Prodrugs of 4'-demethyl-4-deoxypodophyllotoxin: synthesis and evaluation of the antitumor activity.

Article abstract of DOI:10.1016/S0960-894X(02)00758-8

A series of prodrugs of 4'-demethyl-4-deoxypodophyllotoxin (DDPT) including carbamates (3-8), a carbonate (9) and water-soluble amino acid derivatives (10-17) were prepared and tested for their antitumor activity. The carbamate 6 (2-hydroxyethylcarbamoyl-DDPT), carbonate 9 (2-chloroethyloxycarbonyl-DDPT), and most of amino acid prodrugs (12-17) showed enhanced antitumor activity.

Full text of DOI:10.1016/S0960-894X(02)00758-8

Bioorganic & Medicinal Chemistry Letters 12 (2002) 3435–3438
Prodrugs of 40-Demethyl-4-deoxypodophyllotoxin: Synthesis and
Evaluation of the Antitumor Activity
Yong Kim, Young-Jae You, Nguyen-Hai Nam and Byung-Zun Ahn*
College of Pharmacy, Chungnam National University, Taejon 305-764, South Korea
Received 25 December 2001; accepted 19 August 2002
Abstract—A series of prodrugs of 40-demethyl-4-deoxypodophyllotoxin (DDPT) including carbamates (3–8), a carbonate (9) and
water-soluble amino acid derivatives (10–17) were prepared and tested for their antitumor activity. The carbamate 6 (2-hydroxy-
ethylcarbamoyl-DDPT), carbonate 9 (2-chloroethyloxycarbonyl-DDPT), and most of amino acid prodrugs (12–17) showed
enhanced antitumor activity.
# 2002 Elsevier Science Ltd. All rights reserved.
Results and Discussion
Introduction
Chemistry
4-Deoxypodophyllotoxin (DPT, 1, Fig. 1) is a potent
antimitotic agent first isolated from Anthriscus sylves-
tris.1 It was shown to exhibit potent cytotoxicity against
a wide variety of tumor cell lines.2,3 Recently, we reported
the presence of 1 in Pulsatilla koreana, one indigenous
plant in Korea, and demonstrated that this antimitotic
agent completely inhibited the tube-like formation of
human umbilical venous endothelial cells (HUVEC) at a
concentration considerably below the cytotoxic ones.
Moreover, 1 also exhibited significant antitumor activity
in BDF1mice bearing murine Lewis lung carcinoma
(3LL) cells with inhibition rate (IR) of 59%.4
Previously, it had been reported that DPT was abun-
dant in A. sylvestris.6 Thus, we took this advantage and
used this plant as a source of DPT. Briefly, the air-dried
materials (the whole plant, 3 kg) were extracted with
ethyl acetate. The ethyl acetate extract was chromato-
graphed over a silica gel column using cyclohexane/
ethyl acetate (5:1) as an eluent to afford the crude DPT
fraction. Recrystallization of the resulting crude frac-
tion from MeOH afforded DPT (1, 6.8 g), which was
confirmed by direct comparison with an authentic
sample isolated from P. koreana and spectral data
reported previously.4 DPT obtained as such was used
as a starting material for subsequent syntheses of the
prodrugs.
Previously, it was reported that 40-demethyl-4-deoxy-
podophyllotoxin (DDPT, 2) exerted a comparable in
vitro potency with 1.5 However, in vivo experiments
revealed a substantial loss of the antitumor activity of 2
in the same BDF1/3LL model. These results indicate
that the free hydroxy group at the 40 position in DDPT
was not favorable for its antitumor activity. We there-
fore envisioned that transformation of the hydroxy
group into bioreversible functionalities might improve
the in vivo activity of DDPT. In this paper, the synth-
esis and antitumor activity of a series of carbamate,
carbonate, and amino acid prodrugs of DDPT are
presented and discussed.
The general methods employed for the preparation of
DDPT and its derivatives 3–17 are outlined in Scheme 1.
Selective demethylation7 of DPT 1 with trimethylsilyl
iodide (TMS) gave DDPT (2) in 72% yield. Compound
*Corresponding author. Tel.:+82-42-821-5923; fax:+82-42-823-6566;
e-mail: ahnbj@cnu.ac.kr
Figure 1. Structures of DPT (1) and 40-DDPT (2).
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00758-8

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Y. Kim et al. / Bioorg. Med. Chem. Lett. 12 (2002) 3435–3438
Scheme 1. (a) TMSI, CH2Cl2, 0 ꢀC, 5 h, then BaCO3, 30 min; (b) DCC (5 equiv), DMAP (2.5 equiv), CH2Cl2, 0 ꢀC, 1h; (c) 50% TFA/CH 2Cl2, rt, 1h;
(d) TEA, 0 ꢀC, 40 min; (e) DMAP, CH2Cl2, 0 ꢀC, 5 h.
2 was reacted with various isocyanates in the presence
of triethylamine (TEA) to give 40-demethyl-40-O-(carba-
moyl)-DPT derivatives (3–8) in 66–80% yields. In the
presence of 4-dimethylaminopyridine (DMAP) as a base,
DDPT 2 was esterified with 2-chloroethyl chloroformate
to give 40-demethyl-40-O-(2-chloroethylcarbonyloxy)-
DPT (9) in 78% yield. 40-Demethyl-40-O-(aminoacyl)-
DPT derivatives 10–17 were prepared in 73–83% yields
by reacting DDPT 2 with various Boc-protected amino
acids in the presence of dicyclohexylcarbodiimide (DCC)
and DMAP, and successive removal of the Boc protecting
group by trifluoroacetic acid (TFA). All compounds were
unambiguously confirmed by spectroscopic methods.8
pound 2 (AC, 15 nM). The carbamates 3–5 were the
least cytotoxic with approximately 10-fold drops in the
ED50 values relative to that of 2. The results indicate
that under cell culture conditions these carbamates were
not conversed to the parent compound 2 or the rate of
conversion was too slow. Of exception were the com-
pounds 10 and 11, which showed the same potency (AC
values of 14 and 29 nM, respectively) with 2. The potent
cytotoxicity of 10 and 11 suggests that these derivatives
were sufficiently conversed to the parent compound 2
under cell culture conditions. Similar phenomena have
been observed previously.10
The prodrugs were evaluated for the antitumor activity
in BDF1/3LL model. Due to the limited solubility in
solvent system (5% DMSO, 15% Cremophore1 in sal-
ine), the carbamates 3–8 and were evaluated at doses of
0.06 mmol/kg/day and the carbonate 9 was adminis-
tered at 0.03 mmol/kg/day. The amino acid prodrugs
(10–17) were injected at the maximum tolerated doses
(Table 1). Etoposide, a clinical anticancer drug, was
administered at 0.06 mmol/kg/day and used as a posi-
tive control. The results are summarized in Table 1.
Bioactivity
Cytotoxicity of the synthesized compounds was mea-
sured against three cancer cell lines (A549, human lung
carcinoma; SK-MEL-2, human melanoma; and MCF-7,
human breast carcinoma). The results, expressed as
ED50 values (the concentration that produces a 50%
reduction in cell growth), are summarized in Table 1.
For the comparison purpose, the average ED50 values
(AC) of each compound in three cancer cell lines were
calculated and the results are included in Table 1. Gen-
erally, the prodrugs showed decreased cytotoxicity (AC
values up to 171 nM) compared to the parent com-
Our rationale for the preparation of carbamates 3–8
was based on the previous reports that carbamates
could be used as effective prodrugs to obtain prolonged

Y. Kim et al. / Bioorg. Med. Chem. Lett. 12 (2002) 3435–3438
Table 1. Cytotoxicity and antitumor activity of the synthesized prodrugs
3437
Compd
R
Cytotoxicitya (ED50, nM)b
Antitumor activity
IRd
A549
SK-MEL-2
MCF7
ACc
3
4
5
6
7
8
9
10
11
12
Ethylcarbamoyl
Isopropylcarbamoyl
Cyclohexylcarbamoyl
2-Hydroxyethylcarbamoyl
4-Fluorophenylcarbamoyl
4-Methoxyphenylcarbamoyl
2-Chloroethyloxycarbonyl
Alanyl
Glycinyl
Phenylalanyl
3-Aminopropanoyl
4-Aminobutanoyl
6-Aminohexanoyl
8-Aminooctanoyl
4-Aminophenylmethyl
132
153
163
62
86
96
92
22
12
87
97
92
89
74
114
23
15
1900
143
165
152
59
78
89
72
9
34
59
79
9189
77
6169
152
14
142
166
197
46
76
93
88
11
31
72
83
91
72
68
99
11
18
139
161
171
56
80
93
84
14
29
73
NAe (0.06)f
NA (0.06)
NA (0.06)
95 (0.06)
69 (0.06)
NA (0.06)
88 (0.03)
41 (0.3)
33 (0.3)
80 (0.36)
85 (0.3)
85 (0.36)
89 (0.06)
80 (0.42)
80 (0.3)
13
14
15
16
86
79
17
122
16
15
DPT (1)
DDPT (2)
Etoposide
59 (0.06)
13 (0.06)
78 (0.06)
12
aA549, human lung carcinoma; SK-MEL-2, human melanoma; MCF-7, human breast carcinoma.
bDrug concentration required to inhibit the growth of cancer cells by 50%, see ref 14.
cAC; average of ED50 value.
dIR, inhibition rate (%), determined as described in ref 15.
eNA; not active.
fNumbers in parenthesis represents the highest formulable doses (3–9) or maximum tolerated doses (10–17) in mmol/kg/day.
Figure 2. Possible intramolecularly cyclative hydrolysis of 6 leading to the release of the parent compound 2.
duration of action of the parent drug,11 high stability
against unspecific enzymes, or to protect the parent
drugs against hepatic first pass metabolism.12 However,
as shown in Table 1, four carbamates (3–5 and 8) did
not show any antitumor activity. These results again
indicate that the carbamates concerned were not suffi-
ciently conversed to the parent compound 2. Of excep-
tion was the analogue 6 which exhibited very potent
antitumor activity with IR of 95%, much higher than
that of etoposide (IR, 78%). Previously, it was reported
that 3-azido-3-deoxythymidin-5-yl-O-(2-hydroxyethyl)
carbonate showed increased anti-HIV potency com-
pared to its parent drug, zidovuldin (AZT). This was
attributed to the facile release of the parent compound,
AZT through intramolecular cyclic rearrangement of
the prodrug.13 Likewise, it could be postulated that in
the analogue 6, intramolecular cyclic rearrangement of
the hydroxy side chain (Fig. 2) might be an important
factor facilitating the hydrolysis of its 40-carbamate
linkage, resulting in the more effective release of the
cytotoxic compound 2 into foci and thus leading to
more potent antitumor activity of this compound com-
pared to other carbamates. Noteworthy, a carbonate
analogue 9 also exerted potent antitumor activity with
IR of 89%, suggesting that the carbonates might be
more effectively hydrolyzed than the carbamates under
physiological conditions to release the active parent
inside the cancer cell.
Though the two compounds 6 and 9 of the synthesized
carbamates and carbonates showed potent antitumor
activity, their water solubility proved to be relatively
poor for drug formulation for further in vivo evalua-
tion. We envisioned that esterification of the 40-hydroxy

3438
Y. Kim et al. / Bioorg. Med. Chem. Lett. 12 (2002) 3435–3438
group with amino acids would substantially improve the
solubility of the compounds in aqueous system. In
addition, the amino acid derivatives were expected to be
sufficiently reversible, partly due to the folding effects of
the side chain amino group, similar to the case of 6. This
prompted us to synthesize 8 additional amino acid pro-
drugs (10–17) of 2.
Gensler, W. J.; Horwitz, S. B. Cancer Res. 1978, 38, 2688.
Stahelin, H.; Wartburg, A. Progress Drug Res. 1989, 33, 169.
4. Kim, Y.; You, Y. J.; Kim, S. B.; Ahn, B. Z. Planta Med.
2001, 68, 271.
5. Terada, T.; Fujimoto, K.; Nomura, M.; Yamashita, J.;
Kobunai, T.; Takeda, S.; Wierzba, K.; Yamada, Y.; Yama-
guchi, H. Chem. Pharm. Bull. 1992, 40, 2720.
6. Kozawa, M.; Baba, K.; Matsuyama, Y.; Kido, T.; Sakai,
M.; Takemoto, T. Chem. Pharm. Bull. 1982, 30, 2885.
7. Laurent, D.; Yves, G.; Demerseman, P.; Kruczynski, A.;
Etievant, C.; Imbert, T.; Hill, B. T.; Monneret, C. J. Med.
Chem. 1998, 41, 4475.
The synthesized amino acid prodrugs showed excellent
water solubilities. For example, the water solubility of
40-demethyl-40-O-(8-aminohexanoyl)-DPT (15), a repre-
sentative selected from the 40-demethyl-40-O-(amino-
acyl)-DPT compounds (10–17), was 50 times higher
than that of the parent compound DDPT (data not
shown).9 The high water solubility allowed these com-
pounds to be administered at the maximum tolerated
doses. In the BDF1/3LL model, most of the amino acid
prodrugs (12–17) showed potent antitumor activity with
IR of 80–89%, higher than the IR value (78%) of eto-
poside (Table 1). However, compounds 10 and 11
showed relatively weak activity with IR values of 41and
33%, respectively, much lower than that of other amino
acid derivatives (12–17). The weak in vivo activity of 10
and 11 could be due to their premature hydrolysis
caused by the electron-withdrawing effects of the a-
protonated amino group. In contrast, the presence of
the phenyl moiety in 12 might somewhat lower the rate
of hydrolysis of this compound compared to 10 and 11,
resulting in its potent antitumor activity. In summary,
we have reported the synthesis of a series of prodrugs of
compound 2 (40-demethyl-4-deoxypodophylotoxin). Ele-
ven compounds among 15 prodrugs prepared showed
enhanced in vivo antitumor activity compared to the
parent compound 2. The carbamate 6 offered the best
antitumor activity with IR of 95%, followed by the
carbonate 9 (IR value of 89%) and amino acid ester 15
(IR values of 87%). The results demonstrate that the
design and synthesis of the presented prodrugs are ben-
eficial for therapeutic values of the compound 2. The
approaches may be applicable for other antitumor
agents, which possess similar functional features with 2.
8. All newly synthesized compounds gave satisfactory analy-
1
tical and spectroscopic data. 6; H NMR (90 MHz, CDCl3): d
6.68 (s, 1H), 6.53 (s, 1H), 6.39 (s, 2H), 5.93 (s, 2H), 4.90 (br,
1H), 4.60 (d, J=3.80 Hz, 1H), 4.52–4.40 (m, 1H), 3.99–3.62
(m, 9H), 3.25–2.69 (m, 6H). Anal. calcd for C24H25NO9: C,
61.14; H, 5.34; N, 2.97; found: C, 61.03; H, 6.5.38; N, 2.95. 9;
1H NMR (90 MHz, CDCl3): d 6.67 (s, 1H), 6.53 (s, 1H), 6.37
(s, 2H), 5.91(s, 2H), 4.66 (d, J=3.81Hz, 1H), 4.52–4.41(m,
1H), 3.98–3.62 (m, 9H), 3.24–2.72 (m, 6H). Anal. calcd for
C24H23ClO9: C, 58.72; H, 4.72; found: C, 58.62; H, 4.68. 15;
1H NMR (90 MHz, CDCl3): d 7.68 (br, 2H), 6.65 (s, 1H), 6.49
(s, 1H), 6.37 (s, 2H), 5.92 (s, 2H), 4.58 (d, J=3.84 Hz, 1H),
4.44–4.34 (m, 1H), 3.99–3.66 (m, 7H), 3.02–2.54 (m, 8H),
1.90–1.26 (m, 6H). Anal. calcd for C27H31NO8: C, 65.18; H,
6.28; N, 2.82; found: C, 65.00; H, 6.22; N, 2.79.
9. Water solubility was measured by a method described in
literature: Itoh, M.; Hagiwara, D.; Kamiya, T. Bull. Chem.
Soc. Jpn. 1977, 50, 718.
10. Ohisumi, K.; Hatanaka, T.; Nakagawa, R.; Fukuda, Y.;
Morinaga, Y.; Suga, Y.; Nihei, Y.; Ohishi, K.; Akiyama, Y.;
Tsuji, T. Anti-Cancer Drug Des. 1999, 14, 539.
11. Olsson, O. A. T.; Svensson, L. A. Pharm. Res. 1984, 1, 1 9.
12. Thorberg, S. O.; Berg, S.; Lundstorm, J.; Pettersson, B.;
Wijkstorm, A.; Sanchez, D.; Lindberg, P.; Nilsson, J. L. G.
J. Med. Chem. 1987, 30, 2008.
13. Vlieghe, P.; Bihel, F.; Clerc, T.; Pannecouque, C.; Witv-
rouw, M.; Erik, D. C.; Salles, J. P.; Chermann, J. C.; Kraus,
J. L. J. Med. Chem. 2001, 44, 777.
14. Cytotoxicity was determined by a method described pre-
viously. Skehan, P.; Storeng, R.; Scudiero, D.; Monk, A.;
MacMahon, J.; Vistica, D.; Warren, J.; Bokesch, H.; Kenny,
S.; Boyd, M. R. J. Nat. Cancer Inst. 1990, 82, 1107.
15. Lewis lung carcinoma (LLC) cells were transplanted sc
into the auxillary region of the BDF1mice. 24 h after tumor
cell transplantation, compounds tested (0.06 mmol/kg/day)
were intraperitoneally injected on days 1, 5 and 9. The tumor
sizes (width and length) were measured on day 17, and tumor
volume (TV) was calculated according to the following for-
mula: TV (mm3)=L (mm)ꢁW2 (mm2)/2, where L and W
represent the length and the width of the tumor mass, respec-
tively. The inhibition rate (IR) was calculated according to the
following formula: IR (%)=[(mean TV of the control group-
mean TV of the treated group)/mean TV of the control
group]ꢁ100.
References and Notes
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Chem. 1977, 20, 635. Loike, D.; Brewer, G. F.; Streniicht, H.;