Antitumor agents. 280. Multidrug resistance-selective desmosdumotin B analogues

Article abstract of DOI:10.1021/jm100846r

6,6,8-Triethyldesmosdumotin B (2) was discovered as a MDR-selective flavonoid with significant in vitro anticancer activity against a multidrug resistant (MDR) cell line (KB-VIN) but without activity against the parent cells (KB). Additional 2 analogues were synthesized and evaluated to determine the effect of B-ring modifications on MDR-selectivity. Analogues with a B-ring Me (3) or Et (4) group had substantially increased MDR selectivity. Three new disubstituted analogues, 35, 37, and 49, also had high collateral sensitivity (CS) indices of 273, 250, and 100, respectively. Furthermore, 2-4 also displayed MDR selectivity in an MDR hepatoma-cell system. While 2-4 showed either no or very weak inhibition of cellular P-glycoprotein (P-gp) activity, they either activated or inhibited the actions of the first generation P-gp inhibitors verapamil or cyclosporin, respectively.

Full text of DOI:10.1021/jm100846r

J. Med. Chem. 2010, 53, 6699–6705 6699
DOI: 10.1021/jm100846r
Antitumor Agents. 280. Multidrug Resistance-Selective Desmosdumotin B Analogues
Kyoko Nakagawa-Goto,*,† Po-Cheng Chang,†,^ Chin-Yu Lai,†,^ Hsin-Yi Hung,† Tzu-Hsuan Chen,† Pei-Chi Wu,†
Hao Zhu,‡ Alexander Sedykh,‡ Kenneth F. Bastow,*,§ and Kuo-Hsiung Lee*,†,
†Natural Products Research Laboratories, Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill,
North Carolina 27599, ‡Laboratory for Molecular Modeling, Division of Medicinal Chemistry and Natural Products, Eshelman School of
Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, §Division of Medicinal Chemistry and Natural
Products, Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, and
Chinese Medicine Research and Development Center, China Medical University and Hospital, Taichung, Taiwan.
^ These authors contributed equally to the biological work.
Received July 1, 2010
6,6,8-Triethyldesmosdumotin B (2) was discovered as a MDR-selective flavonoid with significant in
vitro anticancer activity against a multidrug resistant (MDR) cell line (KB-VIN) but without activity
against the parent cells (KB). Additional 2 analogues were synthesized and evaluated to determine the
effect of B-ring modifications on MDR-selectivity. Analogues with a B-ring Me (3) or Et (4) group had
substantially increased MDR selectivity. Three new disubstituted analogues, 35, 37, and 49, also had
high collateral sensitivity (CS) indices of 273, 250, and 100, respectively. Furthermore, 2-4 also
displayed MDR selectivity in an MDR hepatoma-cell system. While 2-4 showed either no or very weak
inhibition of cellular P-glycoprotein (P-gp) activity, they either activated or inhibited the actions of the
first generation P-gp inhibitors verapamil or cyclosporin, respectively.
Introduction
mediator of MDR.11-14 Intrinsic or acquired overexpression
of P-gp dramatically reduces drug response, at times to less
than one-quarter, causing poor clinical outcomes following
chemotherapy.15
Incidences of drug resistance still present major and serious
obstacles to the effective chemotherapeutic treatment of cancer,
despite many efforts to overcome it.1,2 Resistance to one drug
often implies simultaneous resistance to structurally and mecha-
nistically diverse anticancer drugs. This efflux phenotype,
called multidrug resistance (MDRa),3,4 is in part mediated
by the overexpression of plasma membrane transporters, such
as P-glycoprotein (P-gp, MDR1, or ABCB1, localized at
7q21.1, a 170 kDa protein),5 MDR-associated proteins
(MRP1 or ABCC1, localized at 16p13.1, a 190 kDa protein,
and MRP2),6 or breast cancer resistant protein (BCRP or
ABCG2, localized at 4q22, a 72 kDa protein).7-9 These three
kinds of proteins belong to the superfamily of ATP-binding-
cassette (ABC) transporters.10 The emergence of MDR pumps
causescancerdrugs tobepumped outof the cell, thusreducing
intracellular drug concentrations below cytotoxic levels.
Because P-gp has broad substrate specificity, tumor cells that
overexpress P-gp show resistance to many classical and newer
molecular-targeted antitumor drugs. The development of
agents targeted toward MDR1 or MRP1 is greatly needed
in order to improve anticancer chemotherapeutic strategies.
MDR1/P-gp is a well-characterized efflux system and a major
To date, the major pharmacological approaches to over-
come MDR have focused on inhibition of the pump function
and/ordown-regulation of pumpoverexpression.16-19 Nume-
rous pump inhibitors (or modulators) have been found and
are generally classified as first, second, or third generation
chemosensitizers. Many second and third generation inhibi-
tors, such as valspodar, zosuquidar, and tariquidar, are more
potent and less toxic than first generation compounds like
verapamil (VERAP) or cyclosporine A (CSA). Although
some of these new generation compounds are still in clinical
trials, the benefits of chemosensitizationremain disappointing
in part because the modulator causes undesirable changes in
drug pharmacokinetics.20
Flavonoids are the most widespread natural compounds
produced in plants, and they exhibit diverse, important bio-
logical activities, including antioxidant, anticarcinogenic, anti-
inflammatory, antiproliferative, antiangiogenic, and anti-
estrogenic effects.21 Many reports have demonstrated that
flavonoids can also interact with ABC transporters, and some
act as P-gp modulators.22-25 We found that the flavonoid
desmosdumotin B (1, Figure 1) exerted unique in vitro antic-
ancer activity against a P-gp expressing MDR tumor cell line
(KB-VIN, ED50 =2.0 μg/mL), resulting in a >20 fold index
of selectivity over the drug sensitive KB parent cell line.26 We
also discovered that 6,8,8-triethyldesmosdumotin B (TEDB, 2)
and its 40-methyl (3) and 40-ethyl (4) analogues were signifi-
cantly more potent and showed MDR-selectivity of >250.27
In contrast, the related 6,8,8-tripropyl and 6,8-diethyl com-
pounds were less optimized for MDR selectivity.
*To whom correspondence should be addressed. For K.N.-G.: phone,
919-843-5209; fax, 919-966-3893; e-mail, goto@email.unc.edu. For K.F.B.:
phone, 919-966-7633; fax, 919-966-0204; e-mail, ken_bastow@unc.edu.
For K.-H.L.: phone, 919-962-0066; fax, 919-966-3893; e-mail, khlee@
unc.edu.
a Abbreviations: ABC, ATP-binding-cassette; ArCHO, substituted
benzaldehyde; BCRP, breast cancer resistant protein; CS, collateral
sensitivity; CSA, cyclosporine A; MDR, multidrug resistance/resistant;
NCI, National Cancer Institute; P-gp, P-glycoprotein; SAR, structure-
activity relationship; TEDB, 6,8,8-triethyldesmosdumotin B; VERAP,
verapamil
r
2010 American Chemical Society
Published on Web 08/25/2010
pubs.acs.org/jmc

6700 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 18
Nakagawa-Goto et al.
The hypersensitivity of drug resistance was first reported
using Escherichia coli in 1952, and the phenomenon was
termed “collateral sensitivity” (CS).28 A significant CS agent
should show at least 2-fold greater activity against the MDR
line than the parental line. Recently, Hall et al. published a
review concerning CS and argued that exploiting CS agents,
whichespecially killMDRcancercells, isanexciting challenge
and opportunity for new drug discovery as well as clinical
development.29 A thiosemicarbazone derivative (NSC73306)
was discovered through the U.S. National Cancer Institute
(NCI) anticancer drug screen as a drug lead for targeting
MDR tumor cell populations, and several derivatives showed
improved MDR selectivity.30,31 While it was toxic toward a
diverse panel of Pgp-expressing tumor cell lines, the CS index
was only 7.3-fold at best. Thus, the MDR selectivity of 2
and its known active analogues is substantially better, but
many questions remain about these unique flavonoids. In
this report, weaddresssomecriticalissuesby lookingateffects
on P-gp function, in vitro anticancer activity using a second
drug-selected (hepatoma) MDR-cell system, and general
mode and mechanism of cytotoxicity. We also explored
structure-activity relationship (SAR) results for this unique
compound class with major focus on the modification of
B-ring substituents.
Chemistry
Generally, analogues 5-55 were synthesized through a
three-step sequence, Claisen-Schmidt condensation of 5626
with a substituted benzaldehyde (ArCHO), cyclization to
form the pyranone A-ring, and C-7 demethylation with
BBr3 as shown in Scheme 1. Because the C-7 methoxy group
was removed more quickly than a phenyl B-ring methoxy
group, treatment with BBr3 was carried out at a low tempera-
ture to retain the latter group. Analogues 10 and 44-46,
which contain hydroxy groups, were obtained from the rela-
ted methoxy (for 10, 44, and 45) or benzyloxy (for 46)
phenylaldehydes. The methyl and benzyl ethers were cleaved
at the final stage to provide the alcohols. 40-Bromomethyl-
phenyl analogue 18 was produced by BBr3 treatment when
40-(methoxymethyl)benzaldehyde, derived from commercially
available [4-(dimethoxymethyl)phenyl]methanol, was used as
the ArCHO. Analogues 2-4 were synthesized previously.
Results and Discussion
All synthesized 2 analogues were evaluated in vitro against
two human tumor cell lines, the KB-VIN cell line, an MDR P-gp
expressing cloned subline stepwise selected using vincristine, and
its parental non-MDR KB cell line. The cytotoxic activity data
including KB/KB-VIN sensitivity are listed in Tables 1 (mono-
substituted phenyl B-ring) and 2 (multisubstituted phenyl
B-ring). Except for 10, 26, and 53, which showed no MDR
selectivity, all 2 analogues demonstrated greater cytotoxicity
against KB-VIN than KB, resulting in CS indices of over 2.0.
Regarding the monosubstituted B-ring analogues (Table1),
40-Me (3) and 40-Et (4) compounds inhibited KB-VIN cell
Figure 1
Scheme 1. Syntheses of TEDB Analoguesa
a Reagents: (a) 50% KOH, EtOH, ArCHO, room temp; (b) I2, DMSO, H2SO4; (c) BBr3.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 18 6701
Table 1. Activity of 3-29 (Monosubstituted Phenyl B-Ring) against
KB and KB-VIN
Table 2. Activity of 30-56 (Multisubstituted Phenyl B-Ring) against
KB and KB-VIN
R
ED50 (μM)a
KB KB-VIN KB/KB-VIN
>236.7 1.07
R
ED50 (μM)a
selectivity
selectivity
KB KB-VIN KB/KB-VIN
compd 20
30
40
50
compd
20
30
40
50
2
>221
460
320
>4
3
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
Me
Me
Me
41.2
35.5
39.0
28.1
8.2
8.52
0.71
2.15
2.95
4.37
2.62
0.10
1.00
0.10
1.37
0.28
7.58
1.57
5.6
5
3
Me
Et
39.2
21.9
0.08
0.07
12.16
1.08
36.95
17.4
10.35
20.6
0.68
7.54
29.17
9.95
1.38
15.70
1.85
27.91
3.40
3.69
5.42
1.22
9.95
3.09
2.41
43.10
3.26
3.93
Me
Cl
F
50
18
10
2
4
Me
Me
5
Pr
>53
8.13
6
iPr
Bu
tBu
Me
Me
Me
Me
7
110.7
29.3
35.7
3.0
2
OMe
OMe
OMe OMe
20.9
28.5
14.7
26.7
24.9
5.43
36.6
9.4
8
8
Me
273
15
250
18
20
5
9
CF3
OH
3.5
1
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
20.6
17.7
F
OMe
OMe
OMe
OMe
OEt
OPr
26
Cl
Me
>52.4
>50.5
36.49
>52.1
>48.1
33.0
>7
>2
4
Me
Me
OMe Me
OCF3
SMe
Br
OMe OMe OMe
6
>38
3
OMe OMe OMe 46.7
Cl
8
OMe OMe 21.8
8.40
5.41
2.75
8.90
0.85
3.41
0.45
4.02
2.51
3.27
31.69
3.24
1.62
3
F
18
OH
Me
Me
Me
OH
OH
OH
10.0
6.5
2
CH2Br
CH2OMe
>46.5
>52.4
49.7
>2
15
Me
2
Me
18.3
3.1
2
Me
14
Me
Me
4
CF3
OMe
OCF3
F
35.7
13.3
6.6
11
Me
Me
14.5
45.5
35.2
4
Me
100
9
>47.4
>56.2
40.9
>5
>18
17
OMe OMe
OMe
OMe
OMe 8.8
OMe 45.2
44.6
4
25
26
27
28
Me
CF3
OMe
F
14
1.4
15
24
45.6
40.8
1
13
OEt OEt
F
Me
Me
47.3
40.5
>56.2
>14
F
a Cytotoxicity as ED50 values for each cell line, the concentration of
compound that caused 50% reduction in absorbance at 562 nm relative
to untreated cells using the sulforhodamine B assay.
a Cytotoxicity as ED50 values for each cell line, the concentration of
compound that caused 50% reduction in absorbance at 562 nm relative
to untreated cells using the sulforhodamine B assay.
growth most strongly with ED50 values of 0.08 and 0.07 μM
and with CS indices of 460 and 320, respectively. Other com-
pounds with significant KB-VIN inhibition (ED50 <2.0 uM)
had the following rank order: 11 (40-OMe, ED50=0.68 μM),
6 (40-iPr, ED50 = 1.08 μM), 22 (30-OMe, ED50 = 1.22 μM),
15 (40-SMe, ED50=1.38 μM), and 17 (40-F, ED50=1.85 μM).
Among of 40-substitued analogues, 2-19, compounds with a
longer side chain (over three carbons), OH, CHO, and CH2Br
at the 40-position or 20-CF3 lost potency against KB-VIN, and
the order of substituents by decreasing selectivity was as follows:
Me, Et>H . SMe, OMe>F>OEt>OPr>CF3, etc.
Compounds 20-28 carry a single substitution on the C20-
or C30-position of the benzene ring. All of them, except 26, exhi-
bited some degree of selectivity; however, they showed lower
activity against KB-VIN compared to the parent unsubsti-
tuted compound.
Regarding multisubstituted B-ring analogues (Table 2), ex-
cept for 54, all analogues showed potent cytotoxicity against
KB-VIN. Among them, 35, 37, and 49 had notable CS indices
of 273, 250, and 100, respectively. Multiple substitutions on
the benzene ring of compounds 29-55 demonstrated that, in
general, increased selectivity and activity toward KB-VIN are
afforded by small hydrophobic groups in C40- and C30-posi-
tions. The decreasing rank order for selectivity was as follows:
3-Me-4-OMe (35), 30-F-40-OMe (37)>30,50-diMe (49)>30,40-
diMe (30)>30-Me-40-F (55)>20,30-diMe-40-OMe (39), > 30-
Cl-40-Me (31)=30-Cl-40-OMe (38)>20-F-50-Me (54)>30,50-
diOMe (52)>30-F-40-Me (32)>20,30-diOMe (50), etc. These
results indicate that substitutions on C30 and C40 significantly
contribute to the KB/KB-VIN sensitivity. Especially, C30-
substitution was the most important for the activity toward
Table 3. Activity of 2-4 against Hep3B and Hep3B-VIN
GI50 (μM)
compd
Hep3B
Hep3B-VIN
Hep3B/Hep3B-VIN
2
3
4
47.57
7.31
6.09
1.57
0.35
0.72
16.17
6.86
30
21
8.5
paclitaxel
vincristine
0.0004
0.0029
KB-VIN (see, for instance, the change in activity for the pairs
of compounds 29 and 30, 34 and 35, 39 and 40, and 48 and 49).
It is unclear to what extent the methoxy and halogen groups
acting as hydrogen bond acceptors contribute to the observed
changes.
The previously synthesized 2-4 were also evaluated in vitro
against a human hepatoma MDR cell system, and the data are
shown in Table 3. Hep3B-VIN cells are P-gp-expressing and
selected using vincristine from the parent non-MDR Hep3B
cellline. The CSindicesmeasuredfor2-4 were 30, 21, and 8.5,
respectively, which are lower than values against KB-VIN cells.
Interestingly, the order of potency was changed to 3 (GI50
0.35 μM)>4 (GI50 0.72 μM) > 2 (GI50 1.57 μM), compared
with that against KB-VIN [4 (ED50 0.068 μM) = 3 (ED50
0.085 μM)>2 (ED50 1.07 μM)] (Tables 1 and 3). However, it
is significant that all three compounds displayed selective in
vitro anticancer action against the hepatoma-derived MDR
cellline becausethisfinding showsthatthe activityis not pecu-
liar to a single MDR cell line.
The effects of 2-4 on P-gp function in KB VIN (MDR) cells
were also investigated as shown in Figure 2. Drug pumping

6702 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 18
Nakagawa-Goto et al.
Figure 2. Effect on P-gp function in KB-VIN cells.
activity and inhibition of P-gp were measured using the
standard calcein-AM loading assay as described under methods.
Figure 2A shows the effects of treatment 2, 3, or 4 compared
with those of first generation competitive inhibitors VERAP
and CSA. The data show that the last two prototype P-gp
inhibitors stimulated calcein-AM influx, while compounds 3
and 4 inhibited the pump but only very weakly over the con-
centration range tested (compared to the action of VERAP
and CSA shown in Figure 2A), and compound 2 was inactive.
These results indicated that 2 had no detectable effect on P-gp
function in KB-VIN cells, while 3 and 4 were weak inhibitors
but only at concentrations much higher than those that
induced CS. The effects of co-treatment with 2, 3, or 4 and a
fixed concentration of an active inhibitor (2.5 μg/mL VERAP
or 4 μg/mL CSA) are shown in Figure 2B-D. Interestingly,
inhibition of P-gp activity was influenced by co-treatment with
each MDR-selective compound and the magnitude and type of
interaction depended on the identity of compound and P-glyco-
protein inhibitor, respectively. All three compounds stimulated
calcein-AM influx upon co-treatment with VERAP but had the
opposite effect when co-treated with CSA. The activation of
VERAP inhibition of Pg-p was clearly concentration-dependent
over the range tested, while the responses were dose-limiting and
more complicated for CSA with compound 3 or 4. Compound 2
possessed the weakest activity, and compounds 3 and 4 were
substantially more potent (compare accumulation of intracellu-
lar fluorescence in Figure 2B-D). A direct correlation between
P-gp functional effects (Figure 2B-D) and in vitro anticancer

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 18 6703
activity against KB-VIN cells (Table 1) was not observed,
although the rank-order of the three compounds’ activity in
both assays is apparent. In conclusion, compounds 2, 3, and 4
are not potent P-gp inhibitors (Figure 2A), although they can
clearly influence interaction of the enzyme with substrates/
inhibitors in MDR cells.
As shown in Figure 3, compound 2 was evaluated against
KB-VIN cells together with nontoxic concentrations of P-gp
modulators VERAP or the allosteric inhibitor flupenthixol.
Co-treatment of 2 with VERAP or flupenthixol (Z-isomer)
partially reversed the in vitro anticancer activity of 2, showing
that the MDR selectivity was dependent in part on P-gp
function and consistent with the effect on P-gp activity mea-
sured using the co-incubation treatment protocol. Similar
results showing partial reversal of 3 and 4 MDR selectivity
were observed (data not shown). Further analysis using suit-
able probe analogues will be needed to determine if the agents
interact directly with P-gp and to map possible interaction
domains.
ELISAPLUS kit (Roche Diagnostics, Mannheim, Germany).
As shown in Figure 4A, 1-day treatment induced a dose-
dependent increase in adsorbance associated with histo-
ne-associated DNA fragments in the cytoplasm. The half-
maximal effect occurred between 1 and 2 μg/mL of 2, consistent
with in vitro anticancer activity (Table 1). Co-treatment with
VERAP reversed the 2-induced DNA fragmentation. At
1 day post-treatment, immunoblot analysis clearly showed
procaspase-3 cleavage, which again was prevented by VERAP
co-treatment (Figure 4B). These data demonstrate that 2 can
induce classical apoptosis in KB-VIN cells, and P-gp activity
is implicated and required because of the antagonistic action
of the pump inhibitor.
Whether these effects are mediated via direct interaction
with the P-gp enzyme (perhaps at an allosteric site resulting in
conformational change) or via indirect action (by affecting
membrane properties and permeation of substrate/inhibitors)
remains to be elucidated. However, the current data are
consistent with 2-4 being novel P-gp actives and suggest that
elucidation of the relationship between the MDR phenotype
and the potent and selective in vitro anticancer action is
warranted and will be worthwhile.
Cell death induced by 2 and effect of co-treatment with
VERAP are shown in Figure 4. The effect of 2 on apoptosis in
KB-VIN cells was measured using a cell death detection
Conclusions
In summary, all analogues demonstrated greater cytotoxi-
city against KB-VIN than KB, resulting in CS indices of over
2.0 except for 10, 26, and 53. Among newly synthesized
analogues, 35, 37, and 49 displayed potent in vitro anticancer
activity against KB-VIN, with CS ratios of 273, 250, and 100,
respectively. These results indicated that substitutions on C30
and C40 significantly contribute to the KB/KB-VIN sensitivity.
Previously synthesized 2-4 also showed MDR selectivity
against the drug-resistant human hepatoma cell line Hep3B-
VIN. While analogues 2-4 did not inhibit or only weakly
inhibited drug efflux P-gp pump activity, they either activated
or inhibited the drug efflux pump activity of prototype P-gp
inhibitors VERAP and CSA, respectively. The flavonoid
compounds clearly influenced interaction of the enzyme with
substrates/inhibitors in MDR cells, although they did not
inhibit P-gp. The MDR selectivity was reversed by co-treat-
ment with diverse P-gp inhibitors, such as VERAP or flu-
penthixol, suggesting dependence on efflux pump activity,
and 2-treatment induced apoptosis in KB-VIN cells.
Figure 3. Chemoprotection of 2 by MDR-1 modulators in KB-
VIN cells. Co-treatment with nontoxic concentrations of VERAP
or flupenthixol partially reversed in vitro anticancer activity of 2
against MDR-1 cell line (measured by use of the anionic protein dye
sulforhodomine B (SRB). These results show that a competitive
substrate-type inhibitor (VERAP) and an allosteric-type pump
inhibitor (Flupent) can negate MDR-selective cytotoxicity.
Figure 4. Effect of 2 on apoptosis in KB-VIN cells was measured using a cell death detection ELISAPLUS kit (Roche Diagnostics, Mannheim,
Germany).

6704 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 18
Nakagawa-Goto et al.
Because of the unique bioactivity of this flavonoid series
against P-gp overexpressing tumor cell lines, we plan to explore
the detailed mechanism of action and preclinical application
studies on their potential as new cancer drugs.
6-CH2CH3), 0.67 (t, 6H, J = 7.4 Hz, 8-CH2CH3). MS (ESIþ)
m/z: 369 (Mþ þ 1). Anal. (C22H24O5) C, H, O.
20,40-Dimethyl-6,8,8-triethyldesmosdumotin B (29). Pale yel-
1
low prisms, mp 129-130 ꢀC (EtOAc-hexane). H NMR (300
MHz, CDCl3): δ 13.14 (s, 1H, 5-OH), 7.35 (d, 1H, J=8.5 Hz, 60-
H), 7.21-7.14 (m, 2H, 30- and 50-H), 6.59 (s, 1H, 3-H), 2.1-2.38
(m, 2H, 6-CH2CH3), 2.43 (s, 3H, 20- or 30-CH3), 2.41 (s, 3H, 20-
or 30-CH3), 2.28-2.12 (m, 2H, 8-CH2CH3), 1.98-1.82 (m, 2H,
8-CH2CH3), 1.04 (t, 3H, J=7.4 Hz, 6-CH2CH3), 0.65 (t, 6H, J=
7.4 Hz, 8-CH2CH3). MS (ESIþ) m/z: 389 (Mþ þ Na). Anal.
(C23H26O4) C, H, O.
Experimental Section
Chemistry. General. All chemicals and solvents were used as
purchased. All melting points were measured on a Fisher-Johns
melting point apparatus without correction. 1H and 13C NMR
spectra were recorded on a Varian Gemini 2000 (300 MHz)
NMR spectrometer with TMS as the internal standard. All
chemical shifts are reported in ppm. NMR spectra were refer-
enced to the residual solvent peak. Chemical shifts δ are in ppm,
and apparent scalar coupling constants J are in Hz. Mass
spectroscopic data were obtained on a TRIO 1000 mass spectro-
meter. Analytical thin-layer chromatography (TLC) was carried
out on Merck precoated aluminum silica gel sheets (Kieselgel
60 F-254). All target compounds were characterized by 1H
NMR, MS, and elemental analyses. The purities of >95% were
determined by 1H NMR and elemental analyses.
General Synthetic Procedures for 1 Analogues. The aryl-
substituted intermediate compound (57) was dissolved in 0.1%
concentrated H2SO4 in DMSO. Then I2 (0.1 equiv mol) was
added and the reaction mixture heated at 90-95 ꢀC for 1 h. The
reaction mixture was quenched with ice-cold aqueous 10%
Na2S2O3 and extracted with EtOAc. The extract was washed
with brine, dried over Na2SO4, and concentrated in vacuo. The
residue was chromatographed on silica gel, eluting with EtOAc-
hexane (1:4 to 1:2, v/v) to afford the 7-methoxy substituted
analogue (58), which was dissolved in anhydrous CH2Cl2. The
mixture was cooled to -78 ꢀC. BBr3 (3 equiv mol, 1.0 M solution
in CH2Cl2) was added to the solution, which was warmed to 0 ꢀC
spontaneously and stirred until the starting material was con-
sumed. After addition of water, the reaction mixture was extrac-
ted three times with CH2Cl2. The extracts were combined, washed
with brine, dried over Na2SO4, and concentrated in vacuo. The
residue was chromatographed on silica gel, eluting with EtOAc-
hexane (1:4) to obtain the target compound (5-55). The physi-
cal data of representative compounds, 5 for 40-substituted
analogue, 20 for 30-substituted analogue, 27 for 20-substituted
analogue, 29 for 20,40-disubstituted analogue, 35 for 30,40-disub-
stituted analogue, and 49 for 30,50-disubstituted analogue, are
described below. Data for other compounds are in the Support-
ing Information.
40-Methoxy-30-methyl-6,8,8-triethyldesmosdumotin
B (35).
Pale yellow prisms, mp 169-170 ꢀC (EtOAc-hexane). 1H
NMR (300 MHz, CDCl3): δ 13.26 (s, 1H, 5-OH), 7.65 (dd,
1H, J=8.2, 2.5 Hz, 60-H), 7.55 (d, 1H, J=2.5 Hz, 20-H), 6.96 (d,
1H, J=8.2 Hz, 50-H), 6.79 (s, 1H, 3-H), 3.93 (s, 3H, 40-OCH3),
2.45 (q, 2H, J = 7.3 Hz, 6-CH2CH3), 2.30 (s, 3H, 30-CH3),
2.33-2.18 (m, 2H, 6- or 8-CH2CH3), 2.06-1.92 (m, 2H, 6- or
8-CH2CH3), 1.04 (t, 3H, J=7.3 Hz, 6-CH2CH3), 0.67 (t, 3H, J=
7.3 Hz, 8-CH2CH3). MS (ESIþ) m/z: 383 (Mþ þ 1). Anal.
(C23H26O5) C, H, O.
30,50-Dimethyl-6,8,8-triethyldesmosdumotin B (49). Pale yel-
1
low prisms, mp 138-139 ꢀC (EtOAc-hexane). H NMR (300
MHz, CDCl3): δ 13.13 (s, 1H, 5-OH), 7.38 (br s, 2H, Ar-H), 7.24
(br s, 1H, Ar-H), 6.87 (s, 1H, 3-H), 3.95 and 3.89 (s, 3H each,
20- and 30-OCH3), 2.45 (q, 2H, J=7.3 Hz, 6-CH2CH3), 2.43 (s,
6H, 30- and 50-CH3), 2.32-2.20 (m, 2H, 8-CH2CH3), 2.06-1.92
(m, 2H, 8-CH2CH3), 1.04 (t, 3H, J=7.4 Hz, 6-CH2CH3), 0.67 (t,
6H, J = 7.3 Hz, 8-CH2CH3). MS (ESIþ) m/z: 399 (Mþ þ Na).
Anal. (C23H26O4) C, H, O.
Cytotoxic Activity Assay. All stock cultures were grown in
T-25 flasks. Freshly trypsinized cell suspensions were seeded in
96-well microtiter plates at densities of 1500-7500 cells per well
with compounds added from DMSO-diluted stock. After 3 days
in culture, attached cells were fixed with cold 50% trichloroa-
cetic acid and then stained with 0.4% sulforhodamine B. The
absorbency at 562 nm was measured using a microplate reader
after solubilizing the bound dye. The mean ED50 is the concen-
tration of agent that reduces cell growth by 50% under the
experimental conditions and is the average from at least three
independent determinations that were reproducible and statis-
tically significant. For the VERAP reversal experiments, cells
were co-treated with VERAP (1 μg/mL). Control experiments
showed this concentration had no effect on the replication of
KB-VIN cells. The following human tumor cell lines were used
in the assay: KB (nasopharyngeal carcinoma) and KB-VIN
(vincristine-resistant KB subline). All cell lines were obtained
from Lineberger Cancer Center (UNC-CH) or from ATCC
(Rockville, MD) except KB-VIN, which was a generous gift of
Professor Y.-C. Cheng, Yale University. Cells were cultured in
RPMI-1640 medium supplemented with 25 mM HEPES, 0.25%
sodium bicarbonate, 10% fetal bovine serum, and 100 μg/mL
kanamycin.
Detection of P-gp Activity (Calcein-AM Loading Assay).
Calcein-AM is a probe substrate of P-gp and is converted into
a fluorescent derivative via cellular enzymes. MDR cells give
low intrinsic fluorescent signals with this probe substrate, unless
P-gp is inhibited, and then relative fluorescence will increase (see
Figure 2A for effects of classical P-gp inhibitors). For the assay,
10 000 KB-VIN (MDR) cells per well were seeded into 96-well
plates with RPMI-1640 medium containing 5% FBS and in-
cubated in a humidified incubator for adhesion and growth.
After 24 h of incubation, the indicated concentrations of test
compounds were added into wells for 30 min at 37 ꢀC. Medium
was aspirated and then replaced with fresh medium containing
1 μM calcein-AM and indicated compounds. After continuing
culture for 30 min at 37 ꢀC in the dark, the medium was removed
and the plates were washed gently and quickly with cold isotonic
(PBS) buffer in the dark. Cells were lysed using hypotonic Tris-
HCl buffer, and fluorescence was detected using an ELISA
40-Propyl-6,8,8-triethyldesmosdumotin B (5). Pale yellow
prisms, mp 169-170 ꢀC (EtOAc-hexane). 1H NMR (300
MHz, CDCl3): δ 13.14 (s, 1H, 5-OH), 7.72 (d, 2H, J = 8.2 Hz,
Ar-H), 7.36 (d, 2H, J=8.2 Hz, Ar-H), 6.87 (s, 1H, 3-H), 2.69 (t,
2H, J = 7.3 Hz, 40-CH2CH2CH3), 2.45 (q, 2H, J = 7.3 Hz,
6-CH2CH3), 2.33-2.19 (m, 2H, 8-CH2CH3), 2.07-1.92 (m, 2H,
8-CH2CH3), 1.76-1.62 (m, 2H, 40-CH2CH2CH3), 1.04 (t, 3H,
J = 7.4 Hz, 6-CH2CH3), 0.97 (t, 3H, J = 7.3 Hz, 40-CH2CH2-
CH3), 0.67 (t, 6H, J=7.3 Hz, 8-CH2CH3). MS (ESIþ) m/z: 381
(Mþ þ 1). Anal. (C24H28O4) C, H, O.
30-Methyl-6,8,8-triethyldesmosdumotin B (20). Pale yellow
prisms, mp 109-110 ꢀC (EtOAc-hexane). 1H NMR (300 MHz,
CDCl3): δ 13.09 (s, 1H, 5-OH), 7.64-7.56 (m, 2H, Ar-H),
7.49-7.39 (m, 1H, Ar-H), 6.89 (s, 1H, 3-H), 2.48 (s, 3H, 30-CH3),
2.46 (q, 2H, J = 7.4 Hz, 6-CH2CH3), 2.34-2.20 (m, 2H,
8-CH2CH3), 2.08-1.93 (m, 2H, 8-CH2CH3), 1.04 (t, 3H, J =
7.4 Hz, 6-CH2CH3), 0.67 (t, 6H, J = 7.4 Hz, 8-CH2CH3). MS
(ESIþ) m/z: 353 (Mþþ1). Anal. (C22H24O4) C, H, O.
20-Methoxy-6,8,8-triethyldesmosdumotin B (27). Pale yellow
prisms, mp 139-140 ꢀC (EtOAc-hexane). 1H NMR (300 MHz,
CDCl3): δ 13.24 (s, 1H, 5-OH), 7.70 (dd, 1H, J = 8.0, 1.6 Hz,
60-H), 7.55 (ddd, 1H, J=8.5, 7.4, 1.6 Hz, 40-H), 7.23 (s, 1H, 3-H),
7.13 (dd, 1H, J=8.0, 7.4 Hz, 50-H), 7.08 (d, 1H, J=8.5 Hz, 30-
H), 2.46 (q, 2H, J = 7.4 Hz, 6-CH2CH3), 2.30-2.16 (m, 2H,
8-CH2CH3),2.04-1.90 (m, 2H, 8-CH2CH3),1.04(t,3H,J=7.4Hz,

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 18 6705
reader (excitation, 494 nm; emission, 517 nm.). Composite data
obtained from two or more independent experiments were
analyzed using Prizm (Graphpad Software, San Diego, CA).
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Acknowledgment. This study was supported by Grant CA-
17625 from the National Cancer Institute, NIH, awarded to
K.-H.L, and bya grantfrom the UniversityResearchCouncil,
awarded to K.N.G.
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~
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Supporting Information Available: Physical data for com-
pounds 6-19, 21-26, 28, 30-34, 36-48, and 50-55; elemental
analysis data for compounds 5-55. This material is available
free of charge via the Internet at http://pubs.acs.org.
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