Antitumor agents. 258. Syntheses and evaluation of dietary antioxidant-taxoid conjugates as novel cytotoxic agents

Article abstract of DOI:10.1016/j.bmcl.2007.06.083

Various dietary antioxidants, including vitamins, flavonoids, curcumin, and a coumarin, were conjugated with paclitaxel (1) through an ester linkage. The newly synthesized compounds were evaluated for cytotoxic activity against several human tumor cell lines as well as the corresponding normal cell lines. Interestingly, most tested conjugates selectively inhibited the growth of 1A9 (ovarian) and KB (nasopharyngeal) tumor cells without activity against other cell lines. Particularly, conjugates 16 and 20 were highly active against 1A9 (ED₅₀ value of 0.005 μg/mL) as well as KB (ED₅₀ values of 0.005 and 0.14 μg/mL, respectively) cells. Compound 22b, the glycinate ester salt of vitamin E conjugated with 1, appears to be a promising lead for further development as a clinical trial candidate as it exhibited strong inhibitory activity against Panc-1 (pancreatic cancer) with less effect on the related E6E7 (normal) cell line.

Full text of DOI:10.1016/j.bmcl.2007.06.083

Bioorganic & Medicinal Chemistry Letters 17 (2007) 5204–5209
Antitumor agents. 258. Syntheses and evaluation of dietary
antioxidant—taxoid conjugates as novel cytotoxic agents
Kyoko Nakagawa-Goto,a Koji Yamada,a Seikou Nakamura,a Tzu-Hsuan Chen,a
Po-Cheng Chiang,a Kenneth F. Bastow,b Shao-Chun Wang,c,d Bill Spohn,c,d
Mien-Chie Hung,c,d Fang-Yu Lee,e Fang-Chen Leee and Kuo-Hsiung Leea,*
aNatural Products Research Laboratories, School of Pharmacy, University of North Carolina, Chapel Hill, NC 27599, USA
bDivision of Medicinal Chemistry and Natural Products, School of Pharmacy, University of North Carolina,
Chapel Hill, NC 27599, USA
cDepartment of Molecular and Cellular Oncology, The University of Texas, MD Anderson Cancer Center,
1515 Holcombe Boulevard Box 108, Houston, TX 77030, USA
dCenter for Molecular Medicine, China Medical University Hospital, Taichung, Taiwan
eYung-Shin Pharmaceutical Co. Ltd, Ta-Chia, Taichung, Taiwan
Received 14 March 2007; revised 21 June 2007; accepted 28 June 2007
Available online 4 July 2007
Abstract—Various dietary antioxidants, including vitamins, flavonoids, curcumin, and a coumarin, were conjugated with paclitaxel
(1) through an ester linkage. The newly synthesized compounds were evaluated for cytotoxic activity against several human tumor
cell lines as well as the corresponding normal cell lines. Interestingly, most tested conjugates selectively inhibited the growth of 1A9
(ovarian) and KB (nasopharyngeal) tumor cells without activity against other cell lines. Particularly, conjugates 16 and 20 were
highly active against 1A9 (ED50 value of 0.005 lg/mL) as well as KB (ED50 values of 0.005 and 0.14 lg/mL, respectively) cells. Com-
pound 22b, the glycinate ester salt of vitamin E conjugated with 1, appears to be a promising lead for further development as a
clinical trial candidate as it exhibited strong inhibitory activity against Panc-1 (pancreatic cancer) with less effect on the related
E6E7 (normal) cell line.
Ó 2007 Elsevier Ltd. All rights reserved.
In current cancer therapy, undesirable side effects due to
toxicity of antitumor drugs on normal tissues present an
important problem to be solved. Therefore, a challeng-
ing research focus in cancer treatment is the discovery
of efficient antitumor drugs with high therapeutic
indexes, which will have selective activity against target
tumors and reduced normal tissue damage. Among
various strategies to improve drug selectivity, conjuga-
tion of cytotoxic drug components has proven to be a
promising approach to enhance the activity as well as
selectivity of an individual lead compound.1 This con-
cept is now accepted as an effective strategy for design-
ing ligands, inhibitors, and other drugs that influence
biological systems.2 On the basis of this theory, some
interesting results have been reported by our group3 as
well as others4 in recent years. In our prior study, we ex-
plored the syntheses and evaluation of heterodimer con-
jugates, which combined two kinds of antitumor drugs
through various linkages, as novel antitumor agents.5
Dietary anti-oxidants, such as flavonoids and vitamins
A, C, and E that are found in various foods, can act
as cancer preventive agents. These compounds are capa-
ble of neutralizing and deactivating reactive oxygen spe-
cies, which can seriously damage DNA and other
cellular molecules, thereby causing tumors.6 Antioxi-
dants act not only as cancer preventive agents, but also
as therapeutic biologic response modifiers, and are able
to directly induce apoptosis in established tumor cells.
In addition, evidence shows that antioxidants can
enhance chemotherapeutic antitumor effects.7
Therefore, conjugation of antitumor drugs with dietary
antioxidants might provide new classes of antitumor
drug candidates with tumor selectivity or activity
Keywords: Conjugation; Paclitaxel; Antioxidant; Cytotoxicity.
*
Corresponding author. Tel.: +1 919 962 0066; fax: +1 919 966
3893; e-mail: khlee@unc.edu
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.06.083

K. Nakagawa-Goto et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5204–5209
5205
against multi-drug resistant cancer cell lines. We chose
paclitaxel (1)8 as the antitumor base compound and var-
ious antioxidants as the conjugate partners as shown in
Figure 1. Vitamins,6 such as retinol (vitamin A, 2a)9,10
and a–tocopherol (vitamin E, 3),9,11 are well known die-
tary antioxidants. Compound 4 was also selected as a re-
lated vitamin E analog. Other known antioxidant
agents, including curcumin (5)6,12 found in turmeric,
dehydrozingerone (6)13 isolated from ginger, and its
analog 7, were also used. In addition, antioxidant flavo-
noids6,14 and coumarins6,15 are widely found in various
vegetables, fruits, nuts, coffee, tea or wine, and represen-
tative compounds [galangin (8), chrysin (9), and 4-meth-
ylumbelliferone (10), respectively] were conjugated with
1 (Scheme 1).
pancreatic cancer cell line with less effect against the re-
lated normal cell line. Therefore, in order to increase
water-solubility, conjugate 13 was converted first to var-
ious amine (22, 27, and 28) or carboxylic (29) esters and
then to the corresponding hydrochloride (22a, 27a, and
28a), methanesulfonate (22b, and 28b), or triethylammo-
nium (29a) salts as shown in Scheme 2.
The newly obtained conjugates were evaluated for cyto-
toxic activity against several human tumor cell lines.
Interestingly, conjugates 12, 14, and 16–20 and the gly-
cinate esters 21 and 23–26 selectively inhibited the
growth of 1A9 and KB cells (data shown in Table 1),
without inhibition of the remaining cell lines [lung carci-
noma (A549), breast cancer (MCF-7), prostate carci-
noma (LN-CAP, PC-3, DU-145) and multi-drug
resistant variant expressing P-glycoprotein KB-VIN].
Conjugates 16 (paclitaxel-dehydrozingerone) and 20
(paclitaxel-coumarin) exhibited the highest potency
against these two cell lines (1A9: ED50 0.005 lg/mL;
KB:ED50 0.005 and 0.14 lg/mL, respectively). More-
over, against KB cells, glycinate ester 26 showed signif-
icantly increased cytotoxic activity (ED50 0.09 lg/mL)
compared to the related parent compound 18 (ED50
0.26 lg/mL), even though glycinate esters 23–25 showed
similar or lower activity compared with the parent com-
pounds 14, 16, and 17.
Paclitaxel (1) was reacted first with succinic anhydride in
pyridine to give 11,16 which has a succinate ester at the
C-20 position. However, esterification of 11 with 2a pro-
vided an unstable compound; therefore, retinoic acid
(2b, vitamin A acid) was reacted directly with 1 to afford
12. Conjugations of 11 with other antioxidants, 3–10,
were carried out by a common esterification method
using EDCI in the presence of DMAP to give 13–20.
Water solubility, which is connected with oral bioavail-
ability, is always of concern for drug discovery and
development. Accordingly, the resulting conjugates,
12–14 and 16–18, were converted to the corresponding
glycinate esters, 21–26. These esters can be converted
to various salts. As discussed later, the paclitaxel-vita-
min E conjugate (13) showed good activity against a
Conjugate 13, its C-7 esters 22 and 27–29, their corres-
ponding salts, and conjugate 15 were evaluated for cyto-
toxic activity against A549, 1A9, colon adenocarcinoma
Figure 1. Structures of paclitaxel (1) and anti-oxidant conjugate partners (2-10).

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K. Nakagawa-Goto et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5204–5209
AcO
O
O
O
O
AcO
O
OH
O
OR
O
Ph
O
Ph
O
2'
2'
7
BzHN
O
BzHN
O
O
OH
pyridine
O
H
H
AcO
HO
1
BzO
AcO
HO
BzO
11: R= R1 =H
13-20: R=H ,
R1 (see below)
EDCI,D MAP,
2b,C H2Cl2,
79%
R1O
O
EDCI, DMAP,
R1OH,( 3-10)
AcO
O
MeNCH2COOH
EDCI, DMAP
OR
O
Ph
O
O
22-26:R =
NMe2
BzHN
O
O
H
AcO
R1 (see below)
HO
BzO
O
O
12: R= H
21:R =
MeNCH2COOH
EDCI, DMAP
NMe2
Conjugate
(R = -H)
Glycinate Ester
R1
(R = -COCH2NMe2)
13
14
22
O
R2 =
R2
O
23
R2 = Me
O
O
15
O
OH
OMe
OMe
4
2
16
17
24
25
R2 = 4-CH=CHCOCH3
R2 = 2-CH=CHCOCH3
R2
O
OMe
O
R3
18
19
26
R2 = OH, R3 = H
R2 = H, R3 = OH
R2
C6H5
O
O
20
O
O
O
Scheme 1. Synthesis of conjugates 12-26.
(HCT-8), epidermoid skin carcinoma (A431), KB, and
KB-VIN cell lines. The data are shown in Table 2. The
ED50 values of 1, 3 or 5, and a 1:1 mixture of 1 with 3 or
5 are also shown for comparison. The 1:1 mixtures of 1
with 3 or 5 showed similar results to 1 itself against
A549, A431 and 1A9 cells but were less potent against
HCT-8 cells. However, the related conjugates 13 and 15
showed unique selectivity. Vitamin E conjugate 13 lost
cytotoxic activity against most cell lines, and inhibited
the growth of only the A431 cell line with an ED50 value
of 0.1 lg/mL. In comparison, curcumin conjugate 15
showed similar potencies to 1 against A549, 1A9, A431,
KB and KB-VIN cells, but lost activity against HCT-8
cells. Among the C-7 esters/salts of conjugate 13 (22 and
27–29), only the glycinate ester 22, succinic ester 29, and
their salts displayed cytotoxic activity against the
A431 cell line with similar ED50 values to the parent
compound 13.

K. Nakagawa-Goto et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5204–5209
5207
Scheme 2. Synthesis of water soluble salts of conjugate 13.
Table 1. Cytotoxic activity data for taxol conjugates 12, 14, 16–21, 23–
26 [ED50 (lg/mL)]a,b
Table 2. Cytotoxic activity data of conjugates 13, 15, and related
selected compounds
Cell line
Compound
ED50 (lg/mL)
1A9c
KBc
A549 1A9 HCT-8a A431a KB KB-VIN
Conjugate
1
3 (Vitamin E)
0.02 0.01 0.06
NAb NA NA
13 (1+3 Conjugate) NA NA NA
0.01
0.75
0.1
0.01 0.2
NA NA
NA NA
0.01 0.1
NA NA
NA NA
NA NA
NA NA
NA NA
NA NA
0.01 0.2
0.01 0.2
12
14
16
17
18
19
20
0.49
0.20
0.005
0.14
0.13
0.20
0.005
0.20
0.94
0.005
0.39
0.26
0.47
0.14
1+3 (1:1 Mixture)
22
22a, 22b
0.02 0.01 0.20
NA NA NA
NA NA NA
0.01
0.2
0.1
27, 27a, 28, 28a, 28b NA NA NA
NA
0.1
29
29a
NA NA NA
NA NA NA
NA NA NA
0.2
Glycinate ester
5 (Curcumin)
0.75
0.03
0.01
21
23
24
25
26
4.47
1.01
0.63
0.19
0.11
7.65
9.79
0.70
0.34
0.09
15 (1+5 Conjugate) 0.03 0.03 NA
1+5 (1:1 Mixture) 0.03 0.01 0.10
a Epidermoid skin carcinoma (A431), colon adenocarcinoma (HCT-8);
for other cell lines, see Table 1.
b NA, not active.
Control
1
0.001
0.002
tumor cells. Glycinate ester salt 22b also showed less
inhibition of E6E7 growth and the highest potency
against Panc-1, compared with the other esters/salts,
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.
b All compounds tested did not reach 50% inhibition against human
lung carcinoma (A549), breast cancer (MCF-7), human prostate
carcinoma (LN-CAP, PC-3, DU-145), and multi-drug resistant KB
variant expressing P-glycoprotein (KB-VIN).
In conclusion, we have synthesized various 1-antioxi-
dant conjugates linked through an ester bond at the
20-position of 1. All conjugates were screened against
various tumor cell lines and showed tumor-selective
activity. Most conjugates selectively inhibited the
growth of 1A9 and KB tumor cells and lacked activity
against other tested cell lines. On the other hand, conju-
gate 13 showed cytotoxic activity only against A431,
while conjugate 17 lacked any activity against HCT-8
while retaining similar activity to 1 against the other tu-
mor cell lines. Salt 22b exhibited inhibitory activity
against Panc-1 with less effect on the related normal cell.
Compound 22b appears to be a promising new lead for
c Human ovarian carcinoma (1A9), human epidermoid carcinoma of
the nasopharynx (KB).
Conjugate 13 and its related esters/salts were also
screened against multiple human tumor as well as the
corresponding normal cell lines. Figure 2 shows selected
data after 48 h. Conjugate 13 demonstrated less growth
inhibition of normal pancreatic cells (E6E7) than related
tumor cells (Panc-1), whereas growths of other normal
cells were inhibited more or as strongly than the related

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K. Nakagawa-Goto et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5204–5209
Paclitaxel (1)
SKHep
150
Chang Liv.
2774
100
IOSE
Panc-1
50
0
E6E7
H1299
WI 38
0um
10um
20um
40um
MCF-7
um
MCF-10A
13
1+3
SKHep
Chang Liv.
2774
SKHep
140
140
120
100
80
60
40
Chang Liv.
2774
120
100
80
60
40
20
0
IOSE
IOSE
Panc 1
E6E7
Panc-1
E6E7
20
0
H1299
WI 38
H 1299
WI 38
0
10
20
40
0um
10um
20um
40um
MCF-7
MCF-10A
MCF-7
MCF-10A
um
um
22b
22
SKHep
Chang Liv.
2774
SKHep
Chang Liv.
2774
140
120
100
80
60
40
150
100
50
IOSE
IOSE
Panc-1
E6E7
Panc 1
E6E7
20
0
H1299
WI 38
H 1299
WI 38
0
0um
10um
20um
40um
0 um
10 um
20 um
40 um
MCF-7
MCF-10A
MCF-7
MCF-10A
um
um
Figure 2. Liver human hepatomacarcinoma (SKHep), normal liver epithelial cell (Chang Liv), human ovarian carcinoma (2774), normal surface
ovarian epithelial cell (IOSE), human pancreatic cancer cell (Panc 1), normal ovarian epithelial cell (E6E7), human lung cancer cell (H1299), normal
human fibroblast (W138), breast cancer (MCF-7) and normal breast epithelial cell (MCF-10A).
3. (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.;
Bastow, K. F.; Tachibana, Y.; Chen, K.; Lee, F. Y.; Lee,
K. H. Bioorg. Med. Chem. 2001, 9, 2999; (d) Ohtsu, H.;
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further development into a clinical trial candidate. In
summary, the conjugation of 1 with dietary antioxidants
enhanced tumor selectivity dependant on the identity of
the partner compound.
Acknowledgment
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This study was supported by Grant CA17525 from the
National Cancer Institute, NIH, awarded to K.H.L.
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