Antitumor agents 210. Synthesis and evaluation of taxoid-epipodophyllotoxin conjugates as novel cytotoxic agents

Article abstract of DOI:10.1016/S0968-0896(01)00206-1

Five compounds composed of a taxoid (paclitaxel or cephalomannine) and a 4′-O-demethyl epipodophyllotoxin derivative joined by an imine linkage were prepared and evaluated as cytotoxic agents and inhibitors of mammalian DNA topoisomerase II. Compounds 12 and 14-16 exhibited comparable or better activity than the unconjugated epipodophyllotoxin derivatives in most tumor cell lines, and 12, 15, and 16 also showed enhanced activity against paclitaxel-resistant cells. Compound 13, which contains an epipodophyllotoxin moiety at both the taxoid 2′ and 7 positions, did not stimulate protein-DNA breaks, but was 2-fold more potent than 12 and 15 and comparable to GL-331 in the topo II inhibitory assay.

Full text of DOI:10.1016/S0968-0896(01)00206-1

Bioorganic & Medicinal Chemistry 9 (2001) 2999–3004
Antitumor Agents 210. Synthesis and Evaluation of Taxoid–
y
Epipodophyllotoxin Conjugates as Novel CytotoxicAgents
Qian Shi,a Hui-Kang Wang,a Kenneth F. Bastow,a Yoko Tachibana,a Ke Chen,a
Fang-Yu Leeb and Kuo-Hsiung Leea,*
aNatural Products Laboratory, School of Pharmacy, University of North Carolina, Chapel Hill, NC 27599, USA
bYung-Shin Pharmaceutical Industrial Company, 1191, Section 1, Chung-Shan Road, Taichia, Taichung, Taiwan
Received 12 March 2001; accepted 29 May 2001
Abstract—Five compounds composed of a taxoid (paclitaxel or cephalomannine) and a 40-O-demethyl epipodophyllotoxin deriva-
tive joined by an imine linkage were prepared and evaluated as cytotoxic agents and inhibitors of mammalian DNA topoisomerase
II. Compounds 12 and 14–16 exhibited comparable or better activity than the unconjugated epipodophyllotoxin derivatives in most
tumor cell lines, and 12, 15, and 16 also showed enhanced activity against paclitaxel-resistant cells. Compound 13, which contains
an epipodophyllotoxin moiety at both the taxoid 20 and 7 positions, did not stimulate protein–DNA breaks, but was 2-fold more
potent than 12 and 15 and comparable to GL-331 in the topo II inhibitory assay. # 2001 Elsevier Science Ltd. All rights reserved.
Introduction
(CPT) derivative and an etoposide (5) analogue.8 Both
compounds induced protein-linked DNA breaks
(PLDB) in a concentration dependent manner in drug
treated cells. The drug induced PLDB could be medi-
ated by both DNA topoisomerases (topo) I and II. The
two conjugates were more active than CPT against
CPT-resistant cells and than etoposide against etopo-
side-resistant cells. Over-expression of the multiple drug
resistant proteins GP170 or MRP had little impact on
the cytotoxicity. The compounds were equally cytotoxic
as CPT in CPT-sensitive or etoposide-resistant cells and
as etoposide in CPT-resistant cells. In vivo, one con-
jugate (3) was more active than both etoposide and CPT
against human KB and DU-145 tumor cells in nude
mice. This novel topo I and II dual inhibitory property
as well as the unique antitumor action of these CPT-
etoposide analogue conjugates suggests that such bi-mole-
cular models should be further explored as anticancer
drugs.
The complex diterpenoid Taxol1 (paclitaxel, 1) is an
exciting new anticancer drug, which is currently in clin-
ical use against ovarian and breast cancer. It acts by an
unusual mechanism: promoting microtubule assembly.
Since the discovery of paclitaxel as an antimitotic anti-
tumor agent, its chemistry and structure–activity rela-
tionships (SAR) have been extensively studied in order
to develop more potent derivatives and to overcome its
low water solubility and drug resistance problems.2,3
Research has focused on modifications at C2, C4, C7,
C9, C10, and the C13 side chain, and the resulting
structure–activity relationships have led to more potent
paclitaxel derivatives. The 2- and 7-positions have been
used frequently for prodrug and analogue modification,
respectively.4 Substitution at these positions led to dif-
ferent tubulin binding.5À7 Also, selective modification of
the 20-hydroxyl led to synthesis of a series of water-
soluble derivatives.4 However, drug resistance is still a
major problem associated with paclitaxel, and novel
derivatives are highly desirable.
In order to develop paclitaxel analogues with multiple
antitumor mechanisms or improved activity against
drug resistant cells, our attention focused on the design
and synthesis of a series of 20-(400-O-demethyl epipodo-
phyllotoxin) taxoids (Scheme 1). These compounds
possess an interesting bi-molecular structure: a topo II
inhibitory 40-O-demethyl epipodophyllotoxin is con-
jugated with an antimitotic taxoid at the 20 or 7 posi-
tion. This report describes the structures, synthesis, and
Previously, we synthesized and evaluated two com-
pounds (3 and 4) that are conjugates of a camptothecin
*Corresponding author. Tel.: +1-919-962-0066; fax: +1-919-966-
3893; e-mail: khlee@unc.edu
yFor part 209 in this series, see ref 1.
0968-0896/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(01)00206-1

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Q. Shi et al. / Bioorg. Med. Chem. 9 (2001) 2999–3004
Scheme 1. Synthesis of taxoid-epipodophyllotoxin conjugates.
preliminary antitumor testing results of these com-
pounds.
(p-aminoanilino)-4-desoxypodophyllotoxin, 8, was syn-
thesized by treating podophyllotoxin with trimethylsilyl
iodide (TMSI) in methylene chloride, followed by sub-
stitution with p-nitroaniline in the presence of barium
carbonate,13 and finally, by hydrogenation of the
resulting nitro compound. Syntheses of the desired
conjugates (12–16) were accomplished by reaction of 9–
11 with 6 or 8 in refluxing benzene to form an imine
linkage (Scheme 1). The structures of these conjugates
were confirmed unambiguously from NMR and mass
spectral data. The detailed assignments of the NMR
signals are listed in Table 1.
Results and Discussion
The 20-hydroxyl of paclitaxel is more reactive than the
sterically hindered 7-hydroxyl.9À11 Therefore, a 40-O-
demethyl epipodophyllotoxin analogue can be intro-
duced selectively at the 20-position of paclitaxel via a
benzaldehyde bridge. The three mono- and bis-(4-for-
myl)benzoyl taxoids (9–11) were synthesized by reacting
paclitaxel (1) and cephalomannine (2) with 4-carboxy-
benzaldehyde in the presence of 1,3-dicyclohexy-
lcarbodiimide (DCC) and 4-dimethylaminopyridine (4-
DMAP) (Scheme 1), respectively. 40-O-Demethyl-4b-
amino-4-desoxypodophyllotoxin, 6, was synthesized
according to a literature method.12 40-O-Demethyl-4b-
The newly synthesized taxoid-40-O-demethyl epipodo-
phyllotoxin conjugates were evaluated in house using
standard cytotoxicity assays in human tumor (HTCL)
and drug-resistant (DRCL) cell lines. Reference com-
pounds were paclitaxel (1), cephalomannine (2), and

Table 1. 1H NMR assignments of taxoids and their conjugates
Proton no.
9
10
11
12
13
14
15
16
Signals belonging to taxoid portion
2
3
5
5.68 (d, 7)
4.20 (d, 8)
4.98 (d, 9)
1.90 (tt, 13, 3)
4.45 (m)
5.80 (d, 7)
4.06 (d, 8)
5.02 (d,9)
1.92 (m)
5.81 (dd, 10, 4)
6.38 (s)
6.25 (t, 9)
2.17 (dd, 15, 9)
2.38 (dd, 15, 9)
1.21 (s)
1.18 (s)
1.97 (s)
1.95 (s)
4.37 (d, 8)
4.25 (d, 8)
5.75 (d, 4)
6.09 (dd, 9, 4)
7.07 (d, 8)
8.13 (d, 8)
7.53 (d, 8)
7.64 (t, 8)
7.93 (d, 8)
8.07 (d, 8)
8.15 (d, 8)
7.97 (d, 8)
7.48 (m)
5.68 (d, 7)
3.81 (d, 7)
4.97 (d, 9)
1.87 (m)
4.46 (m)
6.30 (s)
5.68 (d, 7)
3.81 (d, 7)
4.97 (d, 10)
1.88 (br.t, 13)
4.44 (dd, 10, 7)
6.30 (s)
6.26 (t, 9)
2.14 (dd, 15, 9)
2.33 (dd, 15, 9)
1.23 (s)
5.69 (d, 7)
4.04 (dd, 7, 2)
5.04 (br.d, 9)
1.92 (m)
5.82 (m)
6.38 (s)
6.24 (t, 9)
2.18 (m)
2.36 (m)
1.26 (s)
1.19 (s)
1.97 (s)
1.96 (s)
4.37 (d, 8)
4.27 (d, 8)
5.70 (d, 4)
5.68 (d, 7)
3.81 (d, 7)
4.96 (d, 9)
1.88 (m)
4.44 (m)
6.30 (s)
5.68 (d, 7)
3.82 (d, 7)
4.97 (dd, 8, 2)
1.88 (ddd, 16, 11, 2)
4.45 (dd, 11, 7)
6.30 (s)
6.27 (t, 9)
2.14 (dd, 15, 9)
2.33 (dd, 15, 9)
1.24 (s)
5.68 (d, 7)
3.81 (d, 7)
4.96 (d, 9)
1.88 (br. t, 13 )
4.45 (m)
6.30 (s)
6.26 (br. t, 9)
2.55 (m)
6a, b
7
10
13
14a, b
6.30 (s)
6.27 (t, 9)
2.14 (dd, 15, 9)
2.34 (dd, 15, 9)
1.23 (s)
1.14 (s)
1.97 (s)
1.68 (s)
4.32 (d, 8.4)
4.20 (d, 8)
5.72 (d,4 )
6.08 (dd, 10, 4)
6.97 (d, 9.6)
8.13 (d, 7)
7.52 (t, 7)
7.62 (m)
6.26 (br. t, 9)
2.55 (m)
6.26 (br. t, 9)
2.55 (m)
16-CH3
17-CH3
18-CH3
19-CH3
20a
1.26 (s)
1.15 (s)
1.82 (s)
1.68 (s)
4.30 (d, 8)
4.20 (d, 8)
5.64 (d, 3)
5.94 (dd, 9, 3)
6.55 (d, 9)
8.12 (d, 8)
7.51 (t, 8)
7.62 (t, 8)
—
1.25 (s)
1.14 (s)
1.82 (s)
1.68 (s)
4.30 (d, 8)
4.19 (d, 8)
5.62 (br. s)
5.92 (br. s)
6.56 (d, 9)
8.12 (d, 8)
7.52 (t, 8)
7.62 (t, 8)
—
1.26 (s)
1.15 (s)
1.83 (s)
1.68 (s)
4.29 (d, 8)
4.19 (d, 8)
5.65 (br. s)
5.92 (br. dd, 9, 3)
6.61 (d, 8)
8.11 (d, 8)
7.51 (t, 8)
7.61 (t, 8)
—
1.14 (s)
1.96 (s)
1.68 (s)
1.14 (s)
1.96 (s)
1.68 (s)
4.31 (d, 9)
4.19 (d, 9)
5.70 (d, 4)
6.04 (dd, 9, 4)
7.02 (d, 9)
8.13 (d, 7)
7.52 (d, 7)
7.62 (t, 8)
—
4.31 (d, 9)
4.20 (d, 9)
5.71 (d, 4)
6.05 (dd, 9, 4)
7.06 (d, 9)
8.11 (d, 7.5)
7.52 (d, 7.5)
7.61 (t, 7.5)
—
20b
20
30
6.08 (dd, 9, 4)
30-NH
a
2-OBz (o)
2-OBz (m)
2-OBz (p)
7-OBz (o)
7-OBz (m)
20-OBz (o)
20-OBz (m)
30-Ph (o)
30-Ph (m)
30-Ph (p)
NBz (o)
NBz (m)
NBz (p)
1-OH
8.14 (d, 7)
7.52 (d, 7)
7.64 (t, 7)
7.97 (d, 8)
7.81 (d, 8)
8.08 (d, 8)
7.86 (d, 8)
7.43 (d, 7)
7.47 (t, 7)
7.33 (br.t, 7)
7.75 (d, 7)
7.47 (t, 7)
7.53 (t, 7)
1.97 (s)
—
—
—
—
—
—
—
—
8.14 (d, 8)
7.98 (d, 8)
7.45 (d, 8)
7.41 (t, 8)
7.44 (t, 8)
7.76 (d, 8)
7.52 (br. t, 7)
7.52 (br. t, 7)
1.89 (bt. t, 11, 2)
2.54 (br. s)
—
8.12 (d, 8)
7.97 (d, 8)
7.40 (br.s)
7.40 (br.s)
7.33 (t, 7)
—
8.04 (dd, 7, 2)
7.85 (dd, 7, 2)
7.44 (t, 8)
7.41 (t, 8)
7.43 (d, 8)
7.75 (d, 8)
7.51 (t, 7)
7.52 (t, 7)
1.97 (s)
2.49 (br. s)
—
2.44 (s)
2.23 (s)
—
7.85 (d, 8)
8.01 (d, 8)
7.40 (m)
7.40 (m)
7.32 (m)
—
—
—
—
—
8.15 (d, 8)
7.76 (d, 8)
7.43 (d, 8)
7.41 (t, 8)
7.42 (d, 8)
7.76 (d, 8)
7.51 (t, 7)
7.52 (t, 7)
1.96 (s)
8.11 (br. d, 8)
7.52 (br. d, 8)
7.40 (m)
7.40 (m)
7.32 (m)
—
7.42 (m )
7.35 (dd, 7, 2)
7.77 (br.d, 8)
7.45 (m)
—
—
—
—
—
—
—
—
7.46 (m)
1.92 (br. s)
—
—
2.49 (s)
2.06 (s)
7-OH
20-OH
4-OAc
2.50 (br. s)
—
2.43 (s)
—
—
—
—
2.46 (s)
2.23 (s)
2.43 (s)
2.23 (s)
1.97 (s)
6.43 (br.q, 7)
1.72 (d, 7)
10.11 (s)
2.49 (s)
2.07 (s)
—
2.41 (s)
2.23 (s)
1.96 (br. s)
6.43 (br.q, 7)
1.72 (d, 7)
—
2.41 (s)
2.23 (s)
1.96 (s)
6.45 (br.q, 7)
1.73 (d, 7)
—
10-OAc
200-CH3
300
2.22 (s)
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
400-CH3
CHO
—
10.10 (s), 10.12 (s)
10.13 (s)
S0i0g0 nals belonging to epipodophyllotoxin portion
1000
2
4.61 (br. d, 4)
3.13 (m)
3.11 (m)
4.71 (d, 5)
6.58 (s)
6.39 (s)
4.71 (2H, d, 5)
3.12 (2H, m)
3.05 (2H, m)
4.68 (2H, t, 4)
6.39 (2H, s)
6.60 (2H, s)
4.26 (2H, m)
4.00 (2H, m)
5.98, 5.92 (both 2H,
each br.s)
4.67 (br. s)
3.13 (m)
3.12 (m)
4.71 (d, 5)
6.59 (s)
4.62 (d, 5)
4.61 (br. d, 4)
3.14 (dd, 14, 4)
3.04 (m)
4.73 (br. s)
6.80 (s)
6.54 (s)
3.14 (dd, 15, 5)
3.02 (tdd, 15, 7, 4)
4.73 (dd, 5, 5)
6.80 (s)
6.55 (s)
4.22 (td, 11, 5)
4.00 (td, 11, 5)
3000
4000
5000
8000
11000
6.39 (s)
4.20, 4.27 (t, 9)
4.00, 4.27 (t, 9)
4.01, 4.40 (t, 9)
OCH2O
5.92, 5.98 (br. s)
5.98 (br. s)
5.92, 5.99 (d, 1)
5.96, 5.98 (br. s)
20000,60000
6.39 (s)
3.81 (s)
8.51(s)
—
6.38 (4H, s)
3.81 (6H, s)
8.51 (s), 8.53 (s)
—
6.39 (s)
3.81 (s)
8.51 (s)
—
6.34 (s)
3.80 (s)
8.57 (s)
6.34 (s)
3.80 (s)
8.57 (s)
OCH3Â2
CH¼
NHC6H4–N¼
7.97, 8.07 (d, 9)
7.97, 8.04 (d, 8)
aProton could not be assigned due to complexity of the spectrum.

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Q. Shi et al. / Bioorg. Med. Chem. 9 (2001) 2999–3004
Figure 1. Structures of paclitaxel, cephalomannine, etoposide and its derivatives.
Table 2. Cytotoxicity of 12–16 in selected human tumor cell lines (ED50) (mM)
Compound
KB
A549
HCT-8
CAKI-1
MCF-7
SK-MEL-2
1A9 (OVCAR)
12
13
14
15
0.003
16.5
0.21
NAa
0.70
2.80
3.55
0.48
1.95
0.009
0.006
0.54
NA
2.78
>0.1
>40
3.38
>5
0.047
0.013
>0.25
>4
>5
0.33
>4
1.23
1.86
>5
9.29
>5
0.167
ND
>0.40
NA
8.90
>40
>40
5.95
ND
0.134
ND
0.03
ND
0.03
0.23
1.40
1.86
0.20
0.002
0.016
0.14
0.30
1.12
0.20
0.60
0.003
0.002
>4
16
>10
1.80
2.2
>0.3
NDb
GL-331
Etoposide
Cephalomannine
Paclitaxel
aNA, not active.
bND, not determined.
epipodophyllotoxins [etoposide (5) and GL-331 (7), see
Fig. 1]. The HTCL panel was comprised of KB, A549,
HCT-8, CAKI-1, MCF-7, SK-MEL-2, and IA9 cell
lines. Two paclitaxel-resistant 1A9 sub-lines designated
as PAX-10 and PAX-22 were also tested as part of the
DRCL panel. Other resistant-cell lines were KB deriva-
tives expressing various mechanisms or multi-drug
resistance as described elsewhere.14,15
components, 12, 15, and 16 showed enhanced activity
against paclitaxel-resistant cells (Table 3). Compounds
12, 13, and 15 were selected for further cytotoxicity
evaluation in approximately 60 human tumor cell lines
by NCI. The average log GI50 values over all cell lines
for each tumor type are presented in Table 4. Com-
pound 12 showed the strongest inhibitory effects against
a variety of tumor cell lines, especially leukemia, colon,
and prostate cell lines.
All conjugates except 13 showed significant cytotoxicity
(data in Table 2). Although they were not as active as
paclitaxel and cephalomanine, 12 and 14–16 displayed
comparable or better activity than epipodophyllotoxin
derivatives in most tumor cell lines. Interestingly, the
cytotoxic profile of 15, which is a conjugate of paclitaxel
and 8, was different from those of both precursors,
suggesting that a novel mechanism of cell killing was
achieved through conjugation. Compound 13, which
has an epipodophyllotoxin conjugated at both the tax-
oid 2- and 7-hydroxy groups, was relatively inactive.
Thus, the taxoid 7-hydroxy group may be crucial for
cytotoxic activity; a similar result was found previously
by Mathew et al.4 Compared with the unconjugated
Table 3. Cytotoxicity of 12–16 in drug resistant cell lines (resistance
fold)
Compound
KB-7d KB-VCR KB-CPT PAX-10 PAX-22
12
13
14
15
1
0.6
—
1
—
2
119
—
1
17
—
—
8
—
3
153
—
1
—
—
1
—
1
1
—
1
7
—
>36
>17
17
—
—
a
8
8
16
7.5
—
GL-331
Etoposide
Cephalomannine
Paclitaxel
13
>16
40
>16
—
24
15
24
aND, not determined.

Q. Shi et al. / Bioorg. Med. Chem. 9 (2001) 2999–3004
3003
a
Table 4. Cytotoxicity of 12, 13, and 15 in NCI human tumor cell lines (log GI50
)
Compd
Leukemia
NSCLb
Colon
CNSc
Melanoma
Ovarian
Renal
Prostate
Breast
12
13
15
À7.60
À4.95
À6.02
À6.58
À5.78
À5.50
À7.20
À4.90
À5.12
À6.93
À5.20
À5.54
À6.29
À5.16
À5.01
À6.55
À5.03
À4.95
À6.13
À5.08
À5.88
À7.22
À5.06
À5.06
À6.56
À4.93
À4.96
aThe data were provided by the NCI. GI50 is the concentration that caused 50% inhibition of tumor cell growth.
bNon-small cell lung cancer.
cCentral nervous system cancer.
Table 5. Topoisomerase II inhibitory activities of 12–16
concentrated to afford a white solid, which was further
chromatographed over Si gel with an Elutflash1 flashed
silica gel column and eluted with CH2Cl2/MeOH=10:0
to 20:1 to yield the desired 9, as a white powder.
Compound
Inhibition of Topo II
IC100 (mM)
Cellular protein-DNA
complex formation (%)
12
13
14
15
100
50
NAb
100
NAb
50
100
NAb
NAa
8ꢀ2
NAa
2-(4-Formyl)benzoyl paclitaxel (9). Yield 92% (starting
1
NAb
with 11.7 g of taxol); amorphous; H NMR d (CDCl3)
25ꢀ11
16
NAb
see Table 1; APCI-MS m/z 986.6 (M+H+).
GL-331
Etoposide
Cephalomannine
Paclitaxel
134ꢀ7
100
20,7-Bis(4-formyl)benzoyl paclitaxel (10). Same as gen-
eral procedure except 1 mmol of paclitaxel and 2 mmol
of 4-carboxylbenzaldehyde and DCC were used. Yield
NAb
NAa
aNot active at 50 mM.
1
95% (starting with 19.7 mg of paclitaxel). H NMR d
bNot active at 100 mM.
(CDCl3) see Table 1; APCI-MS m/z 1118.8 (M+H+).
20-(4-Formyl)benzoyl cephalomannine (11). Yield 95%
(starting with 205 mg of cephalomannine); amorphous;
1H NMR d (CDCl3) see Table 1; APCI-MS m/z 964.6
(M+H+).
In the topoisomerase assays, all three conjugates inhib-
ited topo II in vitro but only 12 and 15 were intracel-
lular poisons (Table 5). Compound 13, which contains
two epipodophyllotoxin moieties, did not stimulate
protein-DNA breaks in cells. However, in the topo II
inhibitory assay, 13 was twofold more active than 12
and 15 and comparable to GL-331, suggesting that a
free hydroxy group might play a role in drug transport
into tumor cells.
General procedure for synthesizing target compounds
12–16. To a solution of mono or bis 20-(4-for-
myl)benzoyl taxoid (9–11) in anhydrous benzene was
added an appropriate epipodophyllotoxin derivative (1
or 2 equiv 6 or 8). The mixture was heated to reflux with
a Dean Stark trap for 24 h. The solvent was distilled
and the residue was purified by silica gel column chro-
matography eluting with CH2Cl2/MeOH=20:1 to
afford the desired compounds.
Experimental
Taxol and cephalomannine were kind gifts of Yung-
Shin Pharm. Co., Taiwan. The proton nuclear magnetic
resonance (1H NMR) spectra were measured on a Bru-
ker AC-300 spectrometer or Varian Inova 600 with
Me4Si (TMS) as the internal reference and CDCl3 as
solvent. Atmosphere Pressure Chemical Ionization
(APCI) Mass was determined by PE Sciex API
150 Mass spectrometer. Thin-layer chromatography
(TLC) silica gel plates were purchased from Alltech,
Inc.
Conjugate 12. A yellow powder in 92% yield [starting
with 22.5 mg (0.023 mmol) of 9 and 10.0 mg (0.025 mmol)
of
40-O-demethyl-4b-amino-4-desoxypodophyllotoxin
(6)]; amorphous; 1H NMR d (CDCl3) see Table 1;
APCI-MS m/z 1367.7 (M+H+).
Conjugate 13. A yellow powder in 89% yield [starting
with 7.0 mg (0.006 mmol) of 20,7-bis(4-formyl)benzoyl
paclitaxed (10) and 5.1 mg (0.013 mmol) of 6]; amor-
1
phous; H NMR d (CDCl3) see Table 1; APCI-MS m/z
1864.7 (M+H+).
General procedure for synthesizing (4-formyl)benzoyl
taxoids
Conjugate 14. A yellowish powder in 89% yield [starting
with 39 mg (0.040 mmol) of 20-(4-formyl)benzoyl
Cephalomannine (11) and 16 mg (0.040 mmol) of 6];
amorphous; 1H NMR d (CDCl3) see Table 1; APCI-MS
m/z 1345.8 (M+H+).
To a solution of taxoid in anhydrous CH2Cl2 was added
an equal molar ratio of 4-carboxylbenzaldehyde (2
equivwere used for 10) and a catalytic (ca. 5%) amount
of 4-dimethylaminopyridine (4-DMAP). To the mixture
then was added an equal molar amount of 1,3-dicyclo-
hexylcarbodiimide (DCC). The mixture was stirred
under N2 at room temperature for 24 h with TLC
monitoring. The dicyclohexylurea precipitate was fil-
tered and washed with CH2Cl2. The filtrate was
Conjugate 15. A yellow solid in 70% yield [starting with
20.0 mg ((0.023 mmol) of compound 9 and 10.6 mg
(0.025 mmol) of 8]; amorphous; 1H NMR d (CDCl3) see
Table 1; APCI-MS m/z 1458.8 (M+H+).

3004
Q. Shi et al. / Bioorg. Med. Chem. 9 (2001) 2999–3004
Conjugate 16. A yellow solid in 85% yield [starting with
39 mg (0.040 mmol) of compound 11 and 20 mg (0.040
mmol) of 8]; amorphous; 1H NMR d (CDCl3) see Table 1;
APCI-MS m/z 1437.9 (M+H+).
was supported in part by a grant from the National
Cancer Institute (CA 17625) awarded to K. H. Lee.
Biological assay
References and Notes
The in vitro cytotoxicity assay was carried out accord-
ing to procedures described in Rubinstein et al.16 Drug
stock solutions were prepared in DMSO, and the final
solvent concentration was ꢁ2% DMSO (v/v), a con-
centration without effect on cell replication. The human
tumor cell line panel consisted of epidermoid carcinoma
of the nasopharynx (KB), lung carcinoma (A-549),
ileocecal carcinoma (HCT-8), renal cancer (CAKI-1),
breast cancer (MCF-7), melanoma cancer (SKMEL-2),
and ovarian cancer (1A9). The drug resistant cell line
panel consisted of three KB-derivatives, -7d, -CPT, and
-VCR. The properties of these cells are described else-
where.14,15 The PAX-10 and PAX-22 sub-line deriva-
tives from 1A9 cells are resistant to paclitaxel and have
mutations in b-tubulin.17 Cells were cultured at 37 ꢂC in
RPMI-1640 with 100 mg/mL kanamycin and 10% (v/v)
fetal bovine serum in a humidified atmosphere contain-
ing 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 3 days, and the ED50
value, the drug concentration that reduced the absor-
bance by 50%, was interpolated from dose-response
data. Each test was performed in triplicate, and
absorbance reading varied no more than 5%.
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Protein-linked DNA break and topoisomerase II activity
assays
The procedures were done according to detailed published
methods.8
16. 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.
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Acknowledgements
The PAX-10 and PAX-22 sub-line derivatives were a
generous gift of Dr. P. Giannakakou. This investigation