Anti-AIDS agents 79. Design, synthesis, molecular modeling and structure-activity relationships of novel dicamphanoyl-2′,2′- dimethyldihydropyranochromone (DCP) analogs as potent anti-HIV agents

Article abstract of DOI:10.1016/j.bmc.2010.07.065

In a continued study, 23 3′R,4′R-di-O-(-)-camphanoyl-2′, 2′-dimethyldihydropyrano[2,3-f]chromone (DCP) derivatives (5-27) were synthesized, and screened for anti-HIV activity against both a non-drug-resistant NL4-3 strain and multiple reverse transcriptas

Full text of DOI:10.1016/j.bmc.2010.07.065

Bioorganic & Medicinal Chemistry 18 (2010) 6678–6689
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Anti-AIDS agents 79. Design, synthesis, molecular modeling and structure–
activity relationships of novel dicamphanoyl-20,20-dimethyldihydropyrano-
chromone (DCP) analogs as potent anti-HIV agents
Ting Zhou a, Qian Shi a, Chin-Ho Chen b, Hao Zhu c, Li Huang b, Phong Ho b, Kuo-Hsiung Lee a,d,
*
a Natural Products Research Laboratories, Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, NC 27599, USA
b Medical Center, Box 2926, SORF, Duke University, Durham, NC 27710, USA
c The Laboratory for Molecular Modeling, Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, NC 27599, USA
d 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
a
continued study, 23 30R,40R-di-O-(ꢀ)-camphanoyl-20,20-dimethyldihydropyrano[2,3-f]chromone
Received 28 June 2010
Revised 27 July 2010
Accepted 28 July 2010
Available online 2 August 2010
(DCP) derivatives (5–27) were synthesized, and screened for anti-HIV activity against both a non-drug-
resistant NL4-3 strain and multiple reverse transcriptase (RT) inhibitor-resistant (RTMDR-1) strain, using
2-EDCP (4) and 2-MDCP (35) as controls. New DCP analogs 5, 9, 14, and 22 exhibited potent anti-HIV
activity against HIVNL4-3 with EC50 and therapeutic index (TI) values ranging from 0.036
0.14 M and from 110 to 420, respectively. Compounds 5 and 9 also exhibited good activity against
RTMDR-1 (EC50 0.049 and 0.054 M; TI 310 and 200, respectively), and were twofold more potent than
the leads 4 and 35 (EC50 0.11 and 0.19 M; TI 60 and 58, respectively). Evaluation of water solubility
showed that 5 and 22 were 5–10 times more water soluble than 4. Quantitative structure–activity rela-
tionship (QSAR) modeling results were first performed on this compound type, and the models should aid
in design of future anti-HIV DCP analogs and potential clinical drug candidates.
Ó 2010 Elsevier Ltd. All rights reserved.
lM to
l
Keywords:
l
30R,40R-Di-O-(ꢀ)-camphanoyl-20,
20-dimethyldihydropyrano
[2,3-f]chromone (DCP)
derivatives
l
HIV-1
Reverse transcriptase (RT)
Structure–activity relationship (SAR)
O
O
R
5
5
4
4
1. Introduction
3
6
7
3
2
6
7
2
Although over 30 formulations are now approved by the US FDA
to treat AIDS, drug resistance problems have dramatically reduced
the efficacy of these current anti-HIV agents.1 Therefore, research
to find new anti-HIV agents with either higher potency or novel
mechanisms has attracted great attention to overcome this
problem.2
O
R
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
In our prior studies, 30R,40R-di-O-(ꢀ)-camphanoyl-(+)-cis-khellac-
tone (DCK, 1) and 4-methyl DCK (4-MDCK, 2) showed high potency
against HIV-1IIIB replication in H9 lymphocytes. The EC50 and thera-
O
O
1 DCK, R = H
2 4-Me-DCK,
3 DCP, R = H
4 2-Ethyl-DCP, R = CH2CH3
R = CH3
peutic index (TI) values were reported as 0.049
lM and 328 for DCK,
and, 0.0059
l
M and 6660 for 4-MDCK, respectively (Fig. 1).3,4 More
Figure 1. DCK and DCP analogs.
specifically, preliminary mechanism of action-related studies indi-
cated that 4-MDCK inhibited the activity of HIV-RT through inhibition
of DNA-dependent DNA polymerase activity, in contrast to currently
available NNRTIs that block HIV-RT by inhibiting RNA-dependent
DNA polymerization.5 However, DCK had reduced activity against
the multi-RT inhibitor resistant (RTMDR-1) strain. In the course of
our continuing exploration of DCK analogs as potent anti-HIV agents,
4H-chrom-4-one derivatives (DCPs) were designed and synthesized
as DCK positional isomers (Fig. 1).6,7 Compared with DCKs, DCP ana-
logs not only retained high activity against wild-type HIV, but also
showed potency against RTMDR-1 HIV.7 Among the previously re-
ported DCP derivatives, 2-ethyl DCP (2-EDCP, 4) exhibited the best
anti-HIV activity against both wild-type and drug-resistant strains
with EC50 values of 0.070 and 0.11 lM and TI values of 94 and 60,
respectively. The uniqueness of DCP analogs opens a new avenue for
us to discover a distinct class of potent, effective anti-HIV drugs for
AIDS therapy.
* Corresponding author. Tel.: +919 962 0066; fax: +919 966 3893.
E-mail address: khlee@unc.edu (K.-H. Lee).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.07.065

T. Zhou et al. / Bioorg. Med. Chem. 18 (2010) 6678–6689
6679
The structure–activity relationship (SAR) information provided
from our previous study on the DCP series led to the following con-
clusions. Steric effects of substitutions on position-2, -3, and -6 of
the chromone system could influence the anti-HIV activity. Bulky
substituents at position-3 or -6 dramatically reduced anti-HIV
activity. In addition, appropriate alkyl substitution at position-2
was crucial to maintain high activity against both wild-type and
multi-RT inhibitor-resistant strains. 2-EDCP (4), with an ethyl
group at position-2 of the chromone ring, exhibited the most po-
tent activity against both virus strains.
However, the preliminary SAR information on DCPs was not
extensive enough to establish a feasible pharmacological profile.
Except for the steric effect, prior data could not illustrate how other
factors such as electronic and hydrogen-bond effects might influ-
ence activity. In addition, all active DCP analogs synthesized had
poor water-solubility. Therefore, additional DCP analogs with vary-
ing substituents, particularly different from the prior analogs, are
needed in the search for an optimal anti-HIV-1 drug candidate
from this compound class.
In our present study, DCP analogs with different structural func-
tionalities on the pyranochromone have been synthesized towards
this aim. We first designed and synthesized several 5-alkyl-substi-
tuted DCPs to explore the steric effect at position-5, which was not
a major focus in prior studies. Then, we introduced combinations
of diverse functional groups at position-2, -3, -5 and -6, including
halogen, cyano, and amino groups, to explore electronic and hydro-
gen-bonding effects. Furthermore, we introduced hydrophilic het-
erocyclic amine moieties at position-2 to generate compounds
with better water solubility. All newly synthesized DCPs were eval-
uated for their activity against both wild-type and RTMDR-1
strains. Two of the active and more polar compounds, 5 and 22,
were selected for water solubility analysis in comparison with
the active lead compound 4.
A QSAR molecular modeling study was also performed in this
research using Partial Least Square (PLS) method with QuaSAR-
Model module of MOE 2009 to systematically study the struc-
ture–anti-HIV–activity relationships of DCP-class compounds.
With this study, we aimed not only to establish a pharmacological
profile of DCPs, but also to study the DCP pharmacophores that
play an important role in anti-HIV activity.
Friedel–Crafts reaction in the presence of the Lewis acid ZnCl2 to
afford 30b and 30c, respectively (Scheme 1).8–10
The synthesis of 2,3,5-alkyl substituted DCP analogs is shown in
Scheme 2. Commercially available 1-(2,4-dihydroxyphenyl)etha-
none (30a) and the synthesized 30b–c were converted to 31a–c
by alkylation with 4,4-dimethoxy-2-methyl-2-butanol in pyridine.
This reaction was conducted using a modified method by reaction
of ethanone and butanol in a microwave initiator at 220 °C for
4 h.11 Compounds 4–5, 9–10, and 35–36 were synthesized follow-
ing literature procedures.7 Briefly, reaction of 31a–c with diverse
ethyl alkanoates in the presence of NaH followed by hydrolysis
with Amberlyst 15 resin in isopropanol afforded the chromone ring
closure products (32a–e). 20,20-Dimethyl-3-methylpyaranochr-
mone (32f) was synthesized from propiophenone (30d) in two
steps. Commercially available 30d was treated with methanesulfo-
nyl chloride in dry DMF to afford 7-hydroxy-3-methyl-chromone
(33).12 Compound 33 was converted to the corresponding pyra-
nochromone (32f) by alkylation with 4,4-dimethoxy-2-methyl-2-
butanol in pyridine under microwave conditions. The asymmetric
dihydroxylation of 32a–f was accomplished using a catalytic
Sharpless asymmetric dihydroxylation,13,14 in which K2OsO2(OH)4
served as catalyst and (DHQ)2PYR as chiral auxiliary.14,15 After dry-
ing in vacuo overnight, the diols (34a–f) were reacted with excess
(S)-camphanic chloride in anhydrous dichloromethane in the pres-
ence of excess DMAP at rt for 2 h to afford the target compounds
4–5, 9–10, and 35–36.
Scheme 3 illustrates the synthesis of novel DCP analogs with
various functional groups at position-2. The synthesis of 11 and
12 was accomplished by benzylic bromination with NBS in anhy-
drous carbon tetrachloride in the presence of 3-chloroperbenzoic
acid as a radical initiator. Dibromo-substituted DCP analog (13)
was also obtained during the reaction as previously reported.16
Compound 11 was treated with KCN under mild condition in
DMF to give 15.17 Reaction of 11 with appropriate amine groups
in THF at rt afforded compounds 16 and 25–27.18
The synthesis of novel DCP analogs 17–24 with 3-substitutions
is shown in Scheme 4. Selective bromination of 35 or 4 in acetoni-
trile gave 17 or 18, respectively.19 Compound 18 was subsequently
converted to 20 in the presence of KCN in a mixture of DMF and
95% aq EtOH.17 Reaction of 17 or 18 with 33% aqueous ammonium
solution or methylamine at rt gave 21–24.18 Compound 35 was
treated with I2 in the presence of CF3CO2Ag as catalyst to obtain
19 in almost quantitative yield.20
In this paper, we report and discuss the chemistry and synthesis
of the newly synthesized DCP analogs, the results of anti-HIV activ-
ity evaluation, water solubility analysis, QuaSAR-model and
pharmacophore studies, as well as structure–anti-HIV–activity
relationship conclusions resulting from the studies.
The synthesis of 2-cyano-3-methyl-DCP (14) is given in Scheme
5. Stirring 36 with NBS in dichloromethane and heating to reflux
gave 37, which was further reacted with NaCN to give 14, with a
cyano substituent at position-2,17 rather than displacement of
bromide to give 14a. The postulated Michael addition-elimination
mechanism is illustrated in Scheme 6. Tautomerization of interme-
diate 38 regains resonance stabilization and produces compound
14.
2. Results and discussion
2.1. Chemistry
Scheme 1 illustrates the synthesis of 6-methyl- and 6-ethyl-2,
4-dihydroxyphenyl ethanones (30b–c, respectively). Compounds
29b (commercially available) and 29c [synthesized by reduction
of 30,50-dihydroxyacetophenone (28)] were acylated through a
The synthesis of 6–8 is shown in Scheme 7. Different solvents
were used as mentioned above to selectively generate 6 and
8.16,19 Compound 7 was obtained by using excess NBS.
R
O
O
R
29c
i (for
)
ii
HO
OH
HO
OH
HO
OH
30b R = CH3
30c
28
29b R = CH3
29c
R = CH2CH3
R = CH2CH3
Scheme 1. Synthesis of 30b and 30c. Regents and conditions: (i) Pd/C, H2, 4% HCl, rt; (ii) CH3CN, HCl, ZnCl2, diethyl ether, 0 °C.

6680
T. Zhou et al. / Bioorg. Med. Chem. 18 (2010) 6678–6689
R5
O
O
R5
O
R5
O
i
ii, iii
O
R2
HO
OH
O
OH
32a R2 = CH3, R5 = H
30a R3 = R5 = H
31a R3 = R5 = H
32b
R2 = R5 = CH3
30b
,
31b
,
R3 = H R5 = CH3
R3 = H R5 = CH3
32c R2 = CH2CH3, R5 = H
30c R3 = H, R5 = CH2CH3
31c R3 = H, R5 = CH2CH3
32d R2 = CH2CH3, R5 = CH3
32e
R
2 = R5 = CH2CH3,
O
O
O
iv
i
O
O
HO
OH
HO
O
30d
33
32f
R5
O
O
R5
O
O
R5
O
R3
R2
R3
R2
R3
R2
v
vi
O
O
O
O
OH
OR'
OH
34a
OR'
32a R2 = CH3, R3 = R5 = H
32b
R2 = CH3, R3 = R5 = H
35 R2 = CH3, R3 = R5 = H
R2 = R5 = CH3, R3 = H
32c R2 = CH2CH3, R3 = R5 = H
32d R2 = CH2CH3, R3 = H, R5 = CH3
32e
34b R2 = R5 = CH3, R3 = H
5
R2 = R5 = CH3, R3 = H
34c
34d
R2 = CH2CH3, R3 = R5 = H
4 R2 = CH2CH3, R3 = R5 = H
R2 = CH2CH3, R3 = H, R5 = CH3
9 R2 = CH2CH3, R3 = H, R5 = CH3
R
2 = R5 = CH2CH3, R3 =H
34f R2 = R5 = H, R3 = CH3
10
R2 = R5 = CH2CH3, R3 = H
32f R2 = R5 = H, R3 = CH3
36 R2 = R5 = H, R3 = CH3
R' = (S)-Camphanoyl
Scheme 2. Synthesis of alkyl substituted DCP analogs. Reagents and conditions: (i) 4,4-dimethoxt-2-methyl-2-butanol, pyridine, microwave; (ii) ethyl alkanoates, NaH, THF,
reflux; (iii) Amberlyst 15 resin, isopropanol, reflux; (iv) methanesulfonyl chloride, DMF, (v) K3Fe(CN)6, (DHQ)2PYR; K2OsO2(OH)4, K2CO3, t-butanol/H2O, 0 °C; (vi)
(S)-camphanoyl chloride, DMAP, CH2Cl2.
O
O
O
O
O
O
R
R
Br
i
O
O
+
O
Br
Br
R
OR'
OR'
OR'
OR'
OR'
OR'
11
R = H
13
R = CH3
35 R = H
12 R = CH3
4 R = CH3
O
O
CN
ii
O
O
O
R' = (S)-Camphanoyl
Br
O
O
OR'
OR'
OR'
OR'
15
iii
OR'
O
O
11
16
R
1 = NHCH3
N
N
O
N
25 R1
26
=
=
=
R1
N
R1
27 R1
H
OR'
16, 25-27
N
N
Scheme 3. Synthesis of novel 2-subsituted DCP analogs (11–13, 15, 16, 25–27). Reagents and conditions: (i) NBS, 3-chloroperbenzoic acid, CCl4, reflux; (ii) KCN, DMF, 95% aq
EtOH; (iii) THF, diverse amine.

T. Zhou et al. / Bioorg. Med. Chem. 18 (2010) 6678–6689
6681
O
O
NH2
R2
O
O
O
iv
O
O
Br
OR'
OR'
21
i
O
R2
O
R2
R2 = CH3
OR'
22 R2 = CH2CH3
OR'
OR'
OR'
O
17 R2 = CH3
35 R2 = CH3
NHCH3
R2
v
18 R2 = CH2CH3
4 R2 = CH2CH3
O
O
O
O
OR'
Br
CN
OR'
ii
O
O
O
O
23 R2 = CH3
24 R2 = CH2CH3
OR'
OR'
OR'
OR'
18
20
O
O
O
I
iii
R' = (S)- Camphanoyl
O
O
O
OR'
OR'
OR'
OR'
35
19
Scheme 4. Synthesis of novel 3-subsituted DCP analogs (17–24). Reagents and conditions: (i) NBS, CH2Cl3, reflux; (ii) KCN, DMF, 95% aq EtOH; (iii) I2, CF3COOAg, CH3Cl2, 0 °C;
(iv) NH4OH, THF; (v) NHCH3 H2O, THF.
O
3
CN
O
O 2
OR'
ii
OR'
O
O
O
O
14a
3
Br
i
O
2
O
O
ii
3
OR'
OR'
O
O 2 CN
OR'
OR'
OR'
36
37
R' = (S)-Camphanoyl
OR'
14
Scheme 5. Synthesis of 14. Reagents and conditions: (i) NBS, CH3CN, reflux; (ii) NACN, DMF, 95% aq EtOH.
Br-
O
O
O
O
O
O-
O
CH2
CN
CH3
CN
Br
Br
CN
Tautomerization
O
O
O
O
O
CN-
OR
38
OR
37
OR
OR
14
R = (S)-Camphanoyl
OR
OR
OR
OR
Scheme 6. Speculated mechanism for production of 14.

6682
T. Zhou et al. / Bioorg. Med. Chem. 18 (2010) 6678–6689
O
O
O
O
O
Br
Br
Br
i
O
+
O
O
O
OR'
OR'
OR'
OR'
OR'
OR'
6
5
7
ii
Br
O
O
O
OR'
R' = (S)- Camphanoyl
OR'
8
Scheme 7. Synthesis of compounds 6–8. Reagents and conditions: (i) NBS, CH3CN, reflux; (ii) NBS, 3-chloroperbenzoic acid, CCl4, reflux.
2.2. Biological evaluation
Compounds 5–10 are novel DCP analogs with short alkyl groups
at both position-2 and -5. Compounds 5, 9, and 10 with methyl and
ethyl substituents at these positions exhibited promising anti-HIV
activity against the non-drug-resistant strain HIV-1NL4-3. They also
showed comparable or greater TI values compared with both posi-
tive DCP reference standards 4 and 35. Compound 5 (2-CH3, 5-CH3)
All newly synthesized DCP analogs (5–27) were evaluated for
anti-HIV activity against both HIV-1NL4-3 and HIV-1 RTMDR1, a
multi-RT inhibitor-resistant viral strain, in a single cycle infection
assay using TZM-bl cells. The data are given in Table 1.
Table 1
Anti-HIV activity of DCP analogs 5–27a
R5
O
O
R6
O
R3
R2
OR
R = (S)-Camphanoyl
OR
R5
b
Compd
R2
R3
R6
IC50
(lM)
NL4-3
M)
HIV-1 RTMDR1
EC50
(
l
TI
EC50
(
lM)
TI
5
6
7
8
9
CH3
CH3
CH3
CH3
H
Br
Br
H
H
H
H
H
H
CH3
H
H
Br
CH3
CH3
CH3
CH2Br
CH3
H
H
Br
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
15
0.036
0.59
10
0.81
0.10
0.25
0.55
0.46
1.0
0.14
1.8
0.29
0.55
1.1
0.73
0.55
0.25
0.12
0.30
0.44
420
>24
>1.2
11
110
>120
3.0
7.6
1.8
290
9.4
>100
14
0.049
0.91
>12
1.2
0.054
0.34
0.56
0.24
0.45
1.9
310
>15
1
7.4
200
>88
3.0
15
4.0
21
12
>43
14
>28
8.0
>37
68
>14
>12
8.9
11
>30
1.7
3.5
1.8
40
CH2CH3
CH2CH3
CH2Br
CHBrCH3
C(Br)2CH3
CN
CH2CN
CH2NHCH3
CH3
CH2CH3
CH3
CH2CH3
CH3
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
CH2CH3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
17
1.4
>30
7.7
>27
13
>30
32
23
>60
32
0.69
0.57
0.99
1.6
0.79
0.47
0.31
0.45
1.1
Br
I
>24
18
CN
NH2
NH2
NHCH3
NHCH3
>54
130
190
>200
73
CH2CH3
CH3
CH2CH3
74
>130
29
N
25
26
27
H
H
H
H
H
H
H
H
H
>28
17
2.0
3.7
21
>14
4.5
4.4
7.5
21
>6.3
2.2
O
N
N
N
H
N
>27
>1.3
>1.3
N
4
35
CH2CH3
CH3
H
H
H
H
H
H
6.6
11
0.070
0.10
94
110
0.11
0.19
60
58
a
All data presented in this table were averaged from at least three independent experiments.
Cytotoxicity was determined using a Promega CytoTox-Glo™ assay kit.
b

T. Zhou et al. / Bioorg. Med. Chem. 18 (2010) 6678–6689
6683
had the highest potency (EC50 0.036
compounds, and was two times more potent than
0.07 M, TI 94). However, changing the 5-CH3 in 5 to 5-CH2Br in
8 was unfavorable to anti-HIV activity and also decreased the TI va-
lue (8: 0.81 M, TI 11). Likewise, adding bromine at position-3 (6)
l
M, TI 420) among these six
against drug-resistant virus. Thus, the functional group at posi-
tion-5 of the chromone ring is critical for potent activity against
the drug-resistant strain. Halogen-substituted DCPs (6–8, 11–13,
17–19) showed reduced anti-HIV activity against HIV RTMDR-1
when compared with 4 and 35. Among the halogen-substituted
4
(EC50
l
l
or position-3 and -6 (7) led to decreased potency and TI.
DCPs, 12 showed the best activity with EC50 of 0.24 lM. Amino-
Compounds 11–16 and 25–27 are novel 2-substituted DCPs.
Similarly to 8, bromination of the alkyl group at position-2
(11–13) was unfavorable to anti-HIV activity. The potency of 12
substituted DCP analogs (16, 21–24) showed considerable activity
against wild-type HIV-1Nl4-3, but reduced activity against the drug-
resistant strain. The EC50 values of 16 and 22 against HIV-1RTMDR-1
with bromoethyl substitution (EC50 0.46
lower than that of 4; while 13 with dibromoethyl substitution,
exhibited only mild potency (EC50 1.0 M, TI 1.8). Compound 14
with a cyano group at position-2 (EC50 0.14 M, TI 290) exhibited
l
M, TI 7.6) was six times
were 0.69 and 0.31
three times higher than EC50 against wild-type virus (0.29
and 0.12 M). The SAR analysis of the synthesized DCP derivatives
lM, respectively, which are approximately
lM
l
l
l
against the drug-resistant strain is similar to that for the wild-type
comparable anti-HIV activity and lower cytotoxicity compared
with 4 and 35. However, the anti-HIV activity of 15 with a cyanom-
ethyl group at position 2 decreased significantly. With EC50 of
virus.
2.3. Water solubility (WS) analysis
1.8 lM, 15 was ten times less potent than 14, suggesting that a
slight variation in the substitution at position-2 may result in a sig-
nificant change in anti-HIV activity. It is postulated that expanding
the conjugation of the coumarin core structure by adding a cyano
group at position-2 might contribute to high activity. Compound
16, with 2-CH2NHCH3 substitution, also showed considerable
Because prior active DCP analogs showed poor water solubility,
we were interested in improving this molecular parameter. We se-
lected two active compounds from the preliminary SAR work for
further analysis: 5, which had the best anti-HIV activity against
both virus strains, and 22, which contains a hydrophilic amine
group and maintains high anti-HIV activity against wild-type virus
(Table 1). Both compounds showed lower predicted log P values
than 2-EDCP (4) (Table 2), indicative of increased polarity that
may improve the water solubility. We then performed a WS anal-
ysis with 5 and 22, in comparison to 4. We first established a stan-
dard curve of each tested compound by dissolution of the
compound in acetonitrile at rt at various concentrations. The solu-
bility in water could be determined by HPLC through the correla-
tion between the saturated concentration of each compound in
water and the correlating area detected by HPLC. With a solubility
value less than 0.9 mg/L, 2-EDCP (4) showed the lowest WS among
the three compounds. Compound 5 had an improved WS value
(5.2 mg/L), and compound 22 presented the best WS value of
10.3 mg/L. (Table 2) This latter result confirmed that increasing
the polarity of DCP analogs by introducing polar functional groups
could result in improved water solubility. While both compounds 5
and 22 showed better WS than 2-EDCP (4), 5 also showed more po-
tent anti-HIV activity than 4, and thus, could merit further devel-
opment study as a drug candidate.
anti-HIV activity (EC50 0.29 lM, TI >100). These results suggest
that analogs with polar groups, such as cyano and amino, intro-
duced appropriately at position-2, can maintain anti-HIV activity.
In addition, these groups should increase the compounds’ polarity,
which may improve water solubility. However, 25–27 contain
large hydrophilic moieties at position-2, and showed either very
weak (25 and 26) or no (27) activity.
Compounds 17–24 are novel 3-substitued DCPs. Introduction of
halogens such as bromine (17 and 18) and iodine (19) at position-3
reduced anti-HIV activity and TI. Compounds 17 (3-Br. EC50
0.55
ven times less potent, respectively, than the corresponding non-
brominated 35 (EC50 0.10 M, TI 110). 3-Cyano-2-ethyl-DCP (20)
showed moderate activity (EC50 0.55 M, TI >54), and analogs with
NH2 and NHCH3 substituents at position-3 (21–24) maintained
good anti-HIV activity. With a low EC50 value of 0.12 M, 22
lM, TI 14) and 19 (3-I. EC50 0.73 lM, TI 18) were five and se-
l
l
l
(3-NH2) was equipotent with 4 (3-H) and three times more potent
than 24 (3-NHCH3). The 3-NH2 analogs (21 and 22) showed
comparable or greater potency and TI values than corresponding
3-NHCH3 analogs (23 and 24).
In summary, the influence of position-2 and -3 substituents on
anti-HIV activity was generally equivalent. Electronic and hydro-
gen-bonding effects from halogen, cyano, and amino groups at
these positions could influence both anti-HIV activity and thera-
peutic index. Halogens were not favorable and led to decreased
anti-HIV potency and lower TI, while amino moieties resulted in
both potent anti-HIV activity and high TI values. Analogs with a
cyano substituent, particularly at position-2, maintained good
anti-HIV-1 activity. In addition, the extended conjugation of the
chromone ring system might be important to the high-potency,
and led to the potency difference of 14 and 15. Anti-HIV-1 activity
was quite sensitive to the substituent size at position-2, and large
moieties were not tolerable. Functional groups at position-5 of the
chromone ring are also important for potent anti-HIV activity.
Addition of a methyl group at this position led to increased anti-
HIV activity, as exemplified by 5 and 9. 2,5-Dimethyl DCP (5) had
the highest TI values against both wild-type and drug-resistant
HIV.
2.4. Molecular modeling
2.4.1. Partial least square (PLS) QSAR
The PLS QSAR method was employed in the study using the
QuaSAR-Model module of MOE 2009.21 This method is relatively
less sophisticated among those traditional available QSAR ap-
proaches. It was explored here to test if reliable models could be
built for underlying data sets. A set of 2489 theoretical molecular
descriptors used in this calculation was computed using the soft-
ware Dragon v.5.5.22 The number of components was set to no lim-
it on the degree of the fit. The maximum condition number of the
principal component transform of the correlation matrix S, the
condition limit, was set to be a very large number of 1.0ꢁ106.
We used the structures of the 25 DCP analogs listed in Table 1
and their anti-HIV activities (EC50 in
lM) against both NL4-3 and
Table 2
Log P values and water solubility results of 4, 5 and 2
Most of the new DCP analogs were active against HIV RTMDR-1
strain, but were approximately two to three times less potent than
against wild-type virus. Compounds 5 and 9 showed the most
promising activity against HIV-1RTMDR-1 with EC50 values of 0.049
Compound
Predicted log P valuea
Water solubility (mg/L)
5
22
3.85
2.88
4.01
5.2
10.3
<0.9
4 (2-EDCP)
and 0.054
lM and TI values of 310 and 200, respectively. These
a
Calculated using ACD program.
two compounds were approximately twofold more potent than 4

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T. Zhou et al. / Bioorg. Med. Chem. 18 (2010) 6678–6689
RTMDR1 HIV strains to establish PLS models in the present study.
The activity of each compound was transformed to the commonly
used logarithm format and the log (1/EC50) ranged from ꢀ1.31 to
1.44 for the activity against NL4-3 HIV and from -1.31 to 1.31 for
the activity against RTMDR1 HIV. The leave-one-out cross valida-
tion scheme was used to test the reliability and robustness of the
resulting models. One of the 25 compounds was excluded, and a
PLS model was developed for the remaining 24 compounds. Then
the model was used to predict the anti-HIV activity of the excluded
compound. This procedure was repeated 25 times for each type of
activity until each compound was used as the external test com-
pound. From the leave-one-out cross validation procedure for the
PLS model, the correlation coefficients (R2)/mean absolute errors
(MAE) for the wild type and drug resistant HIV strains were 0.67/
0.30 ( Fig. 2a) and 0.60/0.35, respectively ( Fig. 2b). Compounds
25, 26, and 27 had relatively larger MAE than the remaining
compounds, and compound 26 was a common outlier in both mod-
els. A probable reason is that these compounds have dissimilar R2
substituents compared with the rest of the dataset. The R2 and
MAE values obtained from both models indicated that the newly
established models can reliably be used to screen external chemi-
cal libraries in future studies.
shown in Figure 3 represent the identified pharmacophore. In both
sets of compounds, the planar chromone ring, carbonyl group at
position-4, and the oxygen at position-10 were identified as part
of the corresponding pharmacophore. However, in the most potent
compounds (Fig. 3a), the carbonyl group of the 40-camphanoyl es-
ter, which represents a hydrogen bond acceptor, was identified as a
unique pharmacophore. In the three weakest compounds (Fig. 3b),
the orientation of both camphanoyl groups varied dramatically due
to the introduction of bulky substitutions at position-2, which sug-
gested that the orientations of the 30- and 40-camphanoyl groups
might be critical for maintaining high anti-HIV activity.
3. Conclusions
Our study identified a series of new DCP analogs with high anti-
HIV potency against both wild-type and drug-resistant HIV-1
strains. The following SAR conclusions were drawn from these
results. (1) Position-5 of the DCP chromone ring system is critical
for anti-HIV activity against both wild-type and drug-resistant
HIV-1 strains, and appropriate alkyl groups on this position can
improve anti-HIV activity against both virus strains. (2) Electronic
and hydrogen-bonding effects at position-2 and -3 can influence
the anti-HIV activity as well as therapeutic index. 3. The orienta-
tions of the 30- and 40-camphanoyl groups are critical to maintain
high anti-HIV activity against both virus strain, and the carbonyl
group in the 40 position camphanoyl ester was identified as a po-
tential hydrogen-bond acceptor by pharmacophore analysis. We
also analyzed the water solubility of selected newly synthesized
DCP analogs and confirmed that increasing polarity can dramati-
cally improve the water-solubility of DCP analogs. In addition,
2.4.2. Pharmacophore analysis
To explore DCP pharmacophores, the chemical structures of the
three most potent compounds (4, 5, and 9) and the three weakest
compounds (7, 26 and 27) were energy minimized and superim-
posed using the Flexible Alignment of MOE 2009. Then the phar-
macophore analysis was performed using the Pharmacophore
Query. The results are shown in Figure 3a and b. The yellow balls
Figure 2. The correlation between experimental and predicted EC50 values obtained from leave one out cross validation for (a) NL4-3 HIV strain and (b) RTMDR1 HIV strain.
Figure 3. The pharamacophore analysis of the three most active compounds (a) and three most inactive compounds (b) using MOE 2009. The default setting was used except
the tolerance (neighbor distance) and consensus score threshold (percentage of the compounds containing the pharmacophore) were changed to 0.5% and 100%, respectively.
Atom coloring: gray, carbon; blue, nitrogen; red, oxygen. Pharmacophore schemes: purple, hydrogen bond donor; light blue, hydrogen bond acceptor; yellow, aromatic
center.

T. Zhou et al. / Bioorg. Med. Chem. 18 (2010) 6678–6689
6685
we successfully established reliable PLS QSAR models. These mod-
els should help to predict the EC50 values of newly designed DCP
analogs, which may be a useful tool for design of future new DCP
analogs.
tions. 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. The residue
was purified by column chromatography with hexanes/
EtOAc = 97:3 to afford 31a–c and 32f.
4. Experimental section
4.1. Chemistry
4.1.4.1. 6-Acetyl-2,2-dimethyl-5-hydroxy-2H-chromone (31a).
57.3% yield (starting with 1 g of 30a); MS (ESI+) m/z (%) 219
(M++1, 100); 1NMR d 7.52 (1H, d, J = 8.7 Hz, H-7), 6.72 (1H, d,
J = 7.5 Hz, H-4), 6.33 (1H, d, J = 8.7 Hz, H-8), 6.58 (1H, d,
J = 7.5 Hz, H-3), 2.54 (3H, s, COCH3-1), 1.45 (6H, s, CH3-2,2).
Melting points were measured with a Fisher Johns melting appa-
ratus without correction. The proton nuclear magnetic resonance
(1H NMR) spectra were measured on a 300 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). Optical rotation was measured with
a Jasco Dip-2000 digital polarimeter at 20 °C at the sodium D line.
Thin-layer chromatography (TLC) was performed on PLC Silica Gel
60 F254 plates (0.5 mm, Merck). Biotage Flash and Isco Companion
systems were used as medium-pressure column chromatography.
Shimadzu LC-20AT prominence liquid chromatography was used
as HPLC system. Alltima 2.1 mm ꢂ 100 mm C18 3u was used as
HPLC column. Silica gel (200–400 mesh) from Aldrich, Inc. was used
for column chromatography. All other chemicals were obtained
from Aldrich, Inc. All final compounds are >95% pure on the basis
of the two HPLC conditions.
4.1.4.2. 6-Acetyl-2,2,7-trimethyl-5-hydroxy-2H-chromone (31b).
66.4% yield (starting with 77.2 mg of 30b); mp 56–67 °C; MS (ESI+)
m/z (%) 233 (M++1, 100); 1NMR d 6.69 (1H, d, J = 10.2 Hz, H-4), 6.19
(1H, s, H-8), 5.52 (1H, d, J = 10.2 Hz, H-3), 3.31 (3H, s, COCH3–1),
2.53 (3H, s, CH3-7), 1.43 (6H, s, CH3-2,2).
4.1.4.3. 6-Acetyl-2,2-dimethyl-5-hydroxy-7-ethyl-2H-chromone
(31c). 72.4% yield (starting with 500 mg of 30c); MS (ESI+) m/z
(%) 247 (M++1, 100%); 1H NMR d 6.70 (1H, d, J = 10.2 Hz, H-4),
6.25 (1H, s, H-8), 6.52 (1H, d, J = 10.2 Hz, H-3), 2.86 (2H, q,
J = 7.5 Hz, CH2CH3-7), 2.64 (3H, s, CH3CO-6), 1.42, 1.41 (each 3H,
s, CH3-2,2), 1.26 (3H, t, J = 7.5 Hz, CH2CH3-7).
4.1.4.4. 20,20,3-Trimethyl-pyrano[2,3,f]-chromone (32f). 60%
yield (starting with 120 mg of 33); mp 66–67 °C; MS (ESI+) m/z
(%) 243 (M++1, 100); 1H NMR d 7.98 (1H, d, J = 8.7 Hz, H-5), 7.73
(1H, s, H-2), 6.81 (1H, d, J = 8.7 Hz, H-6), 6.76 (1H, d, J = 9.9 Hz,
H-40), 5.67 (1H, d, J = 9.9 Hz, H-30), 2.00 (3H, s, CH3-3), 1.48 (6H,
s, CH3-20,20).
4.1.1. Preparation of 5-ethylbenzene-1,3-diol (29c)
A reaction mixture of 2 g (13.1 mmol) of 30,50-dihydroxyaceto-
phenone (28), 1 g of Pd/C (10%) and 150 mL aqueous HCl (4%)
was hydrogenated overnight (900 mL of H2). The mixture was
filtered and extracted with three portions of Et2O. The dried solu-
tion was evaporated at reduced pressure. The residue was purified
by column chromatography with hexanes/EtOAc = 10:1 to afford
29c as a white solid. 80% yield; MS (ESI+) m/z (%) 137 (M++1,
100); 1H NMR d 6.26 (2H, s, H-4, 6), 6.18 (1H, s, H-2), 4.90 (2H,
br, OH-1, 3), 2.53 (2H, q, J = 7.5 Hz, CH2CH3-5), 1.20 (3H. t,
J = 7.5 Hz, CH2CH3-5).
4.1.5. General procedure for the preparation of 32a–e
A mixture of 31a–c and ethyl alkanoate in absolute THF was
added slowly to a sodium hydride/THF suspension under nitrogen.
The mixture was warmed to reflux temperature for 2–6 h moni-
tored by TLC, followed by neutralization with 10% aq HCl, and
extraction three times with CH2Cl2. The organic layer was collected
and the solvent evaporated under reduced pressure. The residue
and Amberlyst 15 resin were stirred in isopropanol at reflux tem-
perature to give 2-substituted dimethylpranochromone 32a–e.
4.1.2. Preparation of 1-(2,4-dihydroxy-6-methylphenyl)ethanone
(30b)
4.1.5.1. 2,20,20-Trimethyl-pyrano[2,3,f]-chromone (32a). 56%
yield (starting with 558.2 mg of 31a); mp 123–125 °C; MS (ESI+)
m/z (%) 243 (M++1, 100); 1H NMR d 7.92 (1H, d, J = 8.7 Hz, H-5),
6.80 (1H, d, J = 8.7 Hz, H-6), 6.78 (1H, d, J = 9.9 Hz, H-40), 6.09
(1H, s, H-3), 5.68 (1H, d, J = 9.9 Hz, H-30), 2.36 (3H, s, CH3-2), 1.48
(6H, s, CH3-20,20).
MeCN (0.7 mL, 20 mmol) and dry ZnCl2 (1.36 g, 10 mmol) were
added to a solution of 3,5-dihydroxytoluene 29b (1.24 g, 10 mmol)
in Et2O (5 mL). Hydrogen chloride gas was then bubbled through
the mixture, and the resulting precipitate was filtered off and dis-
solved in water. This solution was neutralized by adding aqueous
ammonia solution (33%) and was subsequently stirred for 30 min
at 100 °C. The crude product was purified by column chromatogra-
phy with hexanes/EtOAc = 7:3 to afford 30b (680 mg). 41% yield;
MS (ESI-) m/z (%) 165 (Mꢀꢀ1, 100); 1NMR d 6.24 (1H, s, H-5),
6.23 (1H, s, H-3), 5.44 (2H, br, OH-2, 4), 2.62 (3H, s, COCH3-1),
2.55 (3H, s, CH3-6).
4.1.5.2. 2,20,20-Trimethyl-5-methylpyrano[2,3,f]-chromone (32b).
38% yield (starting with 770 mg of 31b); mp 128 130 °C; MS (ESI+)
1
m/z (%) 257 (M++1, 100); H NMR d 6.74 (1H, d, J = 9.9 Hz, H-40),
6.56 (1H, s, H-6), 6.00 (1H, s H-3), 5.64 (1H, d, J = 9.9 Hz, H-30),
2.76 (3H, s, CH3-5), 2.31 (3H, s, CH3-2), 1.46 (6H, s, CH3-20,20).
4.1.3. Preparation of 1-(2,4-dihydroxy-6-ethylphenyl)ethanone
(30c)
4.1.5.3. 20,20-Dimethyl-2-ethylpyrano[2,3,f]-chromone (32c).
66% yield (starting with 1.1 g of 31a); mp 97–98 °C; MS (ESI+)
m/z (%) 279 (M++Na, 100); 1H NMR d 7.92 (1H, d, J = 8.7 Hz, H-5),
6.80 (1H, d, J = 8.7 Hz, H-6), 6.77 (1H, d, J = 10.2 Hz, H-40), 6.10
(1H, s, H-3), 5.69 (1H, d, J = 10.2 Hz, H-30), 2.65 (2H, q, J = 7.5 Hz,
CH2CH3-2), 1.44, 1.48 (each 3H, s, CH3-20,20), 1.30 (3H, t,
J = 7.5 Hz, CH2CH3-2).
The procedure was identical to that used for the preparation of
30b. 40% yield (starting with 2.36 g of 29c); MS (ESI+) m/z (%) 181
(M++1, 100); 1NMR d 6.28 (1H, s, H-5), 6.19 (1H, s, H-3), 4.85 (2H,
br, OH-2, 4), 2.91 (2H, q, J = 7.2 Hz, CH2CH3-6), 2.67 (1H, s, COCH3-
1), 1.30 (3H, t, J = 7.2 Hz, CH2CH3-6).
4.1.4. General procedure for the preparation of 31a–c and 32f
A mixture of starting compound 30a–c (1 equiv) or 33, 4,4-dime-
thoxy-2-methyl-2-butanol(1.5–2 equiv) and pyridine (2–3 mL) was
heated at 220 °C for 4 h under high absorption microwave condi-
4.1.5.4. 20,20-Dimethyl-2-ethyl-5-methylpyrano[2,3-f]-chromone
(32d). 45% yield (starting with 64.8 mg of 31b); mp 123–
124 °C; MS (ESI+) m/z (%) 271 (M++1, 100); 1H NMR d 7.75 (1H,

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T. Zhou et al. / Bioorg. Med. Chem. 18 (2010) 6678–6689
d, J = 10.5 Hz, H-40), 6.57 (1H, s, H-6), 6.01 (1H, s, H-3), 5.64 (1H, d,
J = 10.5 Hz, H-30), 2.77 (3H, s, CH3-5), 2.60 (2H, q, J = 7.5 Hz,
CH2CH3-2), 1.59, 1.47 (each 3H, s, CH3-20,20), 1.29 (3H, t,
J = 7.5 Hz, CH2CH3-2).
4.1.7.4. 30R, 40R-Dihydroxy-5,20,20-trimethyl-2-ethylpyrano[2,3-
f]chromone (34d). 40% yield (starting with 272 mg of 32d); mp
114–116 °C; MS (ESI+) m/z (%) 305 (M++1, 100); 1H NMR d 6.62
(1H, s, H-6), 6.06 (1H, s, H-3), 5.15 (1H, dd, J = 3.6, 5.1 Hz, H-40),
3.86 (1H, dd, J = 5.1, 6.9 Hz, H-30), 3.01 (1H, d, J = 6.9 Hz, OH-30),
2.98 (1H, d, J = 3.9 Hz, OH-40), 2.76 (3H, s, CH3-5), 2.64 (2H, q,
J = 7.5 Hz, CH2CH3-2), 1.46, 1.42 (each 3H, s, CH3-20,20), 1.31 (3H,
t, J = 7.5 Hz, CH2CH3-2).
4.1.5.5. 20,20-Dimethyl-2,5-diethylpyrano[2,3,f]chromone (32e).
36% yield (starting with 120 mg of 31c); MS (ESI+) m/z (%) 285
1
(M++1, 100); H NMR d 6.76 (1H, d, J = 10.2 Hz, H-40), 6.61 (1H, s,
H-6), 6.01 (1H, s H-3), 5.64 (1H, d, J = 10.2 Hz, H-30), 3.24 (2H, q,
J = 7.5 Hz, CH2CH3-5), 2.58 (2H, q, J = 7.5 Hz, CH2CH3-2), 1.48 (6H,
s, CH3-20,20), 1.29 (3H, t, J = 7.5 Hz, CH2CH3-5), 1.22 (3H, t,
J = 7.5 Hz, CH2CH3-2).
4.1.7.5. 30R,40R-Dihydroxy-3,20,20-trimethylpyrano[2,3-f]chromone
(34f). 55% yield (starting with 1.3 g of 32f); mp 180–182 °C; MS
(ESI+) m/z (%) 277 (M++1, 100); 1H NMR d 7.98 (1H, d, J = 8.7 Hz, H-
5), 7.74 (1H, d, J = 1.0 Hz, H-2), 6.82 (1H, d, J = 8.7 Hz, H-6), 6.15
(1H, dd, J = 5.4, 3.6 Hz, H-40), 3.85 (1H, dd, J = 5.7, 5.4 Hz, H-30), 3.43
(1H, d, J = 3.6 Hz, OH-40), 3.19 (1H, d, J = 5.7 Hz, OH-30), 1.99 (3H, d,
J = 1.0 Hz, CH3-3), 1.41, 1.42 (each 3H, s, CH3-20,20).
4.1.6. Preparation of 7-hydroxy-3-methylchromone (33)
The commercially available phenol 30d (400 mg, 2.41 mmol) in
dry DMF (6 mL) was heated to 50 °C, and a solution of methanesul-
fonyl chloride (0.5 mL) in dry DMF (1 mL) was added slowly. The
mixture was then reacted at 60 °C for 6 h. After cooling, the reac-
tion mixture was poured into a large volume of ice-cold aqueous
sodium acetate (12 g/100 mL). The crude product was filtered off
and purified by column chromatography with hexanes/EtOAc = 7:3
to afford 33 (120 mg). 28% yield; mp 155–157 °C; MS (ESI+) m/z (%)
199 (M++Na, 100); 1H NMR d 10.72 (1H, s, OH-7), 8.11 (1H, s, H-2),
7.88 (1H, d, J = 9.0 Hz, H-5), 6.89 (1H, dd, J = 9.0, 2.4 Hz, H-6), 6.80
(1H, d, J = 2.4 Hz, H-8), 1.87 (3H, s, CH3-3).
4.1.8. Preparation of 30R,40R-di-O-(ꢀ)-camphanoyl-20,20-dimet-
hyl-3-bromomethyl-dihydropyrano[2,3-f]chromone (37)
The mixture of 36 (40 mg, 0.06 mmol), NBS (18 mg, 0.1 mmol),
and MeCN (2 ml) was heated to reflux for 4 h, monitored by TLC. At
completion, the mixture was concentrated and purified by PTLC
with an eluent of hexanes/EtOAc = 5:4 to afford pure 37 (30 mg):
70% yield; MS-ESI+ (m/z,%) 715 (M++1, 100); 1H NMR d 8.21 (1H,
d, J = 9.0 Hz, H-5), 7.96 (1H, s, H-2), 6.98 (1H, d, J = 9.0 Hz, H-6),
6.72 (1H, d, J = 4.8 Hz, H-40), 5.38 (1H, d, J = 4.8 Hz, H-30), 4.35
(2H, s, CH2Br-3), 2.43, 2.20, 1.93, 1.85 (each 2H, m, camphanoyl
CH2), 1.53, 1.49 (each 3H, s, CH3-20,20), 1.13, 1.10, 1.09, 1.02, 0.98,
0.91 (each 3H, s, camphanoyl CH3).
4.1.7. General procedure for the preparation of 34a–d and 34f
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 methane-
sulfonamide (1 equiv) was added with stirring. After 20 min,
substituted pyranochromone (32a–f) was added. The mixture
was stirred at 0 °C for 1–2 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 = 3:7
to afford the pure substituted (+)-cis-30,40-dihydroxypyranochro-
mones (34a–f).
4.1.9. General procedure for the preparation of 4–5, 9, 35–36
The substituted 30R,40R-dihydroxypyranochromones (34a–f),
(S)-(ꢀ)-camphanic chloride (3 equiv), and DMAP (4 equiv) were
stirred in CH2Cl2 for 1–2 h at rt, monitored by TLC. At completion,
the mixture was diluted with CH2Cl2 and washed by water and
brine. The solvent was then removed under reduced pressure and
the residue was purified by PTLC with hexanes/EtOAc = 3:2 to af-
ford the appropriately alkyl-substituted 30R,40R-di-O-(ꢀ)-campha-
noyl-20,20-dimethyldihydroprano[2,3-f]chromones (4–5, 9, 35–36).
4.1.9.1. 30R,40R-Di-O-(ꢀ)-camphanoyl-2,20,20-trimethyldihydro-
pyrano[2,3-f]chromone (35). 70% yield (starting from 100 mg
of 34a); mp 146–148 °C; MS-ESI+ (m/z,%) 659 (M++Na, 100); 1H
NMR d 8.11 (1H, d, J = 8.8 Hz, H-5), 6.90 (1H, d, J = 8.8 Hz, H-6),
6.75 (1H, d, J = 4.6 Hz, H-40), 6.12 (1H, s, H-3), 5.37 (1H, d,
J = 4.6 Hz, H-30), 2.46, 2.12, 1.92, 1.70 (each 2H, m, camphanoyl
CH2), 2.27 (3H, s, CH3-2), 1.53, 1.46 (each 3H, s, CH3-20), 1.11,
1.10, 1.07, 1.00, 0.97, 0.94 (each 3H, s, camphanoyl CH3); 60% de.
[R]D ꢀ69.6 (c 0.25, CHCl3).
4.1.7.1. 30R,40R-Dihydroxy-2,20,20-trimethylpyrano[2,3-f]chromone
(34a). 66% yield (starting with 1.1 g of 32a); mp 176–178 °C; MS
(ESI+) m/z (%) 276 (M++1, 100); 1H NMR (DMSO) d 7.95 (1H, d,
J = 9.0 Hz, H-5), 6.84 (1H, d, J = 9.0 Hz, H-6), 6.10 (1H, s, H-3), 5.20
(1H, t, J = 4.2, 4.2 Hz, H-40), 3.87 (1H, t, J = 7.2, 4.2 Hz, H-30), 3.44
(1H, d, J = 4.2 Hz, OH-40), 3.18 (1H, d, J = 7.2 Hz, OH-30), 2.40 (3H, s,
CH3-2), 1.50, 1.44 (each 3H, s, CH3-20,20).
4.1.7.2. 30R,40R-Dihydroxy-2, 5, 20,20-tetramethylpyrano[2,3-
f]chromone (34b). 35% yield (starting with 325 mg of 32b); mp
114–116 °C; MS (ESI+) m/z (%) 291 (M++1, 100); 1H NMR d 6.61
(1H, s, H-6), 6.04 (1H, s, H-3), 5.15 (1H, t, J = 3.9, 4.5 Hz, H-40),
3.85 (1H, dd, J = 4.5, 6.6 Hz, H-30), 3.08 (1H, d, J = 3.9 Hz, OH-40),
3.05 (1H, d, J = 6.6 Hz, OH-30), 2.74 (3H, s, CH3-5), 2.35 (3H, s,
CH3-2), 1.46, 1.43 (each 3H, s, CH3-20,20).
4.1.9.2. 30R,40R-Di-O-(ꢀ)-camphanoyl-3,20,20-trimethyldihydro-
pyrano[2,3-f]chromone (36). 75% yield (starting from 200 mg
of 34f); mp 146–148 °C; MS-ESI+ (m/z,%) 659 (M++Na, 100); 1H
NMR d 8.16 (1H, d, J = 9.0 Hz, H-5), 7.61 (1H, s, H-2), 6.91 (1H, d,
J = 9.0 Hz, H-6), 6.70 (1H, d, J = 4.8 Hz, H-40), 5.36 (1H, d,
J = 4.8 Hz, H-30), 2.56, 2.32, 2.24, 1.85 (each 2H, m, camphanoyl
CH2), 2.12 (3H, s, CH3-3), 1.64, 1.59 (each 3H, s, CH3-20), 1.32,
1.24, 1.22, 1.12, 1.10, 1.00 (each 3H, s, camphanoyl CH3); 90% de.
4.1.7.3. 30R,40R-Dihydroxy-20,20-dimethyl-2-ethylpyrano[2,3-f]-
chromone (34c). 28% yield (starting with 120 mg of 32c); mp
153–155 °C; MS (ESI+) m/z (%) 291 (M++1, 100); 1H NMR (DMSO)
d 7.80 (1H, d, J = 9.0 Hz, H-5), 6.83 (1H, d, J = 9.0 Hz, H-6), 6.13
(1H, s, H-3), 4.97 (1H, t, J = 4.8, 4.2 Hz, H-40), 3.64 (1H, t, J = 6.6,
4.8 Hz, H-30), 3.08 (1H, d, J = 4.2 Hz, OH-40), 2.99 (1H, d, J = 6.6 Hz,
OH-30), 2.58 (2H, q, J = 7.5 Hz, CH2CH3-2), 1.38, 1.37 (each 3H, s,
CH3-20,20), 1.26 (3H, t, J = 7.5 Hz, CH2CH3-2).
[
a
]
ꢀ36.2 (c 0.23, CHCl3).
D
4.1.9.3. 30R,40R-Di-O-(ꢀ)-camphanoyl-20,20-dimethyldihydropyr-
ano[2,3-f]chromone (4). 71% yield (starting with 146 mg of
34c); mp 90–92 °C; MS-ESI+ (m/z,%) 645 (M++Na, 100); 1H NMR d
8.15 (1H, d, J = 9.0 Hz, H-5), 7.69 (1H, d, J = 6.3 Hz, H-2), 6.94 (1H,
d, J = 9.0 Hz, H-6), 6.72 (1H, d, J = 4.8 Hz, H-40), 6.32 (1H, d,
J = 6.3 Hz, H-3), 5.37 (1H, d, J = 4.8 Hz, H-30), 2.46, 2.20, 1.90, 1.74

T. Zhou et al. / Bioorg. Med. Chem. 18 (2010) 6678–6689
6687
(each 2H, m, camphanoyl CH2), 1.52, 1.47 (each 3H, s, CH3-20), 1.11,
was extracted with CH2Cl2, and the combined organic layer was
concentrated under reduced pressure to give crude 30R,40R-dihydr-
oxyl-DCP. Without purification, the crude product was stirred with
camphanic chloride (3 equiv) and DMAP (4 equiv) at rt for 2 h to
give 30 mg of 10. 30% yield; mp 108–110 °C; MS-ESI+ (m/z,%) 665
1.10, 1.08, 1.02, 0.99, 0.89 (each 3H, s, camphanoyl CH3); [a]
D
ꢀ95.3 (c 0.17, CHCl3).
4.1.9.4. 30R,40R-Di-O-(ꢀ)-camphanoyl-2,5,20,20-tetramethyldihy-
dropyrano[2,3-f]chromone (5). 54% yield (starting with 290 mg
of 34b); mp 144–145 °C; MS-ESI+ (m/z,%) 645 (M++1, 100); 1H NMR
d 6.74 (1H, d, J = 4.8 Hz, H-40), 6.67 (1H, s, H-6), 6.05 (1H, s, H-3),
5.37 (1H, d, J = 4.8 Hz, H-30), 2.81 (3H, s, CH3-5), 2.50, 2.20, 1.95,
1.85 (each 2H, m, camphanoyl CH2), 2.24 (3H, s, CH3-2), 1.54,
1.47 (each 3H, s, CH3-20,20), 1.14, 1.13, 1.10, 1.01, 1.00, 0.96 (each
1
(M++1, 100); H NMR d 6.70 (1H, d, J = 4.5 Hz, H-40), 6.69 (1H, s,
H-6), 6.04 (1H, s H-3), 5.37 (1H, d, J = 4.5 Hz, H-30), 3.25 (2H, q,
J = 7.5 Hz, CH2CH3-5), 2.50, (2H, q, J = 7.5 Hz, CH2CH3-2), 2.50,
2.15, 1.92, 1.70 (each 2H, m, camphanoyl CH2), 1.53, 1.45 (each
3H, s, CH3-20,20), 1.24 (3H, t, J = 7.5 Hz, CH2CH3-5), 1.21 (3H, t,
J = 7.5 Hz, CH2CH3-2), 1.12, 1.11, 1.07, 1.01, 0.98, 0.96, (each 3H,
3H, s, camphanoyl CH3); [
a]
ꢀ71.2 (c 0.002, CH2Cl2).
s, camphanoyl CH3); [
a]
ꢀ6.5 (c 0.003, CH2Cl2).
D
D
4.1.9.5. 30R,40R-Di-O -(ꢀ)-camphanoyl-20,20-dimethyl-2-ethyl-5-
methyldihydropyrano[2,3-f]chromone (9). 60% yield (starting
with 100 mg of 34d); mp 133–134 °C; MS-ESI+ (m/z,%) 665
4.1.14. Preparation of 30R,40R-di-O-(ꢀ)-camphanoyl-20,20-dime-
thyl-2-bromomethyldihydropyrano[2,3-f]chromone (11)
A
mixture of 35 (200 mg, 0.31 mmol), NBS (60.6 mg,
1
(M++1, 100); H NMR d 6.70 (1H, d, J = 4.5 Hz, H-40), 6.64 (1H, s,
0.34 mmol), and 3-chloroperbenzoic acid (5.4 mg, 0.031 mmol),
dissolved in 2 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 an eluent of hexanes/EtOAc = 1:1 to afford pure 11 (50 mg).
23% yield; mp 180–182 °C; MS-ESI+ (m/z,%) 717 (M++1, 100); 1H
NMR d 8.13 (1H, d, J = 9.0 Hz, H-5), 6.93 (1H, d, J = 9.0 Hz, H-5),
6.74 (1H, d, J = 4.5 Hz, H-40), 6.40 (1H, s, H-3), 5.42 (1H, d,
J = 4.5 Hz, H-30), 4.12 (2H, s, CH2Br-2), 2.50, 2.17, 1.90, 1.74 (each
2H, m, camphanoyl CH2), 1.56, 1.47 (each 3H, s, CH3-20,20), 1.13,
H-6), 6.04 (1H, s H-3), 5.36 (1H, d, J = 4.5 Hz, H-30), 2.78 (3H, s,
CH3-5), 2.50 (2H, q, J = 7.5 Hz, CH2CH3-2), 2.50, 2.14, 1.91, 1.71
(each 2H, m, camphanoyl CH2), 1.52, 1.44 (each 3H, s, CH3-20,20),
1.21 (3H, t, J = 7.5 Hz, CH2CH3-2), 1.11, 1.10, 1.07, 0.99, 0.97, 0.95,
(each 3H, s, camphanoyl CH3); [
a]
ꢀ55.0 (c 0.003, CH2Cl2).
D
4.1.10. Preparation of 30R,40R-di-O-(ꢀ)-camphanoyl-2,5,20,20-
tetramethyl-3-bromodihydropyrano[2,3-f]chromone (6)
A mixture of 5 (80 mg, 0.12 mmol), NBS (32.0 mg, 0.18 mmol)
andMeCN(2 mL)washeatedto110 °C for3 h underhigh-absorption
microwave conditions. At completion, the mixture was concen-
trated and purified by PTLC with an eluent of hexanes/EtOAc = 1:1
to afford pure 6 (28 mg). 32% yield; mp 146–147 °C; MS-ESI+ (m/
z,%) 729 (M+, 100); 1H NMR d 6.72 (1H, d, J = 4.8 Hz, H-40), 6.71 (1H,
s, H-6), 5.36 (1H, d, J = 4.8 Hz, H-30), 2.81 (3H, s, CH3-5), 2.49 (3H, s,
CH3-2), 2.50, 2.15, 1.95, 1.72 (each 2H, m, camphanoyl CH2), 1.53,
1.47 (each 3H, s, CH3-20,20), 1.13, 1.11, 1.09, 1.00, 0.98, 0.95 (each
1.11, 1.09, 1.07, 1.02, 0.98, (each 3H, s, camphanoyl CH3); [a]
D
ꢀ13.9 (c 0.01, CH2Cl2).
4.1.15. Preparation of 30R,40R-di-O-(ꢀ)-camphanoyl-20,20-dime-
thyl-2-(1-bromoethyl)dihydropyrano[2,3-f]chromone (12)
The procedure was identical to that used for the preparation of
11: 25% yield (starting with 50 mg of 4); mp 158–159 °C; MS-ESI+
(m/z,%) 731 (M++1, 100); 1H NMR d 8.12 (1H, d, J = 9.0 Hz, H-5),
6.92 (1H, d, J = 9.0 Hz, H-5), 6.74 (1H, d, J = 4.5 Hz, H-40), 6.34
(1H, s, H-3), 5.42 (1H, d, J = 4.5 Hz, H-30), 4.76 (1H, t, J = 7.2,
CHBrCH3-2), 2.50, 2.19, 1.90, 1.74 (each 2H, m, camphanoyl CH2),
1.57, 1.47 (each 3H, s, CH3-20,20), 1.27 (3H, d, J = 7.2, CHBrCH3-2),
1.12, 1.11, 1.08, 1.05, 1.03, 0.98, (each 3H, s, camphanoyl CH3);
3H, s, camphanoyl CH3); [
a]
D ꢀ65.8 (c 0.018, CH3Cl).
4.1.11. Preparation of 30R,40R-di-O-(ꢀ)-camphanoyl-2,5,20,20-
tetramethyl-3,6-dibromodihydropyrano[2,3-f]chromone (7)
The procedure was identical to that used for the preparation of
6. 10% yield (starting with 80 mg of 5); mp 148–150 °C; MS-ESI+
[a
]
D
ꢀ31.9 (c 0.005, CH2Cl2).
1
(m/z, %) 809 (M+, 100); H NMR d 6.74 (1H, d, J = 4.5 Hz, H-40),
5.39 (1H, d, J = 4.5 Hz, H-30), 3.05 (3H, s, CH3-5), 2.49 (3H, s, CH3-
2), 2.50, 2.16, 1.92, 1.73 (each 2H, m, camphanoyl CH2), 1.56 (6H,
s, CH3-20,20), 1.13, 1.12, 1.09, 1.00, 0.99, 0.96 (each 3H, s, campha-
4.1.16. Preparation of 30R,40R-di-O-(ꢀ)-camphanoyl-20,20-dime-
thyl-2-(1-dibromoethyl)dihydropyrano[2,3-f]chromone (13)
A mixture of 4 (50 mg, 0.08 mmol), NBS (28.5 mg, 0.16 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 an
eluent of hexanes/EtOAc = 1:1 to afford pure 13 (8 mg). 12% yield;
mp 166–168 °C; MS-ESI+ (m/z,%) 809 (M++1, 100); 1H NMR d 8.13
(1H, d, J = 9.0 Hz, H-5), 6.94 (1H, d, J = 9.0 Hz, H-5), 6.80 (1H, d,
J = 4.2 Hz, H-40), 6.67 (1H, s, H-3), 5.42 (1H, d, J = 4.2 Hz, H-30),
2.81 (3H, s, C(Br)2CH3-2), 2.50, 2.20, 1.90, 1.70 (each 2H, m, cam-
phanoyl CH2), 1.57, 1.47 (each 3H, s, CH3-20,20), 1.12, 1.11, 1.08,
noyl CH3); [
a
]
D
ꢀ67.2 (c 0.018, CH3Cl).
4.1.12. Preparation of 30R,40R-di-O-(ꢀ)-camphanoyl-2, 20,20-tri-
methyl-5-bromomethyldihydropyrano[2,3-f]chromone (8)
A mixture of 5 (100 mg, 0.15 mmol), NBS (29.4 mg, 0.17 mmol),
and 3-chloroperbenzoic acid (2.6 mg, 0.015 mmol), dissolved in
2 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 an
eluent of hexanes/EtOAc = 1:1 to afford pure 8 (38 mg). 35% yield;
mp 128–130 °C; MS-ESI+ (m/z,%) 729 (M+, 100); 1H NMR d 6.97 (1H,
s, H-6), 6.72 (1H, d, J = 4.5 Hz, H-40), 6.15 (1H, s, H-3), 5.39 (1H, d,
J = 4.5 Hz, H-30), 5.29 (1H, d, J = 61.2 Hz, CH2Br-5), 5.12 (1H, d,
J = 61.2 Hz, CH2Br-5), 2.50, 2.18, 1.94, 1.71 (each 2H, m, campha-
noyl CH2), 2.26 (3H, s, CH3-2), 1.54, 1.48 (each 3H, s, CH3-20,20),
1.13, 1.11, 1.09, 1.02, 0.99, 0.97 (each 3H, s, camphanoyl CH3);
1.05, 1.03, 0.98, (each 3H, s, camphanoyl CH3); [
0.004, CH2Cl2).
a]
ꢀ48.1 (c
D
4.1.17. Preparation of 30R,40R-di-O-(ꢀ)-camphanoyl-3,20,20-tri-
methyl-2-cyanodihydropyrano[2,3-f]chromone (14)
A solution of sodium cyanide in 95% EtOH (aqueous) was cooled
in an ice-bath. Compound 37 (50 mg, 0.07 mmol) in 0.5 mL DMF
was added slowly to the above solution over a 15–20 min period.
The mixture was stirred at rt and monitored by TLC. At completion,
the mixture was poured into ice-water and extracted with EtOAc
three times. The combined organic layer was washed with brine
and dried over MgSO4. The solvent was evaporated in vacuo and
[
a]
D
ꢀ66.9 (c 0.018, CH3Cl).
4.1.13. Preparation of 30R,40R-di-O-(ꢀ)-camphanoyl-20,20-dime-
thyl-2,5-diethyldihydropyrano[2,3-f]chromone (10)
Compound 32e (50 mg) was dihydroxylated using the identical
procedure described above for 2 days. At completion, the mixture

6688
T. Zhou et al. / Bioorg. Med. Chem. 18 (2010) 6678–6689
the residue purified by PTLC to afford pure 14 (10 mg). 22% yield;
mp 196–198 °C; MS-ESI+ (m/z,%) 684 (M++Na, 100); 1H NMR d
8.13 (1H, d, J = 9.0 Hz, H-5), 6.99 (1H, d, J = 9.0 Hz, H-6), 6.61 (1H,
d, J = 4.5 Hz, H-40), 5.41 (1H, d, J = 4.5 Hz, H-30), 2.55, 2.20, 1.96,
1.85 (each 2H, m, camphanoyl CH2), 2.28 (3H, s, CH3-3), 1.52,
1.48 (each 3H, s, CH3-20,20), 1.13, 1.11, 1.10, 1.09, 1.05, 0.98 (each
1.07, 0.99, 0.98, 0.96, (each 3H, s, camphanoyl CH3); [
(c 0.002, CH2Cl2).
a
]
ꢀ31.5
D
4.1.23. General procedure for the preparation of amino-
substituted DCP derivatives (16, 21–27)
A THF solution of bromo-substituted DCP analogs (11, 17 or 18)
(1 equiv), various amines or aqueous amine solution (2.5 equiv)
was stirred at rt for 3.5 h. The mixture was poured into water (ex-
cess) and extracted with EtOAc. After the usual workup, the crude
product was purified by PTLC with an eluent of hexanes/
EtOAc = 7:1 to afford corresponding amino-substituted DCP ana-
logs (16, 21–27).
3H, s, camphanoyl CH3); [
a]
ꢀ39.0 (c 0.002, CH2Cl2).
D
4.1.18. Preparation of 30R,40R-di-O-(ꢀ)-camphanoyl-20,20-dime-
thyl-2-methylcyanodihydropyrano[2,3-f]chromone (15)
The procedure was identical to that used for the preparation of
14: 27% yield (starting with 20 mg of 11); mp 102–104 °C; MS-ESI+
(m/z,%) 684 (M++Na, 100); 1H NMR d 8.14 (1H, d, J = 9.0 Hz, H-5),
6.97 (1H, d, J = 9.0 Hz, H-6), 6.72 (1H, d, J = 4.2 Hz, H-40), 6.49
(1H, s, H-3), 5.40 (1H, d, J = 4.2 Hz, H-30), 3.63 (2H, t, J = 25.5 Hz,
CH2CN-2), 2.50, 2.10, 1.95, 1.70 (each 2H, m, camphanoyl CH2),
1.56, 1.49 (each 3H, s, CH3-20,20), 1.13, 1.11, 1.09, 1.03, 1.00, 0.98,
4.1.23.1. 30R,40R-Di-O-(ꢀ)-camphanoyl-20,20-dimethyl-2-(meth-
ylamino)methyldihydropyrano[2,3-f]chromone (16). 80% yield
(starting from 50 mg of 11); mp 137–138 °C; MS-ESI+ (m/z,%) 666
(M++1, 100); 1H NMR d 8.14 (1H, d, J = 9.0 Hz, H-5), 6.92 (1H, d,
J = 9.0 Hz, H-6), 6.75 (1H, d, J = 4.5 Hz, H-40), 6.34 (1H, s, H-3),
5.40 (1H, d, J = 4.5 Hz, H-30), 3.60, 3.55 (each 1H, d, J = 8.7 Hz,
CH2NHCH3-2), 2.85 (1H, s, CH2NHCH3-2), 2.45, 2.14, 1.95, 1.71
(each 2H, m, camphanoyl CH2), 2.45 (3H, s, CH2NHCH3-2), 1.55,
1.48 (each 3H, s, CH3-20,20), 1.13, 1.11, 1.08, 1.02, 0.99, 0.97, (each
(each 3H, s, camphanoyl CH3); [
a]
ꢀ27.9 (c 0.002, CH2Cl2).
D
4.1.19. Preparation of 30R,40R-di-O-(ꢀ)-camphanoyl-2,20,20-tri-
methyl-3-bromodihydropyrano[2,3-f]chromone (17)
The procedure was identical to that used for the preparation of
6. 52% yield (starting from 21 mg of 35); mp 160–161 °C; MS-ESI-
(m/z,%) 713 (M- - 1, 100); 1H NMR d 8.16 (1H, d, J = 9.0 Hz, H-5),
6.95 (1H, d, J = 9.0 Hz, H-6), 6.75 (1H, d, J = 4.5 Hz, H-40), 5.38
(1H, d, J = 4.5 Hz, H-30), 2.53 (3H, s, CH3-2), 2.46, 2.16, 1.92, 1.73
(each 2H, m, camphanoyl CH2), 1.54, 1.48 (each 3H, s, CH3-20,20),
1.13, 1.12, 1.09, 1.01, 0.98, 0.96, (each 3H, s, camphanoyl CH3);
3H, s, camphanoyl CH3); [
a]
D
ꢀ35.8 (c 0.003, CH2Cl2).
4.1.23.2. 30R,40R-Di-O-(ꢀ)-camphanoyl-2,20,20-trimethyl-3-amin-
odihydropyrano[2,3-f]chromone (21). 50% yield (starting from
18 mg of 17); mp 137–138 °C; MS-ESI+ (m/z,%) 652 (M++1, 100);
1H NMR d 7.73 (1H, d, J = 8.7 Hz, H-5), 6.75 (1H, d, J = 4.8 Hz, H-
40), 6.70 (1H, d, J = 8.7 Hz, H-6), 5.39 (1H, d, J = 4.8 Hz, H-30), 2.46
(2H, s, NH2-3), 2.45, 2.20, 1.95, 1.85 (each 2H, m, camphanoyl
CH2), 2.17 (3H, s, CH3-2), 1.48, 1.47 (each 3H, s, CH3-20,20), 1.24,
[a
]
D
ꢀ47.7 (c 0.003, CH2Cl2).
4.1.20. Preparation of 30R,40R-di-O-(ꢀ)-camphanoyl-20,20-dime-
thyl-2-ethyl-3-bromodihydropyrano[2,3-f]chromone (18)
The procedure was identical to that used for the preparation of
6. 60% yield (starting from 20 mg of 4); mp 166–168 °C; MS-ESI+
(m/z,%) 730 (M++1, 100); 1H NMR d 8.18 (1H, d, J = 9.0 Hz, H-5),
6.95 (1H, d, J = 9.0 Hz, H-6), 6.74 (1H, d, J = 4.8 Hz, H-40), 5.41
(1H, d, J = 4.8 Hz, H-30), 3.02, 2.85 (each 1H, m, CH2CH3-2), 2.45,
2.10, 1.95, 1.85 (each 2H, m, camphanoyl CH2), 1.55, 1.47 (each
3H, s, CH3-20,20), 1.24 (3H, t, J = 7.5 Hz, CH2CH3-2), 1.13, 1.11,
1.10, 1.07, 0.99, 0.97, 0.86 (each 3H, s, camphanoyl CH3; [a]
ꢀ8.0 (c 0.004, CH2Cl2).
D
4.1.23.3. 30R,40R-Di-O-(ꢀ)-camphanoyl-20,20-dimethyl-2-ethyl-3-
aminodihydropyrano[2,3-f]chromone (22). 40% yield (starting
from 100 mg of 18); mp 145–146 °C; MS-ESI+ (m/z,%) 652 (M++1,
100); 1H NMR
d 7.70 (1H, d, J = 8.7 Hz, H-5), 6.73 (1H, d,
J = 4.5 Hz, H-40), 6.68 (1H, d, J = 8.7 Hz, H-6), 5.38 (1H, d,
J = 4.5 Hz, H-30), 5.0 (2H, br, NH2-3), 2.50 (2H, q, J = 7.5 Hz,
CH2CH3-2), 2.40, 2.20, 1.91, 1.60 (each 2H, m, camphanoyl CH2),
1.52, 1.46 (each 3H, s, CH3-20,20), 1.21 (3H, t, J = 7.5 Hz, CH2CH3-
2), 1.11, 1.10, 1.07, 0.99, 0.97, 0.85, (each 3H, s, camphanoyl
1.081.03, 0.98, 0.98 (each 3H, s, camphanoyl CH3); [
0.006, CH2Cl2).
a
]
D
ꢀ34.1 (c
4.1.21. Preparation of 30R,40R-di-O-(ꢀ)-camphanoyl-2,20,20-tri-
methyl-3-iododihydropyrano[2,3-f]chromone (19)
CH3); [
a]
ꢀ10.0 (c 0.003, CH2Cl2).
D
An anhydrous CH2Cl2 solution of 35 (40 mg, 0.06 mmol) and
CF3CO2Ag (13 mg, 0.06 mmol) was cooled to 0 °C in an ice-bath.
I2 (17.6 mg, 0.07 mmol) was added slowly under N2 protection.
The reaction mixture was stirred at 0 °C for 2 h, monitored by
TLC. At completion, the mixture was concentrated and purified
by PTLC to give pure 19 (43 mg): 90% yield; mp 179–180 °C; MS-
ESI+ (m/z,%) 785 (M++Na, 100); 1H NMR d 8.17 (1H, d, J = 9.0 Hz,
H-5), 6.95 (1H, d, J = 9.0 Hz, H-6), 6.76 (1H, d, J = 4.5 Hz, H-40),
5.39 (1H, d, J = 4.5 Hz, H-30), 2.65 (3H, s, CH3-2), 2.50. 2.15, 1.96,
1,85 (each 2H, m, camphanoyl CH2), 1.55, 1.49 (each 3H, s, CH3-
20,20), 1.13, 1.11, 1.09, 1.02, 0.99, 0.96 (each 2H, s, camphanoyl
4.1.23.4. 30R,40R-Di-O-(ꢀ)-camphanoyl-3,20,20-trimethyl-3-me-
thylaminodihydropyrano[2,3-f]chromone
(23). 80%
yield
(starting from 22.2 mg of 17); mp 122–124 °C; MS-ESI+ (m/z,%)
666 (M++1, 100); 1H NMR d 7.73 (1H, d, J = 8.4 Hz, H-5), 6.76 (1H,
d, J = 4.8 Hz, H-40), 6.70 (1H, d, J = 8.4 Hz, H-6), 5.40 (1H, d,
J = 4.8 Hz, H-30), 3.06 (3H, s, NHCH3-3), 2.54 (1H, s, NHCH3-3),
2.40, 2.20, 1.90, 1.70 (each 2H, m, camphanoyl CH2), 2.20 (3H, s,
CH3-2), 1.52, 1.47 (each 3H, s, CH3-20,20), 1.12, 1.10, 1.06, 0.98,
0.95,0.83 (each 3H, s, camphanoyl CH3); [
CH2Cl2).
a]
ꢀ21.4 (c 0.003,
D
CH3); [
a
]
ꢀ37.5 (c 0.002, CH2Cl2).
D
4.1.23.5. 30R,40R-Di-O-(ꢀ)-camphanoyl-20,20-dimethyl-2-ethyl-3-
methylaminodihydropyrano[2,3-f]chromone (24). 75% yield
(starting from 100 mg of 18); mp 163–164 °C; MS-ESI+ (m/z,%)
680 (M++1, 100); 1H NMR d 7.73 (1H, d, J = 8.4 Hz, H-5), 6.76 (1H,
d, J = 4.5 Hz, H-40), 6.69 (1H, d, J = 8.4 Hz, H-6), 5.39 (1H, d,
J = 4.5 Hz, H-30), 3.08 (3H, s, NHCH3-3), 2.63 (2H, q, J = 7.5 Hz,
CH2CH3-2), 2.48, 2.20, 1.94, 1.77 (each 2H, m, camphanoyl CH2),
1.55, 1.48 (each 3H, s, CH3-20,20),1.13, 1.11, 1.07, 0.99, 0.96,0.87
4.1.22. Preparation of 30R,40R-di-O-(ꢀ)-camphanoyl-20,20-dime-
thyl-2-ethyl-3-cyanodihydropyrano[2,3-f]chromone (20)
The procedure was identical to that used for the preparation of
14: 25% yield (starting with 50 mg of 18); mp 193–194 °C; MS-ESI-
(m/z,%) 674 (Mꢀꢀ1, 100); 1H NMR d 7.75 (1H, d, J = 9.0 Hz, H-5),
6.78 (1H, d, J = 9.0 Hz, H-6), 6.65 (1H, d, J = 4.5 Hz, H-40), 5.38
(1H, d, J = 4.5 Hz, H-30), 2.51 (2H, q, J = 7.5 Hz, CH2CH3-2), 2.40,
2.16, 1.92, 1.74 (each 2H, m, camphanoyl CH2), 1.55, 1.49 (each
3H, s, CH3-20,20), 1.24 (3H, t, J = 7.5 Hz, CH2CH3-2), 1.21, 1.10,
(each 3H, s, camphanoyl CH3), 1.08 (1H, br, NHCH3-3); [
a]
ꢀ24.6
D
(c 0.013, CH2Cl2).

T. Zhou et al. / Bioorg. Med. Chem. 18 (2010) 6678–6689
6689
4.1.23.6.
30R,40R-Di-O-(ꢀ)-camphanoyl-20,20-dimethyl-2-mor-
4.4. Water solubility analysis assay
pholinomethyl-dihydropyrano[2,3-f]chromone (25). 30% yield
(starting from 30 mg of 11); mp 140–142 °C; MS-ESI+ (m/z,%)
722 (M++1, 100); 1H NMR d 8.13 (1H, d, J = 9.0 Hz, H-5), 6.92 (1H,
d, J = 9.0 Hz, H-6), 6.74 (1H, d, J = 4.5 Hz, H-40), 6.48 (1H, s, H-3),
5.38 (1H, d, J = 4.5 Hz, H-30), 3.72 (4H, t, J = 4.5 Hz, 4.8 Hz, morpho-
line CH2), 3.26, 3.42 (each 1H, d, J = 16.5 Hz, CH2), 2.54 (4H, t,
J = 4.5 Hz, 4.8 Hz, morpholine CH2), 2.50, 2.15, 1.95, 1.70 (each
2H, m, camphanoyl CH2), 1.54, 1.48 (each 3H, s, CH3-20,20), 1.13,
Each tested compound was added in excess to 1.5 mL Eppendorf
tubes containing 1 mL of HPLC grade water. The tubes were placed
into Branson 5510 ultrasonic tank at rt for 1 h. The excess solid was
separated from the solution through a PTFE syringe filter (0.2 lM
diameter). The supernatant was dispensed into glass HPLC vials.
The concentration of the samples was determined with HPLC, on
an Alltima C18 3u column (2.1 mm ꢂ 100 mm) and a flow rate of
1.11, 1.08, 1.00, 0.98, 0.95, (each 3H, s, camphanoyl CH3); [
a]
200 lL/min. The samples (5 lL) were injected and run with a solu-
D
ꢀ28.5 (c 0.011, CH2Cl2).
tion of 35% water and 65% MeCN. For each compound, a standard
curve consisting of five concentrations (fivefold stepwise) in MeCN
was established initially.
4.1.23.7. 30R,40R-Di-O-(ꢀ)-camphanoyl-20,20-dimethyl-2-(dimeth-
ylaminopropyl-piperazin1ylmethyl)–dihydropyrano[2,3-f]chro-
mone (26). 15% yield (starting from 30 mg of 11); mp 132–133 °C;
MS-ESI+ (m/z,%) 806 (M+, 100); 1H NMR d 8.13 (1H, d, J = 8.7 Hz, H-
5), 6.92 (1H, d, J = 8.7 Hz, H-6), 6.73 (1H, d, J = 4.5 Hz, H-40), 6.47 (1H,
s, H-3), 5.38 (1H, d, J = 4.5 Hz, H-30), 3.30, 3.44 (each 1H, d,
J = 15.0 Hz, CH2-2), 2.50 (8H, m, piperazine CH2), 2.50, 2.15, 1.95,
1.70 (each 2H, m, camphanoyl CH2), 2.41 (4H, m, amino-propylpip-
erazine CH2), 2.28 (6H, s, dimethylamino-propylpiperazine CH3),
1.70 (2H, m, amino-propylpiperazine CH2), 1.54, 1.48 (each 3H, s,
CH3-20,20), 1.13, 1.11, 1.08, 1.01, 0.98, 0.95, (each 3H, s, camphanoyl
Acknowledgements
This investigation was supported by grant AI033066 from Na-
tional Institute of Allergy and Infectious Disease (NIAID) awarded
to K. H. Lee.
Supplementary data
CH3); [a D
]
ꢀ20.3 (c 0.003, CH2Cl2).
Supplementary data (HPLC conditions and summary of HPLC
purity data for final compounds) associated with this article can
be found, in the online version, at doi:10.1016/j.bmc.2010.07.065.
4.1.23.8. 30R,40R-Di-O-(ꢀ)-camphanoyl-20,20-dimethyl-2-(pyridin-
4-ylmethylamino)methyl-dihydropyrano[2,3-f]chromone
(27). 25% yield (starting from 30 mg of 11); mp 116–118 °C; MS-
ESI+ (m/z,%) 806 (M++1, 100); 1H NMR d 8.55 (2H, d, J = 6.0 Hz, pyr-
idine CH), 8.13 (1H, d, J = 9.0 Hz, H-5), 7.29 (2H, d, J = 6.0 Hz, pyri-
dine CH), 6.92 (1H, d, J = 9.0 Hz, H-6), 6.74 (1H, d, J = 4.5 Hz, H-40),
6.36 (1H, s, H-3), 5.39 (1H, d, J = 4.5 Hz, H-30), 3.85 (1H, s, CH2NH-
2), 3.59, 3.68 (each 1H, d, J = 15.9 Hz, CH2-2), 2.50, 2.15, 1.95, 1.70
(each 2H, m, camphanoyl CH2), 1.55, 1.48 (each 3H, s, CH3-20),
1.13, 1.11, 1.07, 0.99, 0.96, 0.94, (each 3H, s, camphanoyl CH3);
References and notes
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Anti-HIV-1 activity was measured as reductions 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, 4-fold stepwise) were mixed in a total volume of
100
Costar). After 48-h incubation, culture medium was removed from
each well and 100 L of Bright Glo luciferase reagent was added to
lL growth medium in 96-well black solid plates (Corning-
l
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.
4.3. Cytotoxicity assay
21. Montreal, Q., Canada, MOE. Chemical Computing Group, 2007.
22. Italy, T. s. r. l. M., DRAGON for Windows (Software for Molecular Descriptor
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The general procedure was performed according to CytoTox-
GloTM cytotoxicity assay instructions for using product G9290,
G9291 and G9292. (Promega)