Antitumor agents 260. New desmosdumotin B analogues with improved in vitro anticancer activity

Article abstract of DOI:10.1021/jm701208v

Sixteen analogues (3-16, 33, and 48) of the unique flavonoid desmosdumotin B (1) were prepared and evaluated as in vitro inhibitors of the human KB cancer cell line and its MDR subclone, KB-VIN. 6,8,8-Triethyl analogues 10-13 showed enhanced KB-VIN select

Full text of DOI:10.1021/jm701208v

J. Med. Chem. 2008, 51, 3297–3303
3297
Antitumor Agents 260. New Desmosdumotin B Analogues with Improved In Vitro Anticancer
Activity
Kyoko Nakagawa-Goto,*,† Kenneth F. Bastow,*,‡ Tzu-Hsuan Chen,† Susan L. Morris-Natschke,† and Kuo-Hsiung Lee*,†
Natural Products Research Laboratories, School of Pharmacy, UniVersity of North Carolina, Chapel Hill, North Carolina 27599, DiVision of
Medicinal Chemistry & Natural Products, School of Pharmacy, UniVersity of North Carolina, Chapel Hill, North Carolina 27599
ReceiVed September 25, 2007
Sixteen analogues (3-16, 33, and 48) of the unique flavonoid desmosdumotin B (1) were prepared and
evaluated as in vitro inhibitors of the human KB cancer cell line and its MDR subclone, KB-VIN. 6,8,8-
Triethyl analogues 10-13 showed enhanced KB-VIN selectivity. In particular, 4′-alkyl derivatives 11 (4′-
Me) and 12 (4′-Et) showed significant ED50 values of 0.03 and 0.025 µg/mL, respectively, against KB-VIN
with selectivities of >460- and 320-fold compared with that of KB. This report is the first to describe
compounds showing such high activity against MDR cells versus non-MDR cells. The unique activity of
1-analogues is likely MDR-mediated because cotreatment with verapamil, a P-gp inhibitor, partially reversed
the selective toxicity of both 1 and 10. Interestingly, only 1-analogues with a naphthalene B-ring (8 and 14)
showed significant cytotoxic activity against KB and other cancer cell lines. Thus, 1-analogues might be a
new class of potent drug candidates, especially as 11 and 12 express direct selective action against tumors
expressing MDR.
Introduction
Chemotherapy is a useful treatment for cancer. However, its
usefulness is often obstructed by the intrinsic or acquired
resistance of cancer cells to anticancer drugs.1,2 Resistance to
one drug often implies simultaneous resistance to structurally
and mechanistically diverse anticancer drugs, called multidrug
resistance (MDR).3–7 This efflux phenotype is mediated in part
Figure 1. Structures of 1 and 2.
by the overexpression of plasma membrane transporters, includ-
effects, poor solubility, and undesirable changes in pharmaco-
ing P-glycoprotein (P-gp, MDR1 or ABCB1)8 or the multidrug
kinetics present with many marketed antitumor drugs.
resistance-associated protein (MRP1 or ABCC1),9–11 which both
Among many MDR modulator candidates, flavonoids are
belong to the superfamily of ATP-binding-cassette (ABC)
compounds capable of modulating P-gp, hMRP1 transport, and
transporters.12 The emergence of MDR pumps anticancer drugs
out of the cell utilizing the energy of ATP hydrolysis and thus
results in reducing intracellular drug concentrations below
cytotoxic levels. As a result, tumor cells overexpressing MDR
show resistance to most of the currently used antitumor drugs.
The development of agents targeted toward MDR1 or MRP1 is
greatly needed in order to improve anticancer chemotherapeutic
strategies.13–17 MDR1/P-gp is the best characterized factor of
the efflux system and most important mediator of MDR.
Therefore, the major pharmacological approaches to overcome
MDR have been focused on exploring the reversal of P-gp
mediated MDR by inhibiting the function and suppressing the
expression of MDR.13–16 Numerous compounds have been
found and examined as MDR modulators that inhibit P-gp
function and are classified into first, second, and third generation
chemosensitizers. Many second and third generation chemosen-
sitizers, some of which are currently in clinical trials, are more
potent and less toxic than first generation compounds like
verapamil or cyclosporin A, yet they still suffer from adverse
ATPase activities.18–20 We recently found that the flavone
desmosdumotin B (DesB, 1)21 and its possible biosynthetic
intermediate chalcone, desmosdumotin C (DesC, 2) (Figure 1),22
showed higher activity against the P-gp-expressing multidrug-
resistant KB-VIN cell line (vincristine-resistant KB) than its
parental nonresistant KB tumor cell (human epidermoid carci-
noma of the nasopharynx). Interestingly, DesB (1) strongly
inhibited the growth of KB-VIN cells with an ED50 value of
2.0 µg/mL,23 while it showed no activity against the other tested
tumor cell lines, including the parental KB cell line (ED50 >40
µg/mL). The amazing selective activity against only MDR cells,
with a >20-fold selectivity for KB-VIN versus KB, implies that
desmosdumotins act via unusual mechanisms that differ from
those of classical MDR modulators, which are inhibitors of the
efflux pump.
We have previously accomplished the total synthesis of both
123 and 2,24 and the published routes have wide-ranging
application for future analogue syntheses. Focused modification
of 2 and the bioactivity of the resulting analogues have also
been reported.25,26 We have continued the syntheses of 1-ana-
logues and evaluation of in vitro anticancer activity, mainly
focused against non-MDR (KB) and MDR (KB-VIN) cell lines.
Herein, we report the syntheses and bioactivity of novel
1-analogues as well as observed structure-activity relationships
(SAR) in detail.
* Corresponding authors. E-mail: goto@email.unc.edu (K.N.G.);
ken_bastow@unc.edu (K.F.B.); khlee@unc.edu (K.H.L.). Phone: 919-843-
6325 (K.N.G.); 919-966-7633 (K.F.B.); 919-962-0066 (K.H.L.). Fax: 919-
966-3893 (K.N.G.); 919-966-0204 (K.F.B.); 919-966-3893 (K.H.L.).
†
Natural Products Research Laboratories, School of Pharmacy, Univer-
sity of North Carolina.
‡
Chemistry. Fourteen 1-analogues, 3-16, were synthesized
from the related 2-analogues through intramolecular cyclization
Division of Medicinal Chemistry & Natural Products, School of
Pharmacy, University of North Carolina.
10.1021/jm701208v CCC: $40.75
2008 American Chemical Society
Published on Web 05/13/2008

3298 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 11
Nakagawa-Goto et al.
Scheme 1. Syntheses of 1-Analogues
a
a Reaction conditions: (i) RI (3 mol equiv), NaOMe (3 mol equiv), MeOH, reflux. (R ) Me for 35a, R ) Et for 35d, R ) Pr for 35e). (ii) (1) BF3 ·OEt2,
HOAc/Ac2O, (2) MeI, NaOMe, MeOH, reflux, (3) 85% H2SO4, heat, (4) EtI (2 mol equiv), NaOMe (1 mol equiv), MeOH, reflux. (iii) TMSCHN2, -78 to
-40°C. (iv) ArCHO, 50% KOH, EtOH, rt, 1-3 days. (v) I2, DMSO, conc H2SO4, see Tables 1 and 2. (vi) BBr3, CH2Cl2, 0 °C to rt. (vii) (1) PhCHO, 50%
KOH, EtOH, microwave, 90 °C, 20 min; (2) I2, DMSO, microwave, 190 °C, 5 min.
Scheme 2. Synthesis of isoflavone 33a
a (i) EtI (3 mol equiv), NaOMe (3 mol equiv), MeOH, reflux, 50%. (ii) BF3 ·OEt2, PCl5, DMF, 68%.
Table 1. Conditions of Cyclization Step (Method A: Direct Conversion)
and demethylation transformations as shown in Scheme 1. Three
2-analogues, 29-31, were newly synthesized from 2,4,6-
1-2% conc
starting
H2SO4 in
product
trihydroxyacetophenone (34) according to the methodology in
our prior report,24 and 13 2-analogues, 17-28, and 32, were
prepared previously.25,26 As shown in Table 1, compounds 17,
18, 20-23, and 26 were converted directly to the related
1-analogues, 3-8 and 10, by heating at 80-95 °C with catalytic
iodine and 1-2% conc H2SO4 in DMSO for several hours in
moderate yields. However, certain compounds (e.g., 19, 21, and
26) gave unsatisfying low yield of products, even employing a
higher temperature and longer reaction time. Bulkiness in the
aryl ring (2′,6′-dimethylphenyl for 19 and 21) or at the C-8
position (gem-diethyl groups for 26) might hinder the reaction.
Alternatively, a two-step conversion involving cyclization
followed by demethylation was carried out to improve the yield
(Table 2). At this point, a shorter reaction time (less than 1.5 h)
was used for the cyclization step to avoid demethylation and
material (mmol)
DMSO (mL)
temp (°C)
time (h)
(% yield)
17 (0.15)
18 (0.16)
20 (0.17)
21 (0.34)
22 (0.12)
23 (0.13)
26 (0.24)
1.5
1.8
2.0
2.3
1.2
1.8
2.3
90
90
80
80-100
95
90
90-100
5
6
5
24
6
6
3 (52)
4 (48)
5 (44)
6 (10)
7 (51)
8 (45)
10 (10)
7
decomposition. For example, compound 26 was heated at 95
°C for 1.5 h in the presence of catalytic iodine and 1-2% conc
H2SO4 in DMSO to obtain methylated 40 in 68% yield along
with 10 in 19% yield. Demethylation of 40 was performed by
using 3 mol equiv of BBr3 in CH2Cl2 to afford 10 in 82% yield
(79% overall yield for two steps). This improved method was

Antitumor Agents 260. New Desmosdumotin B Analogues
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 11 3299
Table 2. Conditions of Cyclization Step (Method B: Two-Step Conversion)
starting
material (mmol)
1-2% conc
H2SO4 in DMSO (mL)
products in
step 1 (% yield)
product in
overall % yield
in two steps
temp (°C)
time (min)
step 2a (% yield)
25 (0.28)
26 (0.22)
27 (0.19)
28 (0.12)
29 (0.31)
30 (0.11)
31 (0.08)
32 (0.34)
1.8
1.6
1.7
1.2
1.7
1.8
1.7
1.6
95
95
95
95
90
90
90
95
60
90
50
60
90
60
60
90
9 (41)
10 (19)
11 (8)
39 (34)
40 (68)
41 (72)
9 (74)
66
79
62
64
66
82
71
76
10 (82)
11 (76)
12 (-)
13 (-)
14 (-)
15 (-)
16 (90)
w/o separation
w/o separation
w/o separation
w/o separation
16 (2)
46 (75)
a BBr3 in CH2Cl2 rt overnight from 39-46.
Table 3. Activities of 1-Analogues against KB and KB-VIN
compound
ED50 (µg/mL)a
R
R′
Ar
KB
KB-VIN
selectivity KB/KB-VIN
1
3
4
5
6
7
8
9
Me
Me
Ph
>40 (33)
22.54
37.28
53.05
NAb
20.0
0.51
>20 (12)
>80
13.8
8.0
18.0
0.6
5.0
4.0
16.0
2.0
20
4-Me-Ph
2-Me-Ph
3,5-diMe-Ph
2,4,6-triMe-Ph
4-Et-Ph
naphthalen-1-yl
Ph
Ph
4-Me-Ph
4-Et-Ph
2-Me-Ph
naphthalen-1-yl
phenanthren-9-yl
Ph
1.09
1.75
2.91
5.82
1.54
0.16
0.6
0.36
0.03
0.025
0.1
0.3
4.3
0.63
3.5
5.0
20.7
21.3
18.2
>3.4
13.0
3.2
>33
222
460
320
60
2.0
1.2
6.4
4.6
>4
1.4
0.002
Et
Et
10
11
12
13
14
15
16
33
48
51
VINc
Et
Pr
Pr
>20 (18)
10.5
0.015
7.5
7.0
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. b Test compound (20 µg/mL) did not reach 50% inhibition. c Vincristine.
applied to the remaining 2-analogues, 25-32, to give the desired
compounds 9-16 in 62-82% overall yields in two steps through
39-46.
yield. Treatment of 50 with N,N-dimethyl(chloromethylene)-
ammonium chloride, generated by DMF and PCl5, in the
presence of BF3 ·OEt2,27 produced the desired isoflavone 33 in
good yield. The last compound (51) investigated in our study
was reported in our prior papers.23
However, as our synthetic focus was on 1-analogues, we also
designed a synthetic pathway that avoided the corresponding
2-analogues as intermediates. Aldol condensation of 35d with
phenylaldehyde in the presence of 50% aq KOH proceeded only
under microwave conditions (90 °C, 20 min). The resulting aldol
adduct was treated without purification with iodine in DMSO,
again using microwave technology, to provide 10 in 35% overall
yield. The overall yields using the two synthetic pathways (with
or without the 2-analogue intermediate) were almost identical.
However, the direct pathway from 35d to 10 had fewer reaction
steps and required less time. Thus, we have developed an
alternative synthetic pathway to 1-analogues using microwave
technology.
Results and Discussion
All synthesized 1-analogues were evaluated in vitro against
two human tumor cell lines, the KB-VIN cell line, which is an
MDR P-gp-expressing cloned subline stepwise selected using
vincristine, and its parental non-MDR KB cell line. Selected
1-analogues were also evaluated in vitro against four additional
human tumor cell lines, A549 (human lung carcinoma), MCF-7
(breast cancer), HCT-8 (colon adenocarcinoma), and PC-3
(prostate cancer).
Three additional compounds (isoflavone 33, flavone 48, and
flavanone 51) were screened for cytotoxic activity in our SAR
study. During ethylation of 34 to give 35d, 3,5-diethylacetophe-
none (47) was produced as a side product. Compound 47 was
converted to the related flavone 48 by the same reaction
sequence indicated above. In addition, triethylation of 2,4,6-
trihydroxy phenylacetophenone (49) was successfully achieved
using the methods described above to afford 50 in moderate
The cytotoxic activity data for 1 and its analogues 3-16,
33, 48, and 51 are listed in Table 3. Because the KB-VIN
selectivity of 1 was higher than that of 2, we hypothesized that
analogues with the flavone skeleton would show enhanced
inhibition of KB-VIN (MDR) cell growth with less activity
against KB (non-MDR) cells than the related chalcone ana-
logues. In fact, most 1-analogues (3-5, 7, and 9-13) did show
remarkably enhanced inhibition of the growth of KB-VIN cells

3300 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 11
Table 4. Activities of 37, 38, 40, and 46 against KB and KB-VIN
Nakagawa-Goto et al.
ethyl (12) at C-4′ greatly potentiated the inhibitory activity
against KB-VIN cells, resulting in remarkably enhanced ED50
values of 0.03 and 0.025 µg/mL, respectively. Because these
two compounds did not significantly inhibit the parent KB cell
growth, the KB-VIN selectivities for 4′-methyl 11 and 4′-ethyl
12 were 460 and 320, respectively.
compound
ED50 (µg/mL)
Comparison of 12 and 48 showed that an aromatized A-ring
in the latter disturbed both activity and KB-VIN selectivity or,
alternatively, that 6,8,8-trialkyl A-ring substitution was required
for selectivity. Moreover, a C-2,3 double bond in the B-ring
was also necessary because the flavanone 51 showed decreased
activity against and no selectivity for KB-VIN compared to the
related flavone 1. Isoflavonoid analogue 33, which has the B-ring
connected to the C-3 position, also showed only 4.6-fold KB-
VIN selectivity. In addition, as shown in Table 4, putting a
methoxy group at the C-7 position (enol ether) generally resulted
in no inhibitory activity against KB-VIN or KB cells and no
KB-VIN selectivity. The one exception was 38, which showed
potent activity against both KB and KB-VIN cell lines (ED50
1.8 µg/mL). This compound is unsubstituted at the C-6 position.
As discussed above, most 1-analogues tested possessed unique
activity against KB-VIN cells with less or comparable activity
against the parent tumor cell line. The following key SAR
observations were found regarding the KB-VIN selectivity: (1)
A flavone skeleton, as opposed to chalcone or flavanone, was
important for the selectivity. (2) A 6,8,8-trialkyl nonaromatic
A-ring resulted in better selectivity than a 6,8-dialkyl aromatic
ring. (3) The rank order of selectivity for 1-analogues decreased
in the following order: 6,8,8-triethyl > 6-ethyl-8,8-dimethyl >
6,8,8-trimethyl > 6,8,8-tripropyl. (4) A 4′-alkyl group enhanced
the selectivity in triethyl desB analogues and a methyl group
was better than an ethyl group.
R
R′
Ar
KB
KB-VIN
selectivity KB/KB-VIN
37
38
40
46
Me
Me
Et
Me
H
Et
Pr
Ph
Ph
Ph
Ph
38.5
1.8
20.0
8.5
23.0
1.8
15.5
7.0
1.7
1.0
1.3
1.2
Pr
Table 5. Data for Selected of 1-Analogues against Various Cancer Cell
Lines
Ed50 (µg/mL)
cmpd
A549
MCF-7
HCT-8
PC-3
NA
0.5
1
8
>20(16)
0.6
NA
16.0
0.6
8.0
>20(23)
0.6
>20(38)
>20(38)
0.2
4.0
10.0
0.010
NA
0.4
10
11
14
15
33
VIN
NA
15.0
0.4
NA
18.0
0.4
5.6
6.0
18.0
0.004
12.0
0.020
>20(32)
0.010
but were not significantly cytotoxic against KB cells. These
1-analogues had KB-VIN selectivities of 6.4-460 (Table 3),
while tested C-7 methylated analogues (37, 38, 40, and 46) were
not selective (Table 4). Important pharmacophores for the
enhanced KB-VIN activity were a nonaromatic trialkyl A-ring,
2,3-double bond, and monophenyl B-ring, as discussed below.
Monoalkylated B-ring analogues 3, 4, and 7, as well as 3,5-
dimethyl analogue 5, showed almost the same activity and
selectivity as the nonsubstituted parent compound (1). However,
the 2,4,6-trimethyl B-ring analogue 6 displayed decreased
activity against KB-VIN cells. This finding could imply that
the loss of rotational flexibility around the B-C ring axis caused
by 2,6-disubstitution might adversely affect the cytotoxic activity
against MDR cells.28 In addition, analogues 8 and 14, in which
the phenyl B-ring was replaced with a naphthyl ring, strongly
inhibited KB-VIN cell growth with ED50 values of 0.16 and
0.3 µg/mL, although they also inhibited the parental KB cells
with ED50 values of 0.5 and 0.6 µg/mL, respectively. This result
is interesting because 1-analogues without a naphthyl ring did
not show strong cytotoxic activity against KB cells.
Meanwhile, selected analogues (parent compound 1, ana-
logues 10 and 11, which showed high selectivity for MDR (KB-
VIN) versus non-MDR (KB) cells, naphthyl B-ring analogues
8 and 14, which displayed potent cytotoxic activity against both
KB and KB-VIN cell lines, and isoflavonoid 33) were tested
against additional cancer cell lines (Table 5). As was expected,
analogues 8 and 14 showed significant cell growth inhibition
against all tested cell lines, A549, MCF-7, HCT-8, and PC-3,
with ED50 values of 0.2-0.6 µg/mL. In contrast, no cytotoxic
activity was observed with 1, 10, and 11 as well as 33. The
lack of cytotoxic activity against these cell lines suggested that
P-gp might be necessary for the activity of these 1-analogues.
To examine whether functional P-gp was required for the
unique activity of 1-analogues against KB-VIN cells, experi-
ments testing the effect of cotreatment with verapamil, a first
generation P-gp inhibitor, were conducted. The results of this
preliminary approach shown in Figure 2 clearly show that
verapamil significantly reduced the selective toxicity of com-
pounds 1 and 10, suggesting that for these two agents, P-gp
activity was indeed required. Advanced mechanistic investiga-
tions are now in progress, but it is worthwhile noting that the
hyperactivity of 1 and 10-12 is not general for MDR-cell lines
but is restricted to only a few (P. C. Chiang, K. H. Lee, and
K. F. Bastow, unpublished observation). One possible explana-
tion is that P-gp microheterogeneity is important in determining
hypersensitivity. More than 50 single-nucleotide polymorphisms
(SNP) are known, some of which are silent but which can still
radically affect P-gp conformation (any can potentially alter drug
interaction).29,30 Efforts to test the importance of P-gp-micro-
heterogeneity for the unprecedented high collateral sensitivity
of KB-VIN and other MDR cell-lines to 11 are underway, and
mechanistic work will be reported in due course.
Among the A-ring analogues, changing the substituents on
the A-ring caused dramatic effects on KB-VIN selectivity.
Replacing the C-6 methyl group (1) with an ethyl group (9)
resulted in increased cytotoxic activity against KB-VIN (ED50
0.6 versus 2.0 µg/mL) and, correspondingly, in enhanced KB-
VIN selectivity (>33 versus 20). Furthermore, 6,8,8-triethyl-
DesB 10, in which all three A-ring methyl groups have been
replaced with ethyl groups, possessed even greater activity
against KB-VIN cells (ED50 0.36 µg/mL) without activity
against the KB cell line, resulting in greater than 222-fold
selectivity for the KB-VIN cell line. However, extending the
substituent size to three carbons (6,8,8-tripropyl 16) led to
enhanced cytotoxic activity against both cell lines, MDR and
non-MDR, with decreased KB-VIN selectivity of only 6.4.
Although, as discussed above, B-ring alkylation of trimethyl
substituted 1 had no or little effect on activity or selectivity,
B-ring alkylation of triethyl-DesB (10) did have significant
effects. Adding a 2′-Me (13) increased potency against both
cell lines. More notably, introduction of either a methyl (11) or

Antitumor Agents 260. New Desmosdumotin B Analogues
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 11 3301
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 analogues 3-8 and
10.
Method B (Two-Step Conversion). Compounds 25-32 were
dissolved separately in 1% conc H2SO4 in DMSO, and then I2 (0.1
mol equiv) was added and the reaction mixture heated at 90-95
°C for 1 h. The reaction mixture was treated in the same manner
as described above to afford 7-methoxy substituted analogues
39-46, along with a small amount of 9-16. Compounds 39-46
were dissolved separately in anhydrous CH2Cl2, and the mixture
cooled to 0 °C. BBr3 (3 mol equiv, 1.0 M solution in CH2Cl2) was
added to the solution, which was warmed to rt spontaneously and
stirred overnight. After addition of water, the reaction mixture was
extracted 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 analogues 9-16.
Figure 2. Reversal effects of 1 and 10 in KB-VIN cells by verapamil
cotreatment.
In summary, 6,8,8-triethyl-4′-methyl analogue 11 possessed
the most significant and unique activity in this study. It inhibited
KB-VIN cell growth with an ED50 value of 0.03 µg/mL, had
no toxicity against the other tested cancer cells, including KB,
and showed 460-fold KB-VIN selectivity. This surprising
sensitivity of a MDR versus non-MDR cell line has not been
reported since the U.S. National Cancer Institute (NCI) 60 cell
line anticancer drug screen was developed in the late 1980s.31
Currently, a thiosemicarbazone derivative, NSC73306,32 is
undergoing advanced preclinical testing by NCI as a drug lead
for targeting MDR tumor cell populations. It was selectively
toxic to P-gp-expressing tumor cells; however, the sensitivity
of KB-VIN (MDR)/KB-3-1 (non-MDR) was only 7.8-fold. The
proposed mechanisms of action for NSC73306 do not involve
direct interaction with P-gp but are P-gp-dependent and P-gp-
specific. In our study, because 1-analogues showed amazingly
sensitive and potent cytotoxic activity against MDR cells with
no inhibitory activity against non-MDR cells, these compounds
seem to require the existence of P-gp for activity and might
not act as normal MDR modulators, which inhibit the drug efflux
pump and thereby reverse MDR via chemosensitization activity.
Thus, although the mechanism of action of 1-analogues is not
yet clear, it might be unique and different from that of
NSC73306. Triethyl-DesB analogues 10-12 could provide a
new class of promising antitumor drug candidates able to
overcome the current serious MDR problem. In addition,
naphthyl analogues 8 and 14 showed significant cytotoxic
activity against both MDR and non-MDR cell lines and would
be good antitumor drug candidates that act regardless of the
presence of P-gp.
4′-Methyl desmosdumotin B (3). Yellow prisms, mp 201-202
1
°C (CH2Cl2-hexane). H NMR (300 MHz, CDCl3) δ: 13.16 (s,
1H, chelated-OH), 7.70 (d, 2H, J ) 8.2 Hz, 2′- and 6′-H), 7.36 (d,
2H, J ) 8.2 Hz, 3′- and 5′-H), 6.86 (s, 1H, 3-H), 2.46 (s, 3H, 4′-
CH3), 1.88 (s, 3H, 6-CH3), 1.58 (s, 6H, 8-CH3 × 2). MS m/z: 311
(M+ + 1). Anal. (C19H18O4 ·1/4H2O) C, H, O.
2′-Methyl desmosdumotin B (4). Yellow prisms, mp 146-147
1
°C (CH2Cl2-hexane). H NMR (300 MHz, CDCl3) δ: 13.10 (s,
1H, chelated-OH), 7.51-7.44 (m, 2H, Ar-H × 2), 7.40-7.31 (m,
2H, Ar-H × 2), 6.61 (s, 1H, 3-H), 2.47 (s, 3H, 4′-CH3), 1.88 (s,
3H, 6-CH3), 1.52 (s, 6H, 8-CH3 × 2). MS m/z: 311 (M+ + 1).
Anal. (C19H18O4 ·1/8H2O) C, H, O.
3′,5′-Dimethyl desmosdumotin B (5). Colorless prisms, mp
1
112-113 °C (CH2Cl2-hexane). H NMR (300 MHz, CDCl3) δ:
13.16 (s, 1H, chelated-OH), 7.38 (s, 2H, 2′- and 6′-H), 7.23 (s,
1H, 4′-H), 6.85 (s, 1H, 3-H), 2.42 (s, 6H, 3′- and 5′-CH3), 1.88 (s,
3H, 6-CH3), 1.59 (s, 6H, 8-CH3 × 2). MS m/z: 325 (M+ + 1).
Anal. (C20H20O4 ·1/8H2O) C, H, O.
2′,4′,6′-Trimethyl desmosdumotin B (6). Colorless prisms, mp
1
196-197 °C (CH2Cl2-hexane). H NMR (300 MHz, CDCl3) δ:
13.08 (s, 1H, chelated-OH), 6.99 (s, 2H, 3′- and 5′-H), 6.44 (s,
1H, 3-H), 2.36 (s, 3H, 4′-CH3), 2.22 (s, 6H, 2′- and 6′-CH3), 1.88
(s, 3H, 6-CH3), 1.47 (s, 6H, 8-CH3 × 2). MS m/z: 339 (M+ + 1).
Anal. (C21H22O4 ·1/4H2O) C, H, O.
4′-Ethyl desmosdumotin B (7). Colorless prisms, mp 203-204
1
°C (CH2Cl2-hexane). H NMR (300 MHz, CDCl3) δ: 13.16 (s,
1H, chelated-OH), 7.72 (d, 2H, J ) 8.2 Hz, 2′- and 6′-H), 7.38 (d,
2H, J ) 8.2 Hz, 3′- and 5′-H), 6.36 (s, 1H, 3-H), 2.75 (q, 2H, J )
7.4 Hz, 4′-CH2CH3), 1.88 (s, 3H, 6-CH3), 1.58 (s, 6H, 8-CH3 ×
2), 1.29 (t, 3H, J ) 7.4 Hz, 4′-CH2CH3). MS m/z: 323 (M+ - 1).
Anal. (C20H20O4 ·1/8H2O) C, H, O.
2-(Naphthalen-1-yl)desmosdumotin B (8). Colorless prisms, mp
1
164-165 °C (CH2Cl2-hexane). H NMR (300 MHz, CDCl3) δ:
13.10 (s, 1H, chelated-OH), 8.87 (d, 1H, J ) 8.0 Hz, Ar-H),
8.02-7.94 (m, 2H, Ar-H), 7.71-7.67 (m, 1H, Ar-H), 7.65-7.58
(m, 3H, Ar-H), 6.82 (s, 1H, 3-H), 1.91 (s, 3H, 6-CH3), 1.54 (s,
6H, 8-CH3 × 2). MS m/z: 347 (M+ + 1). Anal. (C22H18O4 ·1/8H2O)
C, H, O.
Experimental Section
All chemicals and solvents were used as purchased. All melting
points were measured on a Fisher-Johns melting point apparatus
1
without correction. H and 13C NMR spectra were recorded on a
6,8,8-Triethyl-7-methoxy desmosdumotin B (40). Colorless
prisms, mp 131-132 °C (CH2Cl2-hexane). H NMR (300 MHz,
Varian Gemini 2000 (300 MHz) NMR spectrometer with TMS as
the internal standard. All chemical shifts are reported in ppm. NMR
spectra were referenced to the residual solvent peak, chemical shifts
δ in ppm, apparent scalar coupling constants J in Hz. Mass
spectroscopic data were obtained on a TRIO 1000 mass spectrom-
eter. 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.
1
CDCl3) δ: 7.78-7.72 (m, 2H, Ar-H), 7.58-7.50 (m, 3H, Ar-H),
6.84 (s, 1H, 3-H), 4.01 (s, 3H, 7-OCH3), 2.58 (q, 2H, J ) 7.4 Hz,
6-CH2CH3), 2.26-2.01 (m, 4H, 8-CH2CH3 × 2), 1.15 (t, 3H, J )
7.4 Hz, 6-CH2CH3), 0.71 (t, 6H, J ) 7.4 Hz, 8-CH2CH3 × 2). MS
m/z: 353 (M+ + 1). Anal. (C22H24O4 ·1/8H2O) C, H, O.
6,8,8-Tripropyl-7-methoxy desmosdumotin B (46). Colorless
1
prisms, mp 124-125 °C (CH2Cl2-hexane). H NMR (300 MHz,
General Synthetic Procedures for 1-Analogues. Method A
(Direct Conversion). Compounds 17, 18, 20-23, and 26 were
dissolved separately in 1-2% conc H2SO4 in DMSO, and then I2
(0.1 mol equiv) was added and the reaction mixture heated for
several hours (see below). The reaction mixture was quenched with
CDCl3) δ: 7.78-7.72 (m, 2H, Ar-H), 7.59-7.51 (m, 3H, Ar-H),
6.83 (s, 1H, 3-H), 3.99 (s, 3H, 7-OCH3), 2.54-2.45 (m, 2H,
6-CH2CH2CH3), 2.16-1.92 (m, 4H, 8-CH2CH2CH3 × 2), 1.60-1.46
(m, 2H, 6-CH2CH2CH3), 1.21-1.04 (m, 2H, 8- CH2CH2CH3),
1.02-0.86 (m, 2H, 8- CH2CH2CH3), 0.98 (t, 3H, J ) 7.4 Hz,

3302 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 11
Nakagawa-Goto et al.
6-CH2CH2CH3), 0.83 (t, 6H, J ) 7.4 Hz, 8-CH2CH2CH3 × 2). MS
m/z: 395 (M+). Anal. (C25H30O4 ·1/4H2O) C, H, O.
(t, 3H, J ) 7.3 Hz, 6 6-CH2CH2CH3), 0.79 (t, 6H, J ) 7.8 Hz,
8-CH2CH2CH3 × 2). MS m/z: 379 (M+ - 1). Anal. (C24H26O4) C,
H, O.
6-Ethyl-8,8-dimethyl desmosdumotin B (9). Method B. 66%
two-step overall yield. Pale-yellow prisms, mp 212-213 °C
(CH2Cl2-hexane). 1H NMR (300 MHz, CDCl3) δ: 13.03 (1H,
chelated-OH), 7.84-7.78 (m, 2H, 2′- and 6′-H), 7.64-7.53 (m,
3H, 3′-, 4′- and 5′-H), 6.90 (s, 1H, 3-H), 2.44 (q, 2H, J ) 7.3 Hz,
6-CH2CH3), 1.59 (s, 6H, 8-CH3 × 2), 1.04 (t, 3H, J ) 7.3 Hz,
6-CH2CH3). MS m/z: 310 (M+ - 1). Anal. (C19H18O4) C, H, O.
6,8,8-Triethyl desmosdumotin B (10). Method A. 10% yield.
Pale-yellow prisms, mp 204-205 °C (CH2Cl2). 1H NMR (300 MHz,
CDCl3) δ: 13.06 (1H, chelated-OH), 7.84-7.77 (m, 2H, 2′- and
6′-H), 7.66-7.54 (m, 3H, 3′-, 4′- and 5′-H), 6.91 (s, 1H, 3-H), 2.46
(q, 2H, J ) 7.3 Hz, 6-CH2CH3), 2.34-2.20 (m, 2H, 8- CH2CH3),
2.08-1.94 (m, 2H, 8- CH2CH3), 1.05 (t, 3H, J ) 7.3 Hz,
6-CH2CH3), 0.68 (t, 6H, J ) 7.3 Hz, 8-CH2CH3 × 2). MS m/z:
339 (M+ + 1). Anal. (C21H22O4 ·1/4H2O) C, H, O.
6,8,8-Triethyl-5-hydroxy-3-phenyl-4H-chromene-4,7(8H)-di-
one (33). Colorless prisms, mp 146-147 °C (EtOAc-hexane). 1H
NMR (300 MHz, CDCl3) δ: 13.09 (1H, chelated-OH), 8.08 (s, 1H,
2-H), 7.56-7.44 (m, 5H, Ar-H), 2.46 (q, 2H, J ) 7.4 Hz,
6-CH2CH3), 2.25-2.14 (m, 2H, 8-CH2CH3), 2.00-1.89 (m, 2H,
8-CH2CH3), 1.04 (t, 3H, J ) 7.4 Hz, 6-CH2CH3), 0.67 (t, 6H, J )
7.4 Hz, 8-CH2CH3 × 2). Anal. (C21H22O4 ·1/4H2O) C, H, O.
6,8-Diethyl-2-(4-ethylphenyl)-5,7-dihydroxychromen-4-one
(48). Pale-yellow powder, mp 263-264 °C (CH2Cl2-MeOH). 1H
NMR (300 MHz, 10% CD3OD in CDCl3) δ: 7.83 (d, 2H, J ) 8.2
Hz, 2′- and 6′-H), 7.36 (d, 2H, J ) 8.2 Hz, 3′- and 5′-H), 6.66 (s,
1H, 3-H), 2.91 (q, 2H, J ) 7.3 Hz, 4′-CH2CH3), 2.80-2.66 (m,
4H, 6- and 8-CH2CH3), 1.35-1.22 (m, 6H, 6- and 8-CH2CH3), 1.18
(t, 3H, J ) 7.3 Hz, 4′-CH2CH3). MS m/z: 338 (M+). Anal.
(C21H22O4) C, H, O.
6,8,8-Triethyl-4′-methyl desmosdumotin B (11). Method B.
55% two-step overall yield. Pale-yellow prisms, mp 182-183 °C
(CH2Cl2). 1H NMR (300 MHz, CDCl3) δ: 13.16 (1H, chelated-
OH), 7.70 (d, 2H, J ) 8.1 Hz, 2′- and 6′-H), 7.37 (d, 2H, J ) 8.1
Hz, 3′- and 5′-H), 6.88 (s, 1H, 3-H), 2.47 (s, 3H, 4′-CH3), 2.36-2.18
(m, 2H, 6-CH2CH3), 2.12-1.94 (m, 4H, 8-CH2CH3 × 2), 1.04 (t,
3H, J ) 7.4 Hz, 6-CH2CH3), 0.67 (t, 6H, J ) 7.4 Hz, 8-CH2CH3
× 2). MS m/z: 351 (M+ - 1). Anal. (C22H24O4) C, H, O.
6,8,8-Triethyl-4′-ethyl desmosdumotin B (12). Method B. 66%
two-step overall yield. Colorless prisms, mp 159-160 °C
(CH2Cl2-hexane). 1H NMR (300 MHz, CDCl3) δ: 13.13 (1H,
chelated-OH), 7.72 (d, 2H, J ) 8.2 Hz, 2′- and 6′-H), 7.39 (d, 2H,
J ) 8.2 Hz, 3′- and 5′-H), 6.88 (s, 1H, 3-H), 2.75 (q, 4H, J ) 7.4
Hz, 4′-CH2CH3), 2.45 (q, 4H, J ) 7.4 Hz, 6-CH2CH3), 2.36-2.18
(m, 2H, 8-CH2CH3), 2.08-1.94 (m, 2H, 8-CH2CH3), 1.29 (t, 3H,
J ) 7.4 Hz, 4′-CH2CH3), 1.04 (t, 3H, J ) 7.4 Hz, 6-CH2CH3),
0.67 (t, 6H, J ) 7.4 Hz, 8-CH2CH3 × 2). MS m/z: 367 (M+ + 1).
Anal. (C23H26O4) C, H, O.
6,8,8-Triethyl-2′-methyl desmosdumotin B (13). Method B.
64% two-step overall yield. Pale-yellow prisms, mp 146-147 °C
(CH2Cl2-hexane). 1H NMR (300 MHz, CDCl3) δ: 13.08 (1H,
chelated-OH), 7.52-7.42 (m, 2H, Ar-H), 7.40-7.32 (m, Ar-H),
6.62 (s, 1H, 3-H), 2.46 (s, 3H, 2′-CH3), 2.52-2.42 (m, 2H,
6-CH2CH3), 2.26-2.13 (m, 2H, 8-CH2CH3), 1.98-1.84 (m, 2H,
8-CH2CH3), 1.04 (t, 3H, J ) 7.3 Hz, 6-CH2CH3), 0.67 (t, 6H, J )
7.4 Hz, 8-CH2CH3 × 2). MS m/z: 351 (M+ - 1). Anal. (C22H24O4 ·1/
8H2O) 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% trichloroacetic 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 concentration 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 statistically significant. For the verapamil
reversal experiments, cells were cotreated with verapamil (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: A549 (human lung carcinoma),
MCF-7 (breast cancer), HCT-8 (colon adenocarcinoma), PC-3
(prostate cancer), 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.
Acknowledgment. This study was supported by grant CA-
17625 from the National Cancer Institute, NIH, awarded to
K.H.L.
2-(Naphthalen-1′-yl)-6,8,8-triethyl desmosdumotin B (14).
Colorless prisms, mp 185-186 °C (EtOAc-hexane). 1H NMR (300
MHz, CDCl3) δ: 13.07 (1H, chelated-OH), 8.09 (d, 1H, J ) 7.9
Hz, Ar-H), 8.02-7.91 (m, 2H, Ar-H), 7.69 (dd, 1H, J ) 7.2 and
1.3 Hz, Ar-H), 7.66-7.58 (m, 3H, Ar-H), 6.83 (s, 1H, 3-H), 2.49
(q, 2H, J ) 7.4 Hz, 6-CH2CH3), 2.26-2.12 (m, 2H, 8-CH2CH3),
1.98-1.84 (m, 2H, 8-CH2CH3), 1.07 (t, 3H, J ) 7.4 Hz,
6-CH2CH3), 0.73 (t, 6H, J ) 7.4 Hz, 8-CH2CH3 × 2). Anal.
(C25H24O4) C, H, O.
Supporting Information Available: Elemental analysis data for
compounds 3-16, 33, 40, 46, and 48. This material is available
free of charge via the Internet at http://pubs.acs.org.
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