Anti-AIDS agents 82: Synthesis of seco-(3′R,4′R)-3′,4′-di-O-(S)-camphanoyl-(+)-cis-khellactone (DCK) derivatives as novel anti-HIV agents

Article abstract of DOI:10.1016/j.bmc.2010.04.089

Thirteen novel seco-DCK analogs (4-16) with several new skeletons were designed, synthesized and screened for in vitro anti-HIV-1 activity. Among them, three compounds (5, 13, and 16) showed moderate activity, and compound 9 exhibited the best activity with an EC₅₀ value of 0.058 μM and a therapeutic index (TI) of 1000. The activity of 9 was better than that of 4-methyl DCK (2, EC₅₀: 0.126 μM, TI: 301.2) in the same assay. Additionally, 9 also showed antiviral activity against a multi-RT inhibitor-resistant strain (RTMDR), which is insensitive to most DCK analogs. Compared with 2, compound 9 has a less complex structure, fewer hydrogen-bond acceptors, and a reduced log P value. Therefore, it is likely to exhibit better ADME, and appears to be a promising new lead for further development as an anti-HIV candidate.

Full text of DOI:10.1016/j.bmc.2010.04.089

Bioorganic & Medicinal Chemistry 18 (2010) 4363–4373
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Anti-AIDS agents 82: Synthesis of
seco-(30R,40R)-30,40-di-O-(S)-camphanoyl-(+)-cis-khellactone (DCK)
derivatives as novel anti-HIV agents
a,
b
b
b
Jian Tang a, Keduo Qian b, Bei-Na Zhang a, Ying Chen a, , Peng Xia , Donglei Yu , Yi Xia , Zheng-Yu Yang ,
*
*
Chin-Ho Chen c, Susan L. Morris-Natschke b, Kuo-Hsiung Lee b,
*
a Department of Medicinal Chemistry, School of Pharmacy, Fudan University, Shanghai 201203, China
b Natural Products Research Laboratories, Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, NC 27599-7568, USA
c Department of Surgery, Duke University Medical Center, Durham, NC 27710, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Thirteen novel seco-DCK analogs (4–16) with several new skeletons were designed, synthesized and
screened for in vitro anti-HIV-1 activity. Among them, three compounds (5, 13, and 16) showed moderate
Received 1 March 2010
Revised 23 April 2010
Accepted 25 April 2010
Available online 29 April 2010
activity, and compound 9 exhibited the best activity with an EC50 value of 0.058
lM and a therapeutic index
(TI) of 1000. The activity of 9 was better than that of 4-methyl DCK (2, EC50: 0.126
l
M, TI: 301.2) in the same
assay. Additionally, 9 also showed antiviral activity against a multi-RT inhibitor-resistant strain (RTMDR),
which is insensitive to most DCK analogs. Compared with 2, compound 9 has a less complex structure, fewer
hydrogen-bond acceptors, and a reduced log P value. Therefore, it is likely to exhibit better ADME, and
appears to be a promising new lead for further development as an anti-HIV candidate.
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Seco-DCK analogs
Anti-HIV agents
1. Introduction
at the 20-position is greatly preferable to larger alkyl substituents or
hydrogen atoms.9 (e) 30R,40R-Di-O-(ꢀ)-camphanoyl-20,20-dimethyl-
In our prior studies, 30R,40R-di-O-(ꢀ)-camphanoyl-(+)-cis-khel-
lactone (DCK, 1) and 4-methyl DCK (2) showed remarkable potency
against human immunodeficiency virus type 1 (HIV-1) replication in
H9 lymphocytes.1,2 Subsequent modifications of DCK focused on the
coumarin substituents, ring isomers, and bioisosteric replace-
ments.3 Hundreds of 1-derivatives were designed and synthesized,
and the resulting data led to the following structure–activity rela-
tionship (SAR) conclusions. (a) A 4-methyl is favorable for enhancing
anti-HIV activity and decreasing toxicity.2 (b) Certain bioisosteric
DCK analogs, including 1-thia, 1-aza, 10-thia and 10-carbon DCKs,
show comparable anti-HIV activity with 1.4–7 (c) Two bulky cis-
(S)-(ꢀ)-camphanoyl groups at the 30- and 40-positions are preferred
to other substituents; however, both of them are not essential. The
40R-camphanoyl is more important.8 (d) gem-Dimethyl substitution
dihydropyrano[2,3-f]chromone (DCP) analogs (such as 3), which are
positional ring isomers, also exhibit significant anti-HIV activity
(Fig. 1).8,10 However, development of 1-analogs as effective anti-
AIDS drugs has been hindered by problems of low water solubility
and bioavailability. DCP analogs, with the carbonyl group positioned
differently in ring-A, retained high potency against both non-drug
resistant and multi-RT-inhibitor-resistant viral strains. This result
motivated us to change the skeleton of 1 by opening the A or C ring,
R
O
O
4
6
3
2
O
3
2
A
O
B
O
O
C
8
2' 4'
3'
O
O
Abbreviations: DCK, (30R,40R)-30,40-di-O-(S)-camphanoyl-(+)-cis-khellactone;
HIV-1, human immunodeficiency virus type 1; DCP, 30R,40R0-di-O-(ꢀ)-campha-
noyl-20,20-dimethyldihydropyranol[2,3-f]chromone; RTMDR, multi-RT inhibitor-
resistant strain; ADME, Absorption, distribution, metabolism and elimination;
AZT, zidovudine; SAR, structure–activity relationships; DMF, dimethyl formamide;
AD, asymmetric dihydroxylation; DMAP, 4-(dimethylamino)pyridine; (DHQ)2PHAL,
hydroquinine 1,4-phthalazinediyl diether; TLC, thin-layer chromatography.
* Corresponding authors. Tel.: +86 21 51980116 (P.X.); tel.: +1 919 962 0066; fax:
+1 919 966 3893 (K.H.L.).
O
O
O
O
O
O
O
O
O
O
O
O
O
O
1. R = H, DCK
. R = CH3
3. 2-Et-DCP
2
E-mail addresses: yingchen71@fudan.edu.cn (Y. Chen), pxia@fudan.edu.cn (P.
Xia), khlee@unc.edu (K.-H. Lee).
Figure 1. Structures of previously synthesized DCK analogs (1, 2) and 2-Et-DCP
analog (3).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.04.089

4364
J. Tang et al. / Bioorg. Med. Chem. 18 (2010) 4363–4373
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
6
7
5
4
O
R
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
S
N
O
R
O
O
O
O
O
O
O
O
13
9
14 R = H
8
R = -CH(CH3)2
R = -CH3
R = -CH2CH3
R = -CH2COOCH3
10
11
12
15 R = Me
16 R =
OMe
Figure 2. Structures of newly designed seco-DCK analogs 4–16.
which may represent a new break-through in developing DCK ana-
logs as clinical trial candidates.
with sulfur or nitrogen rather than oxygen linked to the campha-
noyl group of the seco-DCKs.
Therefore, in the current study, we explored a series of simpli-
fied DCK analogs with an opened A or C ring, termed seco-DCKs.
With only two rings, seco-DCK compounds have less complex
structures, fewer hydrogen-bond acceptors, and reduced log P val-
ues, which may result in better ADME than those of 2. Herein, we
report the design, synthesis, and bio-evaluation of these new DCK
analog series (Fig. 2).
2.2. Chemistry
Scheme 1 describes the synthesis of compound 4. Commercially
available 17 was reacted with 3-chloro-3-methyl-1-butyne in DMF
in the presence of K2CO3 and KI to afford 18. The crude product was
heated in refluxing N,N-diethylaniline to yield 19 with a pyrano C
ring. Compound 19 was asymmetrically dihydroxylated using
(DHQ)2-PHAL as a chiral catalyst to afford corresponding 30R,40R-
dihydroxy compound 20. Compound 20 was then reacted with
(ꢀ)-(S)-camphanic chloride at room temperature for 2 days to ob-
tain the target compound 4.
2. Results
2.1. Design
Compound 4, the first A ring-opened derivative (Fig. 2) synthe-
sized, lacks certain structural features found in the A ring of 1–3,
such as a phenoxy oxygen at position-1. Therefore, we also
designed compound 5 (Fig. 2), which contains an ethoxy group that
can freely rotate to adopt an optimal position and orientation for
binding in the target site, as well as an acetyl moiety that can cor-
respond to both the 4-oxo moiety of 3 and the 4-methyl group of 2.
In addition, to investigate whether the integrity of the C ring
impacts antiviral activity, we further designed several additional
seco-C ring DCKs, including 40,8-seco (6), 20,30-seco (7) and bis
(20,30- and 30,40-) seco DCK (8-12) analogs (Fig. 2). Although both
bulky camphanoyl moieties are retained in 6 and 7, their spatial
positions and 3D directions could be changed significantly from
those of 1 due to the absence of the C ring. Only the 40-camphanoyl
moiety is retained in 8–12, because prior SAR demonstrated the
importance of this moiety.8 In addition, 9–12 completely lack a
30-carbon, while 8 has a methyl group at the corresponding posi-
tion of the seco-C ring.
Compound 5 was synthesized from resorcinol via eight steps as
shown in Scheme 2. Treatment of resorcinol 21 with ZnCl2-HOAc
afforded a highly regioselective acetylation product, 1-(2,4-dihy-
droxyphenyl)ethanone (22). Selective protection of the 4-hydroxy
group with benzyl bromide (23) and ethylation of the 2-hydroxy
group with bromoethane gave 1-[2-ethoxy-4-(phenylmeth-
oxy)phenyl]ethanone (24), which then was reacted with BBr3 in
CH2Cl2 to yield 1-(2-ethoxy-4-hydroxyphenyl)ethanone (25). Com-
pound 26 was prepared by reaction of 25 with 3-chloro-3-methyl-
1-butyne. This crude product (26) directly underwent thermal ring
closure upon refluxing in DMF and N,N-diethylaniline to afford the
desired key intermediate 27 as the main product. After Sharpless
asymmetric dihydroxylation (AD) of 27, followed by esterification
of diol 28 with camphanic chloride, target compound 5 was
obtained.
Scheme 3 illustrates the syntheses of C ring-opened compounds
6–9 and 13. 40,8-Seco DCK (6) was prepared by alkylation of
commercially available 4-methyl-7-hydroxycoumarin (29) with 3-
chloro-3-methyl-1-butene, followed by Sharpless AD of 30 and
esterification of the resulting diol with camphanic chloride (31).
Key intermediates (32a and 32b) in the synthetic route to 20,30-seco
and bis (20,30- and 30,40-) seco DCKs (7–9) were obtained from 29 by
In addition, previous bioisosteric replacement study demon-
strated that 1-thia, 10-thia, and 1-aza DCKs maintained potent anti-
viral activity or had better activity against HIV compared with
2.4,5,7 These results motivated us to synthesize compounds 13–16

J. Tang et al. / Bioorg. Med. Chem. 18 (2010) 4363–4373
4365
with ethane-1,2-diol in a toluene solution containing p-toluenesul-
fonic acid. Intermediates 39a–c were obtained from 38 by alkyl-
ation with various halides in the presence of K2CO3 and KI in
acetone, followed by deprotection of the ketal moiety under acidic
conditions to afford 40a–c. Compounds 40a–c were reduced to
41a–c, which were then esterified with camphanic chloride to
yield target compounds 10–12.
O
O
O
i
ii
O
O
HO
17
18
19
The preparations of compounds 14–16 are depicted in Scheme
5. Compound 42 was synthesized in a one-pot reaction by amina-
tion of 33a with (NH4)2CO3 in EtOH, followed by reduction in the
presence of NaBH4 in the same solution. Imines 43 and 45 were
provided by reaction of 33a with methylamine and 4-methoxyan-
iline, respectively, and reduced to give 44 and 46. Three target
compounds 14–16 were obtained from the amidation of 42, 44,
and 46, respectively, with camphanic chloride.
O
O
iii
iv
O
O
OH
20
O
OH
O
O
O
O
O
O
3. Discussion
O
4
Target seco-DCK analogs 4–9 were evaluated for in vitro sup-
pression of HIV-1IIIB replication in the MT-2 cell line in parallel
with AZT and 2. Compounds 6-9 were further tested against HIV-
1NL4-3 and RTMDR, respectively, in MT-4 lymphocytes with 3 as
reference. In addition, compounds 10–16 were screened for antivi-
ral activity against HIV-1NL4-3 with 9 as control. Due to the protocol
differences in the assay systems, 9 showed variable activities in
different screenings. Nevertheless, 9 still showed the most promis-
ing potency in the above assays, especially good activity against
RTMDR. All bioassay data are summarized in Tables 1–3.
Scheme 1. Synthesis of seco-A ring DCK (4). Reagents and conditions: (i) 3-chloro-
3-methyl-1-butyne, K2CO3, KI; (ii) N,N-diethylaniline; (iii) K2OsO2(OH)4, K3Fe(CN)6,
(DHQ)2-PHAL, K2CO3; (iv) camphanoyl chloride, DMAP, CH2Cl2.
Duff formylation (32a) or Fries rearrangement (32b), respectively.
After alkylation of the phenolic hydroxyl in 32 with isopropyl
bromide, the carbonyl in 33 was reduced to a hydroxyl with NaBH4.
Target compounds 8 and 9 were obtained by esterification of 34b
and 34a with camphanic chloride, respectively. Dehydration of
34b with concd H2SO4, followed by AD and esterification gave target
compound 7. Thioester 13 was synthesized by treatment of 34a with
Lawesson reagent, and esterification with camphanic chloride.
Scheme 4 shows the synthetic pathway to compounds 10–12.
Compound 38 was obtained from 32a by a condensation reaction
Initially, we synthesized compound 4. However, it did not show
anti-HIV activity, which implied certain structural features present
in the A ring of 2 or 3, such as the polar oxygen center at position-1,
but absent in the seco-A ring of 4, are necessary for activity.
O
O
iii
i
ii
HO
OH
O
Bn
OH
HO
OH
O
21
22
23
O
O
vi
v
iv
O
OEt
O
Bn
OEt
24
HO
OEt
25
26
O
O
O
vii
viii
O
OEt
O
OEt
OH
O
OEt
O
O
OH
O
27
28
O
O
O
O
O
5
Scheme 2. Synthesis of seco-A ring DCK (5). Reagents and conditions: (i) ZnCl2, HOAc, boiling, 30 min; (ii) K2CO3, KI, BnCl, acetone, reflux, 8 h; (iii) CH3CH2Br, K2CO3, acetone,
reflux; (iv) BBr3, CH2Cl2, ꢀ40 °C; (v) 3-chloro-3-methyl-1-butyne, K2CO3, KI, acetone, reflux; (vi) DMF, N,N-diethylaniline, reflux; (vii) K2OsO2(OH)4, K3Fe(CN)6, (DHQ)2-PHAL,
n-BuOH, H2O, 0 °C; (viii) camphanoyl chloride, DMAP, CH2Cl2.

4366
J. Tang et al. / Bioorg. Med. Chem. 18 (2010) 4363–4373
O
O
O
iii
i
ii
O
O
O
O
O
O
O
O
O
HO
O
O
O
O
OH
O
OH
30
31
29
O
O
6
iv or v
O
O
O
O
vi
iii
vii
O
O
O
O
R
O
O
O
O
HO
O
O
O
O
R
OH
R
O
R
34b
34a R = H;
R = CH3
33a
32a
R = H
33b R = CH3
R = H
9
8
R = H
R = CH3
32b R = CH3
viii
viiii
O
O
O
O
O
O
SH
37
35
iii
ii
O
O
O
O
iii
O
O
O
O
O
O
O
O
O
O
O
O
S
O
OH
O
O
OH
36
13
7
O
Scheme 3. Synthetic routes to seco-C ring DCKs (6–9) and bioisosteric seco DCK 13. Reagents and conditions: (i) 3-chloro-3-methyl-1-butene, K2CO3, KI, acetone, reflux; (ii)
K2OsO2(OH)4, K3Fe(CN)6, (DHQ)2-PHAL, n-BuOH, H2O, 0 °C; (iii) camphanoyl chloride, DMAP, CH2Cl2; (iv) for 32a: hexamethylene tetramine, HOAc, 90 °C; (v) for 32b: (1) Py/
Ac2O, 5 h; (2) AlCl3, 160 °C; (vi) isopropyl bromide, NaH, DMF, reflux; (vii) NaBH4, MeOH; (viii) concd H2SO4, acetone; (viv) Lawesson reagent/N2/toluene, rt, 20 h.
i
ii
HO
O
O
O
R
O
O
HO
O
O
O
O
a: R = -CH3
H
O
O
O
b
c
: R = -CH2CH3
32a
38
39a-c
: R = -CH2COOCH3
O
R
O
O
v
iii
iv
O
O
R
O
O
O
R
O
O
O
CHO
O
O
OH
41a-c
40a-c
10-12
Scheme 4. Synthetic routes to seco-C ring DCKs (10–12). Reagents and conditions: (i) HOCH2CH2OH/TsOH/toluene; (ii) (a) CH3I; (b) CH3CH2Br; (c) BrCH2COOCH3, K2CO3, KI,
acetone, reflux; (iii) 2 N HCl; (iv) NaBH4, MeOH; (v) camphanic chloride, DMAP, CH2Cl2.

J. Tang et al. / Bioorg. Med. Chem. 18 (2010) 4363–4373
4367
O
O
O
iii
NH
O
O
O
O
NH2
O
42
14
O
ia
O
O
O
ii
iii
ib
ic
N
O
O
O
O
O
O
O
O
O
CHO
O
N
43
NH
44
O
33a
15
O
O
O
O
O
O
O
O
O
O
ii
iii
N
N
NH
O
O
O
O
O
O
45
16
46
Scheme 5. Synthesis of bioisosteric seco-DCKs (14–16). Reagents and conditions: (ia) (a) (NH4)2CO3/EtOH/reflux; (b) NaBH4; (ib) CH3NH2, EtOH, rt; (ic) p-CH3C6H4NH2, EtOH,
rt; (ii) NaBH4, MeOH; (iii) camphanic chloride, DMAP, CH2Cl2.
Table 2
Table 1
Anti-HIV data of seco-DCK analogs (7–10) in MT-4 cell linea
Anti-HIV-1IIIB data of seco-DCK analogs (4–9) in MT-2 cell linea
Compd
IC50
(
l
M)
HIV-1NL4-3
M)
RTMDR
M)
Compd
IC50
(l
M)
EC50
(lM)
TI
4
5
6
7
8
9
2
AZT
39.6
39.6
39.2
39.2
56.5
58.4
39.2
NS
0.19
NS
NS
NS
0.058
0.126
0.044
EC50
(
l
TI
EC50
(
l
TI
208.3
6
7
8
9
3
20
20
20
17.27
9.27
20
20
20
0.5
0.08
NS
NS
NS
34.8
116
20
20
20
1.89
NS
NS
NS
9.13
69.9
1000
301.2
42700
0.133
a
1872
All data presented are averages of at least three separate experiments per-
formed by Dr. Chin-Ho Chen, Duke University, NC. CC50: concentration that inhibits
uninfected MT-4 cell growth by 50%. EC50: concentration that inhibits viral repli-
cation by 50%. TI = therapeutic index IC50/EC50. NS: no suppression at the highest
a
All data presented are averages of at least three separate experiments per-
formed by Panacos Pharmaceutical Inc., Gaithersburg, MD. IC50: concentration that
inhibits uninfected H9 cell growth by 50%. EC50: concentration that inhibits viral
replication by 50%. TI = therapeutic index IC50/EC50. NS: no suppression at the
tested concentration (20 lM).
highest tested concentration (20 lM).
other because of the rigid C ring, which benefits the anti-HIV activ-
ity. However, in C ring-opened 6 and 7, the two bulky groups can
rotate more freely and orient differently from each other and the
rest of the molecule, leading to abolished activity. This observation
is consistent with our prior SAR research that only 30R,40R-config-
ured DCK analogs were active; the remaining three 30,40-diastereo-
isomers were inactive.8 These results confirm that proper spatial
orientations of the camphanoyl groups and the remainder of the
molecule are necessary to achieve a proper ‘fit’ into the putative
viral binding site.
Therefore, in compound 5, an ethoxy moiety was incorporated at
this position on the phenyl ring. As we expected, the newly de-
signed compound 5, which has both ether and carbonyl groups
in its seco-A ring, exhibited moderate antiviral activity with an
EC50 value of 0.19 lM and a TI of 208. This result indicates that,
with proper substitutions in the seco-A ring, a modified bicyclic
system with only the coumarin B and C rings can retain moderate
antiviral activity.
Seco-C ring analogs 6 (40,8-seco) and 7 (20,30-seco) contain two
bulky camphanoyl moieties, but were not active in the anti-HIV
screening. These data indicated that the orientation of the two
camphanoyls is important to the antiviral potency. In 1 and 2,
the 30R,40R-dicamphanoyl groups are constrained close to each
Seco-C ring DCK analog 9 (bis 20,30 and 30,40) exhibited the best
anti-HIV activity among the newly designed compounds with an
EC50 value of 0.058
lM and TI of 1000, and was threefold more
potent than 2 (EC50: 0.126
lM, TI: 301.2) in the same antiviral

4368
J. Tang et al. / Bioorg. Med. Chem. 18 (2010) 4363–4373
Table 3
These results indicate that the substituent size at the 7-position
is critical to the compound0s anti-HIV activity, with an isopropoxy
moiety (as in 9) being optimal. The data also confirm our previous
SAR conclusion, based on DCK analogs, that there is an important
region of the binding site corresponding to the 20- and 8-position
of DCKs for viral target interaction.9
Compounds 13–16 with sulfur or nitrogen rather than oxygen
attached to the camphanoyl group were also synthesized, because
bioisosteric replacement is a common medicinal chemistry ap-
proach. Compounds 13 and 16 exhibited moderate anti-HIV activ-
Anti-HIV-1NL4-3 data of seco-DCK analogs (10–16) in MT-4 cell linea
Compd
IC50
(
lM)
EC50
(lM)
TI
10
11
12
13
14
15
16
9
62.4
60.3
54.5
56.24
25
36.61
32.3
44.65
4.67
25
1.71
1.87
1.22
12.04
1
8
15
22.53
56.62
34.72
44.7
5.66
4.69
1.98
ity with EC50 values of 4.67 and 4.69 lM, respectively, indicating
a
All data presented are averages of at least three separate experiments per-
that the antiviral activity of 9 can be retained upon heteroatom
replacement. The inhibitory potency increase from 14 to 16, which
corresponded to the enlarging size of the nitrogen substituent (H,
methyl, p-methoxyphenyl, respectively), suggested that a bigger
moiety is favored to interact with the binding pocket.
formed by Dr. Chin-Ho Chen, Duke University, NC. CC50: concentration that inhibits
uninfected MT-4 cell growth by 50%. EC50: concentration that inhibits viral repli-
cation by 50%. TI = therapeutic index IC50/EC50. NS: no suppression at the highest
tested concentration (20 lM).
In summary, data from our newly designed seco-DCKs led to the
following SAR conclusions. (a) Integrity of the A ring is not essen-
tial for anti-HIV activity; however, both ether (1-oxy) and carbonyl
(4-oxo) features are needed in the seco-A ring. (b) One 40-campha-
noyl moiety is sufficient for anti-HIV activity of DCK analogs, and
the 30-position can be deleted completely. (c) A proper spatial ori-
entation of the 40-camphanoyl with respect to the rest of the mol-
ecule is critical for enhanced anti-HIV activity. (d) A suitably sized
alkoxy group at position-7 is important for enhanced anti-HIV
activity. (e) Converting the 40-oxygen to sulfur or nitrogen can re-
sult in retained inhibitory activity, and a bulkier substituent on
nitrogen is preferred.
replication screening against HIV-1IIIB. Moreover, 9 also showed
marginal activity against the RTMDR strain with EC50 of 1.89
and TI of 9.13, using 3 (EC50: 0.133 M and TI: 69.9) as control.
lM
l
Notably, 9 is the first DCK analog to exhibit anti-HIV replication
activity against a multi-drug resistant viral strain. The removal of
one camphanoyl moiety, with retention of good anti HIV activity,
is important, as the metabolic instability of 2 and its derivatives
is a consequence of 30- and 4-acetoxy camphanoyl ester cleavage
on microsomes.11 Interestingly, compound 8 lost anti-HIV activity
completely in the same assay, even though 8 and 9 differ only by
the presence (8) or absence (9) of a 40-methyl group. The 3D struc-
tures of 2, 8, and 9 were generated using Sybyl (version 7.3) in the
standard energy minimizing condition to predict the orientation of
the 40-camphanoyl group (Fig. 3). The models clearly showed that
the orientation of the 40-camphanoyl in 9 is more similar to that in
2, while the orientation of this group in both stereoisomers of 8 is
very different, perhaps due to repulsion caused by the 40-methyl
group. These results further confirmed that proper interaction of
the 40-camphanoyl group with the viral target is essential to the
anti-HIV potency of DCK analogs. Compound 9, with only one cam-
phanoyl group, has a less complex structure, fewer hydrogen-bond
acceptors, and potentially better ADME than 2. It also has a re-
duced log P value (3.81), which is one log unit lower than that of
2 (log P: 4.96) (calculated by ACD/Log P DB software).
Bis (20,30- and 30,40-) seco modification in the DCK series opens a
significant new research area. Not only can such analogs exhibit
enhanced activity against wild-type HIV strain while gaining some
potency against RTMDR strain, they can also be synthesized and
isolated more easily, due to their simplified structure and deletion
of a chiral center. In addition, they have fewer hydrogen-bond
acceptors and lower log P values, and may have better ADME.
4. Experimental procedures
4.1. Materials
All reagents and solvents used were analytical grade. Melting
points were determined in open capillary tubes and are uncor-
rected. 1H and 13C NMR spectra were recorded in a Bruker-DPX
400 MHz. Mass spectra were measured with HP5973N analytical
mass spectrometers. High resolution mass spectra (HRMS) were
measured on a Shimadzu LCMS-IT-TOF with ESI interface. HPLC
for purity determinations were conducted using a Shimadzu
The promising seco-DCK hit compound 9 was further modified
to build some initial SAR. The 7-isopropoxy group was changed to
other alkoxy groups and the 40-oxygen of 9 was changed to sulfur
or nitrogen. The resulting analogs 10–16 were screened and com-
pared with 9. From the data (Tables 1–3), it can be seen that,
although the activity of 9 varied due to the assay protocol change,
it still showed the best anti-HIV potency among the newly synthe-
sized compounds. Reducing the size of the 7-alkyl ether, as in com-
pounds 10 (methyl) and 11 (ethyl), or enlarging it, as in 12
(methoxycarbonylmethyl), decreased the antiviral potency of the
LCMS-2010 with a Grace Alltima 2.1 ꢁ 150 mm HP C18 5
lM col-
umn and Shimadzu SPD-M20A detector at 254 nm wavelength.
HPLC purity analyses were determined by using two different sol-
vent conditions. The first was 70% MeCN as solvent A and 30% H2O
compounds (EC50 values 36.61, 32.3, and 44.65 lM, respectively).
Figure 3. 3D structures of 2, 8 and 9 by Sybyl (version 7.3) in the standard energy minimizing condition.

J. Tang et al. / Bioorg. Med. Chem. 18 (2010) 4363–4373
4369
as solvent B with 0.2 mL/min flow rate; the second was 80% MeOH
as solvent A and 20% H2O as solvent B with 0.1 mL/min flow rate.
The HPLC model was an isocratic system. All target compounds
had purity greater than 95%. Thin-layer chromatography (TLC)
was performed on PLC Silica Gel 60 F254 plates. Commercially avail-
able silica gel H was used for column chromatography.
27 (1.2 g) as a colorless oil in 92% yield. 1H NMR d 1.39 (t, 3H,
5-CH3), 1.41 (s, 6H, 2 ꢁ –CH3), 2.58 (s, 3H, 6-CH3), 3.89 (q, 2H,
5-CH2), 5.66 (d, J = 10.17 Hz, 1H, 3-H), 6.58 (d, J = 10.17 Hz, 1H,
4-H), 6.58 (d, J = 8.22 Hz, 1H, 7-H), 7.54 (d, J = 8.61 Hz, 1H, 8-H).
MS-EI+ (m/z, %): 246 (M+, 21.92) 231 (100) 203 (58.19) 149 (39.83).
4.3.2. 1-[(3R,4R)-Dihydroxy-5-ethoxy-2,2-dimethyl-3,4-
4.2. Synthesis of seco-A ring DCK (4)
dihydro-2H-chromen-6-yl]ethanone (28)
A mixture of K3Fe(CN)6 (5 equiv), K2CO3 (5 equiv), (DHQ)2PHAL
(5 equiv %), and K2OsO2(OH)4 (5 equiv %) in t-BuOH/H2O (v/v, 1:1)
was cooled to ꢀ5 to 0 °C. Under stirring, compound 27 (268 mg,
1.01 mmol) was added. The mixture was stirred for 14 h at 0 °C,
and then the reaction temperature was gradually raised to rt over
24 h. When TLC monitoring showed the reaction was completed,
an excess of aqueous Na2S2O3 was added and stirring continued
for 0.5 h. The mixture was extracted with EtOAc, the combined or-
ganic layer was dried over Na2SO4, and the solvent was removed.
The residue was purified by chromatography on a silica column
with petroleum ether/acetone = 10:1 as eluent to afford pure 28
as a clear oil (119 mg, 39%). 1H NMR d 1.32 (s, 3H, 2-CH3), 1.48
(s, 3H, 2-CH3), 1.48 (t, 3H, 5-CH3), 2.58 (s, 3H, 6-CH3), 3.16 (d,
J = 3.62 Hz, 1H, 3-CH), 3.81 (dd, J = 4.63, 3.63 Hz, 1H, 4-CH2), 3.94
(m, 1H, 5-CH2), 4.13 (m, 1H, 5-CH2), 4.35 (s, 1H, 3-OH), 5.04 (d,
J = 4.70 Hz, 1H, 4-OH), 6.67 (d, J = 8.96 Hz, 1H, 4-H), 7.59 (d,
J = 8.75 Hz, 1H, 7-H). MS-EI+ (m/z, %): 280 (M+, 9.76) 263 (12.69)
209 (36.00) 181 (45.31) 163 (67.42) 137 (33.42) 43 (100).
4.2.1. 2,2,5-Trimethyl-6-acetyl-2H-chromene (19)
Commercially available 17, K2CO3 (2.5 equiv), KI (1 equiv), and
excess 3-chloro-3-methyl-1-butyne (2–3 equiv) in dry DMF were
heated to 70–80 °C with stirring until the reaction was complete
as monitored by TLC. After filtering the solid, the filtrate was con-
centrated in vacuo. The residue 18, without purification, was di-
rectly heated to reflux in 10 mL of N,N-diethylaniline for 4–6 h.
The reaction mixture was cooled to rt, diluted with EtOAc, and
washed with 10% aqueous HCl, water, and brine. The organic layer
was separated and concentrated. The residue was purified by col-
umn chromatography with an eluant of hexane/EtOAc = 5:1 to af-
ford intermediate 19 as a brown oil; 60% yield (starting with
450 mg of 17); 1H NMR d 7.47 (1H, d, J = 8.4 Hz, H-7), 6.40 (1H, d,
J = 8.4 Hz, H-8), 6.60 (1H, d, J = 10.2 Hz, H-4), 5.69 (1H, d,
J = 10.2 Hz, H-3), 2.50 (3H, s, –COCH3), 2.49 (3H, s, CH3-5), 1.40
(6H, s, 2 ꢁ CH3-2).
4.2.2. 3R,4R-Dihydroxy-2,2,5-trimethyl-6-acetyl-chroman (20)
A mixture of K3Fe(CN)6 (3 equiv), K2CO3 (3 equiv), (DHQ)2-PYR
or (DHQ)2PHAL (2 equiv %), and K2OsO2(OH)4 (2 equiv %) was dis-
solved in t-BuOH/H2O (v/v, 1:1) at rt. The solution was cooled to
0 °C and methanesulfonamide (1 equiv) was added with stirring.
When the solution turned from light yellow to orange, 19 was
added. The mixture was stirred for 1–2 days at 0 °C and monitored
by TLC. At completion, Na2S2O5 (excess), water, and CHCl3 were
added, and stirring continued 0.5 h at rt. The mixture was ex-
tracted with CHCl3 three times, the combined organic layer was
dried over K2SO4, and then solvent was removed. The residue
was separated by column chromatography (hexane/EtOAc = 3:1)
to afford pure dihydroxy compound with 58% yield (starting with
108 mg of 19). Mp 106–108 °C; 1H NMR d 7.53 (1H, d, J = 8.5 Hz,
H-7), 6.69 (1H, d, J = 8.5 Hz, H-8), 4.84 (1H, d, J = 4.8 Hz, H-4),
3.75 (1H, d, J = 4.8 Hz, H-3), 2.54 (3H, s, COCH3-6), 2.50 (3H, s,
CH3-5), 1.38, 1.36 (3H each, s, CH3-2).
4.3.3. 1-[(3R,4R-Di-O-(ꢀ)-camphanoyloxy)-5-ethoxy-2,2-
dimethyl-3,4-dihydro-2H-chromen-6-yl)]-ethanone (5)
A mixture of compound 28 (1 equiv), (S)-(ꢀ)-camphanic chlo-
ride (2 equiv), and DMAP (3 equiv) was stirred for 2 h in CH2Cl2
at rt and monitored by TLC. After the reaction was completed,
the mixture was purified by chromatography on silica column with
petroleum ether/EtOAc = 2:1 as eluent to afford 5 (white powder)
(146 mg, 75%). Mp 230–232 °C. 1H NMR d 7.64 (1H, d, J = 9.0 Hz,
H-8), 6.07 (1H, d, J = 9.0 Hz, H-7), 6.48 (1H, d, J = 4.69 Hz, CHO-4),
5.51 (1H, d, J = 5.09 Hz, CHO-3), 3.94 (1H, m, CH2O-5), 3.82 (1H,
m, CH2O-5), 2.54 (3H, s, CH3CO-6), 2.42, 2.38, 1.90, 1.69 (each
2H, m, camphanoyl CH2), 1.45, 1.43 (each 3H, s, CH3-2) 1.11,
1.10, 1.09, 1.09, 0.97, 0.95 (each 3H, s, camphanoyl CH3). MS-ESI+
(m/z, %) 663 (M++Na, 100).
4.4. Synthetic routes to seco-C ring DCKs (6–9) and bioisosteric
seco DCK 13
4.2.3. 3R,4R-Dicamphanoyloxy-2,2,5-trimethyl-6-acetyl-2H-
chroman (4)
Compound 20, (S)-(ꢀ)-camphanic chloride (3 equiv), and DMAP
(0.1–1 equiv) were reacted 1–2 days in CH2Cl2 at rt and monitored
by TLC. At completion, the reaction mixture was concentrated and
the residue was purified by PTLC with an eluant of hexane/
4.4.1. 7-(2-Methylbut-3-en-2-yloxy)-4-methyl-2H-chromen-2-
one (30)
A mixture of 29 (2.50 g, 14.1 mmol), K2CO3 (20.00 g, 0.15 mol),
KI (2.55 g, 9.59 mmol), and 3-chloro-3-methyl-1-butene in acetone
(100 mL) was refluxed for 4 h. After filtering, the residue was
recrystallized from EtOAc to yield 30 as colorless crystals (2.68 g,
77%), mp 94–97 °C. 1H NMR d 7.48 (1H, d, J = 8.61 Hz, H-5), 6.86
(1H, dd, J = 8.61, 2.35 Hz, H-6), 6.81 (1H, d, J = 2.34 Hz, H-8), 6.12
(1H, s, H-3), 5.47 (1H, m, CHCH2-7), 4.57 (2H, d, J = 6.65 Hz,
CH2CH-7), 2.39 (3H, s, CH3-4), 1.80, 1.76 (each 3H, s, (CH3)2C-7).
MS-EI+ (m/z, %) 244 (M+, 4.65), 176 (Mꢀ68 100).
EtOAc = 3:1 to afford the final compound 4 with yield 39% (starting
from 55 mg of 20); mp 88–90 °C; MS-ESI+ (m/z, %): 628 (M++NH4
,
+
100); 1H NMR d 7.66 (1H, d, J = 8.7 Hz, H-7), 7.77 (1H, d, J = 8.7 Hz,
H-8), 6.43 (1H, d, J = 4.8 Hz, H-4), 5.30 (1H, d, J = 4.8 Hz, H-3), 2.52
(3H, s, COCH3-6), 2.29 (3H, s, CH3-5), 2.42, 2.17, 1.90, 1.68 (2H each,
m, camphanoyl CH2), 1.43, 1.39 (3H each, s, CH3-2), 1.08, 1.07, 1.05,
1.04, 0.97, 0.93 (3H each, camphanoyl CH3).
4.4.2. 7-(2R,3-Dihydroxy-1,1-dimethylpropoxy)-4-methyl-
chromen-2-one (31)
4.3. Synthesis of seco-A ring DCK (5)
Same synthetic procedure as for 28 but starting from 30. Com-
pound 31 was crystallized from EtOAc as white crystals (266 mg,
58%). Mp 172–173 °C. 1H NMR d 7.67 (1H, d, J = 8.55 Hz, H-5),
6.96 (1H, dd, J = 8.55 2.56 Hz, H-6), 6.98 (1H, d, J = 2.56 Hz, H-8),
6.19 (1H, d, J = 0.85 Hz, H-3), 5.10 (1H, d, J = 5.56 Hz, HOCH-7),
4.48 (1H, s, HOCH2-7), 4.32 (1H, dd, J = 1.71 10.25 Hz, CHCH2OH-
7), 3.90 (1H, dd, J = 8.12, 9.73 Hz, CHCH2OH-7), 3.55 (1H, m,
4.3.1. 1-(5-Ethoxy-2,2-dimethyl-2H-chromen-6-yl)ethanone
(27)
Intermediate 26 is a known compound and was prepared from
resorcinol as shown in Scheme 2. The crude product 26 (1.3 g,
5.3 mmol) was added to DMF/N,N-diethylaniline (15 mL/1 mL),
and the mixture heated at reflux under nitrogen for 5 h to give

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J. Tang et al. / Bioorg. Med. Chem. 18 (2010) 4363–4373
HOCHCH2-7), 2.39 (3H, s, CH3-4), 1.14, 1.08 (each 3H, s, (CH3)2C-7).
MS-EI+ (m/z, %) 278 (M+, 20.04), 176 (Mꢀ102 100).
CH2O), 4.73 (1H, m, (CH3)2CHO-7), 2.57 (1H, broad, OH), 2.40
(3H, d, J = 1.17 Hz, CH3-4), 1.42, 1.42 (each 3H, d, J = 5.87 Hz,
CH3CH-7). MS-EI+ (m/z, %) 248 (M+, 10.76), 188 (Mꢀ60, 100).
4.4.3. 7-(2R,3-Di-O-(ꢀ)-camphanoyloxy-1,1-dimethylpropoxy)-
4-methyl-2H-chromen-2-one (6)
4.4.7. 8-[O-(ꢀ)-Camphanoyloxymethyl]-7-isopropoxy-4-
methyl-2H-chromen-2-one (9)
Same synthetic procedure as for 5 but starting from 31. Chro-
matography (CHCl3–MeOH, 50:1) to yield 6 as a white solid
(410 mg, 93%). Mp 255–260 °C. 1H NMR d 7.56 (1H, d, J = 8.77 Hz,
H-8), 6.86 (1H, dd, J = 2.63 8.77 Hz, H-7), 6.76 (1H, d, J = 2.63 Hz,
H-8), 6.16 (1H, s, CH-3), 5.65 (1H, dd, J = 2.63 8.63 Hz, CHO-7),
4.41 (1H, dd, J = 2.63, 10.52 Hz, CH2O-7), 4.23 (1H, dd, J = 8.33,
10.53 Hz, CH2O-7), 2.45, 2.02, 1.92, 1.69 (each 2H, m, camphanoyl
CH2), 2.39 (3H, s, CH3-4), 1.11, 1.10, 1.06, 1.06, 1.00, 0.94 (each 3H,
s, camphanoyl CH3), 1.62 (each 3H, s, CH3C-7). 13C NMR d: 9.67,
16.59, 16.66, 16.74, 16.93, 18.62, 22.36, 22.84, 28.75 (2C), 30.43,
30.71, 54.17, 54.38, 54.87, 66.38, 75.84, 83.19, 91.00 (2C), 102.05,
111.57, 112.41, 114.30, 126.04, 152.37, 155.08, 160,64, 160.98,
166.10, 166.83, 178.10 (2C). MS-ESI+ (m/z, %) 639 (M++1, 100).
HRMS calcd for C35H42O11+Na 661.2619, found 661.2592 (M+Na).
Same synthetic procedure as for 5 but starting from 34a. White
solid (yield, 100%); mp 64–65 °C. 1H NMR d 7.58 (1H, d, J = 9.00 Hz,
H-6), 6.88 (1H, d, J = 8.61 Hz, H-5), 6.15 (1H, d, J = 1.17 Hz, H-3),
5.51 (2H, dd, J = 11.35 19.56 Hz, CH2O), 4.70 (1H, m, (CH3)2CHO-
7), 2.43, 2.03, 1.89, 1.65 (each H, m, camphanoyl CH2), 2.39 (3H,
s, CH3-4), 1.08, 1.02, 0.97 (each 3H, s, camphanoyl CH3), 1.36,
1.37 (each 3H, d, J = 4.70 Hz, CH3C-7). 13C NMR d 9.67, 16.69,
18.70, 21.43, 21.98, 28.97, 30.61, 54.26, 54.71, 56.15, 66.02,
71.47, 91.29, 109.10, 111.44, 112.11, 113.46, 126.29, 152.39,
153.84, 159.67, 160.49, 167.37, 178.35. MS-ESI+ (m/z, %) 429
(M++1, 100). HRMS calcd for C24H28O7: H, 427.1762, found
427.1748 (MꢀH).
4.4.8. 8-Acetyl-7-hydroxy-4-methyl-2H-chromen-2-one (32b)
Compound 29 (5 g, 22.9 mmol) and AlCl3 (4.59 g, 34.4 mmol)
was stirred vigorously for 3 h, then poured into 2 N HCl and ex-
tracted with EtOAc four times. The organic layer was dried over
Na2SO4, filtered, and the solvent removed. The residue was purified
by column chromatography on silica gel (hexane–EtOAc, 3:1) to
provide 32b as a light yellow solid (1.2 g, 24%), mp: 207–209 °C.
4.4.4. 7-Hydroxy-4-methyl-2-oxo-2H-chromene-8-carbaldehyde
(32a)
A reaction mixture of 29 (20 g, 114 mmol) and hexamethylene-
tetramine (40 g, 285 mmol) in HOAc (150 mL) was stirred for 5.5 h
at 95 °C. Thereafter, aq HCl solution (300 mL, concd HCl/
H2O = 84:100, v/v) was added, and the reaction mixture was stirred
for 0.5 h at 70 °C. After cooling, the reaction mixture was poured
into ice-water (1.5 L) and extracted with EtOAc four times. The
combined organic layer was dried over Na2SO4, and the solvent
was removed. The residue was recrystallized from EtOAc to pro-
vide 32a as a light yellow solid (5.23 g, 22%). Mp 120–122 °C.
4.4.9. 8-Acetyl-7-isopropoxy-4-methyl-chromen-2-one (33b)
Same synthetic procedure as for 33a but starting from
32b.White crystalline needles (yield, 47%); mp 139–140 °C. 1H
NMR d 7.53 (1H, d, J = 8.79 Hz, H-6), 6.87 (1H, d, J = 8.79 Hz, H-5),
6.13 (1H, d, J = 1.10 Hz, H-3), 4.67 (1H, m, CHO-7), 2.56 (3H, s,
CH3-4), 1.36, 1.36 (each 3H, d, J = 10.55 Hz, CH3CH-7). MS-EI+
(m/z, %) 260 (M+, 8.46), 203 (Mꢀ57, 100).
4.4.5. 7-Isopropoxy-4-methyl-2-oxo-2H-chromen-8-carbaldehyde
(33a)
Compound 32a (1 g, 5 mmol), ethane-1,2-diol (435 mg,
7.02 mmol), and p-toluenesulfonic acid (30 mg, 0.17 mmol) were
dissolved in toluene (40 mL). The reaction mixture was refluxed
for 2 h with removal of water. After adjusting the pH to 7–8 with
triethylamine and washing with brine, the organic layer was dried
over Na2SO4, filtered, and evaporated in vacuo. The residue was
recrystallized from EtOAc to give a light yellow solid (630 mg,
51%, mp 214–218 °C). A mixture of this intermediate (500 mg,
2.02 mmol), K2CO3 (836 mg, 6.05 mmol), KI (50 mg, 0.30 mmol),
and 2-bromopropane in acetone (50 mL) was refluxed for 6 h. After
filtering and removing the solvent, the residue was recrystallized
from EtOAc to yield a white solid (473 mg, 80%, mp: 150–
152 °C). This solid was added to 2 N HCl (30 mL), stirred for 5 h
at rt, extracted with EtOAc four times, and dried over Na2SO4. Fil-
tration and removal of the solvent in vacuo gave 33a as a white so-
lid (390 mg, 97%). Mp 166–168 °C. 1H NMR d 10.64 (1H, s, CHO),
7.71 (1H, d, J = 9.00 Hz, H-6), 6.94 (1H, d, J = 9.00 Hz, H-5), 6.20
(1H, d, J = 1.17 Hz, H-3), 4.77 (1H, m, (CH3)2CHO-7), 2.41 (3H, d,
J = 0.78 Hz, CH3-4), 1.44, 1.44 (each 3H, d, J = 6.65 Hz, CH3CH-7).
MS-EI+ (m/z, %) 246 (M+, 6.48), 148 (Mꢀ98, 100).
4.4.10. 8-(1-Hydroxyethyl)-7-isopropoxy-4-methyl-2H-
chromen-2-one (34b)
Same synthetic procedure as for 34a but starting from 33b.White
crystals (yield, 100%); mp 126–127 °C. 1H NMR d 7.46 (1H, d,
J = 8.79 Hz, H-6), 6.88 (1H, d, J = 8.97 Hz, H-5), 6.14 (1H, d,
J = 1.10 Hz, H-3), 5.54 (1H, m, CH3CHOH), 4.76 (1H, m, (CH3)2CHO-
7), 3.71 (1H, d, J = 1.91 Hz, OH), 2.39 (3H, d, J = 1.10 Hz, CH3-4), 1.58
(3H, d, J = 6.59 Hz, CH3CH-8), 1.43, 1.43 (each 3H, d, J = 5.86 Hz,
CH3CH-7). MS-EI+ (m/z, %) 262 (M+, 11.08), 202 (Mꢀ60, 100).
4.4.11. 8-[2-O-(ꢀ)-Camphanoyloxyethyl]-7-isopropoxy-4-
methyl-2H-chromen-2-one (8)
Same synthetic procedure as for 5 but starting from 34b. White
oil (yield, 100%). 1H NMR d 7.50 (1H, d, J = 8.67 Hz, H-6), 6.87 (1H,
dd, J = 8.94, 2.89 Hz, H-5), 6.67 (1H, dd, J = 6.71 13.54 Hz, CH3CHO-
8), 6.13 (1H, s, CH-3), 4.71 (1H, m, (CH3)2CHO-7), 2.49, 2.00, 1.91,
1.66 (each 1H, m, camphanoyl CH2), 2.39 (3H, s, CH3-4), 1.74 (3H, d,
J = 6.57 Hz, R:S 1:1, CH3CH-8), 1.15, 1.09, 1.07, 1.01, 0.96, 0.87 (each
1.5H, s, R:S 1:1, camphanoyl CH3). MS-ESI+Na (m/z, %) 465 (M++Na,
100). HRMS calcd for C25H30O7+Na 465.1884, found 465.1870
(M+Na).
4.4.6. 8-Hydroxymethyl-7-isopropoxy-4-methyl-2H-chromen-
2-one (34a)
A mixture of 33a (200 mg, 0.81 mmol) and NaBH4 (116 mg,
3.07 mmol) in MeOH (10 mL) was stirred for 1.5 h at rt. After the
pH was adjusted to 3–4 using 2 N HCl, MeOH was removed in va-
cuo. The residue was extracted with EtOAc three times and the or-
ganic layer was dried over Na2SO4. After filtration and removal of
the solvent, 34a was obtained as a colorless oil (240 mg, 100%).
Crystals of 34a were obtained by recrystallization from EtOAc,
mp: 118–121 °C. 1H NMR d 7.51 (1H, d, J = 9.00 Hz, H-6), 6.89
(1H, d, J = 9.00 Hz, H-5), 6.14 (1H, d, J = 1.17 Hz, H-3), 4.96 (2H, s,
4.4.12. 7-Isopropoxy-4-methyl-8-vinyl-2H-chromen-2-one (35)
A solution of 34b (75 mg, 0.29 mmol) and two drops of concd
H2SO4 in acetone (5 mL) was refluxed for 20 min. The reaction mix-
ture was neutralized with 2 N aqueous Na2CO3. After removal of
the solvent and extraction with EtOAc, the organic layer was dried
over Na2SO4 and concentrated in vacuo. The residue was purified
by column chromatography with an eluent of petroleum ether/
EtOAc = 8:1 to provide pure 35 as a white crystalline solid in 17%
yield; mp 102–104 °C. 1H NMR d 7.43 (1H, d, J = 9.00 Hz, H-6),

J. Tang et al. / Bioorg. Med. Chem. 18 (2010) 4363–4373
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7.08 (1H, dd, J = 12.13, 18.00 Hz, CHCH2-8), 6.88 (1H, d, J = 9.00 Hz,
H-5), 6.33 (1H, dd, J = 2.35, 18.00 Hz, CH2CH-8), 6.15 (1H, s, H-3),
5.61 (1H, dd, J = 2.35, 12.13 Hz, CH2CH-8), 4.71 (1H, m,
(CH3)2CHO-7), 2.40 (3H, d, J = 1.17 Hz, CH3-4), 1.41, 1.40 (each
3H, s, CH3CH-7). MS-EI+ (m/z, %) 244 (M+, 24.98), 174 (Mꢀ70, 100).
toluene (40 mL) was refluxed 2 h with removal of water. The reac-
tion mixture was cooled to rt and the pH adjusted to 7–8 with tri-
ethylamine. After being washed with brine, the organic layer was
dried over anhydrous Na2SO4 and concentrated under reduced
pressure to afford a yellow solid, which was recrystallized from
EtOH to yield a light yellow solid (630 mg, 51%): mp 214–218 °C.
1H NMR d 2.39 (s, 3H, 4-CH3), 4.14, 4.26 (m, 4H, 2 ꢁ –CH2), 6.13
(d, J = 1.18 Hz, 1H, 3-H), 6.40 (m, 1H, 8-CH), 6.85 (d, J = 8.60 Hz,
1H, 5-H), 7.52 (d, J = 8.60 Hz, 1H, 6-H), 9.15 (s, 1H, 7-OH). MS-EI+
(m/z, %) 248 (M+, 46.01).
4.4.13. 8-(1R,2-Dihydroxyethyl)-7-isopropoxy-4-methyl-2H-
chromen-2-one (36)
Same synthetic procedure as for 28 but starting from 35. Crystal-
line needles (yield, 70%); mp: 102–104 °C. 1H NMR d 7.50 (1H, d,
J = 9.00 Hz, H-6), 6.88 (1H, d, J = 9.00 Hz, H-5), 6.14 (1H, d,
J = 1.17 Hz, H-3), 5.46 (1H, dd, J = 4.27 8.54 Hz, HOCH2CH-8), 4.75
(1H, m, (CH3)2CHO-7), 3.94 (1H, dd, J = 8.85 11.29 Hz, HOCHCH2-8),
3.83 (1H, m, HOCH2-8), 3.72 (1H, dd, J = 4.27, 11.29 Hz, HOCH2CH-
8), 2.39 (3H, d, J = 1.22 Hz, CH3-4), 1.79 (1H, s, HOCH-8), 1.42, 1.41
(each 3H, d, J = 5.60 Hz, CH3CH-7). MS-EI+ (m/z, %) 278 (M+, 100).
4.5.2. Synthesis of intermediates 41a–c
Compound 38 (1 equiv), K2CO3 (5 equiv), and halide (3 equiv)
were added into acetone (50 mL) and refluxed for 6 h. After
filtration, the solvent was removed in vacuo to give crude product
(39a–c), which was stirred for 5 h in 2 N HCl solution (30 mL) at rt.
The reaction mixture was extracted with EtOAc four times. The
organic layer was dried over Na2SO4 and the solvent distilled to
give a white solid (40a–c). The intermediate (1 equiv) and NaBH4
(1.5 equiv) in MeOH (10 mL) were allowed to react for 1 h at rt,
then acidified to pH 3–4 with 2 N HCl. After removal of MeOH,
the residue was extracted with EtOAc three times. The organic
layer was dried over Na2SO4 and concentrated to provide the
desired product.
4.4.14. 8-(1R,2-Di-O-(ꢀ)-camphanoyloxyethyl)-7-isopropoxy-4-
methyl-2H-chromen-2-one (7)
Same synthetic procedure as for 5 but starting from 36. White
solid (yield, 100%); mp 64–66 °C. 1H NMR
d 7.57 (1H, d,
J = 9.00 Hz, H-6), 6.88 (1H, d, J = 9.00 Hz, H-5), 6.89 (1H, dd,
J = 8.61 3.91 Hz, CH2CHO-8), 6.15 (1H, d, J = 1.17 Hz, H-3), 5.02
(1H, dd, J = 11.74, 8.61 Hz, CHCH2O-8), 4.71 (1H, m, (CH3)2CHO-
7), 4.69 (1H, dd, J = 11.73, 3.91 Hz, CHCH2O-8), 2.46, 2.02, 1.91,
1.66, (each 2H, m, camphanoyl CH2), 2.40 (3H, d, J = 1.18 Hz, CH3-
4), 1.44, 1.43 (each 3H, d, J = 6.26 Hz, (CH3)2CHO-7), 1.09, 1.07,
1.02, 1.01, 0.91, 0.87 (each 3H, s, camphanoyl CH3). 13C NMR d
9.68, 16.61, 16.69, 18.84, 21.90, 21.96, 28.95, 30.66 (2C), 54.17,
54.79 (2C), 64.63, 67.10, 71.96, 91.00 (2C), 109.44, 111.42,
112.16, 113.54, 126.62, 152.41, 153.33, 159.17, 159.88, 166.60,
167.06, 177.94, 178.14. MS-ESI+ (m/z, %) 639 (M++1, 100). HRMS
calcd for C35H42O11: H, 637.2654, found 637.2634 (MꢀH).
4.5.2.1. 8-Hydroxymethyl-7-methoxy-4-methyl-2H-chromen-2-
one (41a). Light yellow crystals from EtOAc, MS-ESI+, (m/z, %)
221.0 (M++1, 100).
4.5.2.2. 8-Hydroxymethyl-7-ethoxy-4-methyl-2H-chromen-2-
one (41b). Light yellow crystals from MeOH, yield 68%, mp
169–172 °C. 1H NMR d 7.52 (1H, d, J = 8.99 Hz, H-6), 6.88 (1H, d,
J = 9.00 Hz, H-5), 6.15 (1H, d, J = 0.78 Hz, H-3), 4.98 (2H, d,
J = 6.65 Hz, 8-CH2), 4.19 (2H, dd, J = 6.65, 7.05 Hz, 7-CH3CH2O),
2.58 (1H, t, J = 3.52, 3.13 Hz, 8-OH), 2.40 (3H, d, J = 1.17 Hz, CH3-
3), 0.83 (3H, t, J = 6.05, 7.04 Hz, 7-CH3).
4.4.15. 7-Isopropoxy-8-(mercaptomethyl)-4-methyl-2H-
chromen-2-one (37)
Under nitrogen, a mixture of compound 34a (150 mg, 0.61 mmol)
and Lawesson reagent (367 mg, 0.91 mmol) in toluene (10 mL) was
stirred for 20 h and monitored by TLC (hexane:EtOAc/1:1). After fil-
tration, the solvent was removed in vacuo. The residue was purified
by silica gel chromatography (eluent: hexane/EtOAc = 1/1) to obtain
37 as a yellow solid (43 mg), yield: 27%; mp 113–116 °C. 1H NMR d
7.45 (1H, d, J = 9.00 Hz, H-6), 6.86 (1H, d, J = 8.99 Hz, H-5), 6.15 (1H,
s, H-3), 4.73 (1H, m, 7-CH), 3.93 (2H, d, J = 8.59 Hz, 8-CH2), 2.40 (3H,
s, 4-CH3), 2.13 (1H, t, J = 8.6 Hz, 8-OH), 1.42 (6H, d, J = 5.87 Hz,
2 ꢁ 7-CH3). MS-ESI+ (m/z, %) 265 (M++1, 100).
4.5.2.3. Methyl 2-(8-hydroxymethyl-4-methyl-2-oxo-2H-chro-
men-7-yloxy)acetate (41c).
White crystals from MeOH, yield
79%, mp 142–144 °C. 1H NMR d 7.52 (1H, d, J = 9.00 Hz, H-6), 6.78
(1H, d, J = 8.60 Hz, H-5), 6.18 (1H, d, J = 1.17 Hz, H-3), 5.02 (2H, s,
7-CH2), 4.81 (2H, d, 8-CH2), 3.81 (3H, s, 7-CH3), 2.55 (1H, br s,
8-OH), 2.40 (3H, d, J = 1.17 Hz, CH3-3).
4.5.3. Synthesis of target compounds 10–12
Same synthetic procedure as for 5 but starting from 41a–c,
respectively.
4.4.16. 8-[S-(ꢀ)-Camphanoyl)mercaptomethyl]-7-isopropoxy-4-
methyl-2H-chromen-2-one (13)
4.5.3.1. 8-[O-(ꢀ)-Camphanoyloxymethyl]-7-methoxy-4-methyl-
Same synthetic procedure as for 5 but starting from 37. Light
yellow crystals (yield, 91%); mp 166–168 °C. 1H NMR d 7.47 (1H,
d, J = 8.99 Hz, H-6), 6.83 (1H, d, J = 9.00 Hz, H-5), 6.13 (1H, s, H-
3), 4.68 (1H, m, 7-CH), 4.47 (2H, s, 8-CH2), 2.50, 1.95, 1.68, 1.37
(each H, m, camphanoyl CH2), 2.39 (3H, s, CH3-4), 1.09, 1.09, 0.96
(each 3H, s, camphanoyl CH3), 1.37, 1.37 (each 3H, d, J = 2.47
3.13 Hz, 2 ꢁ CH3C-7). 13C NMR d 9.69, 16.53, 16.65, 18.69, 20.89,
22.01, 28.94, 31.08, 54.59, 55.44, 71.28, 96.30, 108.85, 112.09,
112.76, 113.53, 124.61, 152.39, 152.97, 158.86, 160.62, 177.95,
195.75. MS-ESI+ (m/z, %) 445 (M++1).
2H-chromen-2-one (10).
White solid from hexane/acetone =
1/1, yield 78%, mp 185–188 °C. 1H NMR d 7.61 (1H, d, J = 8.61 Hz,
H-6), 6.90 (1H, d, J = 9.00 Hz, H-5), 6.16 (1H, s, H-3), 5.51 (2H, dd,
J = 13.7, 14.08 Hz, 8-CH2), 3.93 (3H, s, 7-CH3), 2.45, 2.03, 1.88,
1.66 (each H, m, camphanoyl CH2), 2.42 (3H, s, 4-CH3), 1.62, 1.08,
1.03, 0.97 (each 3H, s, camphanoyl CH3). 13C NMR d 9.66, 16.53,
16.61, 18.71, 28.96, 30.55, 54.30, 54.72, 55.95, 56.16, 91.29,
107.07, 110.60, 112.29, 113.90, 126.54, 152.38, 153.51, 160.35,
160.95, 167.37, 178.33. MS-ESI+ (m/z, %) 401 (M++1, 100). HRMS
calcd for C22H24O7+Na 423.1414, found 423.1402(M+Na).
4.5. Synthetic routes to seco-C ring DCKs (10–12)
4.5.3.2. 8-[O-(ꢀ)-Camphanoyloxymethyl]-7-ethoxy-4-methyl-
2H-chromen-2-one (11).
White solid from hexane/
4.5.1. 8-(1,3-Dioxolan-2-yl)-7-hydroxy-4-methyl-2H-chromen-
2-one (38)
Using a water separator, a mixture of 32a (1 g, 5 mmol), ethane-
1,2-diol (335 mg, 5.4 mmol) and p-toluenesulfonic acid (30 mg) in
EtOAc = 1/1, yield 48%, mp 161–163 °C. 1H NMR d 7.59 (1H, d,
J = 9.00 Hz, H-6), 6.88 (1H, d, J = 9.00 Hz, H-5), 6.15 (1H, d,
J = 1.17 Hz, H-3), 5.53 (2H, dd, J = 1.34 17.60 Hz, 8-CH2), 4.16 (2H,
dd, J = 7.04 13.69 Hz, 7-CH2), 2.45, 2.04, 1.89, 1.65 (each H, m, cam-

4372
J. Tang et al. / Bioorg. Med. Chem. 18 (2010) 4363–4373
phanoyl CH2), 2.41 (3H, s, CH3-4), 1.08, 1.02, 0.97 (each 3H, s, cam-
phanoyl CH3), 1.45 (3H, t, J = 7.94, 7.07 Hz, 7-CH3). 13C NMR d 9.66,
14.65, 16.60, 16.65, 18.70, 28.96, 30.59, 54.28, 54.72, 56.03, 64.70,
91.29, 107.89, 110.63, 112.16, 113.70, 126.46, 152.42, 153.60,
160.44, 167.38, 178.35. MS-ESI+ (m/z, %) 415 (M++1, 100). HRMS
calcd for C23H26O7+Na 437.1571, found 437.1470 (M+Na).
4.6.3.2.
7-Isopropoxy-8-[(4-methoxyphenylimino)methyl]-4-
methyl-2H-chromen-2-one (45). Yield 100%, mp 157–159 °C.
1H NMR d 10.62, 8.84 (1H, s, 8-CH), 7.70, 7.59 (1H, d, J = 9.00 Hz,
H-6), 7.30 (1H, d, J = 8.99 Hz, H-5), 7.28 (1H, d, J = 7.44 Hz, 8-Ar-
H), 6.95, 6.74, 6.65 (3H, m, 8-Ar-H), 6.18 (1H, s, H-3), 4.73 (1H,
m, 7-CH), 3.84 (3H, s, 8-OCH3), 2.42 (3H, s, CH3-4), 1.41 (6H, s,
2 ꢁ CH3C-7). MS (EI) (m/z, %) 351 (M++1, 14.23), 229 (100).
4.5.3.3. 8-[O-(ꢀ)-Camphanoyloxymethyl]-7-(methoxycarbonyl-
methoxy)-4-methyl-2H-chromen-2-one (12). White solid from
EtOAc, yield 83%, mp 155–157 °C. 1H NMR d 7.59 (1H, d, J = 8.60 Hz,
H-6), 6.76 (1H, d, J = 8.60 Hz, H-5), 6.19 (1H, d, J = 1.18 Hz, H-3),
5.58 (2H, dd, J = 14.09, 13.69 Hz, 8-CH2), 4.78 (2H, s, 7-CH2), 3.80
(3H, s, 7-CH3), 2.47, 2.04, 1.89, 1.65 (each H, m, camphanoyl
CH2), 2.41 (3H, d, J = 1.18 Hz, CH3-4), 1.08, 1.04, 0.96 (each 3H, s,
camphanoyl CH3). 13C NMR d 9.64, 16.52, 16.60, 18.68, 28.95,
30.55, 52.42, 54.28, 54.71, 55.91, 65.71, 91.28, 107.87, 111.55,
112.85, 114.75, 126.43, 152.15, 153.60, 159.14, 160.07, 167.28,
168.26, 178.32. MS-ESI+ (m/z, %) 459 (M++1, 100). HRMS calcd for
C24H26O9+Na 481.1469, found 481.1468 (M+Na).
4.6.4. Synthesis of 44 and 46
A mixture of imine (1 equiv) and NaBH4 (2.3 equiv) in MeOH
(10 mL) was stirred for 30 min at rt. After removal of the solvent,
the residue was extracted with EtOAc three times. The organic
layer was dried over anhydrous Na2SO4, and concentrated to get
a light yellow solid, which was recrystallized from EtOAc to pro-
vide white crystals.
4.6.4.1.
2H-chromen-2-one (44).
7-Isopropoxy-4-methyl-8-[(N-methyl)aminomethyl]-
Yield 98%, mp 128–130 °C. 1H
NMR d 7.46 (1H, d, J = 9.00 Hz, H-6), 6.86 (1H, d, J = 9.00 Hz, H-5),
6.13 (1H, s, H-3), 4.68 (1H, m, 7-CH), 4.02 (2H, s, 8-CH2), 2.41
(3H, s, CH3-3), 2.39 (3H, d, J = 0.78 Hz, 8-CH3), 1.73 (1H, broad,
8-NH), 1.40, 1.39 (each 3H, d, CH3CH-7). MS-EI+ (m/z, %) 261 (M+,
8.05), 246 (100).
4.6. Synthesis of bioisosteric seco-DCKs (14–16)
4.6.1. 8-Aminomethyl-7-isopropoxy-4-methyl-2H-chromen-2-
one (42)
4.6.4.2. 7-Isopropoxy-8-[(N-4-methoxyphenyl)aminomethyl]-4-
methyl-2H-chromen-2-one (46). Yield 96%: mp 137.5–140 °C.
1H NMR d 7.43 (1H, d, J = 9.00 Hz, H-6), 6.84 (1H, d, J = 9.00 Hz, H-
5), 6.74 (4H, dd, J = 9.00 Hz, 8-Ar-H), 6.13 (1H, s, H-3), 4.73 (1H, m,
7-CH), 4.56 (2H, s, 8-CH2), 3.71 (3H, s, 8-OCH3), 2.37 (3H, s, CH3-3),
2.39 (3H, d, J = 0.78 Hz, 8-CH3), 1.59 (1H, br, 8-NH), 1.44, 142 (each
3H, d, CH3CH-7). MS (EI) (m/z, %) 353 (M+, 44.59), 310 (5.53), 231
(19.07), 189 (100).
A
mixture of 33a (246 mg, 1 mmol) and (NH4)2CO3 (1 g,
10.4 mmol) in EtOH (50 mL) was refluxed for 48 h, then NaBH4
(50 mg, 1.32 mmol) was added, and reflux continued for 15 min.
After removal of the solvent, the residue was separated by silica
gel chromatography to afford a yellow oil (164 mg). Light yellow
crystals were obtained from hexane/acetone = 1/1, yield 66%: mp
196–198.5 °C. 1H NMR (400 MHz, DMSO-d6)
d 7.60 (1H, d,
J = 9.00 Hz, H-6), 7.07 (1H, d, J = 8.70 Hz, H-5), 6.19 (1H, d,
J = 1.8 Hz, H-3), 4.78 (1H, m, 7-CH), 3.78 (2H, s, 8-CH2), 2.37 (3H,
s, CH3-4), 1.54 (2H, br s, NH2), 1.29 (6H, d, J = 6.00 Hz, 2 ꢁ CH3C-
7). MS (ESI) (m/z, %) 495(2M++1, 100).
4.6.5. Synthesis of 15 and 16
Same synthetic procedure as for 5 but starting from 44 and 46,
respectively.
4.6.2. 8-[(N-(ꢀ)-Camphanoyl)aminomethyl]-7-isopropoxy-4-
methyl-2H-chromen-2-one (14)
4.6.5.1. 8-[(N-Methyl-N-(ꢀ)-camphanoyl)aminomethyl]-7-iso-
propoxy-4-methyl2H-chromen-2-one(15). Yield 86%. 1H NMR
d 7.50 (1H, d, J = 9.00 Hz, H-6), 6.84 (1H, d, J = 8.61 Hz, H-5), 6.09
(1H, d, J = 1.17 Hz, H-3), 5.09, 4.72 (2H, dd, J = 11.35 19.56 Hz,
CH2N-8), 4.65 (1H, m, (CH3)2CHO-7), 2.91 (3H, s, CH3N), 2.39,
2.03, 1.89, 1.62 (each H, m, camphanoyl CH2), 2.37 (3H, s, CH3-4),
1.07, 1.05, 1.02 (each 3H, s, camphanoyl CH3), 1.31, 1.23 (each
3H, d, J = 6.30 Hz, CH3C-7). MS-ESI+ (m/z, %) 442 (M++1, 100).
Same synthetic procedure as for 5 but starting from 42. Light
yellow crystals (yield 87%); mp 236–237.5 °C. 1H NMR (400 MHz,
CDCl3) d 7.30 (1H, d, J = 9.16 Hz, Ar-H), 7.28 (1H, d, J = 9.17 Hz,
Ar-H), 6.86 (1H, d, J = 9.17 Hz, Ar-H), 6.85 (1H, d, J = 9.16 Hz, Ar-
H), 5.80 (1H, s, H-3), 5.76 (1H, s, H-3), 4.68 (5H, m), 4.52 (1H, d,
J = 14.36 Hz), 2.58, 2.33, 1.94, 1.72 (each H, m, camphanoyl CH2),
2.22, 2.21 (3H, s, 2 ꢁ CH3-4), 1.51, 1.48, 1.42, 1.37 (each 3H, d,
4 ꢁ –CH3), 1.30, 1.16, 1.25 (each 3H, s, camphanoyl CH3). 13C
NMR d 9.73, 17.47, 17.94, 18.49, 21.97, 22.14, 22.60, 29.50, 31.32,
37.51, 39.61, 53.81, 55.47, 70.80, 93.11, 108.75, 111.02, 112.05,
124.75, 152.34, 153.32, 160.15, 167.37, 179.06. MS-ESI+ (m/z, %)
428 (M++1). HRMS calcd for C24H29NO6+Na 450.1887, found
450.1874 (M+Na).
4.6.5.2. 8-[(N-4-Methoxyphenyl-N-(ꢀ)-camphanoyl)aminomethyl]-
7-isopropoxy-4-methyl-2H-chromen-2-one (16). Yield 100%,
colorless oil. 1H NMR d 7.39 (1H, d, J = 8.61 Hz, H-6), 6.72 (1H,
d, J = 8.61 Hz, H-5), 7.00–6.49 (5H, m, Ar-H), 5.97 (1H, s, H-3),
5.46 (1H, d, J = 3.3 Hz, CH2N-8), 4.91 (1H, d, J = 3.59 Hz, CH2N-8),
4.60 (1H, m, (CH3)2CHO-7), 3.68 (3H, s, CH3O), 1.75,1.55, 1.40,
1.10 (each H, m, camphanoyl CH2), 2.33 (3H, s, CH3-4), 1.20, 1.03,
0.97 (each 3H, s, camphanoyl CH3), 1.33, 1.25 (each 3H, d,
J = 5.87 Hz, CH3C-7). MS-ESI+ (m/z, %) 534 (M++1, 100).
4.6.3. Synthesis of 43 and 45
Compound 33a (1 equiv) and amine (1.1 equiv) were dissolved
in EtOH (10 mL). The mixture reaction was stirred for 2 h at rt and
monitored by TLC (hexane/acetone = 2/1). After removal of the sol-
vent, yellow crystals were obtained from EtOAc.
4.7. Anti-HIV replication assay against wild-type HIV-1IIIB in MT-
2 cell lines
4.6.3.1. 7-Isopropoxy-4-methyl-8-[(methylimino)methyl]-2H-
chromen-2-one (43).
This assay was performed by Panacos Pharmaceuticals, Inc as
follows. The human T-cell line, MT-2, was maintained in continu-
Yield 91%, mp 160–161 °C. 1H NMR d
8.71 (1H, s, 8-CH), 7.03 (1H, d, J = 8.55 Hz, H-6), 6.15 (1H, d,
J = 8.55 Hz, H-5), 5.97 (1H, s, H-3), 4.59 (1H, m, 7-CH), 3.44 (3H,
s, 8-CH3), 2.49 (3H, d, J = 4.89 Hz, CH3-4), 1.36, 1.35 (6H, s,
2 ꢁ CH3C-7). MS-EI+ (m/z, %) 260 (M++1, 65.87), 232 (76.43), 190
(100).
ous culture with -glutamine at 5% CO2 and 37 °C. Test samples
were first dissolved in dimethyl sulfoxide. The following were
the final drug concentrations routinely used for screening 100,
L
20, 4 and 0.8
lg/mL. For agents found to be active, additional dilu-
tions were prepared for subsequent testing so that an accurate EC50

J. Tang et al. / Bioorg. Med. Chem. 18 (2010) 4363–4373
4373
value could be determined. Test samples were prepared, and to
Xia, and Grant AI-33066 from the National Institute of Allergies
and Infectious Diseases awarded to K. H. Lee. We also appreciate
the National Ministry of Science and Technology, China, for support
from a grant (2009zx09301-011) for a large platform of integrated
drug discovery and technical development.
each sample well was added 90
l
L of media containing H9 cells
at 3 ꢁ 105 cells/mL and 45
lL of virus inoculum (HIV-1 IIIB isolate)
containing 125 TCID50. Control wells containing virus and cells
only (no drug) and cells only (no virus or drug) were also prepared.
A second set of samples was prepared identical to the first and
were added to cells under identical conditions without virus (mock
infection) for toxicity determinations (IC50 defined below). In addi-
tion, AZT and 2 were also assayed during each experiment as a po-
sitive drug control. On days 1 and 4 post-infection (PI), spent media
was removed from each well and replaced with fresh media. On
day 6 PI, the assay was terminated and cultured supernatants were
harvested for analysis for virus replication by p24 antigen capture.
The compound toxicity was determined by XTT using the mock-in-
fected sample wells. If a test sample inhibited virus replication and
Supplementary data
Supplementary data (additional information on compound pur-
ity, high-resolution mass spectral data, and HPLC analysis results of
the seco DCK analogs 4–9) associated with this article can be
found, in the online version, at doi:10.1016/j.bmc.2010.04.089.
References and notes
was not toxic, its effects were reported in the following terms: IC50
,
the concentration of test sample that was toxic to 50% of the mock-
infected cells; EC50, the concentration of test sample that was able
1. Huang, L.; Kashiwada, Y.; Cosentino, L. M.; Fan, S.; Chen, C. H.; McPhail, A. T.;
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12
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4.8. Anti-HIV replication assay against HIV-1NL4-3 and RTMDR
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Acknowledgments
This research was supported by grants from the National Natu-
ral Science Foundation of China awarded to P. Xia (No. 20272010)
and Y. Chen (No. 30200348 and 30873164), respectively. It was
also supported by a research grant (2002046069) for Ph D program
from the National Education Administration of China awarded to P.