Antitumor agents 259. Design, syntheses, and structure-activity relationship study of desmosdumotin C analogs

Article abstract of DOI:10.1021/jm0702534

Desmosdumotin C (1) and its analogs previously showed potent, selective in vitro anticancer activity. To explore structure-activity relationships of 1 and further increase potency and selectivity, 15 novel analogs (7-15 and 21-26) were synthesized and eva

Full text of DOI:10.1021/jm0702534

3354
J. Med. Chem. 2007, 50, 3354-3358
Antitumor Agents 259. Design, Syntheses, and Structure-Activity Relationship Study of
Desmosdumotin C Analogs
Kyoko Nakagawa-Goto,† Tzu-Hsuan Chen,† Chieh-Yu Peng,† Kenneth F. Bastow,‡ Jiu-Hong Wu,§ and Kuo-Hsiung Lee*,†
Natural Products Research Laboratories, School of Pharmacy, UniVersity of North Carolina, Chapel Hill, North Carolina 27599, DiVision of
Medicinal Chemistry and Natural Products, School of Pharmacy, UniVersity of North Carolina, Chapel Hill, North Carolina 27599, 306
Hospital of PLA, Department of Pharmacy, Beijing 100101, China
ReceiVed March 6, 2007
Desmosdumotin C (1) and its analogs previously showed potent, selective in vitro anticancer activity. To
explore structure-activity relationships of 1 and further increase potency and selectivity, 15 novel analogs
(7-15 and 21-26) were synthesized and evaluated for cytotoxity against several human tumor cell lines,
as well as inhibition of human endothelial (HUVEC) replication. 4-Bromo-3′,3′,5′-tripropyl analog 26 showed
significant cytotoxity against A549, A431, 1A9, and HCT-8 with ED50 values of 1.0, 1.2, 0.9, and 1.3
µg/mL, respectively. Compound 26 also strongly inhibited the growth of matched tumor cells, KB-VIN and
its parent cell KB. Furthermore, analogs 13 and 21 were over 5-fold more potent against KB-VIN than KB.
Bromination of ring-B and tripropyl functionalization of ring-A enhanced activity, while alkylation of ring-B
promoted KB-VIN/KB selectivity. 2-Furyl analog 16 showed selective activity against HUVEC, suggesting
that it may have potential as a new prototype for angiogenesis inhibition.
Introduction
Natural products are a significant source of drug candidates.
An impressive number of modern drugs have been developed
from natural sources, especially from plants used as traditional
folk medicines.1 Thus, drug discovery from medicinal plants
plays an important role in the treatment and prevention of
various human diseases, and the continuous importance of
natural products in modern medicine has been highlighted in
several recent reviews and reports.2-6 Our research interest is
the discovery and development of novel anticancer drugs from
natural plants. Currently, about three-quarters of anticancer drugs
come from either natural products or their derivatives.7 Cancer
is a leading cause of death worldwide accounting for 13 percent
of all deaths in 2005,8 even though many effective and diverse
cancer treatments have been approved and are used. Major
problems associated with cancer chemotherapy include undesir-
able toxic side effects and multidrug resistance. Therefore, a
pressing need to develop more effective antitumor drugs still
remains.
Desmos dumosus (Roxb.) is a climber plant found in alluvial
forests in southern Asia and has been used in Chinese folk
medicine as an antimalarial, insecticidal, antirheumatic, antis-
pasmodic, and analgesic agent.9 Recently, some novel bioactive
flavonoids named desmosdumotins B and C were isolated from
the plant root.10,11 Desmosdumotin C (1; Figure 1) has a
distinctive chalcone skeleton with an unusual nonaromatic A
ring possessing a gem-dimethyl group on C-3′ and a methyl
group on C-5′. It showed significant and selective in vitro
cytotoxicity against 1A9 (ovarian cancer) and A549 (human lung
carcinoma) cell lines, with IC50 values of 4.0 and 3.5 µg/mL,
respectively.10 In addition, it was more active against drug-
resistant KB-VINa cells than against the parent KB (epidermoid
nasopharyngeal carcinoma) cell line. Thus, 1 is a promising new
Figure 1. Structure of 1.
lead for further new antitumor analog development. We previ-
ously achieved a simple first total synthesis of 111 as well as
some modifications of the A- and B-rings and reported the
cytotoxic activity data against four tumor cell lines, 1A9, A549,
KB, and KB-VIN.12 Among the tested compounds, 4-bromodes-
mosdumotin C (2) showed 2- to 3-fold enhanced activity
compared with 1. This promising result encouraged us to
continue the modification of this series to develop novel
anticancer drug candidates. In this paper, we describe further
modifications of the A- and B-rings as well as evaluation of
newly and previously synthesized analogs against seven human
tumor cell lines, A549, 1A9, KB, KB-VIN, A431 (epidermoid
skin carcinoma), HCT-8 (colon adenocarcinoma), and PC-3
(prostate cancer), as well as HUVEC.
Chemistry
The simplicity of our accomplished synthesis11 of 1 allows
easy modification of the A-ring, by using another alkyl iodide
rather than methyl iodide in the first step, and the B-ring, by
using a different aromatic aldehyde from benzaldehyde in the
final step (Scheme 1). First, nine B-ring modified analogs, 7-15,
were synthesized from intermediate 29 using 4-fluorophenyla-
ldehyde, o- and p-tolualdehyde, 4-ethylbenzaldehyde, 2,6- and
3,5-dimethylbenzaldehyde, 2,4,6-trimethylbenzaldehyde, 2-vi-
nylbenzaldehyde, and 1-naphthaldehyde under basic aldol
conditions. Second, modification of the A-ring to introduce an
ethyl or propyl group at the C-3 and C′-5′ positions was
accomplished by the following method. Treatment of trihy-
droxyacetophenone 28 with 3 mol equiv of ethyl or propyl
iodide in the presence of sodium methoxide provided the
corresponding trialkylacetophenones (32 and 33, respectively,
Scheme 2). Similarly, treatment of 3012 with 1 mol equiv of
ethyl iodide afforded 31. Selective methylation of the resulting
* To whom correspondence should be addressed. Phone: (919) 962-
0066. Fax: (919) 966-3893. E-mail: khlee@unc.edu.
† Natural Products Research Laboratories, University of North Carolina.
‡ Division of Medicinal Chemistry and Natural Products, University of
North Carolina.
§ 306 Hospital of PLA.
a Abbreviations: HUVEC, human umbilical vein endothelial cell; KB-
VIN, vincristine-resistant KB, expressing P-glycoprotein, cell subline.
10.1021/jm0702534 CCC: $37.00 © 2007 American Chemical Society
Published on Web 06/15/2007

Study of Desmosdumotin C Analogs
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 14 3355
Table 1. Cytotoxic Activity Data for 1-Analogs
ED50a (µg/mL)
Scheme 1. Syntheses of 1-Analogs: B-Ring Modifications
cmpd A549 A431 1A9 HCT-8 PC-3 KB KB-VIN HUVEC
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
3.5
1.4
3.0
2.8
2.4
3.1
3.3
4.3
4.6
6.1
8.3
4.3
8.0
2.2
3.6
10.0
4.5
3.5
>5
5.1 3.5
9.0 1.1
9.2 2.8
4.5 2.9
6.6 2.5
14.9 2.9
4.1 NTc
5.1 NT
5.2 NT
12.0 5.3
10.1 NT
4.0 NT
20.0 6.2
2.6 NT
4.1 NT
14.7 7.9
10.8 4.0
6.0 3.5
10.5 4.6
7.0 3.6
13.8 3.8
5.4 2.1
10.3 4.3
9.8 3.8
1.9 1.5
1.2 0.9
14.0 8.0
3.7
7.4
5.6
2.9
4.1
14.8
3.6
4.1
4.0
10.5
7.8
2.9
8.4
2.1
2.3
17.5
8.6
5.1
5.0
3.3
9.4
2.8
9.4
8.5
1.4
1.3
15.8
11.1
17.7
18.2
14.3
15.6
NAb
7.2
8.2
9.0
15.1
17.1
8.8
4.0
1.7
3.6
3.3
3.3
3.8
3.7
3.9
4.1
6.9
7.1
3.1
3.0
1.9
2.9
2.4
2.8
3.8
2.7
1.9
1.6
1.9
2.1
1.6
1.84
1.1
1.3
8.8
3.4
3.0
3.8
2.9
0.3
1.2
1.0
2.6
0.8
0.9
6.5
2.1
3.6
2.5
1.6
1.7
4.0
2.0
2.4
2.3
NT
3.0
2.5
NT
1.1
1.8
2.4
2.5
1.6
2.0
NA
NT
NT
NT
NT
NT
NT
2.1
NA NA
4.6
9.0
1.9
2.8
9.9
5.5
4.3
7.3
4.1
4.3
2.8
3.0
3.6
0.6
0.9
20.6
18.2
10.8
11.1
14.2
18.9
7.7
15.0
15.0
3.3
20 >5 (48)
21
22
23
24
25
26
27
5.5
3.4
4.6
3.8
1.4
1.0
9.0
2.3
17.8 10.5
a See Experimental “Cytotoxic Activity Assay” for cell line descriptions.
b Not active: >20 µg/mL. c Not tested.
a See ref 11. b Based on recovered starting material. c See ref 12.
tripropyl 25, which also were quite potent against A549, 1A9,
KB, and KB-VIN cell lines. Notably, 25 strongly inhibited the
growth of KB and KB-VIN cells with ED50 values of 0.6 and
0.8 µg/mL, respectively.
Interestingly, while most analogs, including 2, were generally
less active against PC-3 replication, three compounds, 14, 25,
and 26, displayed enhanced activity against PC-3 with ED50
values of 4.6, 3.3, and 2.3 µg/mL, respectively. These three
analogs also inhibited the growth of A431 cells with ED50 values
of 2.6, 1.9, and 1.2 µg/mL, respectively. Furthermore, in the
PC-3, A431, and HCT-8 cell lines, bromination at C-4 (2)
decreased cytotoxic activity relative to 1, while fluorination (7)
increased activity.
trialkylacetophenones, 31-33, with an excess of TMSCHN2 at
low temperature, followed by aldol reaction with benzaldehyde,
gave 21, 22, and 25. The aldol reaction of 35 and 36 with
4-methyl, 4-ethyl, or 4-bromobenzaldehyde gave A- and B-ring
analogs, 23, 24, and 26, respectively. All synthesized com-
pounds, except for 7, 12, and 15, exist as a mixture of two
tautomeric isomers as discussed in our prior papers.12,13
Compounds 1-6, 16-19, 20, and 27 were synthesized previ-
ously.12
Results and Discussion
Together with 1 and previously synthesized analogs, all newly
synthesized compounds were evaluated for in vitro anticancer
activity against several human tumor cell lines, A549, A431,
1A9, HCT-8, PC-3, KB, and KB-VIN, and against HUVEC, a
normal cell useful for assessing anti-angiogenic potential. The
average ED50 values (µg/mL) are listed in Table 1.
Compound 1 showed activity against A549, 1A9, HCT-8,
and HUVEC with ED50 values of 3.5, 3.5, 3.7, and 2.1 µg/mL,
respectively, but was less active against A431 and PC-3 cells.
Compound 1 was also 1.3-fold more active against KB-VIN
cells (ED50 3.0 µg/mL) than the parent KB cell line. Except for
14, 25, and 26, most 1-analogs showed similar activity patterns,
as described later.
From a comparison of the data for 1 with that of 3-5 and
8-9, the insertion of a methoxy or methyl group on the phenyl
B-ring had either no effect on or slightly decreased activity,
regardless of the substitution position. The 4-ethyl (10), 2,6-
dimethyl (11), and trimethyl (13) derivatives also showed
decreased activity against all cell lines, although 1 and the 3,5-
dimethyl derivative (12) had comparable potencies. However,
it is noteworthy that the introduction of an alkyl group on the
B-ring enhanced the growth inhibition of the KB-VIN cell line
resulting in a 2- to 5-fold ratio of KB/KB-VIN selectivity (see
8-14). However, the insertion of a hetero aromatic ring rather
than the phenyl ring did not affect the cytotoxic activity (see
16-18).
Significantly enhanced cytotoxic activities were observed with
2, 2-vinyl desmosdumotin C (14), 3′,3′,5′-tripropyl desmosdu-
motin C (25), and 4-bromo-3′,3′,5′-tripropyl desmosdumotin C
(26). Especially, 26 displayed enhanced in vitro antitumor
activity against all cell lines with ED50 values of 0.9-2.3 µg/
mL. In addition, 26 was not affected by multidrug resistant
expressing P-glycoprotein, because it showed similar and potent
cell growth inhibition of KB and KB-VIN cell replication (ED50
0.9 µg/mL). The strong activity of 26 could be predicted because
it combines the structural features of 4-bromo 2 and 3′,3′,5′-
For the A-ring analogs, tetramethyl analog 19, dimethyl
analog 20 (ceroptene),14 3′,3′-dimethyl-5′-ethyl analog 21, and
4′-demethyl analog 27 were less potent against all cell lines
than the parent compound 1. However, 21 showed a remarkably
high KB/KB-VIN selectivity ratio of 14.3. Among the triethyl
compounds, 23 and 24 were less active than 1, while 22, without
a substituent on the B-ring, showed slightly enhanced activity
against most cell lines, except for A549 and A431.
Angiogenesis is necessary for tumor growth and metastasis;
therefore, it presents another target for cancer treatment. Selected

3356 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 14
Scheme 2. Syntheses of 1-Analogs: A/B-ring Modifications
Nakagawa-Goto et al.
The extract was washed with brine, dried over Na2SO4, and
concentrated in vacuo. The residue was chromatographed on silica
gel with CH2Cl2-hexane as eluent to afford the target compound,
which was crystallized from CH2Cl2-hexane.
compounds were also evaluated in a standard anti-angiogenesis
assay using HUVEC. All tested compounds, except 20, showed
potent activity with ED50 values less than 4.0 µg/mL. Compound
14 showed the highest potency with an ED50 value of 1.1 µg/
mL. However, this compound also showed strong activity
against other tumor cell lines, suggesting that the activity against
HUVEC may be nonspecific. However, analogs 16-18, in
which the phenyl B-ring was replaced with five-membered
heteroaromatic rings, demonstrated significant activity against
HUVEC with less activity against tumor cell replication. In
particular, 2-furyl analog 16 possessed the highest differential,
with an ED50 value of 2.4 µg/mL against HUVEC compared to
ED50 values of 7.9-20.6 µg/mL against the other tumor cell
lines. This selective activity suggests that 16 may have potential
as a new prototype as an angiogenesis inhibitor.
In summary, the preliminary SAR studies led to the following
observations: (1) a bromide function on the B-ring enhanced
activity (1 vs 2 and 25 vs 26); (2) the order of potency for C-3′
and -5′ functionalities was 3′,3′,5′-tripropyl > 3′,3′,5′-trimethyl
> 3′,3′,5′-triethyl > 3′,3′-dimethyl-5′-ethyl > 3′,3′-dimethyl >
3′,3′,5′,5′-tetramethyl; (3) an alkyl group on the B-ring promoted
KB-VIN/KB selectivity; (4) a C-4′ methoxy group is required
for activity; (5) five-membered hetero B-rings increased the
differential angiogenesis activity. From all results, 26 showed
significant promise as a new antitumor drug candidate because
of its potent cytotoxity against tumor cell lines and lack of
multidrug cross resistance. Focused studies on the KB-VIN/
KB selectivity of desmosdumotin series are currently ongoing,
and the results will be reported elsewhere in the near future.
4-Fluoro Desmosdumotin C (7). Yellow prisms, mp 109-
110 °C (CH2Cl2-hexane). 1H NMR (CDCl3): δ 19.18 (s, 1H,
chelated-OH), 8.27 (d, 1H, J ) 15.6 Hz, trans-olefinic proton),
7.89 (d, 1H, J ) 15.6 Hz, trans-olefinic proton), 7.71-7.60 (m,
2H, Ar-2,6-H), 7.12-7.00 (m, 2H, Ar-3,5-H), 3.96 (s, 3H, OCH3),
2.00 (s, 3H, 5′-CH3), 1.38 (s, 6H, 3′-CH3 × 2). MS m/z 331 (M+
+ 1). Anal. (C19H19FO4) C, H, O.
4-Methyl Desmosdumotin C (8). Yellow prisms, mp 110-
1
111 °C (CH2Cl2-hexane). H NMR (300 MHz, CDCl3): δ 19.32
and 18.92 (3:1, each s, 1H, chelated-OH), 8.52 and 8.31 (1:3, each
d, 1H, J ) 15.7 Hz, olefin), 7.95 and 7.93 (1:3, each d, 1H, J )
15.7 Hz, olefin), 7.62-7.54 (m, 2H, Ar-2′, 6′-H), 7.24-7.17 (m,
2H, Ar-3, 5-H), 3.96 and 3.89 (3:1, each s, 3H, OCH3), 2.00 and
1.96 (3:1, each s, 3H, 5′-CH3), 1.47 and 1.38 (1:3, each s, 6H,
3′-CH3 × 2). MS m/z 327 (M+ + 1). Anal. (C20H22O4) C, H, O.
2-Methyl Desmosdumotin C (9). Yellow prisms, mp 93-
1
94 °C (CH2Cl2-hexane). H NMR (300 MHz, CDCl3): δ 19.32
and 18.62 (4:1, each s, 1H, chelated-OH), 8.34 (s, 2H), 8.05-7.94
(m, 1H), 7.51-7.25 (m, 3H, Ar-H), 4.10 and 4.03 (4:1, each s,
3H, OCH3), 2.63 (s, 3H, Ar-CH3), 2.15 and 2.01 (4:1, each s, 3H,
5′-CH3), 1.52 (s, 6H, 3′-CH3 × 2). MS m/z 327 (M+ + 1). Anal.
(C20H22O4) C, H, O.
4-Ethyl Desmosdumotin C (10). Yellow prisms, mp 90-91 °C
(CH2Cl2-hexane). 1H NMR (300 MHz, CDCl3): δ 19.19 and 18.80
(2:1, each s, 1H, chelated-OH), 8.52 and 8.32 (1:2, each d, 1H, J
) 15.9 Hz, olefin), 7.96 and 7.94 (1:2, each d, 1H, J ) 15.9 Hz,
olefin), 7.62 and 7.60 (1:2, each d, 2H, J ) 7.9 Hz, Ar-2, 6-H),
7.23 and 7.22 (1:2, each d, 2H, J ) 7.9 Hz, Ar-3, 5-H), 3.95 and
3.89 (2:1, each s, 3H, OCH3), 2.68 (q, 2H, J ) 7.7 Hz, Ar-CH2-
CH3), 2.00 and 1.96 (2:1, each s, 3H, 5′-CH3), 1.47 and 1.38 (1:2,
each s, 6H, 3′-CH3 × 2), 1.25 (t, 3H, J ) 7.7 Hz, Ar-CH2CH3).
MS m/z 339 (M+ - 1). Anal. (C21H24O) C, H, O.
Experimental Section
Materials and Methods. Melting points were measured with a
Fisher Johns melting apparatus without correction. Proton nuclear
magnetic resonance (1H NMR) spectra were measured on a 300
MHz Varian Gemini 2000 spectrometer using TMS as internal
standard. All chemical shifts are reported in ppm. Mass spectra
were measured on a PE-SCIEX API 3000 instrument with turbo
ion spray source or Agilent-1100, LC/MSD-Trap. Thin-layer
chromatography (TLC) and preparative TLC were performed on
precoated silica gel GF plates purchased from Merck, Inc. Biotage
Flash or Isco Companion systems were used for medium-pressure
column chromatography. Silica gel (200-400 mesh) from Aldrich,
Inc., was used for column chromatography. All other chemicals
were obtained from Aldrich, Inc.
1
2,6-Dimethyl Desmosdumotin C (11). Yellow oil. H NMR
(300 MHz, CDCl3): δ 19.14 and 18.80 (2:1, each s, 1H, chelated-
OH), 8.17 (d, 1H, J ) 15.7 Hz, olefin), 7.87 (d, 1H, J ) 15.7 Hz,
olefin), 7.20-7.01 (m, 3H, Ar-3,4,5-H), 3.95 and 3.88 (2:1, each
s, 3H, OCH3), 2.44 (s, 6H, Ar-2,6-CH3), 2.00 and 1.92 (2:1, each
s, 3H, 5′-CH3), 1.48 and 1.35 (1:2, each s, 6H, 3′-CH3 × 2). MS
m/z 341 (M+ + 1). Anal. (C21H24O4) C, H, O.
3,5-Dimethyl Desmosdumotin C (12). Yellow prisms, mp 105-
1
106 °C (CH2Cl2-hexane). H NMR (300 MHz, CDCl3): δ 19.20
(s, 1H, chelated-OH), 8.31 (d, 1H, J ) 15.7 Hz, olefin), 7.91 (d,
1H, J ) 15.7 Hz, olefin), 7.30 (s, 2H, Ar-2′, 6′-H), 7.03 (s, 1H,
Ar-4-H), 3.96 (s, 3H, OCH3), 2.34 (s, 6H, Ar-3,5-CH3), 2.00 (s,
3H, 5′-CH3), 1.39 (s, 6H, 3′-CH3 × 2). MS m/z 341 (M+ + 1).
Anal. (C21H24O4) C, H, O.
General Procedures for the Aldol Reactions. A solution of
acetyl compound (29 or 34-36) in EtOH-50% aq KOH (1:1, v/v)
and an appropriate aldehyde (excess) was stirred at room temper-
ature. After the reaction was complete by TLC analysis, the mixture
was poured into ice-cold 1 N HCl and then extracted with CH2Cl2.
2,4,6-Trimethyl Desmosdumotin C (13). Yellow prisms, mp
87-88 °C (AcOEt-hexane). 1H NMR (300 MHz, CDCl3): δ 19.17

Study of Desmosdumotin C Analogs
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 14 3357
and 18.48 (3:1, each s, 1H, chelated-OH), 8.21-8.09 (m, 1H,
olefin), 7.96 and 7.90 (1:3, each d, 1H, J ) 15.5 Hz, olefin), 6.92
and 6.90 (1:3, each s, 2H, Ar-3, 5-H), 3.95 and 3.88 (3:1, each s,
3H, OCH3), 2.43 and 2.41 (3:1, each s, 6H, Ar-2,6-CH3), 2.30 and
2.29 (1:3, each s, 3H, Ar-4-CH3), 2.00 and 1.92 (3:1. each s, 3H,
5′-CH3), 1.47 and 1.35 (1:3, each s, 6H, 3′-CH3 × 2). MS m/z 355
(M+ + 1). Anal. (C22H26O4‚1/8H2O) C, H.
3′,3′,5′-Tripropyl Desmosdumotin C (25). Yield 76%; yellow
oil. 1H NMR (300 MHz, CDCl3): δ 19.29 and 18.90 (2:1, each s,
1H, chelated-OH), 8.49 and 8.42 (1:2, each d, 1H, J ) 15.6 Hz,
olefin), 7.95 and 7.92 (1:2, each d, 1H, J ) 15.6 Hz, olefin), 7.74-
7.65 (m, 2H, Ar-2,6-H), 7.44-7.34 (m, 3H, Ar-3,4,5-H), 4.00 and
3.92 (2:1, each s, 3H, OCH3), 2.56-2.42 (m, 2H, 5′-CH2Et), 2.00-
1.65 (m, 4H, 3′-CH2Et × 2), 1.65-1.48 (m, 2H, 3′-CH2CH2CH3),
1.16-0.96 (m, 7H, 5′-CH2CH2CH3 × 2 and 3′-CH2CH2CH3), 0.90-
0.77 (m, 6H, 3′-CH2CH2CH3 × 2). MS m/z 395 (M+ - 1). Anal.
(C25H32O4) C, H, O.
3-Vinyl Desmosdumotin C (14). Yellow prisms, mp 76-77 °C
(CH2Cl2-hexane). 1H NMR (300 MHz, CDCl3): δ 19.19 and 18.79
(3:1, each s, 1H, chelated-OH), 8.54 and 8.34 (1:3, each d, 1H, J
) 15.9 Hz, olefin), 7.96 and 7.93 (1:3, each d, 1H, J ) 15.9 Hz,
olefin), 7.67 and 7.65 (1:3, each br s, 1H, Ar-2-H), 7.60 and 7.58
(1:3, each d, 1H, J ) 7.4 Hz, Ar-4 or 6-H), 7.47 and 7.45 (1:3,
each d, 1H, J ) 7.4 Hz, Ar-4 or 6-H), 7.37 and 7.36 (1:3, each t,
1H, Ar-5-H), 6.74 (dd, 1H, J ) 10.8 and 17.4 Hz, Ar-CHdCH2),
5.81 (d, 1H, J ) 17.4 Hz, Ar-CHdCH2), 5.31 (d, 1H, J ) 10.8
Hz, Ar-CHdCH2), 3.96 and 3.89 (3:1, each s, 3H, OCH3), 2.00
and 1.96 (3:1, each s, 3H, 5′-CH3), 1.48 and 1.39 (1:3, each s, 6H,
3′-CH3 × 2). MS m/z 359 (M+ + 1). Anal. (C21H22O4) C, H, O.
2-[1-Hydroxy-3-(naphthalen-1-yl)allyidene]-5-methoxy-4,6,6-
trimethylcyclohex-4-ene-1,3-dione (15). Yellow prisms, mp 128-
4-Bromo-3′,3′,5′-tripropyl Desmosdumotin C (26). Yield 38%;
1
yellow prisms, mp 114-115 °C. H NMR (300 MHz, CDCl3): δ
19.28 and 18.81 (2:1, each s, 1H, chelated-OH), 8.48 and 8.40 (1:
2, each d, 1H, J ) 15.6 Hz, olefin), 7.86 and 7.84 (1:2, each d,
1H, J ) 15.6 Hz, olefin), 7.60-7.47 (m, 5H, Ar-H), 4.00 and 3.92
(2:1, each s, 3H, OCH3), 2.58-2.41 (m, 2H, 5′-CH2Et), 2.00-1.80
(m, 4H, 3′-CH2Et × 2), 1.80-1.46 (m, 2H, 3′-CH2CH2CH3), 1.18-
0.93 (m, 7H, 5′-CH2CH2CH3 × 2 and 3′-CH2CH2CH3), 0.93-0.77
(m, 6H, 3′-CH2CH2CH3 × 2). MS m/z 475 and 477 (M+ - 1).
Anal. (C25H31O4Br) C, H, O.
Synthesis of Intermediates 31-36. 2-Acetyl-6-ethyl-3,5-dihy-
droxy-4,4-dimethylcyclohexa-2,5-dienone (31). A solution of
2-acetyl-3,5-dihydroxy-4,4-dimethylcyclohexa-2,5-dienone (30, 713
mg, 3.6 mmol) and sodium methoxide (2.0 mL, 9.3 mmol, 25%
MeOH solution) in anhydrous MeOH (3 mL) containing ethyl
iodide (0.3 mL, 3.8 mmol) was refluxed for 4 h. After removal of
the volatile solvent, the residue was partitioned between EtOAc
and 1 N aqueous HCl. The water phase was extracted with EtOAc
(×3). The combined organic layers were dried over Na2SO4 and
concentrated in vacuo. The residue was chromatographed on silica
gel with EtOAc-hexane (1:9 to 1:4, v/v) as an eluent to provide
31 (196 mg, 24%) along with recovered starting material. Colorless
1
129 °C (CH2Cl2-hexane). H NMR (300 MHz, CDCl3): δ 19.2
(s, 1H, chelated-OH), 8.83 (d, 1H, J ) 15.4 Hz, olefin), 8.42 (d,
1H, J ) 15.4 Hz, olefin), 8.28 (d, 1H, J ) 7.5 Hz, naphthyl-2 or
8-H), 8.04 (d, 1H, J ) 7.5 Hz, naphthyl-2 or 8-H), 7.96-7.84 (m,
2H, naphthyl-H), 7.64-7.46 (m, 3H, naphthyl-H), 7.37 and 7.36
(1:3, each t, 1H, Ar-5-H), 6.74 (dd, 1H, J ) 10.8 and 17.4 Hz,
Ar-CHdCH2), 5.81 (d, 1H, J ) 17.4 Hz, Ar-CHdCH2), 5.31 (d,
1H, J ) 10.8 Hz, Ar-CHdCH2), 3.96 and 3.89 (3:1, each s, 3H,
OCH3), 2.00 and 1.96 (3:1, each s, 3H, 5′-CH3), 1.48 and 1.39
(1:3, each s, 6H, 3′-CH3 × 2). MS m/z 363 (M+ + 1). Anal.
(C23H22O4) C, H, O.
1
prisms, mp 153-154 °C (EtOAc-hexane). H NMR (300 MHz,
3′,3′-Dimethyl-5′-ethyl Desmosdumotin C (21). Yield 75%;
yellow oil. 1H NMR (300 MHz, CDCl3): δ 19.37 and 18.90 (2:1,
each s, 1H, chelated-OH), 8.67 and 8.47 (1:2, each d, 1H, J )
15.9 Hz, olefin), 8.10 and 8.07 (1:3, each d, 1H, J ) 15.9 Hz,
olefin), 7.89-7.76 (m, 2H, Ar-2,6-H), 7.59-7.48 (m, 3H, Ar-3,4,5-
H), 4.12 and 4.06 (2:1, each s, 3H, OCH3), 2.65 and 2.62 (2:1,
each q, J ) 7.4 Hz, 2H, 5′-CH2CH3), 1.62 and 1.53 (1:2, each s,
6H, 3′-CH3 × 2), 1.32 and 1.29 (2:1, each t, J ) 7.4 Hz, 3H, 5′-
CH2CH3). MS m/z 325 (M+ - 1). Anal. (C20H22O4) C, H, O.
3′,3′,5′-Triethyl Desmosdumotin C (22). Yield 57%; yellow
oil. 1H NMR (300 MHz, CDCl3): δ 19.30 and 18.89 (3:2, each s,
1H, chelated-OH), 8.52 and 8.44 (2:3, each d, 1H, J ) 15.3 Hz,
olefin), 7.96 and 7.93 (2:3, each d, 1H, J ) 15.3 Hz, olefin), 7.73-
7.66 (m, 2H, Ar-2,6-H), 7.43-7.36 (m, 3H, Ar-3,4,5-H), 4.03 and
3.96 (3:2, each s, 3H, OCH3), 2.60 and 2.56 (3:2, each q, 2H, J )
7.4 Hz, 5′-CH2CH3), 2.04-1.76 (m, 4H, 3′-CH2CH3 × 2), 1.20
and 1.16 (3:2, each t, 3H, J ) 7.4 Hz, 5′-CH2CH3), 0.71 and 0.70
(2:3, each t, 3H, J ) 7.4 Hz, 3′-CH2CH3 × 2). MS m/z 355 (M+
+ 1). Anal. (C22H26O4) C, H, O.
4-Methyl-3′,3′,5′-triethyl Desmosdumotin C (23). Yield 63%;
yellow oil. 1H NMR (300 MHz, CDCl3): δ 19.30 and 18.91 (3:2,
each s, 1H, chelated-OH), 8.50 and 8.41 (2:3, each d, 1H, J )
15.3 Hz, olefin), 7.96 and 7.93 (2:3, each d, 1H, J ) 15.3 Hz,
olefin), 7.64-7.55 (m, 2H, Ar-2,6-H), 7.25-7.14 (m, 2H, Ar-3,5-
H), 4.03 and 3.95 (3:2, each s, 3H, OCH3), 2.60 and 2.56 (3:2,
each q, 2H, J ) 7.4 Hz, 5′-CH2CH3), 2.38 and 2.35 (3:2, each s,
3H, Ar-4-CH3), 2.07-1.90 and 1.90-1.72 (3:2, each m, 4H, 3′-
CH2CH3 × 2), 1.30-1.21 (m, 3H, 5′-CH2CH3), 0.70 (t, 6H, J )
7.4 Hz, 3′-CH2CH3 × 2). MS m/z 367 (M+ - 1). Anal. (C22H24O4)
C, H.
DMSO-d6): δ 19.00 (br s, 1H, chelated-OH), 2.48 [s, 3H, C(O)-
CH3], 2.35 (q, 2H, J ) 7.2 Hz, CH2CH3), 1.29 (s, 6H, 4-CH3 × 2),
0.95 (t, 3H, 6-CH2CH3). MS m/z 223 (M+ - 1). Anal. (C12H16O4‚
1/8H2O) C, H, O.
2-Acetyl-4,4,6-triethyl-3,5-dihydroxycyclohexa-2,5-dienone (32).
A solution of 2,4,6-trihydroxyacetophenone (1.117 g, 6.7 mmol)
and sodium methoxide (4.8 mL, 22.2 mmol, 25% MeOH solution)
in anhydrous MeOH (5 mL) containing ethyl iodide (1.65 mL, 20.6
mmol) was refluxed for 7 h. The reaction mixture was cooled to 0
°C and acidified with 1 N aqueous HCl and then extracted with
EtOAc (×3). The combined organic layers were dried over Na2-
SO4 and concentrated in vacuo. The residue was chromatographed
on silica gel with EtOAc-hexane (1:9 to 1:4, v/v) as an eluent to
provide 32 (723 mg, 43%). Colorless prisms, mp 149-150 °C
(EtOAc-hexane). 1H NMR (300 MHz, DMSO-d6): δ 18.98 (br s,
1H, chelated-OH), 2.51 [s, 3H, C(O)CH3], 2.42 (q, 2H, J ) 7.2
Hz, 6-CH2CH3), 1.81 (q, 4H, J ) 7.4 Hz, 4-CH2CH3 × 2), 0.97 (t,
3H, J ) 7.2 Hz, 6-CH3CH3), 0.57 (t, 3H, J ) 7.2 Hz, 4-CH2CH3
× 2). MS m/z 251 (M+ + 1). Anal. (C21H22O4‚1/16H2O) C, H, O.
2-Acetyl-3,5-dihydroxy-4,4,6-tripropylcyclohexa-2,5-di-
enone (33). The procedure was identical to that described above
except with propyl iodide instead of ethyl iodide to provide 33
(41%). Colorless prisms, mp 95-96 °C (EtOAc-hexane). 1H NMR
(300 MHz, CD3OD): δ 2.53 [s, 3H, C(O)CH3], 2.42 (t, 2H, J )
7.5 Hz, 6-CH2Et), 1.94-1.74 (m, 4H, 4-CH2Et × 2), 1.54-1.38
(m, 2H, 6-CH3CH2CH3), 1.16-0.90 (m, 4H, 4-CH3CH2CH3 × 2),
0.94 (t, 3H, J ) 7.4 Hz, 6-CH3CH2CH3), 0.81 (t, 6H, J ) 7.1 Hz,
6-CH3CH2CH3 × 2). MS m/z 293 (M+ - 1). Anal. (C17H26O4) C,
H, O.
4-Methyl-3′,3′,5′-triethyl Desmosdumotin C (24). Yield 61%;
yellow oil. 1H NMR (300 MHz, CDCl3): δ 19.30 and 18.93 (2:1,
each s, 1H, chelated-OH), 8.50 and 8.42 (1:2, each d, 1H, J )
15.3 Hz, olefin), 7.97 and 7.93 (1:2, each d, 1H, J ) 15.3 Hz,
olefin), 7.68-7.56 (m, 2H, Ar-2,6-H), 7.30-7.18 (m, 2H, Ar-3,5-
H), 4.03 and 3.95 (2:1, each s, 3H, OCH3), 2.55-2.50 (m, 4H,
CH2CH3 × 2), 2.08-1.72 (m, 4H, CH2CH3 × 2), 1.33-1.10 (m,
6H, CH2CH3 × 2), 0.80-0.64 (m, 6H, CH2CH3 × 2). MS m/z 381
(M+ - 1). Anal. (C24H30O4) C, H, O.
2-Acetyl-6-ethyl-3-hydroxy-5-methoxy-4,4-dimethylcyclohexa-
2,5-dienone (34). To a solution of 31 (124 mg, 0.55 mmol) in
anhydrous EtOAc-MeOH (5:1, 1.8 mL), a solution of TMSCHN2
(2 mL, 4.0 mmol, 2 M solution in diethyl ether) was added slowly
at -78 °C under an argon atmosphere, and the mixture was stirred
for 3 h. Acetic acid was then added to destroy the excess
TMSCHN2. The mixture was concentrated in vacuo, and the residue
was purified by silica gel column chromatography with EtOAc-
hexane (1:9 to 1:4 v/v) as an eluent to obtain 34 (117 mg, 89%);

3358 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 14
Nakagawa-Goto et al.
yellow oil. 1H NMR (300 MHz, CDCl3): δ 18.97 and 18.14 (2:1,
each s, 1H, chelated-OH), 3.97 and 3.90 (2:1, each s, 3H, OCH3),
2.71 and 2.61 [1:2, each s, 3H, C(O)CH3], 2.48 and 2.43 (2:1, each
q, 2H, J ) 7.4 Hz, 6-CH2CH3), 1.45 and 1.34 (1:2, each s, 6H,
4-CH3), 1.16 and 1.11 (2:1, each t, 3H, 6-CH2CH3 × 2). MS m/z
237 (M+ - 1).
72 h at 37 °C. After 72 h, plates were stained with 0.5% crystal
violet in 20% MeOH, rinsed with water, and air-dried. The stain
was eluted with 1:1 solution of EtOH/0.1 M sodium citrate, and
absorbance was measured at 540 nm with an ELISA reader.
Acknowledgment. This work was supported by a NIH Grant
CA17625 from the National Cancer Institute, awarded to K.-
H.L. and Grant No. 30572213 from the National Natural Science
Foundation of China, awarded to J.-H.W.
2-Acetyl-4,4,6-triethyl-3-hydroxy-5-methoxycyclohexa-2,5-di-
enone (35). The same procedure was employed to obtain 35 (80%)
1
from 32; yellow oil. H NMR (300 MHz, CDCl3): δ 19.00 and
18.12 (3:2, each s, 1H, chelated-OH), 4.02 and 3.93 (3:2, each s,
3H, OCH3), 2.71 and 2.64 [2:3, each s, 3H, C(O)CH3], 2.57 and
2.51 (3:2, each q, 2H, J ) 7.4 Hz, 6-CH2CH3), 2.03-1.72 (m, 4H,
4-CH2CH3 × 2), 1.18 and 1.12 (3:2, each t, 3H, 6-CH2CH3), 0.67
and 0.65 (2:3, each t, 6H, 4-CH2CH3 × 2). MS m/z 267 (M+ + 1).
2-Acetyl-3-hydroxy-5-methoxy-4,4,6-tripropylcyclohexa-2,5-
dienone (36). The same procedure was employed to obtain 36
(53%) from 33 along with recovered starting material (32%); yellow
oil. 1H NMR (300 MHz, CDCl3): δ 18.99 and 18.15 (3:2, each s,
1H, chelated-OH), 3.98 and 3.90 (3:2, each s, 3H, OCH3), 2.67
and 2.63 [2:3, each s, 3H, C(O)CH3], 2.53-2.40 (m, 2H, 6-CH2-
Et), 1.95-1.42 (m, 6H, 6-CH2CH2CH3 and 4-CH2Et × 2), 1.10-
0.95 (m, 7H, 6-CH2CH2CH3 and 4-CH2CH2CH3 × 2), 0.83 and
0.80 (3:2, each t, 6H, 4-CH2CH2CH3 × 2). MS m/z 307 (M+ - 1).
Cytotoxic Activity Assay.15 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. The following human
tumor cell lines were used in the assay: A549 (human lung
carcinoma), A431 (epidermoid skin carcinoma), 1A9 (human
ovarian carcinoma), HCT-8 (colon adenocarcinoma), PC-3 (prostate
cancer), KB (nasopharyngeal carcinoma), KB-VIN (vincristine-
resistant KB subline), and HUVEC (human endothelial). All cell
lines were obtained from the Lineberger Cancer Center (UNC-CH)
or from ATCC (Rockville, MD) and were cultured in RPMI-1640
medium supplemented with 25 mM HEPES, 0.25% sodium
bicarbonate, 10% fetal bovine serum, and 100 µg/mL kanamycin.
Angiogenesis Assay. The method is according to the NCI’s
Angiogenesis Resource Center protocol. HUVEC were purchased
from Cambrex Bio-science. HUVEC (1.5 × 103) were plated in a
96-well plate in 100 µL of EGM-2 (Clonetec # CC3162). After 24
h (day zero), the test compound (100 µL) was added to each well
at 2× the desired concentration in EGM-2 medium. One plate was
stained with 0.5% crystal violet in 20% MeOH for 10 min, rinsed
with water, and air-dried. The remaining plates were incubated for
Supporting Information Available: Elemental analysis data
for compounds 7-15, 21-26, and 31-33. This material is available
free of charge via the Internet at http://pubs.acs.org.
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