Anti-tumor agents 255: Novel glycyrrhetinic acid-dehydrozingerone conjugates as cytotoxic agents

Article abstract of DOI:10.1016/j.bmc.2007.06.027

Esterification of glycyrrhetinic acid (GA) with dehydrozingerone (DZ) resulted in a novel cytotoxic GA-DZ conjugate. Based on this exciting finding, we conjugated eleven different DZ analogs with GA or other triterpenoids, including oleanoic acid (OA) or ursolic acid (UA). In an in vitro anti-cancer assay using nine different human tumor cell lines, most of the GA-DZ conjugates showed significant potency. Particularly, compounds 5, 29, and 30 showed significant cytotoxic effects against LN-Cap, 1A9, and KB cells with ED₅₀ values of 0.6, 0.8, and 0.9 μM, respectively. Similar conjugates between DZ and OA or UA were inactive suggesting that the GA component is critical for activity. Notably, although GA-DZ conjugates showed potent cytotoxic activity, the individual components (GA and DZ analogs) were inactive. Thus, GA-DZ conjugates are new chemical entities and represent interesting hits for anti-cancer drug discovery and development.

Full text of DOI:10.1016/j.bmc.2007.06.027

Bioorganic & Medicinal Chemistry 15 (2007) 6193–6199
Anti-tumor agents 255: Novel glycyrrhetinic
acid–dehydrozingerone conjugates as cytotoxic agents
Jin Tatsuzaki,a Masahiko Taniguchi,a Kenneth F. Bastow,b Kyoko Nakagawa-Goto,a
Susan L. Morris-Natschke,a Hideji Itokawa,a Kimiye Babac and Kuo-Hsiung Leea,*
aNatural Products Research Laboratories, School of Pharmacy, University of North Carolina, Chapel Hill, NC 27599-7360, USA
bDivision of Medicinal Chemistry and Natural Products, School of Pharmacy, University of North Carolina,
Chapel Hill, NC 27599-7360, USA
cDepartment of Pharmacognosy, Osaka University of Pharmaceutical Sciences, 4-20-1 Nasahara, Takatsuki, Osaka 569-1094, Japan
Received 26 February 2007; revised 31 May 2007; accepted 12 June 2007
Available online 14 June 2007
Abstract—Esterification of glycyrrhetinic acid (GA) with dehydrozingerone (DZ) resulted in a novel cytotoxic GA–DZ conjugate.
Based on this exciting finding, we conjugated eleven different DZ analogs with GA or other triterpenoids, including oleanoic acid
(OA) or ursolic acid (UA). In an in vitro anti-cancer assay using nine different human tumor cell lines, most of the GA–DZ con-
jugates showed significant potency. Particularly, compounds 5, 29, and 30 showed significant cytotoxic effects against LN-Cap, 1A9,
and KB cells with ED50 values of 0.6, 0.8, and 0.9 lM, respectively. Similar conjugates between DZ and OA or UA were inactive
suggesting that the GA component is critical for activity. Notably, although GA–DZ conjugates showed potent cytotoxic activity,
the individual components (GA and DZ analogs) were inactive. Thus, GA–DZ conjugates are new chemical entities and represent
interesting hits for anti-cancer drug discovery and development.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
Conjugation of two bioactive compounds is now ac-
cepted as an effective strategy for designing ligands,
inhibitors, and other drugs.8 Our group has imple-
mented the conjugation approach in drug discovery,
which has led to promising results with varying com-
pound classes.9 Therefore, based on the diverse bioactiv-
ities of the above-mentioned terpenoids as well as DZ,
we initiated a structure activity relationship (SAR) study
of terpenoid–DZ conjugation. Herein, we report the
syntheses of terpenoid–DZ analogs and their unique
cytotoxic activities (Fig. 1).
Triterpenoids, including glycyrrhetinic acid (GA) (1),
oleanoic acid (OA) (2), and ursolic acid (UA) (3), are
widely distributed in the plant kingdom worldwide and
show various pharmacological activities. Structurally,
OA and UA have a carboxylic acid on the C17 position
rather than the C20 position as in GA and also do not
have a ketone at C11 as does GA. GA (1) is the main
constituent in the roots of the medicinal plant licorice
(Glycyrrhiza glabra L), which is broadly used as a
flavoring and sweetening agent in food products. The
wide-ranging biological activities of GA include anti-
inflammatory,1 anti-viral,2 anti-allergic,3,4 and anti-tu-
mor promoting effects.5 OA (2) and its regioisomer
UA (3) also have interesting biological activities.6 In
addition, a well-known phenolic natural product, dehy-
drozingerone (DZ) (4), possesses anti-inflammatory,
anti-oxidant, and anti-tumor promoting activities.7
2. Results and discussion
Conjugation of DZ with the terpene acids GA, OA, and
UA was achieved by a well-known esterification proce-
dure using 1-(3-dimethylaminopropyl)-3-ethylcarbodii-
mide hydrochloride (EDCI) as condensation reagent in
the presence of 4-dimethylaminopyridine (DMAP).
Among the three initial conjugates, GA–DZ (5), OA–
DZ (6), and UA–DZ (7), only compound 5 displayed
significant cytotoxic activity as described later. This
exciting finding prompted us to synthesize novel
Keywords: Glycyrrhetinic acid; Dehydrozingerone; Conjugation;
Cytotoxicity.
*
Corresponding author. Tel.: +1 919 962 0066; fax: +1 919 966
3893; e-mail: khlee@unc.edu
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.06.027

6194
J. Tatsuzaki et al. / Bioorg. Med. Chem. 15 (2007) 6193–6199
Figure 1. Structures of GA, OA, UA, DZ, and their conjugates.
GA–DZ analogs 29–38 as shown in Scheme 1. DZ (4)
and its analogs (8–17) were derived from the corre-
sponding substituted benzaldehyde (18–28) with acetone
or acetophenone via an aldol reaction. The conjugated
compounds 29–38 were obtained from the resulting
aldol products and GA using the same manner as
described above.
All GA–DZ analogs 29–38 as well as GA–DZ (5) itself
were tested against nine human tumor cell lines: KB,
KB-VIN, lung (A549), ovarian (1A9), colon (HCT-8),
breast (ZR-751), and prostate (PC-3, DU-145, and
LN-Cap).10–13 Doxorubicin was used as a positive
anti-tumor drug control, and GA and DZ were tested
for comparison with the conjugates. The bioassay re-
sults are shown in Table 2.
Conjugates 5–7 were first evaluated against nasophar-
ynx (KB) and multi-drug resistant KB sub-line
expressing P-glycoprotein (KB-VIN) cancer cell lines
as shown in Table 1. Interestingly, only GA–DZ (5)
showed significant activity against these two cell lines
with ED50 values of 1.7 and 2.8 lM, respectively,
whereas its individual components (GA and DZ) were
not active. OA–DZ (6) and UA–DZ (7) were also
inactive. These results suggested that not only was
conjugation important, but that a ketone on C11
and/or the position of carboxylic acid on C20 rather
than C17 might be important factors for the activity.
This exciting result prompted us to explore additional
novel GA–DZ analogs 29–38.
These results indicate that, while conjugation between
GA and DZ is necessary for significant cytotoxicity,
the different substitution patterns on the DZ portion
obviously affected the selectivity as well as activity
against the nine tested cancer cell lines. Generally, con-
jugate 31, with a 2-GA ester and 4-OMe, was remark-
ably less potent than 5, 29, and 30, which showed
significant cytotoxic effects against most of the cell lines
with ED50 values less than 3.0 lM. This result suggested
that an ortho relation between the methoxy and GA es-
ter groups might be required for strong activity, possibly
related to protein binding. Although both 29 and 31
bear the GA ester on the 2-position, 29 with 3-OMe
Scheme 1. Syntheses of GA–DZ conjugates 29–38.

J. Tatsuzaki et al. / Bioorg. Med. Chem. 15 (2007) 6193–6199
Table 1. Cytotoxicity screening of DZ–terpenoid conjugates
6195
3. Conclusions
Compound
ED50 (lM)a/cell lineb
KB-VIN
In summary, we found that conjugates between GA and
DZ analogs showed significant cytotoxic activity,
although the individual components, GA and DZ, and
similar conjugates between DZ and similar terpenoids,
OA and UA, did not. Novel GA–DZ analogs 5, 29,
and 30, in which the methoxy and GA ester groups have
an ortho relationship, showed significant cytotoxic activ-
ity against most of the tested cell lines. More interest-
ingly, different DZ substitution patterns generated
various cancer cell selectivities, with the highest potency
KB
5 (GA–DZ)
6 (OA–DZ)
7 (UA–DZ)
DZ
1.7
2.8
NAc
10.5
NA
NA
NA
8.3
NA
NA
NA
NA
NA
8.1
GA
OA
UA
a Cytotoxicity as ED50 values for each cell line, the concentration of
compound that caused 50% reduction in absorbance at 562 nm rel-
ative to untreated cells using the sulforhodamine B assay. The
average value is from two independent determinations and variation
(SEM) was no greater than 10%.
b Human epidermoid carcinoma of the nasopharynx (KB), multi-drug
resistant KB sub-line expressing P-glycoprotein (KB-VIN).
c Not active.
against
LN-Cap
(ED50 = 0.6 lM)
and
1A9
(ED50 = 0.9 lM) for 5, KB (ED50 = 0.8 lM) and 1A9
(ED50 = 0.8 lM) for 29, KB (ED50 = 0.9 lM) for 30.
Thus, GA–DZ conjugates represent new hits for anti-
cancer drug discovery and development.
4. Experimental
4.1. General
showed broad spectrum cytotoxicity, while 31 with 4-
OMe was selective against LN-Cap (EC50 = 1.9 lM)
and HCT-8 (EC50 = 2.6 lM) cell lines. Regarding cell
line selectivity, conjugate 5 with 4-GA ester and 3-
OMe showed its highest potency (ED50 = 0.9 and
0.6 lM) against 1A9 and LN-Cap cells, respectively.
Comparatively, conjugate 29 with 2-GA ester and 3-
OMe displayed stronger activities against KB
(EC50 = 0.8 lM), 1A9 (EC50 = 0.8 lM), and PC-3
(EC50 = 1.1 lM) than the remaining cell lines, and con-
jugate 30 with 3-GA ester and 4-OMe showed its stron-
gest activity against KB cells with an ED50 value of
0.9 lM.
The proton nuclear magnetic resonance (1H NMR)
spectra were measured on a Varian Gemini 2000
(300 MHz) NMR spectrometer with TMS as the inter-
nal standard. All chemical shifts are reported in ppm.
Mass spectra were obtained on a Hitachi M-4100H mass
spectrometer. Analytical thin layer chromatography
(TLC) was performed on Merck pre-coated aluminum
silica gel sheets (Kieselgel 60 F 254). Column chroma-
tography was performed on a CombiFlash Companion
system using RediSep normal phase silica columns
(ISCO, Inc., Lincoln, NE). All other chemicals were ob-
tained from Aldrich, Inc. unless otherwise noted.
Replacing the methoxy (5 and 29) with ethoxy (32 and
33) caused decreased activity against all cell lines, with
loss of activity against KB-VIN, A549, ZR-751, and
DU-145 cells. To a lesser extent, a similar trend was
found with a meta-fluoro group (compare 29 and 34).
Replacing the methyl group at R2 with phenyl abolished
the cytotoxic activity (see 35–38).
4.2. General procedure for aldol reaction (4 and 8–17)
The aldol intermediates were obtained by using the same
procedure described by Elias and Rao.14 For 4 and 8–
13, acetone with 1 N NaOH was used, and for 14–17,
Table 2. Data for GA–DZ conjugates against human tumor cell replication
Compound
ED50 (lM)/cell linea
KB
KB-VIN
A549
1A9
HCT-8
ZR-751
PC-3
DU-145
LN-Cap
5
29
1.6
0.8
0.9
6.2
1.8
2.9
3.0
NA
9.9
NA
>14
>21
NA
0.1
2.5
2.8
2.0
2.2
0.9
0.8
1.7
1.9
2.8
3.0
1.4
1.1
3.1
3.6
0.6
2.8
30
31
1.9
2.8
1.6
5.9
2.0
2.6
1.9
2.8
7.4
9.9
6.5
1.9
>15
1.7
15.5
1.7
>15
5.2
>15
5.8
32
33
1.1
1.8
2.7
4.9
3.3
3.5
1.1
6.8
13.2
8.7
3.0
3.2
8.8
2.7
>15
2.7
34
35
1.3
2.2
1.6
4.4
NA
NA
NA
>14
>21
NA
4.97
>14
>14
NA
NA
NA
>52
0.18
>14
13.3
>14
NA
>21
33.9
0.02
>14
>14
>14
>14
19.5
>52
1.20
NA
>14
NA
NA
NA
>52
0.04
>14
14.1
14.1
>14
>21
>52
0.26
>14
>14
>14
13.0
>21
>52
0.15
>14
14.1
14.1
>14
>21
51.0
0.04
36
37
38
GA
DZ
DOXb
a Human epidermoid carcinoma of the lung (A549), ovarian (1A9), colon (HCT-8), breast (ZR-751), prostate (PC-3, DU-145, LN-Cap).
b Doxorubicin.

6196
J. Tatsuzaki et al. / Bioorg. Med. Chem. 15 (2007) 6193–6199
acetophenone in MeOH with 5 N KOH was used. Final
compounds were purified with CombiFlash chromato-
graphy (EtOAc–hexane gradient).
38.47 (C-400). 37.00 (C-1000), 33.79 (CH2), 33.26 (CH3),
33.00 (CH2), 32.56 (CH2), 31.63 (CH2), 30.67 (C-2000),
28.11 (CH3), 27.59 (CH2), 27.45 [–C(O)CH3], 27.19
(CH2), 25.81 (CH3), 23.61 (CH3), 23.44 (CH2), 23.08
(CH2), 18.33 (CH2), 17.21 (CH3), 15.45 (CH3), 15.30
(CH3); MS m/z 631 [M+H]+; HR-SIMS m/z 669.4005
[M+K]+ (calcd for C41H58O5K, 669.3921).
4.3. General procedure for esterification (5 and 29–38)
A CH2Cl2 solution of the same mol ratio of the corre-
sponding terpenoid and DZ analog (4 and 8–17) with
a twofold mol ratio of EDCI and DMAP was stirred
at room temperature under inert atmosphere overnight.
The crude mixture was extracted with CH2Cl2, and the
organic phase was washed with brine, dried over NaSO4,
and concentrated in vacuo to obtain the product as a so-
lid. The crude solid was purified with CombiFlash chro-
matography (EtOAc–hexane gradient).
4.3.3. DZ–UA conjugate (7). 1H NMR (300 MHz,
CDCl3): d 7.46 (d, 1H, J = 16.3 Hz, 1-H), 7.06 (dd,
1H, J = 8.1, 1.8 Hz, 60-H), 7.09 (br s, 1H, 20-H), 6.96
(d, 1H, J = 8.1 Hz, 50-H), 6.60 (d, 1H, J = 16.3 Hz,
2-H), 5.31 (t, 1H, J = 3.9 Hz, 1200-H), 3.82 (s, 3H,
OCH3), 3.21 (br s, 1H, 300a-H), 2.35 (d, 1H,
J = 11.1 Hz, 18-H), 2.38 (s, 3H, 4-CH3), 1.13 (s, 3H,
CH3), 1.00 (s, 3H, CH3), 0.97 (d, 3H, J = 6.3 Hz,
CH3), 0.94 (s, 3H, CH3), 0.89 (d, 3H, J = 4.5 Hz,
CH3), 0.85 (s, 3H, CH3), 0.79 (s, 3H, CH3); 13C NMR
(300 MHz, CDCl3) d 198.22 (C-3, C@O in DZ), 175.22
(C-3000, –C(O)O–DZ), 151.86 (C-30 or 40), 142.89
[–CH@CHC(O)Me in DZ], 142.21 (C-40 or 30), 137.70
(@CH–), 132.97 (C-10), 127.09 [–CH@CHC(O)Me in
DZ], 126.02 (@CH–), 123.41 (@CH–), 121.52 (@CH–),
111.21 (C-20), 79.03 (C-300, CH–OH in UA), 55.80
(–OCH3 in DZ), 55.25 (C-500, CH), 52.95 (C-1800 or 900,
CH), 48.78 (C-1800 or 900, CH), 47.60 (C-1700), 42.26 (C-
1400), 39.70 (C-800), 39.19 (C-1900 or 2000, CH), 38.83 (C-
1900 or 2000, CH), 38.76 (C-100, CH2), 38.68 (C-4000), 37.00
(C-1000), 36.54 (CH2) 33.26 (CH3), 30.79 (CH2 ), 28.15
(CH3), 27.46 [–C(O)CH3], 27.25 (CH2), 24.37 (CH2),
23.40 (CH2), 21.18 (CH3), 18.35 (CH2), 17.53 (CH3),
16.99 (CH3), 15.63 (CH3), 15.52 (CH3); HR-SIMS m/z
669.3967; [M+K]+ (calcd for C41H58O5K, 669.3921).
MS m/z 631 [M+H]+.
4.3.1. DZ–GA conjugate (5). 1H NMR (300 MHz,
CDCl3) d 7.47 (d, 1H, J = 16.3 Hz, 1-H), 7.17–7.12 (m,
2H, 20-H and 60-H), 7.01 (d, 1H, J = 8.1 Hz, 50-H),
6.60 (d, 1H, J = 16.3 Hz, 2-H), 5.70 (br s, 1H, 1200-H),
3.87 (s, 3H, OCH3), 3.22 (br s, 1H, 3b-H), 2.78 (br d,
1H, J = 13.5 Hz, 100b-H), 2.44–2.38 (m, 1H, 100a-H),
2.39 (s, 3H, 4-H), 2.35 (s, 1H, 900a-H), 1.40 (s, 3H,
CH3), 1.35 (s, 3H, CH3), 1.15 (s, 3H, CH3), 1.14 (s,
3H, CH3), 1.01 (s, 3H, CH3), 0.88 (s, 3H, CH3), 0.81
(s, 3H, CH3); 13C NMR (300 MHz, CDCl3) d 200.22
(C1100, C@O in GA), 198.18 (C-3, C@O in DZ),
174.33 [C-3000, GA–C(O)O–DZ], 169.14 (C-1300,
–C@C– in GA), 151.57 (C-30) 142.76 [–CH@CHC(O)Me
in DZ], 141.74 (C-40), 133.29 (C-10), 128.55
[–CH@CHC(O)Me in DZ], 127.29 (@CH–), 123.21
(@CH–), 121.49 (@CH–), 111.25 (@CH–), 78.78 (C-300,
CH–OH in GA), 61.83 (C-900, CH), 55.74 (–OCH3 in
DZ), 54.97 (C-500, CH), 48.10 (C-1800, CH), 45.36,
44.44, and 43.21 (C-80, 1400, and 2000), 41.27 (C-1900,
CH2), 39.12 (C-100 or 2100, CH2), 37.46 (C-2100 or 100,
CH2), 37.08 (C-1000 or C-400), 32.79 (C-700, CH2), 31.87
(C-400, or C1000), 31.21 (C-700 or 2100, CH2), 28.54 (C-
2800, CH3), 28.30 (C-2400, CH3), 28.11 (C-2700, CH3),
27.47 [C-1, –C(O)CH3], 27.31 (C-1500 or C-1600, CH2),
26.47 (C-1600 and C-1500, CH2 · 2), 23.40 (C-2900, CH3),
18.73 (CH3), 17.47 (C-600, CH2), 16.35 (C-2500, CH3),
15.56 (C-2300, CH3); HR-SIMS m/z 645.4159 [M+H]+
(calcd for C41H57O6, 645.4152).
4.3.4. DZ–GA conjugate (29). 1H NMR (300 MHz,
CDCl3) d 7.56 (d, 1H, J = 16.4 Hz, 1-H), 7.26–7.19
(m, 2H, 40-H and 60-H), 7.02–6.99 (m, 1H, 50-H)
6.68 (d, 1H, J = 16.4 Hz, 2-H), 5.69 (s, 1H, 1200-H),
3.84 (s, 3H, OCH3), 3.23 (dd, 1H, J = 10.2, 6.3 Hz,
300a-H), 2.77 (ddd, 1H, J = 13.7, 3.6, 3.6 Hz, 100b-H),
2.53 (br d, 1H, J = 13.7 Hz, 100a-H) 2.35 (s, 1H, 900a-
H), 2.33 (s, 3H, 4-H), 1.45 (s, 3H, CH3), 1.41 (s,
3H, CH3), 1.15 (s, 3H, CH3), 1.13 (s, 3H, CH3), 1.01
(s, 3H, CH3), 0.91 (s, 3H, CH3), 0.81 (s, 3H, CH3);
13C NMR (300 MHz, CDCl3) d 200.16 (C1100, C@O
in GA), 197.74 (C-3, C@O in DZ), 174.11 (C-3000,
–C(O)O–DZ), 169.11 (C-1300, –C@C– in GA), 161.61
(C-30 or 20), 138.96 (C-20 or 30), 136.23
[–CH@CHC(O)Me in DZ], 129.22 (@CH–), 128.63
(@CH–), 128.54 (C-10), 126.60 (@CH–), 118.38
(@CH–), 113.79 (@CH–), 78.75 (C-300, CH–OH in
GA), 61.82 (C-900, CH), 55.80 (–OCH3 in DZ), 54.95
(C-500, CH), 47.77 (C-1800, CH), 45.37, 44.68, and
43.20 (C-80, 1400 or 2000), 41.27 (C-1900, CH2), 39.12
(C-100 or 2200, CH2), 37.08 (C-1000), 32.78 (C-700 or
2100, CH2), 31.88 (C-400), 31.30 (C-700 or 2100, CH2),
28.62 (CH3), 28.54 (CH3), 28.09 (CH3), 27.50
[–C(O)CH3], 27.29 (CH2), 26.53 (CH2), 26.40 (C-1600
or -1500, CH2), 23.42 (C-2900, CH3), 18.71 (CH3),
17.49 (C-600, CH2), 16.33 (C-2500, CH3), 15.56 (C-2300,
CH3); HR-SIMS m/z 645.4160 [M+H]+ (calcd for
C41H57O6, 645.4152).
4.3.2. DZ–OA conjugate (6). 1H NMR (300 MHz,
CDCl3): d 7.46 (d, 1H, J = 16.3 Hz, 1-H), 7.16–7.10
(m, 2H, 20-H and 60-H), 6.97 (d, 1H, J = 8.1 Hz, 50-H),
6.60 (d, 1H, J = 16.3 Hz, 2-H), 5.32 (br s, 1H, 1200-H),
3.83 (s, 3H, OCH3), 3.22 (br s, 1H, 300a-H), 2.89 (m,
1H, 1800-H), 2.38 (s, 3H, 4-CH3), 1.19 (s, 3H, CH3),
1.00 (s, 3H, CH3), 0.94 (s, 3H, CH3), 0.92 (s, 3H,
CH3), 0.90 (s, 3H, CH3), 0.85 (s, 3H, CH3), 0.79 (s,
3H, CH3); 13C NMR (300 MHz, CDCl3) d 198.23 (C-
3, C@O in DZ), 175.48 (OA–C(O)O–DZ), 172.88 (C-
1300, –C@C– in OA), 151.85 (C-30), 143.20
[–CH@CHC(O)Me in DZ], 142.90 (C-40), 142.20
(@CH–), 132.97 (C-10), 127.09 [–CH@CHC(O)Me in
DZ], 123.45 (@CH–), 123.04 (@CH–), 122.73 (@CH–),
111.20 (C0-2), 79.00 (C-300, CH–OH in OA), 55.080 1
(–OCH3 in DZ), 55.23 (C-500, CH), 48.35 (C-17 ),
47.62 (C-900, CH), 46.55 (C-1900, CH2), 41.80 (C-1400),
41.32 (C-1800, CH), 39.43 (C-800), 38.75 (C-100, CH2),

J. Tatsuzaki et al. / Bioorg. Med. Chem. 15 (2007) 6193–6199
6197
4.3.5. DZ–GA conjugate (30). 1H NMR (300 MHz,
CDCl3) d 7.44 (d, 1H, J = 16.3 Hz, 1-H), 7.40 (dd, 1H,
J = 8.5, 2.2 Hz, 60-H), 7.20 (d, 1H, J = 2.2 Hz, 20-H),
6.98 (d, 1H, J = 8.5 Hz, 50-H) 6.59 (d, 1H, J = 16.3 Hz,
2-H), 5.70 (s, 1H, 1200-H), 3.87 (s, 3H, OCH3), 3.22 (m,
1H, 300a-H), 2.78 (ddd, J = 13.5, 3.5, 3.5 Hz, 100b-H),
2.42 (br d, 1H, J = 13.5, 2.8 Hz, 100a-H), 2.37 (s, 3H, 4-
H), 2.35 (s, 1H, 900a-H), 1.40 (s, 3H, CH3), 1.37 (s, 3H,
CH3), 1.15 (s, 3H, CH3), 1.14 (s, 3H, CH3), 1.01 (s,
3H, CH3), 0.89 (s, 3H, CH3), 0.81 (s, 3H, CH3); 13C
NMR (300 MHz, CDCl3) d 200.24 (C1100, C@O in
GA), 198.21 (C-3, C@O in DZ), 174.42 (C-3000,
–C(O)O–DZ), 169.19 (C-1300, –C@C– in GA), 155.12
(C-30 or 40), 142.34 [–CH@CHC(O)Me in DZ], 140.08
(C-40 or 30), 128.52 (@CH–), 127.77 (C-10), 127.48
(@CH–), 125.94 (@CH–), 121.99 (@CH–), 112.40
(@CH–), 78.76 (C-300, CH–OH in GA), 61.83 (C-900,
CH), 55.86 (–OCH3 in DZ), 54.94 (C-500, CH), 48.07
(C-1800, CH), 45.39, 44.42, and 43.20 (C-80, 1400 or 2000),
41.25 (C-1900, CH2), 39.13 (C-100 or 2200, CH2), 37.45
(C-1000), 37.08 (C-100 or 2200, CH2), 32.78 (C-700 or 2100,
CH2), 31.88 (C-400), 31.22 (C-700 or 2100, CH2), 28.58
(CH3), 28.34 (CH3), 28.09 (CH3), 27.44 [–C(O)CH3],
27.30 (CH2), 26.53 (CH2), 26.44 (C-1600 or -1500, CH2),
23.40 (C-2900, CH3), 18.71 (CH3), 17.49 (C-600, CH2),
16.36 (C-2500, CH3), 15.57 (C-2300, CH3); HR-SIMS m/z
645.4157 [M+H]+ (calcd for C41H57O6, 645.4152).
3.4, 3.4 Hz, 100b-H), 2.35–2.40 (m, 1H, 100a-H), 2.39 (s,
3H, 4-H), 2.35 (s, 1H, 900a-H), 1.40 (t, 3H, J = 6.9 Hz,
OCH2CH3), 1.40 (s, 3H, CH3), 1.37 (s, 3H, CH3), 1.14
(s, 3H, CH3), 1.13 (s, 3H, CH3), 1.01 (s, 3H, CH3),
0.87 (s, 3H, CH3), 0.81 (s, 3H, CH3); 13C NMR
(300 MHz, CDCl3) d 200.14 (C1100, C@O in GA),
198.25 (C-3, C@O in DZ), 174.24 (C-3000, –C(O)O–
DZ), 169.02 (C-1300, –C@C– in GA), 151.00 (C-40 or
30), 142.88 [–CH@CHC(O)Me in DZ], 141.71 (C-40 or
30), 133.22 (C-10), 128.63 (@CH–), 127.19 (@CH–),
123.67 (@CH–), 121.24 (@CH–), 111.96 (@CH–),
108.46 (@CH–), 78.75 (C-300, CH–OH in GA), 64.23
(–OCH2CH3 in DZ), 61.82 (C-900, CH), 54.94 (C-500,
CH), 48.11 (C-1800, CH), 45.39, 44.40, and 43.18 (C-80,
1400 or 2000), 41.11 (C-1900, CH2), 39.12 (C-100 or 2200,
CH2), 37.62 (C-100 or 2200, CH2), 37.09 (C-1000), 32.75
(C-700 or 2100, CH2), 31.92 (C-400), 31.14 (C-700 or 2100,
CH2), 28.49 (CH3), 28.38 (CH3), 28.08 (CH3), 27.45
[–C(O)CH3], 27.29 (CH2), 26.50 (CH2), 26.43 (CH2),
23.45 (CH3), 18.68 (CH3), 17.47 (C-600, CH2), 16.33 (C-
2500, CH3), 15.55 (C-2300, CH3), 14.85 (OCH2CH3 in
DZ); HR-SIMS m/z 659.4315 [M+H]+ (calcd for
C42H59O6, 659.4309).
4.3.8. DZ–GA conjugate (33). 1H NMR (300 MHz,
CDCl3) d 7.59 (d, 1H, J = 16.5 Hz, 1-H), 7.23–7.17 (m,
2H, 50-H and 60-H), 6.99 (dd, 1H, J = 6.8, 2.9 Hz, 40-
H), 6.68 (d, 1H, J = 16.5 Hz, 2-H), 5.70 (s, 1H, 1200-H),
4.10 (q, 2H, J = 6.9 Hz, OCH2CH3), 3.23 (m, 1H, 300a-
H), 2.78 (ddd, J = 9.9, 3.6, 3.6 Hz, 100b-H), 2.49 (m,
1H, 100a-H), 2.33 (s, 3H, 4-H), 2.35 (s, 1H, 900a-H),
1.39 (t, 3H, J = 6.9 Hz, OCH2CH3), 1.46 (s, 3H, CH3),
1.41 (s, 3H, CH3), 1.14 (s, 3H, CH3), 1.23 (s, 3H,
CH3), 1.01 (s, 3H, CH3), 0.91 (s, 3H, CH3), 0.81 (s,
3H, CH3); 13C NMR (300 MHz, CDCl3) d 200.07
(C1100, C@O in GA), 197.66 (C-3, C@O in DZ),
174.03 (C-3000, –C(O)O–DZ), 168.97 (C-1300, –C@C– in
GA), 150.98 (C-20 or 30), 138.96 (C-20 or 30), 136.35
(@CH–), 129.11 (@CH–), 128.78 (@CH–), 128.69
(@CH–), 126.56 (@CH–), 118.07 (@CH–), 114.38
(@CH–), 78.76 (C-300, CH–OH in GA), 64.28
(–OCH2CH3 in DZ), 61.83 (C-900, CH), 54.95 (C-500,
CH), 47.69 (C-1800, CH), 45.39, 44.64, and 43.17 (C-80,
1400 or 2000), 41.07 (C-1900, CH2), 39.12 (C-100 or 2200,
CH2), 37.80 (C-100 or 2200, CH2), 37.09 (C-1000), 32.75
(C-700 or 2100, CH2), 31.96 (C-400), 31.27 (C-700 or 2100,
CH2), 28.92 (CH3), 28.43 (CH3), 28.09 (CH3), 27.72
[–C(O)CH3], 27.30 (CH2), 26.50 (CH2), 26.36 (CH2),
23.54 (CH3), 18.70 (CH3), 17.49 (C-600, CH2), 16.34
(C-2500, CH3), 15.56 (C-2300, CH3), 14.92 (OCH2CH3 in
DZ); HR-SIMS m/z 659.4311 [M+H]+ (calcd for
C42H59O6, 659.4309).
4.3.6. DZ–GA conjugate (31). 1H NMR (300 MHz,
CDCl3) d 7.63 (d, 1H, J = 8.8 Hz, 60-H), 7.53 (d, 1H,
J = 16.2 Hz, 1-H), 6.84 (dd, 1H, J = 8.8, 2.5 Hz, 50-H)
6.56 (d, 1H, J = 2.5 Hz, 30-H), 6.61 (d, 1H,
J = 16.2 Hz, 2-H), 5.67 (s, 1H, 1200-H), 3.85 (s, 3H,
OCH3), 3.23 (ddd, 1H, J = 9.9, 6.3, 6.3 Hz, 300a-H),
2.78 (ddd, J = 13.7, 3.5, 3.5 Hz, 100b-H), 2.30 (br d,
1H, J = 13.7, 3.5 Hz, 100a-H), 2.31 (s, 3H, 4-H), 2.35 (s,
1H, 900a-H), 1.45 (s, 3H, CH3), 1.40 (s, 3H, CH3), 1.14
(s, 3H, CH3), 1.13 (s, 3H, CH3), 1.01 (s, 3H, CH3),
0.89 (s, 3H, CH3), 0.81 (s, 3H, CH3); 13C NMR
(300 MHz, CDCl3) d 199.99 (C1100, C@O in GA),
197.63 (C-3, C@O in DZ), 174.63 (C-3000, –C(O)O–
DZ), 168.40 (C-1300, –C@C– in GA), 162.22 (C-40
or 20), 150.98 (C-40 or 20), 136.01 (@CH–), 128.80
(@CH–), 128.11 (@CH–), 126.48 (@CH–), 120.00
(C-10), 112.48 (@CH–), 108.46 (@CH–), 78.76 (C-300,
CH–OH in GA), 61.84 (C-900, CH), 55.65 (–OCH3 in
DZ), 54.96 (C-500, CH), 48.24 (C-1800, CH), 45.39,
44.63, and 43.22 (C-80, 1400 or 2000), 41.04 (C-1900,
CH2), 39.12 (C-100 or 2200, CH2), 37.91 (C-100 or 2200,
CH2), 37.11 (C-1000), 32.79 (C-700 or 2100, CH2), 31.98
(C-400), 31.24 (C-700 or 2100, CH2), 28.57 (CH3), 28.30
(CH3), 28.10 (CH3), 27.64 [–C(O)CH3], 27.31 (CH2),
26.46 (CH2), 26.39 (CH2), 23.46 (C-2900, CH3), 18.70
(CH3), 17.50 (C-600, CH2), 16.34 (C-2500, CH3), 15.55
(C-2300, CH3); HR-SIMS m/z 645.4145 [M+H]+ (calcd
for C41H57O6, 645.4152).
4.3.9. DZ–GA conjugate (34). 1H NMR (300 MHz,
CDCl3) d 7.53 (d, 1H, J = 16.5 Hz, 1-H), 7.45–7.17 (m,
3H, 40, 50, and 60-H), 6.72 (d, 1H, J = 16.5 Hz, 2-H),
5.68 (s, 1H, 1200-H), 3.23 (m, 1H, 300a-H), 2.78 (ddd,
J = 9.6, 6.0, 6.0 Hz, 100b-H), 2.38 (m, 1H, 100a-H), 2.35
(s, 3H, 4-H), 2.35 (s, 1H, 900a-H), 1.45 (s, 3H, CH3),
1.40 (s, 3H, CH3), 1.14 (s, 3H, CH3), 1.13 (s, 3H,
CH3), 1.01 (s, 3H, CH3), 0.89 (s, 3H, CH3), 0.81 (s,
3H, CH3); 13C NMR (300 MHz, CDCl3) d 200.11
(C1100, C@O in GA), 197.36 (C-3, C@O in DZ),
4.3.7. DZ–GA conjugate (32). 1H NMR (300 MHz,
CDCl3) d 7.47 (d, 1H, J = 16.2 Hz, 1-H), 7.13 (dd, 1H,
J = 8.0, 1.9 Hz, 60-H), 7.11 (d, 1H, J = 1.9 Hz, 20-H),
6.98 (d, 1H, J = 8.0 Hz, 50-H) 6.59 (d, 1H, J = 16.2 Hz,
2-H), 5.71 (s, 1H, 12-H), 4.10 (q, 2H, J = 6.9 Hz,
OCH2CH3), 3.23 (m, 1H, 300a-H), 2.78 (ddd, J = 13.5,

6198
J. Tatsuzaki et al. / Bioorg. Med. Chem. 15 (2007) 6193–6199
173.75 (C-3000, –C(O)O–DZ), 168.61 (C-1300, –C@C– in
GA), 156.26 (C-30 or 20), 137.54 (C-30 or 20), 134.99
(@CH–), 129.97 (C-10), 129.76 (@CH–), 128.76 (@CH–),
126.93 (@CH–), 122.29 (@CH–), 118.18 (@CH–), 78.77
(C-300, CH–OH in GA), 61.83 (C-900, CH), 54.96 (C-500,
CH), 47.98 (C-1800, CH), 45.38, 44.82, and 43.18 (C-80,
1400 or 2000), 41.16 (C-1900, CH2), 39.13 (C-100 or 2200,
CH2), 37.57 (C-100 or 2200, CH2), 37.10 (C-1000), 32.78
(C-700 or 2100, CH2), 31.89 (C-400), 31.29 (C-700 or 2100,
CH2), 28.42 (CH3), 28.38 (CH3), 28.10 (CH3), 27.79
[–C(O)CH3], 27.31 (CH2), 26.49 (CH2), 26.41 (CH2),
23.43 (CH3), 18.70 (CH3), 17.49 (C-600, CH2), 16.34 (C-
2500, CH3), 15.61 (C-2300, CH3); HR-SIMS m/z 633.3949
[M+H]+ (calcd for C40H54O5F5, 633.3953).
(C-1800, CH), 45.37, 44.68, and 43.20 (C-80, 1400 or 2000),
41.30 (C-1900, CH2), 39.12 (C-100 or 2200, CH2), 37.47
(C-100 or 2200, CH2), 37.08 (C-1000), 32.79 (C-700 or 2100,
CH2), 31.85 (C-400), 31.31 (C-700 or 2100, CH2), 28.55
(CH3), 28.51 (CH3), 28.09 (CH3), 27.30 (CH2), 26.55
(CH2), 26.42 (CH2), 23.39 (CH3), 18.71 (CH3), 17.50
(C-600, CH2), 16.34 (C-2500, CH3), 15.56 (C-2300, CH3);
HR-SIMS m/z 707.4315 [M+H]+ (calcd for C46H59O6,
707.4309).
4.3.12. DZ–GA conjugate (37). 1H NMR (300 MHz,
CDCl3) d 8.02–7.45 (m, 5H, 2000-6000-H) 7.74 (d, 1H,
J = 15.7 Hz, 1-H), 7.39 (d, 1H, J = 15.7 Hz, 2-H), 7.49
(dd, 1H, J = 8.3 Hz, 60-H), 7.31 (d, 1H, J = 1.9 Hz, 20-
H), 7.00 (d, 1H, J = 8.5 Hz, 50-H), 5.72 (s, 1H, 1200-H),
3.89 (s, 3H, OCH3), 3.23 (m, 1H, 300a-H), 2.78 (dm,
1H, J = 13.7 Hz, 100b-H), 2.44 (dm, 1H, J = 13.7 Hz,
100a-H), 2.35 (s, 1H, 900a-H), 1.40 (s, 3H, CH3), 1.39 (s,
3H, CH3), 1.16 (s, 3H, CH3), 1.14 (s, 3H, CH3), 1.01
(s, 3H, CH3), 0.89 (s, 3H, CH3), 0.81 (s, 3H, CH3);
13C NMR (300 MHz, CDCl3) d 200.21 (C1100, C@O in
GA), 190.53 (C-3, C@O in DZ), 174.45 (C-3000,
–C(O)O–DZ), 169.19 (C-1300, –C@C– in GA), 153.17
(C-40 or 30), 143.85 [–CH@CHC(O)–Ph in DZ], 140.10
(C-40 or 30), 138.33 (C-4), 132.61 (@CH–), 128.57
(C · 2 in Ph), 128.47 (C · 2 in Ph), 128.28 (@CH–),
128.15 (@CH–), 128.03 (C-10), 121.93 (@CH–), 120.89
(@CH–), 112.38 (@CH–), 78.76 (C-300, CH–OH in
GA), 61.82 (C-900, CH), 55.86 (–OCH3 in DZ), 54.94
(C-500, CH), 48.00 (C-1800, CH), 45.39, 44.42, and 43.20
(C-80, 1400 or 2000), 41.28 (C-1900, CH2), 39.12 (C-100 or
2200, CH2), 37.47 (C-100 or 2200, CH2), 37.08 (C-1000),
32.77 (C-700 or 2100, CH2), 31.89 (C-400), 31.25 (C-700 or
2100, CH2), 28.58 (CH3), 28.39 (CH3), 28.09 (CH3),
27.30 (CH2), 26.53 (CH2), 26.45 (CH2), 23.41 (CH3),
18.71 (CH3), 17.49 (C-600, CH2), 16.36 (C-2500, CH3),
15.57 (C-2300, CH3); HR-SIMS m/z 707.4305 [M+H]+
(calcd for C46H59O6, 707.4309).
4.3.10. DZ–GA conjugate (35). 1H NMR (300 MHz,
CDCl3) d 8.03–7.49 (m, 5H, 2000-6000-H) 7.77 (d, 1H,
J = 15.8 Hz, 1-H), 7.46 (d, 1H, J = 15.8 Hz, 2-H), 7.26
(dd, 1H, J = 8.3 Hz, 60-H), 7.21 (d, 1H, J = 1.9 Hz, 20-
H), 7.04 (d, 1H, J = 8.3 Hz, 50-H), 5.71 (s, 1H, 1200-H),
3.90 (s, 3H, OCH3), 3.23 (dd, 1H, J = 10.0, 5.9 Hz,
300a-H), 2.78 (ddd, 1H, J = 13.5, 3.2, 3.2 Hz, 100b-H),
2.41 (br d, 1H, J = 13.5 Hz, 100a-H), 2.35 (s, 1H, 900a-
H), 1.40 (s, 3H, CH3), 1.37 (s, 3H, CH3), 1.16 (s, 3H,
CH3), 1.14 (s, 3H, CH3), 1.01 (s, 3H, CH3), 0.89 (s,
3H, CH3), 0.81 (s, 3H, CH3); 13C NMR (300 MHz,
CDCl3) d 200.48 (C1100 C@O in GA), 190.80 (C-3,
C@O in DZ), 174.58 (C-3000, –C(O)O–DZ), 169.40 (C-
1300, –C@C– in GA), 151.78 (C-40 or 30), 144.51
[–CH@CHC(O)Ph in DZ], 141.95 (C-40 or 30), 138.43
(C-4), 133.99 (C-10), 133.03 (@CH–), 128.87 (C · 2 in
Ph), 128.76 (C · 2 in Ph), 123.44 (@CH–), 122.57
(@CH–), 121.62 (@CH–), 112.03 (@CH–), 79.01 (C-300,
CH–OH in GA), 62.07 (C-900, CH), 56.03 (–OCH3 in
DZ), 55.19 (C-500, CH), 48.33 (C-1800, CH), 45.63,
44.68, and 43.45 (C-80, 1400 or 2000), 41.52 (C-1900,
CH2), 39.36 (C-100 or 2200, CH2), 37.69 (C-100 or 2200,
CH2), 37.32 (C-1000), 33.02 (C-700 or 2100, CH2), 32.12
(C-400), 31.46 (C-700 or 2100, CH2), 28.80 (CH3), 28.54
(CH3), 28.32 (CH3), 27.55 (CH2), 26.69 (CH2), 23.64
(CH2), 18.95 (CH3), 17.73 (C-600, CH2), 16.60 (C-2500,
CH3), 15.80 (C-2300, CH3); HR-SIMS m/z 707.4306
[M+H]+ (calcd for C46H59O6, 707.4309).
4.3.13. DZ–GA conjugate (38). 1H NMR (300 MHz,
CDCl3) d 7.99–7.45 (m, 5H, Ar–H) 7.82 (d, 1H,
J = 15.7 Hz, 1-H), 7.40 (d, 1H, J = 15.7 Hz, 2-H), 7.77
(dd, 1H, J = 8.5 Hz, 60-H), 6.87 (dd, 1H, J = 8.8,
2.5 Hz, 50-H), 6.59 (d, 1H, J = 2.5 Hz, 30-H), 5.66 (s,
1H, 1200-H), 3.87 (s, 3H, OCH3), 3.22 (dd, 1H,
J = 10.0, 6.2 Hz, 300a-H), 2.76 (ddd, 1H, J = 13.5, 3.3,
3.3 Hz, 100b-H), 2.29 (br dd, 1H, J = 13.5, 4.6 Hz, 100a-
H), 2.34 (s, 1H, 900a-H), 1.41 (s, 3H, CH3), 1.39 (s, 3H,
CH3), 1.12 (s, 3H, CH3), 1.12 (s, 3H, CH3), 1.00 (s,
3H, CH3), 0.86 (s, 3H, CH3), 0.80 (s, 3H, CH3); 13C
NMR (300 MHz, CDCl3) d 200.02 (C1100, C@O in
GA), 190.27 (C-3, C@O in DZ), 174.74 (C-3000,
–C(O)O–DZ), 168.57 (C-1300, –C@C– in GA), 162.26
(C-40 or 20), 151.29 (C-40 or 20), 138.33 (C-4), 137.61
(@CH–), 132.55 (@CH–), 128.73 (@CH–), 128.55
(C · 2 in Ph), 128.42 (C · 2 in Ph), 128.00 (@CH–),
121.61 (@CH–), 120.56 (C-10), 112.53 (@CH–), 108.40
(@CH–), 78.77 (C-300, CH–OH in GA), 61.80 (C-900,
CH), 55.68 (–OCH3 in DZ), 54.93 (C-500, CH), 48.22
(C-1800, CH), 45.38, 44.63, and 43.20 (C-80, 1400 or 2000),
41.04 (C-1900, CH2), 39.12 (C-100 or 2200, CH2), 37.80
(C-100 or 2200, CH2), 37.07 (C-1000), 32.78 (C-700 or 2100,
CH2), 31.95 (C-400), 31.24 (C-700 or 2100, CH2), 28.53
4.3.11. DZ–GA conjugate (36). 1H NMR (300 MHz,
CDCl3) d 8.00–7.46 (m, 5H, 2000-6000-H) 7.84 (d, 1H,
J = 15.9 Hz, 1-H), 7.48 (d, 1H, J = 15.9 Hz, 2-H), 7.38
(dd, 1H, J = 8.1, 1.5 Hz, 60-H), 7.25 (dd, 1H, J = 8.1,
8.1 Hz, 50-H), 7.03 (dd, 1H, J = 8.1, 1.5 Hz, 40-H), 5.69
(s, 1H, 1200-H), 3.85 (s, 3H, OCH3), 3.23 (m, 1H, 300a-
H), 2.77 (ddd, 1H, J = 13.5, 3.4, 3.4 Hz, 100b-H), 2.77
(ddd, 1H, J = 13.5, 3.4, 3.4 Hz, 100a-H), 2.34 (s, 1H,
900a-H), 1.40 (s, 3H, CH3), 1.40 (s, 3H, CH3), 1.14 (s,
3H, CH3), 1.13 (s, 3H, CH3), 1.01 (s, 3H, CH3), 0.88
(s, 3H, CH3), 0.80 (s, 3H, CH3); 13C NMR (300 MHz,
CDCl3) d 200.17 (C1100, C@O in GA), 190.24 (C-3,
C@O in DZ), 174.24 (C-3000, –C(O)O–DZ), 151.74 (C-
1300, –C@C– in GA), 139.27 (C-20 or 30), 138.05 (C-20
or 30), 137.73 (@CH–), 132.74 (@CH–), 129.17 (C-4),
128.60 (C · 2 in Ph), 128.50 (C · 2 in Ph), 126.63
(@CH–), 124.47 (@CH–), 118.30 (@CH–), 113.81
(@CH–), 78.76 (C-300, CH–OH in GA), 61.80 (C-900,
CH), 55.84 (–OCH3 in DZ), 54.95 (C-500, CH), 47.79

J. Tatsuzaki et al. / Bioorg. Med. Chem. 15 (2007) 6193–6199
6199
Bastow, K. F.; Tachibana, Y.; Chen, K.; Lee, F. Y.; Lee,
K. H. Bioorg. Med. Chem. 2001, 9, 2999; (d) Ohtsu, H.;
Nakanishi, Y.; Bastow, K. F.; Lee, F. Y.; Lee, K. H.
Bioorg. Med. Chem. 2003, 11, 1851; (e) Nakagawa-Goto,
K.; Nakamura, S.; Bastow, K. F.; Nyarko, A.; Peng, C.
Y.; Lee, Y. F.; Lee, F. C.; Lee, K. H. Bioorg. Med. Chem
Lett. 2007, 17, 2894–2898; (f) Nakagawa-Goto, K.;
Yamada, K.; Nakamura, S.; Chen, T. H.; Bastow, K. F.;
Wang, S. C.; Hung, M. C.; Lee, F. Y.; Lee, F. C.; Lee, K.
H. Bioorg. Med. Chem. Lett., in press.
(CH3), 28.29 (CH3), 28.09 (CH3), 27.30 (CH2), 26.46
(CH2), 26.38 (CH2), 23.44 (CH3), 18.69 (CH3), 17.49
(C-600, CH2), 16.35 (C-2500, CH3), 15.56 (C-2300, CH3);
HR-SIMS m/z 707.4320 [M+H]+ (calcd for C46H59O6,
707.4309).
Acknowledgment
This investigation was supported in part by a grant from
the National Cancer Institute (CA 17625) awarded to K.
H. Lee.
10. The in vitro cytotoxicity assay was carried out according
to procedures described in Rubinstein et al.11 Drug stock
solutions were prepared in DMSO, and the final solvent
concentration was <1% DMSO (v/v), a concentration
without effect on cell replication. The human tumor cell
line panel consisted of epidermoid carcinoma of the
nasopharynx (KB), a multi-drug resistant KB sub-line
expressing P-glycoprotein (KB-VIN), lung (A549), ovar-
ian (1A9), colon (HCT-8), breast (ZR-751), and prostate
(PC-3, DU-145, and LN-Cap). The KB-VIN tumor cell
line is cross-resistant to doxorubicin (Table 1). Detailed
characterization of this cell is described elsewhere.10,11 Cells
were cultured at 37 ꢁC in RPMI-1640 with 100 lg/mL
kanamycin and 10% (v/v) fetal bovine serum in a
humidified atmosphere containing 5% CO2. Initial seeding
densities varied among the cell lines to ensure a final
absorbance of 1–2.5 A562 units. Drug exposure was for 2
days and the ED50 value, the drug concentration that
reduced the absorbance by 50%, was interpolated from
dose–response data. Each test was performed in duplicate,
and absorbance readings varied no more than 5% between
replicates.
References and notes
1. Finney, R. S.; Tarnoky, A. L. J. Pharm. Pharmacol. 1960,
12, 49.
2. Pompei, R.; Flore, O.; Marccialis, M. A.; Pani, A.; Loddo,
B. Nature 1979, 281, 689.
3. Tanaka, S.; Uno, C.; Akimoto, M.; Tabata, M.; Honda,
C.; Kamisako, W. Planta Med. 1991, 57, 527.
4. Ichikawa, Y.; Mizoguchi, Y.; Kioka, K.; Kobayashi, K.;
Tomekawa, K.; Morosawa, S.; Yamamoto, S. Arerugi
1989, 38, 365.
5. Okamoto, H.; Yoshida, D.; Mizusaki, S. Cancer Lett.
1983, 19, 47.
6. (a) Liu, J. J. Ethnopharm. 1995, 49, 57; (b) Liu, J.
J. Ethnopharm. 2005, 100, 92, and references therein.
7. Motohashi, N.; Yamagami, C.; Tokuda, H.; Konoshima,
T.; Okuda, Y.; Okuda, M.; Mukainaka, T.; Nishino, H.;
Saito, Y. Cancer Lett. 1998, 134, 37.
11. Rubinstein, L. V.; Shoemaker, R. H.; Paull, K. D.; Simo,
R. M.; Tosini, S.; Skehan, P.; Scudiero, P. A.; Monks, A.;
Boyd, M. R. J. Natl. Cancer Inst. 1990, 82, 1113.
8. Choi, S. K. Synthetic Multivalent Molecules; Wiley-Inter-
science: New York, 2004.
12. Ferguson, P. J.; Fisher, M. H.; Stephenson, J.; Li, D. H.;
Zhou, B. S.; Cheng, Y. C. Cancer Res. 1988, 48, 5956.
13. Beidler, D. R.; Chang, J. Y.; Zhou, B. S.; Cheng, Y. C.
Cancer Res. 1996, 56, 345.
9. (a) Bastow, K. F.; Wang, H. K.; Cheng, Y. C.; Lee, K. H.
Bioorg. Med. Chem. 1997, 5, 1481; (b) Chang, J. Y.; Guo,
X.; Chen, H. X.; Jiang, Z.; Fu, Q.; Wang, H. K.; Bastow,
K. F.; Zhu, X. K.; Guan, J.; Lee, K. H.; Cheng, T. C.
Biochem. Pharm. 2000, 59, 497; (c) Shi, Q.; Wang, H. K.;
14. Elias, G.; Rao, M. N. A. Eur. J. Med. Chem. 1988, 23, 379.