.05, 1.02, 0.98, 0.95
from 10 mg of 35k); white solid; mp 155e157 ꢁC; 1H NMR
d 12.94
(1H, s, OH-8), 7.43 (1H, t, J ¼ 8.0, 8.4 Hz, H-10), 6.78 (1H, d,
J ¼ 4.8 Hz, H-1), 6.74 (1H, d, J ¼ 8.0 Hz, H-9), 6.67 (1H, d, J ¼ 8.4 Hz,
H-11), 6.31 (1H, s, H-5), 5.36 (1H, d, J ¼ 4.8 Hz, H-2), 3.99 (3H, s,
OCH3-6), 2,45, 2.18, 1.90, 1.65 (each 2H, m, camphanoyl-CH2), 1.52,
1.45 (each 3H, s, CH3-3,3), 1.10, 1.09, 1.00, 0.96, 0.91, 0.88 (each 2H, s,
(each 3H, s, camphanoyl-CH3); [
a
]
ꢀ30.0ꢁ (c ¼ 0.0012, CHCl3);
D
HRMS for (Mþ þ Na): calcd m/z 750.2527, found: 750.2523.
camphanoyl-CH3); [
a
]
ꢀ62.5ꢁ (c ¼ 0.0018, CHCl3); HRMS for
6.1.7.19. 1R,2R-(ꢀ)-Dicamphanoyl-3,3-dimethyl-6-methoxy-9-cyano-
1,2-dihydropyrano[2,3-c]xanthen-7(1H)-one (29). 65% Yield (starting
D
(Mþ þ Na): calcd m/z 741.2523, found: 741.2528.
from 30 mg of 37); white solid; mp 165e167 ꢁC; 1H NMR
d 8.59 (1H,
6.1.7.13. 1R,2R-(ꢀ)-Dicamphanoyl-3,3-trimethyl-6-methoxy-11-fluoro-
1,2-dihydropyrano[2,3-c]xanthen-7(1H)-one (20). 80% Yield (starting
s, H-8), 7.84 (1H, d, J ¼ 8.8 Hz, H-10), 7.37 (1H, d, J ¼ 8.8 Hz, H-11),
6.84 (1H, d, J ¼ 4.8 Hz, H-1), 6.39 (1H, s, H-5), 5.40 (1H, d, J ¼ 4.8 Hz,
H-2), 4.02 (3H, s, OCH3-6), 2.50, 2.10, 1.90, 1.70 (each 2H, m, cam-
phanoyl-CH2), 1.54, 1.50 (each 3H, s, CH3-3,3), 1.14, 1.12, 1.05,
from 45 mg of 35l); white solid; mp 183e184 ꢁC; 1H NMR
d 8.05 (1H,
d, J ¼ 8.0 Hz, H-8), 7.39 (1H, t, J ¼ 8.0, 8.4 Hz, H-9), 7.28 (1H, d,
J ¼ 8.4 Hz, H-10), 6.76 (1H, d, J ¼ 4.4 Hz, H-1), 6.36 (1H, s, H-5), 5.43
(1H, d, J ¼ 4.4 Hz, H-2), 4.01 (3H, s, OCH3-6), 2.51, 2.20, 1.92, 1.75 (each
2H, m, camphanoyl-CH2), 1.58, 1.48 (each 3H, s, CH3-3,3), 1.13, 1.13,
1.00, 0.93, 0.92 (each 3H, s, camphanoyl-CH3);
[a]
ꢀ51.0ꢁ
D
(c ¼ 0.0023, CH2Cl2); HRMS for (Mþ þ Na): calcd m/z 750.2527,
found: 750.2526.

96
T. Zhou et al. / European Journal of Medicinal Chemistry 47 (2012) 86e96
6.1.8. 1R,2R-(ꢀ)-Dicamphanoyl-3,3-dimethyl-6-methoxy-1,2-
dihydropyrano[2,3-c]xanthen-7(1H)-one oxime (8)
yield; 1H NMR
d
13.62 (1H, s, OH-6), 8.20 (1H, d, J ¼ 2.0 Hz, H-8),
7.74 (1H, dd, J ¼ 8.8, 2.0 Hz, H-10), 7.48 (1H, d, J ¼ 8.8 Hz, H-11), 6.89
(1H, s, H-5), 5.30, 5.30 (each 1H, s, H-1,2), 2.44 (2H, s, CH2Br-9), 2.40,
2.15, 1.90, 1.70 (each 2H, m, camphanoyl CH2), 1.56, 1.55 (each 3H, s,
CH3-3,3), 1.11, 1.10, 1.04, 1.03, 0.98, 0.85 (each 3H, s, camphanoyl
CH3); HRMS for (Mþ þ Na): calcd m/z 817.1836, found: 817.1822.
Compound 7 (32 mg, 0.045 mmol) and NH2OH HCl (5 mg,
0.07 mmol) were dissolved in anhydrous pyridine (1 mL) and stirred
at 100 ꢁC, monitored by TLC. At completion, the reaction mixture was
poured into water and extracted with EtOAc. The organic layer was
collected and the crude product was purified by PTLC with
hexane:EtOAc ¼ 1:1 to yield 8 as a white solid. 80% yield; mp
6.2. HIV-1 infectivity assay
160e162 ꢁC; 1H NMR
d
8.99 (1H, d, J ¼ 8.4 Hz, H-8), 7.41 (1H, t, J ¼ 7.2,
8.4 Hz, H-10), 7.21 (1H, t, J ¼ 7.2, 8.4 Hz, H-9), 7.15 (1H, d, J ¼ 8.4 Hz, H-
11), 6.76 (1H, d, J ¼ 4.8 Hz, H-1), 6.33 (1H, s, H-5), 5.35 (1H, d,
J ¼ 4.8 Hz, H-2), 3.98 (3H, s, OCH3-6), 2.50, 2.20,1.90,1.70 (each 2H, m,
camphanoyl-CH2),1.52,1.45 (each 3H, s, CH3-3,3),1.13,1.12,1.03, 0.99,
Anti-HIV-1 activity was measured as reduction in Luc reporter
gene expression after a single round of virus infection of TZM-bl
cells. HIV-1 at 200 TCID50 and various dilutions of test samples
(eight dilutions, four-fold stepwise) were mixed in a total volume of
0.91, 0.89 (each 3H, s, camphanoyl-CH3); [
a]
D
ꢀ141.3ꢁ (c ¼ 0.0023,
100
Costar). After 48-h incubation, culture medium was removed from
each well and 100 L of Bright Glo luciferase reagent was added to
mL growth medium in 96-well black solid plates (Corning-
CH2Cl2); HRMS for (Mþ þ Na): calcd m/z 740.2683, found: 740.2714.
m
6.1.9. 1R,2R-(ꢀ)-Dicamphanoyl-3,3-dimethyl-6-hydroxy-1,2-
dihydropyrano[2,3-c]xanthen-7(1H)-one (9)
each culture well. The luciferase activity in the assay wells was
measured using a Victor 2 luminometer. The 50% inhibitory dose
(IC50) was defined as the sample concentration that caused a 50%
reduction in Relative Luminescence Units (RLU) compared to virus
control wells after subtraction of background RLU.
Compound 7 (20 mg, 0.028 mmoL) was heated with 48% HBr
(2.0 mL) at reflux temperature for 12 h. The reaction mixture was
allowed to cool, diluted with water, and filtered and then the residue
was washed thoroughly with water. The crude product was purified
by PTLCwithhexane:EtOAc ¼ 1:1 toyield9 asawhitesolid. 63% yield;
mp 165e167 ꢁC; 1H NMR
d
13.67 (1H, s, OH-6), 8.25 (1H, d, J ¼ 7.2 Hz,
6.3. Cytotoxic activity assay
H-8), 7.74 (1H, d, J ¼ 8.8, 8.4 Hz, H-10), 7.46 (1H, d, J ¼ 8.4 Hz, H-11),
7.41 (1H, d, J ¼ 8.8, 7.2 Hz, H-10), 6.63 (1H, d, J ¼ 4.8 Hz, H-1), 6.44 (1H,
s, H-5), 5.36 (1H, d, J ¼ 4.8 Hz, H-2), 2.50, 2.20, 1.90, 1.70 (each 2H, m,
camphanoyl CH2), 1.50, 1.47 (each 3H, s, CH3-3,3), 1.17, 1.11, 1.08, 1.07,
The general procedure was performed according to CytoTox-
GloÔ cytotoxic activity assay instructions for using product G9290,
G9291 and G9292 (Promega).
0.98, 0.97 (each 3H, s, camphanoyl CH3); [
a]
D
ꢀ70.0ꢁ (c ¼ 0.0023,
CH2Cl2); HRMS for (Mþ þ NH4): calcd m/z 706.2864, found: 706.2896.
Acknowledgments
6.1.10. 1R,2R-(ꢀ)-Dicamphanoyl-3,3-dimethyl-5-bromo-6-methoxy-
1,2-dihydropyrano[2,3-c]xanthen-7(1H)-one (25)
This investigation was supported by grant AI033066 from
National Institute of Allergy and Infectious Disease (NIAID) awar-
ded to K. H. Lee. This study was also supported in part by the
Department of Health Clinical Trial and Research Center of Excel-
lence (DOH-100-TD-B-111-004).
A mixture of 7 (50 mg, 0.071 mmoL), NBS (17.0 mg, 0.10 mmol)
andCH2Cl2 (2 mL)washeated to 100 ꢁC for2 h underhigh-absorption
microwave conditions. At completion, the mixture was concentrated
and purified by PTLC with hexane:EtOAc ¼ 3:2 to afford pure 25 as
a white solid (43.7 mg). 78.9% yield; mp 139e140 ꢁC; 1H NMR
d 8.29
Appendix. Supplementary data
(1H, d, J ¼ 8.0 Hz. H-8), 7.65 (1H, t, J ¼ 7.6, 7.6 Hz, H-10), 7.39 (1H, t,
J ¼ 8.0, 7.6 Hz, H-9), 7.29 (1H, d, J ¼ 7.6 Hz, H-11), 6.90 (1H, d,
J ¼ 4.8 Hz, H-1), 5.44 (1H, d, J ¼ 4.8 Hz, H-2), 4.05 (3H, s, OCH3-6), 2.53,
2.21, 1.95, 1.75 (each 2H, m, camphanoyl CH2), 1.14, 1.13, 1.05, 1.01,
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.ejmech.2011.10.025.
0.94, 0.93 (each 3H, s, camphanoyl CH3); [
a
]
ꢀ51.3ꢁ (c ¼ 0.0023,
D
CHCl3); HRMS for (Mþ þ Na): calcd m/z 803.1679, found: 803.1680.
References
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6.1.11. 1R,2R-(ꢀ)-Dicamphanoyl-3,3,9-trimethyl-5-bromo-6-
methoxy-1,2-dihydropyrano[2,3-c]xanthen-7(1H)-one (26)
The procedure was identical to that used for the preparation of 25.
56% yield (starting from 50 mg of 12); white solid; mp 170e171 ꢁC; 1H
NMR
d
8.06 (1H, s, H-8), 7.45 (1H, d, J ¼ 8.4 Hz, H-10), 7.19 (1H, d,
J ¼ 8.4 Hz, H-11), 6.88 (1H, d, J ¼ 4.4 Hz, H-1), 5.43 (1H, d, J ¼ 4.4 Hz, H-
2), 4.04 (3H, s, OCH3-6), 2.44 (3H, s, CH3-9), 2.50, 2.20,1.90,1.70 (each
2H, m, camphanoyl CH2),1.58,1.58(each 3H, s, CH3-3,3), 1.41, 1.13, 1.04,
1.01, 0.93, 0.91 (each 3H, s, camphanoyl CH3); [
a
]D ꢀ10.8ꢁ (c ¼ 0.0078,
CHCl3); HRMS for (Mþ þ Na): calcd m/z 817.1836, found: 817.1826.
6.1.12. 1R,2R-(ꢀ)-Dicamphanoyl-3,3-dimethyl-6-hydroxy-9-
bromomethyl-1,2-dihydropyrano[2,3-c]xanthen-7(1H)-one (27)
A mixture of 12 (50 mg, 0.07 mmoL), NBS (18.6 mg, 0.105
mmoL), and 3-chloroperbenzoic acid (2 mg, 0.01 mmoL), dissolved
in 1 mL of anhydrous CCl4 was heated to 100 ꢁC for 5 h under high-
absorption microwave conditions. At completion, the mixture was
concentrated and the residue was purified by PTLC with
hexane:EtOAc ¼ 1:1 to afford pure 27 (31.3 mg, white solid). 56.1%