Synthesis of novel rhinacanthins and related anticancer naphthoquinone esters

Article abstract of DOI:10.1021/jm030323g

Rhinacanthin-M, -N and -Q, natural products isolated from the medicinal plant Rhinacanthus nasutus, and 39 novel naphthoquinone esters have been synthesized in excellent yield by esterification of naphthoquinone-3-(propan- 3′-ols) with benzoic or naphthoi

Full text of DOI:10.1021/jm030323g

J . Med. Chem. 2004, 47, 4427-4438
4427
Syn th esis of Novel Rh in a ca n th in s a n d Rela ted An tica n cer Na p h th oqu in on e
Ester s
Ngampong Kongkathip,*,† Suwaporn Luangkamin,† Boonsong Kongkathip,† Chak Sangma,† Ronald Grigg,‡
Palangpon Kongsaeree,§ Samran Prabpai,§ Narathip Pradidphol,† Suratsawadee Piyaviriyagul,# and
Pongpun Siripong#
Natural Products and Organic Synthesis Research Unit (NPOS), Department of Chemistry, Faculty of Science,
Kasetsart University, Bangkok, 10900, Thailand, Molecular Innovation, Diversity and Automated Synthesis (MIDAS) Centre,
School of Chemistry, Leeds University, Leeds, LS2 9J T, U.K., Department of Chemistry, Faculty of Science,
Mahidol University, Bangkok, 10400, Thailand, and Natural Products Research Section, Research Division,
National Cancer Institute, Bangkok, 10400, Thailand
Received J uly 2, 2003
Rhinacanthin-M, -N and -Q, natural products isolated from the medicinal plant Rhinacanthus
nasutus, and 39 novel naphthoquinone esters have been synthesized in excellent yield by
esterification of naphthoquinone-3-(propan-3′-ols) with benzoic or naphthoic acids. Almost all
the naphthoquinone esters that contain a C-3 hydroxy group showed significant cytotoxicities
against KB, HeLa, and HepG2 cell lines. In contrast, ester derivatives lacking the C-3 hydroxy
group were inactive to the cancer cell lines. Two methyl substituents on the C-2′ of propyl
chain conferred more potent cytotoxicity than when there is only one or no methyl group.
Naphthoate esters exhibited greater cytotoxicity than benzoate esters. Computer modeling has
been done to obtain a first look at the mode of action in connection with these observations.
In tr od u ction
Rhinacanthins1-3 are naphthoquinone ester deriva-
tives isolated from the methanolic extract of the roots
of the medicinal plant Rhinacanthus nasutus (Acan-
thaceae). Some of these compounds have been reported2
to exhibit cytotoxicity against P388, A-549, HT-29, and
HL-60 cell lines. In Thailand, the roots and leaves of R.
nasutus are used for the treatment of cancer.4 There
has been no reported synthesis of rhinacanthins. We
now report the synthesis of rhinacanthin-M, -N, -Q and
39 related naphthoquinone esters (Figure 1) together
with their cytotoxicities against human carcinoma cell
lines, KB (oral human epidermoid carcinoma), HeLa
(human cervical carcinoma), and HepG2 (human hepa-
tocellular carcinoma). We also conducted preliminary
studies on the mode of action of some of these naph-
thoquinone esters by computer modeling.
Ch em istr y
Rhinacanthins and related naphthoquinone esters
were obtained by esterification of naphthoquinone al-
cohols with benzoic or 2-naphthoic acids based on the
disconnection shown in Figure 2. Naphthoquinone al-
cohols (51, 52) with a gem-dimethyl group on the propyl
chain were prepared from 1-hydroxy-2-naphthoic acid,3,5,6
whereas naphthoquinone alcohols (45, 46, 49, and 50)
were prepared from 2-allyl or 2-methylallyl-1-naphthol7-9
via hydroboration followed by two successive oxidations
using Fremy’s salt followed by DDQ. Finally, hydrolysis
F igu r e 1. Structures of rhinacanthin-M, -N, -Q and 39 related
novel naphthoquinone esters.
* To whom correspondence should be addressed. Phone: +66-2-942-
8900, extension 211. Fax: +66-2-5793955. E-mail: fscinpk@ku.ac.th
of 47 and 48 under basic condition provided 49 and 50
in 86% and 94% yield, respectively (Scheme 1).
Preparation of rhinacanthin-Q (3) and its analogues
(14, 15, 18, 19, 21, 23, 24, and 26) was achieved in 58-
and fscinpk@yahoo.com.
† Kasetsart University.
‡ Leeds University.
§ Mahidol University.
#
National Cancer Institute.
10.1021/jm030323g CCC: $27.50 © 2004 American Chemical Society
Published on Web 08/04/2004

4428 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 18
Kongkathip et al.
Sch em e 1. Synthesis of
Naphthoquinone-3-(propan-3′-ols) (45, 46, 49, and 50)a
F igu r e 2. Retrosynthetic analysis of rhinacanthins and their
derivatives.
87% yield by esterification of naphthoquinone alcohols
(49-51) with various 2-naphthoic acids in the presence
of 1,1′-carbodiimidazole (CDI)10 (Table 1). The corre-
sponding diester was also produced in trace amounts.
Rhinacanthin-M (1) and its analogues (4, 6, 9, 10, and
12) were prepared in 69-98% yield by esterification of
naphthoquinone alcohols (50 and 51) with a range of
benzoic acids in the presence of dicyclohexylcarbodiim-
ide (DCC) and 4-(dimethylamino)pyridine (DMAP)11
(Table 2). Low yields of esters were encountered when
1,1′-carbodiimidazole was used for coupling.
Naphthoquinone esters (28, 29, 31, 32, 34, 35, and
37) lacking a C-3 hydroxy group were prepared accord-
ing to the procedure used to synthesize rhinacanthin-M
and its analogues (Table 2).
Selective demethylation of rhinacanthin-Q (3) and
naphthoquinone esters (4, 6, 10, 12, 15, 19, 21, 24, 26,
29, 32, 35, and 37) by treatment with boron tribromide12
in dichloromethane at -78 °C for 15 min afforded
a
Conditions: (a) BH3‚THF and then H2O2/OH-; (b) Fremy’s
salt, MeOH-DMF, 1 M NaOAc, room temp, 12 h; (c) DDQ,
p-TsOH, benzene, reflux, 20 min; (d) 1% aqueous NaOH, reflux, 2
h.
rhinacanthin-N (2) in 95% yield and naphthoquinone
esters (5, 7, 11, 13, 16, 20, 22, 25, 27, 30, 33, 36, and
38) in good yield (Table 3). In the case of naphtho-
quinone esters (8 and 17), yields of 72% and 73%,
respectively, were obtained by demethylation at both
methoxy groups of naphthoquinone ester (6) and rhi-
nacanthin-Q (3) with boron tribromide in dichlo-
romethane at room temperature for 2 h (Table 3).
Naphthoquinone esters (39-42) containing a C-3
methoxy group were synthesized in good yield by
Ta ble 1. Carbodiimidazole (CDI) Mediated Esterification of Naphthoquinone Alcohol

Rhinacanthins and Naphthoquinone Esters
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 18 4429
Ta ble 2. DCC/DMAP Mediated Esterification of Naphthoquinone Alcohol
methylation of naphthoquinone esters (1, 6, 14, and 15)
with methyl iodide (Table 4).
the cancer cell lines KB, HeLa, and HepG2 as well as
against the normal Vero cell line. The results14 showed
that the naphthoate (2) (Table 6) inhibits the cancer cell
lines more effectively than the benzoate (7) (Table 5)
with IC50 in the range <0.22 to 0.38 ( 0.06 and 1.18 (
0.26 to 3.33 ( 0.37 µM while they were much less toxic
to the Vero cell line (12.65 and 11.49 µM). Other
naphthoates (3, 14, 15, 16, and 17) exhibit more potent
cytotoxicity (Table 6) against the cancer cell lines than
the benzoates having the same substitution (1, 4, 5, 6,
and 8). The substituents on the naphthoquinone ring
(R5), propyl chain (R1 and R2), and benzene or naph-
thalene ring (R3 and R4) affect the activity. The presence
of a C-3 hydroxy group (R5 ) OH) on the naphtho-
quinone ring resulted in significantly greater cytotox-
icity of 3; KB, HeLa, and HepG2 inhibited with IC50
values of 0.35 ( 0.16, 1.09 ( 0.11, and 0.97 ( 0.10 µM,
respectively, compared to 37 (R5 ) H), which is inactive.
Biologica l Activity
Compounds 1-42 (Figure 1) were subjected to cyto-
toxic evaluation against KB (human epidermoid carci-
noma), HeLa (human cervical carcinoma), and HepG2
(human hepatocellular carcinoma) cell lines as well as
against the normal Vero cell line employing the MTT
colorimetric method.13 Adriamycin, which exhibits cy-
totoxicity against KB, HeLa, and HepG2 cell lines, was
used as reference. The results are summarized in Tables
5 and 6. Parent naphthoquinone alcohols and acids were
tested for cytotoxicity against the KB cell line as shown
in Table 7.
Resu lts a n d Discu ssion
Rhinacanthin-M, -N, -Q and naphthoquinone esters
(4-42) (Figure 1) were tested for cytotoxicity against

4430 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 18
Ta ble 3. Boron Tribromide Demethylation of Aryl Ethers
Kongkathip et al.
With regard to the propyl chain of the naphtho-
quinone ring, methyl substituents at the C-2′ position
affected cytotoxicity. Two methyl groups at the C-2′
position of the propyl chain conferred more potent
cytotoxicity against cancer cell lines than when there
is only one methyl or no methyl group (see compounds
15, 19, and 24 in Table 6). For example, compound 15
showed better cytotoxicity against the KB cell line than
19 and 24 with IC50 values of <0.22, 0.66 ( 0.19, and
33.66 ( 6.58 µM, respectively. In addition, the R3 and
R4 substituents on the naphthalene ring affect cytotox-
icity. Thus, 14 (NKPSL4, R3, R4 ) H), 2 (rhinacanthin-
N, R3 ) OH, R4 ) OMe), and 3 (rhinacanthin-Q, R3, R4
) OMe) inhibit KB, HeLa, and HepG2 cell lines differ-
ently. Rhinacanthin-N showed better cytotoxicity against
the KB cell line (Table 6) than rhinacanthin-Q and
NKPSL4 with IC50 values of <0.22, 0.35 ( 0.16, and
1.59 ( 0.37 µM, respectively. Moreover, NKPSL3 (22)
showed greater cytotoxicity against the cancer cell lines
(IC50 values in the range 2.16 ( 0.94 to 3.14 ( 0.45 µM)
than NKPSL2 (21) (IC50 values in the range 3.52 ( 0.18
to 3.80 ( 0.38 µM). The presence of a hydroxy group at
C-1 and a methoxy group at C-4 of the naphthalene ring
resulted in stronger cytotoxicity than the presence of
methoxy groups at both of these positions. For example,
in Table 6, rhinacanthin-N (2) showed greater cytotox-
icity against the KB, HeLa, and HepG2 cell lines (IC50
values of <0.22, 0.30 ( 0.05, and 0.38 ( 0.06 µM,
respectively) than rhinacanthin-Q (3) (IC50 values of
0.35 ( 0.16, 1.09 ( 0.11, and 0.97 ( 0.10 µM, respec-
tively). This might be due to either the better electron-
donating capacity of the hydroxy group or the better
hydrogen-bonding ability of the hydroxy group and
carbonyl group which facilitates better binding.
The X-ray crystallographic analyses of rhinacan-
thin-N (2) and rhinacanthin-Q (3) crystals revealed that
rhinacanthin-N and rhinacanthin-Q crystals have folded
structures in the crystalline state whereas NKPSL4 (14)

Rhinacanthins and Naphthoquinone Esters
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 18 4431
Ta ble 4. Methylation of 3-Hydroxynaphthoquinone Esters
Ta ble 5. Cytotoxicities of Rhinacanthin-M (1) and Naphthoquinone Esters (4-13, 41, and 42) against Human Carcinoma Cell Lines
(KB, HeLa, and HepG2) and Vero Cell Linea
cancer cell lines, IC50 (µM)b
Vero cell
compd
R1
R2
R3
R4
R5
KB
HeLa
HepG2
IC50 (µM)b
1
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
H
H
H
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OMe
OMe
1.53 ( 0.34
1.86 ( 0.45
4.65 ( 0.91
3.38 ( 0.76
1.18 ( 0.26
64.40 ( 8.38
4.57 ( 1.27
1.64 ( 0.32
4.65 ( 1.57
37.56 ( 1.34
4.04 ( 0.27
42.99 ( 2.53
14.94 ( 2.80
0.033
3.02 ( 0.58
4.04 ( 0.99
4.34 ( 0.22
3.42 ( 0.46
3.33 ( 0.37
45.48 ( 0.00
4.76 ( 0.29
4.12 ( 0.32
10.29 ( 1.88
41.87 ( 3.24
4.55 ( 0.41
40.56 ( 2.73
38.81 ( 2.50
0.33
4.85 ( 1.14
4.72 ( 0.43
5.44 ( 0.79
3.54 ( 0.39
1.61 ( 0.31
47.56 ( 4.90
8.43 ( 0.16
4.04 ( 0.49
11.62 ( 0.62
53.05 ( 7.55
4.96 ( 0.26
46.74 ( 4.32
40.71 ( 2.25
0.40
48.64
8.98
4
OMe
OH
OMe
OH
OH
H
H
5
H
>135.58
6
OMe
OMe
OH
H
9.11
7
11.49
77.05
2.31
8
9
10
11
12
13
41
42
H
OMe
OH
OMe
OH
H
H
H
29.79
75.85
4.05
14.02
34.79
13.39
23.94
H
H
OMe
OMe
H
H
Me
Me
OMe
OMe
adriamycinc
a
KB ) human epidermoid carcinoma. HeLa ) human cervical carcinoma. HepG2 ) human hepatocellular carcinoma. Vero cell line )
African green monkey kidney cell. The results are the mean of six replicate determinations ( SD. c Used as reference.
b
showed an extended structure (Figures 3-5). In 2 and
3, the R3 and R4 substituents are electron-donating
groups that enhance the electron density of the naph-
thalene moiety and provide π-π interaction with the
naphthoquinone moiety (intramolecular charge trans-
fer), leading to the folded conformation with an average
distance between the two planes of 3.65 Å. The crystal
conformations of the other naphthoates and benzoates
in the series are also being investigated by X-ray
crystallography.
For studying the mode of action of the naphtho-
quinone esters, we have done a preliminary test for
topoisomerase II (Topo II) inhibitory activity of the two
most active compounds (2 and 3) by the electrophoresis
method, as shown in Figure 11. In this figure, special
interest is focused on the relaxed DNA of row a. By
comparison with lane 3, lanes 4-7 indicated that
rhinacanthin-N and -Q inhibit Topo II activity by
relaxing supercoiled DNA. So both compounds inhibited
topoisomerase II. Therefore, Topo II was chosen as a
target enzyme based on this preliminary result and
some reports15-17 that showed that 1,2-naphthoquinone
and 1,4-naphthoquinone inhibit Topo II. Our cytotoxicity
results revealed that the naphthoquinone ester contain-
ing an -OH group at C-3 of the quinone ring showed
high toxicity against the cancer cell lines tested, whereas
the naphthoquinone esters with a methoxy group or
without an -OH group at that position showed no
activity or much less cytotoxicity. So we proposed that
the naphthoquinone esters with the C-3 hydroxyl group
on the quinone ring could exist in two tautomers that
are 1,4-naphthoquinone (A) and 1,2-naphthoquinone (B)
as shown in Figure 7. We first tried docking some of
our naphthoquinone esters, compared to that of adria-
mycin18,19 (doxorubicin) (Figure 6). Adriamycin, a com-
monly used anthracycline, is of primary importance in
the treatment of patients suffering from lymphomas,
breast cancer, and sarcomas. Topoisomerase II is the

4432 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 18
Kongkathip et al.
Ta ble 6. Cytotoxicities of Rhinacanthin-N (2), Rhinacanthin-Q (3), and Naphthoquinone Esters (14-40) against Human Carcinoma
Cell Lines (KB, HeLa, and HepG2) and Vero Cell Linea
cancer cell lines, IC50 (µM)b
Vero cell
compd
R1
R2
R3
R4
R5
KB
<0.22
HeLa
HepG2
IC50 (µM)b
2
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
H
Me
Me
Me
Me
Me
Me
H
OH
OMe
H
OMe
OH
OH
H
OMe
OH
OMe
OH
H
OMe
OH
OMe
OH
H
OMe
OMe
H
H
H
OH
H
H
H
OMe
OMe
H
H
H
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
H
0.30 ( 0.05
1.09 ( 0.11
1.17 ( 0.21
0.23 ( 0.02
<0.23
0.38 ( 0.06
0.97 ( 0.10
1.30 ( 0.21
0.24 ( 0.05
0.32 ( 0.02
12.93 ( 0.76
3.70 ( 0.27
1.04 ( 0.10
1.00 ( 0.08
3.65 ( 0.24
3.14 ( 0.45
15.54 ( 0.00
45.67 ( 2.15
16.16 ( 1.36
35.87 ( 1.83
4.28 ( 0.35
45.23 ( 3.55
14.41 ( 0.96
48.31 ( 1.71
27.30 ( 5.26
39.45 ( 2.50
42.08 ( 1.88
10.22 ( 1.67
54.17 ( 4.08
>259.07
12.65
32.70
1.50
17.60
1.00
3
0.35 ( 0.16
1.59 ( 0.37
<0.22
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
<0.23
4.22 ( 0.10
3.58 ( 0.20
0.66 ( 0.19
1.06 ( 0.23
3.52 ( 0.18
2.16 ( 0.94
14.40 ( 1.27
33.66 ( 6.58
12.32 ( 0.67
12.78 ( 1.00
4.09 ( 0.35
32.04 ( 2.41
12.50 ( 0.98
38.24 ( 3.59
28.99 ( 9.83
26.45 ( 2.33
38.56 ( 5.24
13.74 ( 1.66
50.00 ( 0.00
>259.07
12.44 ( 0.76
4.25 ( 0.42
0.38 ( 0.03
3.97 ( 0.40
3.80 ( 0.38
2.83 ( 1.15
15.80 ( 0.52
42.87 ( 0.98
15.09 ( 0.54
32.28 ( 6.46
4.24 ( 0.54
41.21 ( 3.41
36.92 ( 4.49
45.89 ( 0.00
38.20 ( 2.69
39.45 ( 3.64
46.25 ( 2.09
12.92 ( 0.82
47.50 ( 1.58
>259.07
15.42
1.80
H
0.97
H
0.70
H
5.91
H
<0.22
23.96
120.19
124.39
75.38
40.89
33.29
30.37
90.82
43.40
20.24
37.92
14.86
40.00
>259.07
>232.56
48.48
47.90
5.96
H
H
H
H
H
H
H
OMe
OMe
H
H
H
Me
Me
Me
Me
Me
Me
H
Me
Me
Me
H
OMe
OH
H
OMe
OH
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OMe
OH
OMe
OH
H
H
H
H
H
H
H
H
H
OMe
OMe
H
H
>232.56
>232.56
>232.56
H
H
H
93.75 ( 13.60
91.51 ( 10.29
10.08 ( 0.76
0.033
45.40 ( 5.23
44.86 ( 1.05
9.47 ( 0.33
0.33
64.42 ( 5.21
127.10 ( 10.13
11.50 ( 1.09
0.40
Me
Me
Me
Me
OMe
OMe
OMe
H
adriamycinc
23.94
a
KB ) human epidermoid carcinoma. HeLa ) human cervical carcinoma. HepG2 ) human hepatocellular carcinoma. Vero cell line )
African green monkey kidney cell. The results are the mean of six replicate determinations ( SD. c Used as reference.
b
F igu r e 4. ORTEP plot of the X-ray crystal structure of
F igu r e 3. ORTEP plot of the X-ray crystal structure of
rhinacanthin-Q (3).
rhinacanthin-N (2).
(residues 500-990) and human (residues 512-999)
(accession numbers M13814 and NP-001058). The pro-
gram AUTODOCK, version 3.05,22 was used to dock
separately adriamycin (reference drug), four active
compounds (2, 3, 26, and 27), and three inactive
compounds (37-39) into one of the best binding site
according to PASS23 (Figures 8-10). We have found that
all active compounds have the quinone ring lying on the
right-hand side and the propyl chain pointing down
(Figure 9), whereas the inactive compounds bind in a
different direction with the quinone ring lying on the
right and the propyl chain pointing up (Figure 10),
which supports the cytotoxicity result. The quinone ring
of adriamycin lies on the same plane as that of our
active compounds but not that of the inactive com-
primary cellular target for a number of clinically
important antitumor agents, including adriamycin.
Frydman’s group15 (1997) also reported the mechanism
of inhibition of topoisomerase II by â-lapachone (1,2-
naphthoquinone) and 1,4-naphthoquinone (R-lapachone)
(Figure 6). They reported â-lapachone was 10-fold more
reactive than 1,4-naphthoquinone. Therefore, we de-
cided to carry out molecular modeling or docking experi-
ments on Saccharomyces cereviseae topoisomerase II as
the target enzyme for our cytotoxic naphthoquinone
esters. There is no human Topo II crystal to do docking,
but the amino acid sequences20 of Topo II especially in
their DNA-binding domains are highly conserved in
eukaryotes. The DNA-binding21 domains show 50.1%
homology + 80.4% similarity between S. cereviseae

Rhinacanthins and Naphthoquinone Esters
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 18 4433
F igu r e 5. ORTEP plot of the X-ray crystal structure of
NKPSL4 (14).
Ta ble 7. Cytotoxicity of Parent Naphthoquinone Alcohols and
Acids
F igu r e 6. Structures of adriamycin, â-lapachone, and R-lapa-
chone.
F igu r e 7. Two isomers of naphthoquinone esters having a
C-3 hydroxy group.
F igu r e 8. Adriamycin (doxorubicin) in yellow, docked into the
proposed binding site.
cell lines KB, HeLa, and HepG2 than the benzoate
esters. Some naphthoate esters such as rhinacanthin-N
(2), rhinacanthin-Q (3), and NKPSL5 (15) promise to
possess great cytotoxicity against the said cancer cell
lines and to cause little harm to the normal Vero cell
line. Almost all the naphthoquinone esters that contain
a C-3 hydroxy group on the naphthoquinone ring
showed significant cytotoxicities against KB, HeLa, and
HepG2 cell lines, whereas esters with a methoxy group
or without a hydroxy group at that position showed no
activity or much less toxicity. In addition, two methyl
substituents on the C-2′ propyl chain of the esters
conferred more potent cytotoxicity against the cancer
cell lines than one methyl group or without a methyl
group on that position. Substituents on C-1 and C-4
positions of the naphthalene ring showed some effects
on the cytotoxicity. Therefore, varying the substituents
on the naphthalene moiety of the naphthoate ester
supported by molecular modeling would be valuable to
our investigation on the synthesis and cytotoxicity
testing of rhinacanthins. In addition, we are synthesiz-
a
The results are the mean of six replicate determinations. KB
b
) human epidermoid carcinoma. Used as reference.
pounds (see Figures 9 and 10). The details of how the
naphthoquinone esters interact with topoisomerase II
and/or the DNA substrate are being investigated by our
group.
In addition, it is cautioned that our naphthoquinone
esters might be hydrolyzed in vitro and in vivo by
carboxylate esterases to the parent carboxylic acids and
alcohols. It was found that the esters were not hydro-
lyzed in vitro, which were proved by cytotoxicity testing
of parent naphthoquinone alcohols and naphthoic acids.
The results (Table 7) showed that both the alcohols and
acids exhibited less or no toxicity against the KB cell
line.
Con clu sion
Our report shows that naphthoquinone naphthoate
esters exhibit a stronger cytotoxicity against the cancer

4434 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 18
Kongkathip et al.
F igu r e 9. Four active compounds (a1 ) compound 2, a2 ) compound 3, a3 ) compound 26, and a4 ) compound 27) docked at
the same site as that of adriamycin (in yellow).
F igu r e 10. Three inactive compounds (b1 ) compound 37, b2 ) compound 38, and b3 ) compound 39) docked at the same site
as that of adriamycin (in yellow).
new anticancer naphthoquinone esters including their
mechanism of action challenges us to work relentlessly.
Exp er im en ta l Section
Gen er a l Rem a r k s. Melting points were determined on a
Fisher J ohn apparatus and are uncorrected. The IR spectra
were recorded on an FTIR Perkin-Elmer system 2000. Mass
spectral data were obtained on the GCMS-QP-5050A. Nuclear
magnetic resonance spectra were recorded at 400 MHz on a
Brucker Advance DPX-400. Chemical shifts are given in parts
per million (δ) downfield from tetramethylsilane (TMS) as
internal standard. Coupling constants are given in hertz (Hz).
The following abbreviations are used: s ) singlet, d ) doublet,
t ) triplet, q ) quartet, m ) multiplet, br s ) broad singlet,
dd ) doublet of doublet, dt ) doublet of triplet. Column
chromatography was performed with flash silica gel (Merck
F igu r e 11. Inhibition of topoisomerase II. Supercoiled p-
bluescript DNA was relaxed with Topo II in the presence or
absence of the inhibitors rhinacanthin-Q (Rhi Q) and rhina-
canthin-N (Rhi N) for 30 or 60 min. The figure shows inhibitor-
free control (lanes 2 and 3), Rhi Q at 10 µM (lane 4) and at 20
µM (lane 7), and Rhi N at 10 µM (lane 5) and at 20 µM (lane
6).
9385).
2-(3-Hyd r oxyp r op yl)n a p h th a len -1-ol (43). A 1 M solu-
tion of borane in tetrahydrofuran (7.4 mL, 7.4 mmol) was
added dropwise to a stirred solution of 2-allylnaphthalen-1-ol
(1.36 g, 7.4 mmol) in anhydrous tetrahydrofuran (15 mL) at
room temperature under nitrogen. After the mixture was
stirred at room temperature for 4 h, water (0.74 mL) was
added dropwise, followed by 3 M sodium hydroxide (1 mL).
Then hydrogen peroxide (40%, 1 mL) was added at such a rate
that the temperature of the reaction mixture stayed between
30 and 50 °C. Following the addition, stirring was continued
ing naphthoquinone esters with aliphatic moiety as well
as testing their cytotoxicity. Acquiring knowledge on

Rhinacanthins and Naphthoquinone Esters
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 18 4435
for 4 h at room temperature. Diethyl ether (15 mL) was added
to the reaction mixture, and the mixture was washed with
brine and water. After being dried over anhydrous sodium
sulfate, filtered, and concentrated in vacuo, the residue was
purified by flash column chromatography, eluting with 9:1 v/v
hexane-ethyl acetate to afford the product 43 (71%) as a
phase was washed with brine and water, dried over anhydrous
sodium sulfate, filtered, and concentrated in vacuo. The
residue was purified by flash column chromatography.
Rh in a ca n th in -Q (3). Flash column chromatography elut-
ing with 17:3 v/v hexanes-ethyl acetate afforded the product
rhinacanthin-Q (3) (71%) as an orange amorphous powder,2
mp 117-118 °C. IR (KBr) cm-1: 3374, 1705, 1652, 1595, and
1276.
colorless amorphous powder, mp 86-87 °C. IR (KBr) cm-1
:
3481, 1513, and 1272.
2-(3-Hydr oxy-2-m eth ylpr opyl)n aph th alen -1-ol (44). The
method for preparation of 44 is the same as described for 43.
The product 44 was obtained in 96% yield as a colorless
amorphous powder, mp 89-90 °C. IR (KBr) cm-1: 3376, 1384,
and 1016.
Na p h th a len e-2-ca r boxylic Acid 3-(3-Hyd r oxy-1,4-d i-
oxo-1,4-d ih yd r on a p h th a len -2-yl)-2,2-d im eth ylp r op yl Es-
ter (14). Flash column chromatography eluting with 9:1 v/v
hexane-ethyl acetate afforded the product 14 (87%) as a
yellow amorphous powder, mp 114-115 °C. IR (KBr) cm-1
:
3352, 1711, 1665, 1589, 1273, and 1224.
2-(3-Hyd r oxyp r op yl)-[1,4]-n a p h th oqu in on e (45). To a
stirred solution of compound 43 (0.2 g, 0.99 mmol) in methanol-
dimethylformamide (MeOH-DMF) (3:1) (13 mL), a solution
of Fremy’s salt (2.7 g) in water (52 mL) and 1 M aqueous
sodium acetate solution (1.44 mL) was added. After being
stirred for 12 h at room temperature, the reaction mixture was
extracted with diethyl ether (3 × 20 mL). The combined
organic phase was washed with water and brine, dried over
anhydrous sodium sulfate, and concentrated in vacuo. The
residue was purified by flash column chromatography, eluting
with 4:1 v/v hexane-ethyl acetate to afford the product 45
(71%) as a yellow solid, mp 66-67 °C. IR (KBr) cm-1: 3249,
1663, and 1305.
1-Meth oxyn aph th alen e-2-car boxylic Acid 3-(3-Hydr oxy-
1,4-d ioxo-1,4-d ih yd r on a p h th a len -2-yl)-2,2-d im eth ylp r o-
p yl Ester (15). Flash column chromatography eluting with
17:3 v/v hexane-ethyl acetate afforded the product 15 (82%)
as a yellow oil. IR (neat) cm-1: 3363, 1712, 1650, 1593, 1280,
and 1084.
Na p h th a len e-2-ca r boxylic Acid 3-(3-Hyd r oxy-1,4-d i-
oxo-1,4-d ih yd r on a p h t h a len -2-yl)-2-m et h ylp r op yl E st er
(18). Flash column chromatography eluting with 17:3 v/v
hexanes-ethyl acetate provided the product 18 (83%) as a
yellow amorphous powder, mp 164-166 °C. IR (KBr) cm-1
3363, 1715, 1649, 1589, 1275, and 1224.
:
2-(3-H yd r oxy-2-m et h ylp r op yl)-[1,4]-n a p h t h oq u in on e
(46). Compound 46 was prepared from compound 44 in the
same manner as for compound 45. The product 46 was
obtained in 85% yield as a yellow oil. IR (neat) cm-1: 3403,
1662, 1589, and 1029.
3,4-Dih yd r o-2H-ben zo[h ]ch r om en e-5,6-d ion e (47). A
mixture of compound 45 (0.2 g, 0.9 mmol), dichlorodicyanoben-
zoquinone (DDQ) (0.3 g, 1.4 mmol), and p-toluenesulfonic acid
monohydrate (0.02 g, 0.09 mmol) in dry benzene (4 mL) was
stirred for 30 min under reflux. Then the reaction mixture was
cooled to room temperature, filtered, washed with dichlo-
romethane, and concentrated in vacuo. The residue was
filtered through aluminum oxide to afford the product 47 (95%)
as an orange amorphous powder, mp 184-185 °C. IR (KBr)
cm-1: 1687, 1642, 1564, and 1258.
1-Meth oxyn aph th alen e-2-car boxylic Acid 3-(3-Hydr oxy-
1,4-dioxo-1,4-dih ydr on aph th alen -2-yl)-2-m eth ylpr opyl Es-
ter (19). Flash column chromatography eluting with 7:1 v/v
hexanes-ethyl acetate afforded the product 19 (73%) as a
yellow amorphous powder, mp 85-86 °C. IR (KBr) cm-1: 3347,
1728, 1646, 1589, 1276, and 1225.
1,4-Dim eth oxyn a p h th a len e-2-ca r boxylic Acid 3-(3-Hy-
d r oxy-1,4-d ioxo-1,4-d ih yd r on a p h t h a len -2-yl)-2-m et h yl-
p r op yl Ester (21). Flash column chromatography eluting
with 17:3 v/v hexanes-ethyl acetate afforded the product 21
(73%) as a yellow amorphous powder, mp 113-115 °C. IR
(KBr) cm-1: 3295, 1727, 1648, 1592, 1276, and 1223.
Na p h th a len e-2-ca r boxylic Acid 3-(3-Hyd r oxy-1,4-d i-
oxo-1,4-d ih yd r on a p h th a len -2-yl)p r op yl Ester (23). Flash
column chromatography eluting with 17:3 v/v hexanes-ethyl
acetate provided the product 23 (73%) as a yellow amorphous
powder, mp 120-121 °C. IR (KBr) cm-1: 3362, 1714, 1645,
1590, 1278, and 1226.
3-Me t h yl-3,4-d ih yd r o-2H -b e n zo[h ]ch r om e n e -5,6-d i-
on e (48). Compound 48 was prepared from compound 46 in
the same manner as for compound 47. The product 48 was
obtained in 95% yield as an orange amorphous powder, mp
148-149 °C. IR (KBr) cm-1: 1688, 1645, 1256, and 1168.
2-H yd r oxy-3-(3-h yd r oxyp r op yl)-[1,4]-n a p h t h oq u in -
on e (49). A solution of compound 47 (0.2 g, 0.93 mmol) in 1%
aqueous sodium hydroxide solution (6 mL) was refluxed for 2
h. Then the reaction mixture was cooled to room temperature,
acidified with acetic acid, and extracted with dichloromethane
(3 × 30 mL). The combined organic phase was washed with
water, dried over anhydrous sodium sulfate, filtered, and
concentrated in vacuo. The residue was recrystallized from
hexane-dichloromethane to give the product 49 (86%) as a
1-Meth oxyn aph th alen e-2-car boxylic Acid 3-(3-Hydr oxy-
1,4-d ioxo-1,4-d ih yd r on a p h th a len -2-yl)p r op yl Ester (24).
Flash column chromatography eluting with 9:1 v/v hexanes-
ethyl acetate provided the product 24 (77%) as a yellow
amorphous powder, mp 125-126 °C. IR (KBr) cm-1: 3356,
1704, 1643, 1589, 1276, and 1132.
1,4-Dim eth oxyn a p h th a len e-2-ca r boxylic Acid 3-(3-Hy-
d r oxy-1,4-d ioxo-1,4-d ih yd r on a p h th a len -2-yl)p r op yl Es-
ter (26). Flash column chromatography eluting with 17:3 v/v
hexanes-ethyl acetate provided the product 26 (58%) as a
yellow amorphous powder, mp 146-147 °C. IR (KBr) cm-1
:
yellow amorphous powder, mp 106-107 °C. IR (KBr) cm-1
3342, 1640, 1638, 1365, and 1272.
:
3372, 1727, 1650, 1593, 1235, and 1097.
1H NMR, 13C NMR, MS, and elemental analysis of naph-
thoquinone esters 3, 14, 15, 18, 19, 21, 23, 24, and 26 are in
Supporting Information.
2-H yd r oxy-3-(3-h yd r oxy-2-m et h ylp r op yl)-[1,4]-n a p h -
th oqu in on e (50). Compound 50 was prepared from compound
48 in the same manner as for compound 49. The product 50
was obtained in 94% yield as a yellow amorphous powder, mp
146-147 °C. IR (KBr) cm-1: 3485, 1656, 1371, and 1215.
1H NMR, 13C NMR, MS, HR-MS, and elemental analysis of
compounds 43-50 are in Supporting Information.
Gen er a l P r oced u r e for Syn th esis of Na p h th oqu in on e
Ester s 1, 4, 6, 9, 10, 12, 28, 29, 31, 32, 34, 35, a n d 37. To a
stirred solution of acid (0.26 mmol) and 4-(dimethylamino)-
pyridine (DMAP) (0.06 mmol) in dry dichloromethane (2 mL),
a
solution of naphthoquinone alcohol (0.2 mmol) in dry
Gen er a l P r oced u r e for Syn th esis of Na p h th oqu in on e
Ester s 3, 14, 15, 18, 19, 21, 23, 24, a n d 26. To a stirred
solution of acid (0.3 mmol) in tetrahydrofuran (THF) (3 mL),
a solution of 1,1′-carbodiimidazole (CDI) (0.3 mmol) in THF
(2 mL) was added. After the mixture was stirred for 3 h at
room temperature, a solution of quinone (0.2 mmol) in THF
(5 mL) was added. Then it was continuously stirred for 7 h at
room temperature. The reaction mixture was quenched with
saturated ammonium chloride solution (30 mL) and extracted
with dichloromethane (3 × 30 mL). The combined organic
dichloromethane (2 mL) was added. The reaction mixture was
stirred at room temperature for 5 min, and then a solution of
dicyclohexylcarbodiimide (DCC) (0.26 mmol) in dry dichlo-
romethane (3 mL) was added to the reaction mixture. After
that, it was continuously stirred overnight at room tempera-
ture. Filtered off was the precipitate of dicyclohexyl urea, and
then the organic solution was washed twice with saturated
ammonium chloride solution and water, dried over anhydrous
sodium sulfate, filtered, and concentrated in vacuo. The
residue was purified by flash column chromatography.

4436 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 18
Kongkathip et al.
Rh in a ca n th in -M (1). Flash column chromatography elut-
ing with 9:1 v/v hexanes-ethyl acetate afforded the product
rhinacanthin-M (1) (91%) as a yellow amorphous powder,1 mp
100-101 °C. IR (KBr) cm-1: 3240, 1719, 1639, 1586, and 1271.
2-Meth oxyben zoic Acid 3-(3-Hyd r oxy-1,4-d ioxo-1,4-d i-
h ydr on aph th alen -2-yl)-2,2-dim eth ylpr opyl Ester (4). Flash
column chromatography eluting with 22:3 v/v hexane-ethyl
acetate afforded the product 4 (98%) as a yellow amorphous
powder, mp 122-123 °C. IR (KBr) cm-1: 3243, 1687, 1640,
1590, 1303, and 1215.
2,5-Dim eth oxyben zoic Acid 3-(3-Hyd r oxy-1,4-d ioxo-
1,4-dih ydr on aph th alen -2-yl)-2,2-dim eth ylpr opyl Ester (6).
Flash column chromatography eluting with 17:3 v/v hexane-
ethyl acetate afforded the product 6 (92%) as a yellow
amorphous powder, mp 109-110 °C. IR (KBr) cm-1: 3269,
1673, 1500, 1277, and 1219.
Gen er a l P r oced u r e for Syn th esis of Na p h th oqu in on e
Ester s 2, 5, 7, 11, 13, 16, 20, 22, 25, 27, 30, 33, 36, a n d 38.
To a solution of naphthoquinone ester (0.1 mmol) in dry
dichloromethane (5 mL), 1 M boron tribromide in dichlo-
romethane (0.2 mmol) was added dropwise at -78 °C, and the
reaction mixture was stirred for 15 min. Then cold water was
added, and the reaction mixture was extracted with dichlo-
romethane (3 × 30 mL). The combined organic phase was
washed with water, dried over anhydrous sodium sulfate,
filtered, and concentrated in vacuo. The residue was purified
by flash column chromatography.
Rh in a ca n th in -N (2). Flash column chromatography elut-
ing with 23:2 v/v hexanes-ethyl acetate afforded the product
rhinacanthin-N (2) (95%) as an orange amorphous powder,1
mp 124-125 °C. IR (KBr) cm-1: 3235, 1663, 1638, 1592, and
1243.
2-Hyd r oxyben zoic Acid 3-(3-Hyd r oxy-1,4-d ioxo-1,4-d i-
h ydr on aph th alen -2-yl)-2,2-dim eth ylpr opyl Ester (5). Flash
column chromatography eluting with 47:3 v/v hexane-ethyl
acetate afforded the product 5 (90%) as a yellow amorphous
powder, mp 110-111 °C. IR (KBr) cm-1: 3358, 1670, 1641,
1589, 1363, and 1275.
2-Hyd r oxy-5-m eth oxyben zoic Acid 3-(3-Hyd r oxy-1,4-
dioxo-1,4-dih ydr on aph th alen -2-yl)-2,2-dim eth ylpr opyl Es-
ter (7). Flash column chromatography eluting with 9:1 v/v
hexane-ethyl acetate afforded the product 7 (89%) as a yellow
amorphous powder, mp 92-93 °C. IR (KBr) cm-1: 3319, 1665,
1612, 1492, 1283, and 1232.
2-Hyd r oxyben zoic Acid 3-(3-Hyd r oxy-1,4-d ioxo-1,4-d i-
h yd r on a p h th a len -2-yl)-2-m eth ylp r op yl Ester (11). Flash
column chromatography eluting with 93:7 v/v hexane-ethyl
acetate afforded the product 11 (70%) as a yellow amorphous
powder, mp 121-122 °C. IR (KBr) cm-1: 3354, 1674, 1641,
1482, and 1222.
2-Hyd r oxy-5-m eth oxyben zoic Acid 3-(3-Hyd r oxy-1,4-
d ioxo-1,4-d ih yd r on a p h th a len -2-yl)-2-m eth ylp r op yl Ester
(13). Flash column chromatography eluting with 22:3 v/v
hexane-ethyl acetate afforded the product 13 (83%) as a
yellow solid, mp 142-143 °C. IR (KBr) cm-1: 3357, 1677, 1645,
1492, 1273, and 1224.
Ben zoic Acid 3-(3-Hyd r oxy-1,4-d ioxo-1,4-d ih yd r on a p h -
th a len -2-yl)-2-m eth ylp r op yl Ester (9). Flash column chro-
matography eluting with 9:1 v/v hexane-ethyl acetate afforded
the product 9 (69%) as a yellow amorphous powder, mp 144-
145 °C. IR (KBr) cm-1: 3351, 1719, 1651, 1589, and 1276.
2-Meth oxyben zoic Acid 3-(3-Hyd r oxy-1,4-d ioxo-1,4-d i-
h yd r on a p h th a len -2-yl)-2-m eth ylp r op yl Ester (10). Flash
column chromatography eluting with 22:3 v/v hexane-ethyl
acetate afforded the product 10 (93%) as a yellow amorphous
powder, mp 109-110 °C. IR (KBr) cm-1: 3350, 1730, 1657,
1242, and 1085.
2, 5-Dim et h oxyb en zoic Acid 3-(3-Hyd r oxy-1,4-d ioxo-
1,4-d ih yd r on a p h th a len -2-yl)-2-m eth ylp r op yl Ester (12).
Flash column chromatography eluting with 4:1 v/v hexane-
ethyl acetate afforded the product 12 (69%) as a yellow
amorphous powder, mp 144-145 °C. IR (KBr) cm-1: 3343,
1728, 1643, 1585, 1277, and 1216.
Na p h th a len e-2-ca r boxylic Acid 3-(1,4-Dioxo-1,4-d ih y-
d r on a p h th a len -2-yl)-1,2-d im eth ylp r op yl Ester (28). Flash
column chromatography eluting with 47:3 v/v hexane-ethyl
acetate afforded the product 28 (74%) as a yellow solid, mp
93-94 °C. IR (KBr) cm-1: 1711, 1657, 1591, 1281, and 1224.
1-Meth oxyn a p h th a len e-2-ca r boxylic Acid 3-(1,4-Dioxo-
1,4-d ih yd r on a p h t h a len -2-yl)-2,2-d im et h ylp r op yl E st er
(29). Flash column chromatography eluting with 47:3 v/v
hexane-ethyl acetate afforded the product 29 (98%) as a
yellow solid, mp 66-68 °C. IR (KBr) cm-1: 1706, 1662, 1597,
1275, and 1133.
Na p h th a len e-2-ca r boxylic Acid 3-(1,4-Dioxo-1,4-d ih y-
d r on a p h th a len -2-yl)-2-m eth ylp r op yl Ester (31). Flash
column chromatography eluting with 19:1 v/v hexane-ethyl
acetate afforded the product 31 (83%) as a yellow oil. IR (neat)
cm-1: 1716, 1663, 1595, 1280, and 1094.
1-Meth oxyn a p h th a len e-2-ca r boxylic Acid 3-(1,4-Dioxo-
1,4-d ih yd r on a p h th a len -2-yl)-2-m eth ylp r op yl Ester (32).
Flash column chromatography eluting with 47:3 v/v hexane-
ethyl acetate afforded the product 32 (90%) as a yellow oil. IR
(neat) cm-1: 1721, 1664, 1595, 1274, and 1134.
Na p h th a len e-2-ca r boxylic Acid 3-(1,4-Dioxo-1,4-d ih y-
d r on a p h th a len -2-yl)p r op yl Ester (34). Flash column chro-
matography eluting with 9:1 v/v hexane-ethyl acetate afforded
the product 34 (86%) as a yellow amorphous powder, mp 114-
116 °C. IR (KBr) cm-1: 1712, 1660, 1589, and 1289.
1-Meth oxyn a p h th a len e-2-ca r boxylic Acid 3-(1,4-Dioxo-
1,4-d ih yd r on a p h th a len -2-yl)p r op yl Ester (35). Flash col-
umn chromatography eluting with 23:2 v/v hexane-ethyl
acetate afforded the product 35 (85%) as a yellow amorphous
powder, mp 95-96 °C. IR (KBr) cm-1: 1718, 1657, 1589, 1276,
1237, and 1141.
1,4-Dim eth oxyn a p h th a len e-2-ca r boxylic Acid 3-(1,4-
Dioxo-1,4-dih ydr on aph th alen -2-yl)pr opyl Ester (37). Flash
column chromatography eluting with 9:1 v/v hexane-ethyl
acetate afforded the product 37 (82%) as a yellow amorphous
powder, mp 121-122 °C. IR (KBr) cm-1: 1698, 1662, 1594,
1242, and 1104.
1H NMR, 13C NMR, MS, and elemental analysis of naph-
thoquinone esters 1, 4, 6, 9, 10, 12, 28, 29, 31, 32, 34, 35, and
37 are in Supporting Information.
1-Hydr oxyn aph th alen e-2-car boxylic Acid 3-(3-Hydr oxy-
1,4-d ioxo-1,4-d ih yd r on a p h th a len -2-yl)-2,2-d im eth ylp r o-
p yl Ester (16). Flash column chromatography eluting with
9:1 v/v hexane-ethyl acetate afforded the product 16 (80%)
as a yellow solid, mp 162-163 °C. IR (KBr) cm-1: 3327, 1661,
1580, 1257, and 1162.
1-Hydr oxyn aph th alen e-2-car boxylic Acid 3-(3-Hydr oxy-
1,4-dioxo-1,4-dih ydr on aph th alen -2-yl)-2-m eth ylpr opyl Es-
ter (20). Flash column chromatography eluting with 9:1 v/v
hexane-ethyl acetate afforded the product 20 (81%) as a
yellow amorphous powder, mp 191-192 °C. IR (KBr) cm-1
3363, 1658, 1593, 1259, and 1166.
:
1-Hyd r oxy-4-m eth oxyn a p h th a len e-2-ca r boxylic Acid
3-(3-H yd r oxy-1,4-d ioxo-1,4-d ih yd r on a p h t h a len -2-yl)-2-
m eth ylp r op yl Ester (22). Flash column chromatography
eluting with 23:2 v/v hexane-ethyl acetate afforded the
product 22 (80%) as an orange solid, mp 149-151 °C. IR (KBr)
cm-1: 3354, 1660, 1592, 1234, and 1152.
1-Hydr oxyn aph th alen e-2-car boxylic Acid 3-(3-Hydr oxy-
1,4-d ioxo-1,4-d ih yd r on a p h th a len -2-yl)p r op yl Ester (25).
Flash column chromatography eluting with 23:2 v/v hexane-
ethyl acetate afforded the product 25 (85%) as a yellow
amorphous powder, mp 137-138 °C. IR (KBr) cm-1: 3350,
1658, 1585, 1254, and 1162.
1-Hyd r oxy-4-m eth oxyn a p h th a len e-2-ca r boxylic Acid
3-(3-Hyd r oxy-1,4-d ioxo-1,4-d ih yd r on a p h th a len -2-yl)p r o-
p yl Ester (27). Flash column chromatography eluting with
9:1 v/v hexane-ethyl acetate afforded the product 27 (79%)
as an orange solid, mp 164-165 °C. IR (KBr) cm-1: 3365, 1663,
1590, 1245, and 1092.
1-Hyd r oxyn a p h th a len e-2-ca r boxylic Acid 3-(1,4-Dioxo-
1,4-d ih yd r on a p h t h a len -2-yl)-2,2-d im et h ylp r op yl E st er
(30). Flash column chromatography eluting with 24:1 v/v
hexane-ethyl acetate afforded the product 30 (80%) as a

Rhinacanthins and Naphthoquinone Esters
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 18 4437
yellow amorphous powder, mp 151-152 °C. IR (KBr) cm-1
:
2,5-Dim eth oxyben zoic Acid 3-(3-Meth oxy-1,4-d ioxo-
1,4-d ih yd r on a p h t h a len -2-yl)-2,2-d im et h ylp r op yl E st er
(42). Flash column chromatography eluting with 9:1 v/v
hexane-ethyl acetate afforded the product 42 (93%) as a
yellow oil. IR (neat) cm-1: 1701, 1667, 1599, 1499, 1217, and
1046.
1663, 1589, 1251, and 1161.
1-Hyd r oxyn a p h th a len e-2-ca r boxylic Acid 3-(1,4-Dioxo-
1,4-d ih yd r on a p h th a len -2-yl)-2-m eth ylp r op yl Ester (33).
Flash column chromatography eluting with 24:1 v/v hexane-
ethyl acetate afforded the product 33 (84%) as a yellow
amorphous powder, mp 130-131 °C. IR (KBr) cm-1: 3492,
1716, 1662, 1461, 1273, and 1133.
1-Hyd r oxyn a p h th a len e-2-ca r boxylic Acid 3-(1,4-Dioxo-
1,4-d ih yd r on a p h th a len -2-yl)p r op yl Ester (36). Flash col-
umn chromatography eluting with 24:1 v/v hexane-ethyl
acetate afforded the product 36 (92%) as a yellow amorphous
powder, mp 173-175 °C. IR (KBr) cm-1: 1662, 1593, 1254,
and 1165.
1-Hyd r oxy-4-m eth oxyn a p h th a len e-2-ca r boxylic Acid
3-(1,4-Dioxo-1,4-dih ydr on aph th alen -2-yl)pr opyl Ester (38).
Flash column chromatography eulting with 19:1 v/v hexanes-
ethyl acetate afforded the product 38 (46%) as an orange
amorphous powder, mp 129-130 °C. IR (KBr) cm-1: 3440,
1662, 1593, and 1240.
1H NMR, 13C NMR, MS, and elemental analysis of naph-
thoquinone esters 2, 5, 7, 11, 13, 16, 20, 22, 25, 27, 30, 33,
36, and 38 are in Supporting Information.
Gen er a l P r oced u r e for Syn th esis of Na p h th oqu in on e
Ester s 8 a n d 17. To a solution of naphthoquinone ester (0.05
g, 0.1 mmol) in dichloromethane (5 mL) at room temperature,
boron tribromide (0.05 mL, 0.5 mmol) was added dropwise,
and the solution was stirred for 1 h at room temperature. Then
water was added and a portion was extracted with dichlo-
romethane (3 × 30 mL). The combined organic phase was
washed with water, dried over anhydrous sodium sulfate,
filtered, and concentrated in vacuo. The residue was purified
by flash column chromatography.
1H NMR, 13C NMR, MS, and elemental analysis of naph-
thoquinone esters 39-42 are in Supporting Information.
X-r a y Cr ysta llogr a p h y of Com p ou n d s 2, 3, a n d 14.
Crystals of compounds 2, 3, and 14, suitable for an X-ray
structure analysis, were obtained by slow evaporation from
EtOAc-hexane. For each sample, data were collected at room
temperature on a Bruker-Nonius κCCD diffractometer with
Mo KR radiation (λ ) 0.710 73 Å). The crystal structures were
solved by direct methods using SHELXS-97, and then all
atoms except hydrogen atoms were refined anisotropically on
F2 using SHELXL-97. Atomic coordinates, bond lengths, bond
angles, and thermal parameters have been deposited with the
Cambridge Crystallographic Data Center (CCDC 211195-
211197). Copies of the data can be obtained free of charge on
application to the Director, CCDC, 12 Union Road, Cambridge,
CB2 1EZ, U.K. (e-mail: deposite@ccdc.cam.ac.uk).
Sin gle-Cr ysta l X-r a y Diffr a ction An a lysis of Rh in a -
ca n th in -N (2). C27H24O7, MW 460.482, monoclinic, P21/c, a )
9.7885(3) Å, b ) 24.3474(7) Å, c ) 12.7679(4) Å, â ) 130.005-
(1)°, V ) 2330.82(12)) Å3, Z ) 4, µ ) 0.095 mm-1, Dx ) 1.313
g‚cm-3, R ) 0.0446 (Rw ) 0.1107 for 2692 observed reflections).
Sin gle-Cr ysta l X-r a y Diffr a ction An a lysis of Rh in a -
ca n th in -Q (3). C28H26O7, MW 474.509, triclinic, P1h, a )
8.3524(4) Å, b ) 9.8595(7) Å, c ) 15.2588(13) Å, R ) 96.791-
(3)°, â ) 105.678(4)°, γ ) 94.520(5)°, V ) 1193.24(1) Å3, Z )
2, µ ) 0.095 mm-1, Dx ) 1.321 g‚cm-3, R ) 0.0507 (Rw ) 0.1253
for 2356 observed reflections).
Sin gle-Cr ysta l X-r a y Diffr a ction An a lysis of Com -
p ou n d 14. C26H22O5, MW 414.457, monoclinic, P21/c, a )
18.8100(15) Å, b ) 6.1294(3) Å, c ) 22.6710(15) Å, â ) 126.356-
(3)°, V ) 2104.9(2) Å3, Z ) 4, µ ) 0.090 mm-1, Dx ) 1.308
g‚cm-3, R ) 0.0454 (Rw ) 0.1128 for 1937 observed reflections).
Other crystal data and structure refinements of rhinacan-
thin-N (2), rhinacanthin-Q (3), and compound 14 are in
Supporting Information.
Cytotoxicity Assa y. Th e MTT Color im etr ic Meth od .13
Compounds 1-42 dissolved in dimethyl sulfoxide (DMSO)
were subjected to cytotoxic evaluation against KB (human
epidermoid carcinoma), HeLa (human cervical carcinoma), and
HepG2 (human hepatocellular carcinoma) cell lines employing
the colorimetric method. Adriamycin was used as the reference
drug which exhibits cytotoxicity against KB, HeLa, and HepG2
cell lines.
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide (Sigma Chemical Co.) was dissolved in saline to make a
concentration of 5 mg/mL as a stock solution. Cancer cells (3
× 103 cells) suspended in 100 µg/well of MEM medium
containing 10% fetal calf serum (FCS, Gibco BRL, Life
Technologies, NY) were seeded onto a 96-well culture plate
(Costar, Corning Incorporated, NY 14831). After 24 h of
preincubation at 37 °C in a humidified atmosphere of 5% CO2/
95% air to allow cell attachment, various concentrations of test
solution (10 µL/well) as listed in Tables 5 and 6 were added
and then incubated for 48 h under the above conditions. At
the end of the incubation, 10 µL of tetrazolium reagent was
added to each well and then incubated at 37 °C for 4 h. The
supernatant was decanted, and DMSO (100 µL/well) was
added to allow formosan solubilization. The optical density
(OD) of each well was detected by a Microplate reader (Bio-
Rad, Benchmark Microplate reader) at 550 nm and for
correction at 595 nm. Each determination represents the mean
of six replicates. The 50% inhibition concentration (IC50) was
determined by curve fitting.
2,5-Dih yd r oxyben zoic Acid 3-(3-Hyd r oxy-1,4-d ioxo-1,4-
d ih yd r on a p h t h a len -2-yl)-2,2-d im et h ylp r op yl E st er (8).
Flash column chromatography eluting with 21:4 v/v hexane-
ethyl acetate afforded the product 8 (72%) as an orange
amorphous powder, mp 147-149 °C. IR (KBr) cm-1: 3365,
1670, 1488, and 1213.
1,4-Dih yd r oxyn a p h th a len e-2-ca r boxylic Acid 3-(3-Hy-
d r oxy-1,4-d ioxo-1,4-d ih yd r on a p h th a len -2-yl)-2,2-d im eth -
ylp r op yl Ester (17). Flash column chromatography eluting
with 17:3 v/v hexane-ethyl acetate afforded the product 17
(73%) as an orange amorphous powder, mp 160-162 °C. IR
(KBr) cm-1: 3372, 1662, 1634, 1593, and 1250.
1H NMR, 13C NMR, MS, and elemental analysis of com-
pounds 8 and 17 are in Supporting Information.
Gen er a l P r oced u r e for Syn th esis of Na p h th oqu in on e
Ester s 39-42. A mixture of naphthoquinone ester (0.1 mmol),
potassium carbonate (0.2 mmol), acetone (3 mL), and
iodomethane (1.0 mmol) was refluxed for 3 h. Then the reaction
mixture was cooled to room temperature. Water was added to
the reaction mixture, and the sample was then extracted with
dichloromethane, washed with water, dried over anhydrous
sodium sulfate, filtered, and concentrated in vacuo. The
residue was purified by flash column chromatography.
Na p h th a len e-2-ca r boxylic Acid 3-(3-Meth oxy-1,4-d i-
oxo-1,4-d ih yd r on a p h th a len -2-yl)-2,2-d im eth ylp r op yl Es-
ter (39). Flash column chromatography eluting with 19:1 v/v
hexane-ethyl acetate afforded the product 39 (75%) as a
yellow oil. IR (neat) cm-1: 1715, 1667, 1597, 1462, 1275, and
1222.
1-Meth oxyn a p h th a len e-2-ca r boxylic Acid 3-(3-Meth -
oxy-1,4-d ih yd r on a p h th a len -2-yl)-2,2-d im eth ylp r op yl Es-
ter (40). Flash column chromatography eluting with 19:1 v/v
hexane-ethyl acetate afforded the product 40 (92%) as a
yellow oil. IR (neat) cm-1: 1716, 1666, 1599, 1461, 1271, and
1135.
Electr op h or esis Meth od for Top oisom er a se II In h ibi-
tion . The method is in Supporting Information.
Ben zoic Acid 3-(3-Meth oxy-1,4-d ioxo-1,4-d ih yd r on a p h -
th a len -2-yl)-2,2-d im eth ylp r op yl Ester (41). Flash column
chromatography eluting with 97:3 v/v hexane-ethyl acetate
afforded the product 41 (91%) as a yellow oil. IR (neat) cm-1
:
Ack n ow led gm en t. S.L. is a Ph.D. student under the
1718, 1668, 1599, 1454, 1269, and 1114.
Royal Golden J ubilee program, the Thailand Research

4438 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 18
Kongkathip et al.
A. J .; Appelt, K.; Davies, J . F., II; Wu, B. W.; Musick, L.
Structure-based design and synthesis of substituted 2-butanols
as nonpeptidic inhibitors of HIV protease: Secondary amide
series. J . Med. Chem. 1996, 39, 2781-2794.
Fund (TRF). We are indebted to the National Research
Council of Thailand (NRCT), Kasetsart University
Research and Development Institute (KURDI), and the
Thailand Research Fund for financial support. The
Biodiversity Research and Training Program (BRT) is
acknowledged for supporting in part the bioactivity
tests. The HRMS data were provided by the BRT and
the School of Chemistry, Leeds University, U.K. Thanks
are also extended to the Postgraduate Education and
Research Program in Chemistry, Mahidol University,
for supporting the X-ray facility. We are also indebted
to Dr. Somchai Pornbanlualap, Department of Biochem-
istry, Faculty of Science, Kasetsart University, for
preliminary testing of rhinacanthin-N and rhinacan-
thin-Q for topoisomerase II inhibition.
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Liu, A. A.; Wang, H. M.; Mao, Y.; Wu, H. Y.; Sanders, M. M.;
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Su p p or tin g In for m a tion Ava ila ble: 1H NMR, 13C NMR,
MS, HR-MS, and elemental analysis data for compounds 1-50,
crystal data, atomic coordinates, equivalent isotropic displace-
ment parameters, anisotropic displacement parameters, bond
lengths and angles, torsion angles, hydrogen coordinates,
isotropic displacement parameters, and observed hydrogen
bonds for 2, 3, and 14, and electrophoresis method for
topoisomerase II inhibition. This material is available free of
charge via the Internet at http://pubs.acs.org. In addition,
tables of coordinates, bond distances, bond angles, and aniso-
tropic thermal parameters have been deposited with the
Crystallographic Data Centre, Cambridge, CB2, 1EW,
England.
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