Antitumor agents. 256. Conjugation of paclitaxel with other antitumor agents: Evaluation of novel conjugates as cytotoxic agents

Article abstract of DOI:10.1016/j.bmcl.2007.02.051

Sixteen different taxoid conjugates were prepared by linking various anticancer compounds, including camptothecin (CPT), epipodophyllotoxin (EP), colchicine (COL), and glycyrrhetinic acid (GA), at the 2′- or 7-position on paclitaxel (TXL, 1) through an ester, imine, amine, or amide bond. Newly synthesized conjugates were evaluated for cytotoxic activity against replication of several human tumor cell lines. Among them, TXL-CPT conjugates, 8-10, were more potent than TXL itself against the human prostate carcinoma cell line PC-3 (ED₅₀ = 14.8, 3.1, 19.4 nM compared with 55.5 nM), and conjugate 10 was also 8-fold more active than TXL against the LN-CAP prostate cancer cell line. These compounds also possessed anti-angiogenesis ability as well as lower inhibitory effects against a normal cell line (MRC-5). Thus, conjugates 8-10 are possible antitumor drug candidates, particularly for prostate cancer.

Full text of DOI:10.1016/j.bmcl.2007.02.051

Bioorganic & Medicinal Chemistry Letters 17 (2007) 2894–2898
Antitumor agents. 256. Conjugation of paclitaxel with other
antitumor agents: Evaluation of novel conjugates
as cytotoxic agents
Kyoko Nakagawa-Goto,a Seikou Nakamura,a Kenneth F. Bastow,b Alexander Nyarko,a
Chieh-Yu Peng,a Fang-Yu Lee,c Fang-Chen Leec 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
cYung-Shin Pharmaceutical Co., Ltd, Ta-Chia, Taichung, Taiwan
Received 30 January 2007; accepted 20 February 2007
Available online 27 February 2007
Abstract—Sixteen different taxoid conjugates were prepared by linking various anticancer compounds, including camptothecin
(CPT), epipodophyllotoxin (EP), colchicine (COL), and glycyrrhetinic acid (GA), at the 20- or 7-position on paclitaxel (TXL, 1)
through an ester, imine, amine, or amide bond. Newly synthesized conjugates were evaluated for cytotoxic activity against replica-
tion of several human tumor cell lines. Among them, TXL–CPT conjugates, 8–10, were more potent than TXL itself against the
human prostate carcinoma cell line PC-3 (ED50 = 14.8, 3.1, 19.4 nM compared with 55.5 nM), and conjugate 10 was also 8-fold
more active than TXL against the LN-CAP prostate cancer cell line. These compounds also possessed anti-angiogenesis ability
as well as lower inhibitory effects against a normal cell line (MRC-5). Thus, conjugates 8–10 are possible antitumor drug candidates,
particularly for prostate cancer.
Ó 2007 Elsevier Ltd. All rights reserved.
Paclitaxel (TXL, 1, Fig. 1), a plant derived antimitotic
agent,1,2 is currently in clinical use against ovarian and
breast cancer. It promotes the irreversible assembly of
tubulin into microtubules by binding to and stabilizing
microtubules. This mechanism of action is unique
among the established antitumor drugs, including the
vinca alkaloids, vincristine and vinblastine, which pre-
vent microtubule assembly by microtubule binding.3
Other natural products, including camptothecin
(CPT),4 which is approved for clinical use in the United
State, epipodophyllotoxin (EP),5 and colchicine (COL),6
also possess potent antitumor activities with different
mechanisms of action3 from TXL. Some EP derivatives,
such as etoposide (2d), topotecan, and irinotecan, are
approved by the FDA for cancer treatment, although
their therapeutic use is often limited by undesired side
effects, such as myelosuppression, multi-drug resistance,
and poor water solubility. In addition, glycyrrhetinic
acid (GA),7 which is a well-known natural compound
isolated from plants, shows antitumor-promoter
activity, as well as anti-allergic, anti-inflammatory, and
anti-ulcer effects.
Conjugation of two antitumor agents with different
mechanisms of action can possibly augment the potency
of both compounds or reduce the side effects and drug-
resistance development. We have already reported the
unique antitumor activities of such conjugates, including
TXL–CPT,8 TXL–EP,9 and CPT–EP.10 In our continu-
ous effort to develop new classes of antitumor agents
based on TXL, we investigated the syntheses and evalu-
ation of TXL conjugates with the above-mentioned anti-
tumor or antitumor-promoter compounds through
various linkages at the TXL C-20 and C-7 positions as
reported herein.
Newly synthesized conjugates are shown in Figure 2. In
conjugates 6–15, EP, CPT, COL, and GA are connected
to the TXL C-20 position through various linkages,
while conjugates 16–21 contain EP, COL, and GA
connected to the C-7 position of TXL. As shown in
Keywords: Paclitaxel; Conjugation; Cytotoxicity; Prostate cancer.
*
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.2007.02.051

K. Nakagawa-Goto et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2894–2898
2895
Figure 1. Structures of paclitaxel (1) and other antitumor agents used for conjugation.
Scheme 1, a linking group was introduced directly at the
TXL C-20 position using the appropriate anhydride or
carboxylic acid to provide 22–25.11 For the C-7 linked
conjugates, the TXL C-20 hydroxyl group was first pro-
tected with a Cbz or TBS group, the linking carboxylic
acid was added at C-7, and the protecting group was then
removed to afford 26–28. Compounds 22–24, 26, and 27
were conjugated with EP derivative 2c,12 COL deriva-
tives 4b–d,13 and GA (5), respectively, by using EDCI
in the presence of DMAP to provide 6, 7, 11–13,
15–17, and 21. Heating intermediates 22 and 23 with
7-formyl-CPT (3b)14 in benzene produced the corre-
sponding imines 8 and 10, respectively. In the same
way, imines 14, 18, and 20 were prepared from 25 with
4d and 28 with 2b or 4d, respectively. Hydrogenation
of 8 and 18 produced the corresponding amines 9 and 19.
expressing P-glycoprotein (KB-VIN) and against the
normal cell line, human embryonic fibroblast (MRC-5).
The results are shown in Table 1 with the values for
TXL (1), EP derivatives 2b–c, etoposide (2d), CPT (3a),
7-formyl-CPT (3b), 2-demethyl-COL (4b), 2-demethyl-
thio-COL (4c), and GA (5) for comparison. All conju-
gated compounds showed better activity than the
partner compounds, although most of them were less
potent than TXL itself. However, TXL–CPT conjugates
8–10 displayed different patterns of inhibition against the
LN-CAP and PC-3 prostate cancer cell lines, with less
effect on the normal cell line, MRC-5. Imine 10 had an
ED50 value of 0.34 nM and was 7.7-fold more potent
than TXL against LN-CAP cells, while compound 9
had an ED50 value of 3.1 nM and was 18-fold more
potent than TXL against PC-3 cells.
All synthesized compounds were evaluated for cytotoxic
activity against replication of several human tumor cell
lines, human ovarian carcinoma (1A9), human lung car-
cinoma (A549), breast cancer (MCF-7), human prostate
carcinoma (LN-CAP, PC-3, DU-145), human epider-
moid of the nasopharynx (KB), and multi-drug resistant
Cytotoxic activity was somewhat dependent on the con-
jugated position on TXL as well as the type of linkage.
From comparison of the TXL–EP conjugates 6, 7, 16,
and 17, the conjugates linked at the C-20 position (6,
7) showed better activity than conjugates linked at the
C-7 position (16, 17) against all cell lines. Moreover,

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K. Nakagawa-Goto et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2894–2898
Figure 2. Structures of conjugates.
the linkage with two methylenes (6, 16) gave better
results than the one with three methylenes (7, 17). How-
ever, TXL–COL conjugates 14 (20-linkage) and 20(7-
linkage) showed similar potency against most cell lines.
A phenyl imino linkage was better than the linear amido
linkage, as TXL–COL conjugate 14 was 4- to 9-fold
more potent than 13 against 1A9, A549, and KB cells.
The results of selected compounds in an anti-angiogenesis
assay are shown in Table 2. Compared with the other con-
jugates, imines 8 and 10 possessed significant activity with
ED50 values of 0.73 and 0.98 lg/mL, respectively.
In conclusion, we have synthesized 16 different
conjugates between paclitaxel (TXL) and other

K. Nakagawa-Goto et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2894–2898
2897
Scheme 1. Synthesis of conjugates.
Table 1. Cytotoxic activity data of taxol conjugates
a
ED50 (nM)
Compound
ED50 (lM)
KB-VIN MRC-5
1A9
1.0
739
A549
2.3
MCF-7
LN-CAP
PC-3
55.5
DU-145
KB
1
1.1
686
2.6
680
1.3
3107
1600
10.9
7.7
1.8
311
2050
14447
15
NTa
NT
2b
2d
3a
3b
4b
4c
5
1074
1609
1234
9452
111.2
1365
20.9
80.6
146.1
47.9
>20b
8.4
284.9
3.2
4.3
11659
3.2
4.3
530.1
2.0
4.3
>30
NT
6.9
16.7
12.8
950.6
99.8
>20b
18.4
475.3
112.2
>20b
25
NT
NT
183.7
43.6
>20b
361.0
59.9
>20b
6.4
324.7
59.9
>20b
4.2
611.9
113.5
>20b
8.6
28977
7426
>20b
9964
6231
NT
NT
6
2.9
8.1
10.1
23.7
2.6
12.1
42.8
14.8
3.1
>10b
6174
177.7
6133
243.6
NT
7
22.1
26.7
2.7
10.2
3.0
32.5
3.7
13.9
1.3
8
1.6
1.5
62.4
2050
56.3
>20b
>20b
>20b
308
>20b
>20b
NA
9
10
2.6
1.9
1.2
2.3
1.9
1.9
1.5
1.0
34.1
26.5
24.7
29.2
NT
0.34
12.9
19.5
23.7
NT
19.4
50.7
71.8
70.4
11
12
13
14
15
16
17
18
19
20
21
10.6
10.3
14.0
2.3
27.3
23.2
28.5
3.8
31.9
29.8
34.4
NT
19.0
17.0
21.57
5.4
NT
NT
NT
NT
NT
45.3
20.7
77.4
45.6
38.7
43.9
125.0
48.6
68.2
NT
46.2
22.1
NT
103.6
86.3
NT
68.3
83.4
NT
53.6
31.6
347.2
53.8
87.4
2.3
3279
NT
104
1319
60.1
75.7
2.3
111.0
123.5
3.8
90.1
128.5
NT
145.3
191.5
NT
139.1
167.5
NT
6220
5200
308
>20b
NT
NT
NT
NT
154.3
224.8
129.8
192.7
652.1
311.0
147.3
Human ovarian carcinoma (1A9), human lung carcinoma (A549), breast cancer (MCF-7), human prostate carcinoma (LN-CAP, PC-3, DU-145),
human umbilical vein endothelial cell (HUVEC), human epidermoid carcinoma of the nasopharynx (KB), multi-drug resistant expressing
P-glycoprotein (KB-VIN), and human embryonic fibroblast (MRC-5).
a Cytotoxicity as ED50 values for each cell line, the concentration of compound that caused 50% reduction in absorbance at 562 nm relative to
untreated cells using the sulforhodamine B assay.
b Test compound (20 lg/mL or 10 lg/mL) did not reach 50% inhibition.

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K. Nakagawa-Goto et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2894–2898
Table 2. Anti-angiogenesis assay data of selected compounds in
HUVECs
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Acknowledgment
This study was supported by Grant CA-17625 from
the National Cancer Institute, NIH, awarded to
K.H.L.