Molecules 2006, 11, 849-857
Synthesis and Biological Evaluation of New 4β-5-Fu-substituted
Fu-Min Zhang 1,†, Xiao-Jun Yao 1,‡, Xuan Tian 1, 2,* and Yong-Qiang Tu 1,*
1 State Key Laboratory of Applied Organic Chemistry & Department of Chemistry, Lanzhou
University, Lanzhou, 730000, P. R. China. Tel: (+86) 931-8912410; Fax: (+86) 931-8912582;
E-mails: †email@example.com; ‡firstname.lastname@example.org
2 Xinjiang Production & Construction Corps Key Laboratory of Protection and Utilization of
Biological Resources in Tarim Basin, Tarim, Xinjiang, P.R. China.
* Authors to whom correspondence should be addressed. E-mails: email@example.com or
Received: 27 September 2006; in revised form: 26 October 2006 / Accepted: 27 October 2006 /
Published: 2 November 2006
Abstract: A series of new 4β-5-Fu-substituted 4'-demethylepipodophyllotoxin
derivatives were synthesized and evaluated, together with some previously prepared ones,
for their cytotoxic activities against four tumor cell lines (HL60, P388, A549 and
BEL7402). Three of these compounds exhibited superior in vitro anticancer activity
against P388 and A549 than the reference compound etoposide. In addition, the partition
coefficients (P) of all the new and previously synthesized derivatives were determined.
4'-Demethylepipodophyllotoxin, 5-fluorouracil, anticancer activities,
Podophyllotoxin (1, Figure 1) exhibits high cytotoxic activity against various cancer cell lines, but
its severe toxic side-effects have prevented it from being used directly as a therapeutic agent and this
has prompted the search for derivatives with a greater therapeutic window [1,2]. Etoposide (VP-16, 2),
teniposide (VM-26, 3) and etopophos (4) are three semisynthetic derivatives currently in clinical use as
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antineoplastic agents . Unfortunately, several drawbacks, such as myelosuppression, anemia,
metabolic inactivation, development of drug resistance, severe gastrointestinal side effects,
cytotoxicity towards normal cells and poor bioavailability, still exist during the administration of these
drugs, so extensive structural modifications of podophyllotoxin at various positions have been
undertaken in many laboratories to discover and develop more potent and less toxic anticancer agents
[4-11], and some of these derivatives, such as NK-611 (5), GL-331 (6), TOP-53 (7), are currently
being tested in phase I or II clinical trials for treatment of various cancers [12-14].
Figure 1. Podophyllotoxin and related compounds.
5-Fluorouracil (5-FU, 9, Scheme 1), an important clinically useful anticancer drug, was first
synthesized in 1957 . Combination chemotherapy including 5-FU has been used extensively in the
treatment of a wide range of solid tumors , but its negative effects, such as mucositis, nausea,
vomiting and cardiotoxicity have often been observed. To tackle these problems, numerous
modifications of the 5-FU structure have also been performed. The N-1 or/and N-3 substituted
derivatives, in particular, have exhibited improved pharmacological and pharmacokinetic properties,
including increased bioactivity, selectivity, metabolic stability, absorption and lower toxicity [17-22].
In recent years, nearly one hundred podophyllotoxin derivatives have been designed and
synthesized and their biological activities against various cancers have been evaluated in the author’s
laboratory [23-28]. As a result some less toxic derivatives have been discovered, for example, GP-11
(8) . As a part of our ongoing effort to find derivatives with improved anticancer activity and water
solubility, we first designed a new series of derivatives seeking to combine the different anticancer
mechanisms of 4'-demethyepipodophyllotoxin and 5-fluorouracil. In a previous communication ,
we reported the synthesis of seven novel derivatives and the evaluation of their activity against only
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two matrix metalloproteinases (MMP1 and MMP3). Based on the above encouraging activity results,
we report herein the synthesis of another three new derivatives, the anticancer activities of all ten
derivatives against four cancer lines (HL60, P388, A549 and BEL7402) and the determination of their
Results and Discussion
Drug resistance is the most important challenge in cancer treatment research. Clinically, the cancer
cells can’t be totally “killed” by using a single drug for a long period of time, as they will become
resistant to this drug and other drugs with a similar mechanism of action. In order to ensure efficacy,
the optimal administration schedule involves a combination of two or more drugs, especially drugs
with different mechanisms of action . Based on the combination principle of drug design, the two
drugs may be connected directly or by means of a linker. This technique can be used to overcome
many problems including poor solubility, absorption, patient acceptability, instability and toxicity, and
especially drug resistance. Natural L-amino acids are good pharmacophore carriers as well as good
kinetophores, so we sought to combine demethyepipodophyllotoxin (an inhibitor of topoisomerase II)
and 5-FU (a nucleoside antimetabolite) through a peptide bond derived from a natural L-amino acid.
Scheme 1. The synthesis  and structures of compounds 1.1-1.10.
1.1 n = 1; 1.2 n = 2; 1.3 R = H; 1.4 R = CH2CHMe2; 1.5 R = CHMeCH2Me; 1.6 R =
CH2CH2SMe; 1.7 R = CH2Ph; 1.8 R = Me; 1.9 R = CHMe2; 1.10 R = CH(OH)Me.
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At the same time, the significance of a drug’s lipophilicity has widely been recognized by many
researchers working in the drug discovery and drug design fields [31, 32]. The absorption, distribution,
metabolism, excretion and toxicity of a drug are closely related with its lipophilicity, so this property
must be considered in the rational design of anticancer drugs [33, 34]. The normal standard for
expressing lipophilicity is the partition coefficient (P) in the immiscible n-octanol/water binary solvent
system. Biologically active compounds, whose log P approaches zero either from the negative or
positive side, should be ideal drugs, since these compounds possess appropriate hydrophilicity and
lipophilicity. With this in mind our other aim in connecting these two types of anticancer drugs was to
change demethyepipodophyllotoxin’s hydrophilicity and 5-fluorouracil’s lipophilicity, respectively, in
the hope of producing derivatives that might be good drug candidates.
The three new compounds 1.8-1.10 were synthesized according to a previously published method
 (Scheme 1).
Biological activity and partition coefficients
The cytotoxicities of compounds 1.1-1.10 were tested in vitro against four tumor cell lines (HL60,
P388, A549 and BEL7402) [35, 36]. The assay results were then used to obtain the corresponding
inhibition rates, from which IC50 values were calculated (Table 1). These synthetic 4׳-demethyl-
epipodophyllotoxin derivatives exhibited an interesting in vitro anticancer activity. Some of the
derivatives were shown to be nearly equipotent or more potent than etoposide (2) and 5-Fu (9), two
clinical drugs, in particular the compounds 1.8, 1.9, 1.10, which exhibited highly potent in vitro
anticancer activity against all four cancer cell lines.
Table 1. Cytotoxic Activity of 1.1-1.10 in vitro (IC50, uM) and their partition coefficients (P).
1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 1.10 2 9
HL-60 a -c - c 3.57 5.38 5.80 5.45 4.80 0.9811.37 0.209 0.404 - c
P388 a 2.21 2.27 0.388 2.57 2.06 2.14 3.13 0.0473 0.0102 0.386 6.13 15.8
A549 b 3.56 10.5 1.74 2.85 3.25 3.74 4.07 0.036 0.522 0.0857 0.738 4.56
BEL7402 b - c - c 4.35 - c - c - c - c 0.5691.34 0.478 1.23 1.66
11.6 2.28 0.59 7.25 10.7 3.04 3.04 2.71 3.64 3.16 3.71 ND d
a MTT method, drug exposure was for 48h; b SRB method, drug exposure was for 72h.
c IC50 >100 uM. d ND: not determined.
The partition coefficients (P) of these derivatives were determined according to the published
method , and the results are included in Table 1 (row five). The data showed that the logarithms of
the partition coefficient of compounds 1.2, 1.3, 1.8 are all closer to zero than that of etoposide.
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Structure-activity relationship (SAR) analysis
A simple structure-activity relationship (SAR) analysis was undertaken to study the influence of
the different substituents on the cytotoxic potency of the synthesized compounds. The results can be
summarized as follows: 1) compounds 1.1 and 1.2, with no amino acid moiety, were less active than
the other eight compounds, so we may conclude that the peptide bond is important for anticancer
activity; 2) the relationship between the anticancer activities and the different substituents on the α-
carbon of the amino acids appear to indicate that moderately sized hydrophobic substituents such as
Me, CHMe2 and CH(OH)Me increase the anticancer activity against HL60, A549 and P388, so in
order to obtain anticancer derivatives with good activity it seems essential to consider such substituents
at those positions; 3) for the anticancer activity it is also important to find a balance between the size of
substituents and the partition coefficients.
In summary, we have synthesized some 4'-demethylepipodophyllotoxin derivatives that display
more potent in vitro anti-cancer than the reference standard etoposide. Of special interest is the fact
that we think that the activity of these new derivatives against four cancer cell lines may be synergistic.
A study of the in vivo anticancer activities of compounds 1.8, 1.9 and 1.10 is in progress.
Melting points were determined on an X4 melting point apparatus and are uncorrected. The 1H-
NMR spectra were recorded in CD3COCD3 solutions containing TMS as an internal reference on a
Bruker AM 400MHz spectrometer. IR spectra were recorded on a NIC-DX IR spectrometer. High-
resolution mass spectra were recorded on a Bruker Daltonics APEX II 49e spectrometer using the ESI
technique. Optical rotations were measured on a Perkin Elmer Model 341 digital polarimeter.
Compounds 1.8, 1.9 and 1.10 were prepared according to the previously published method  and
their analytical data are given below.
4'-O-Demethyl-4β-N-(5-FU acetyl-L-alanine acylamine)-4-desoxypodophyllotoxin (1.8). Yield: 83%;
m.p. 192-194 oC; 1H-NMR: 7.68 (d, J = 6.4 Hz, 5-FU ring H-6), 6.60 (s, 1H, H-5), 6.51 (s, 1H, H-8),
6.34 (s, 2H, H-2', 6'), 5.98 (d, J = 2.8 Hz, OCH2O), 5.28 (d, J = 3.2 Hz, H-4), 4.52 (s, 2H, 5-Fu ring N-
substituted CH2), 4.48 (m, 1H, amino acid α-CH), 4.25 (m, 1H, H-11), 3.96 (m, 1H, H-11'), 3.67 (s,
6H, OCH3), 3.10 (m, 1H, H-2), 3.03 (m, 1H, H-3), 1.26 (d, J = 7.2 Hz, 3H, CH3). IR (KBr) υ cm-1,
3383, 1773, 1513, 1482, 1232, 931; HRMS (FAB) C30H29O11N4FNa (M+Na)+: calcd. 663.1709, found
4'-O-Demethyl-4β-N-(5-FU acetyl-L-valine acylamine)-4-desoxypodophyllotoxin (1.9). Yield: 84%;
m.p. 214-216 oC; 1H-NMR: 7.80 (d, J = 6.5 Hz, 1H, 5-Fu ring H-6), 6.74 (s, 1H, H-5), 6.50 (s, 1H, H-
D = -72.4 ° (c = 0.5, CH3COCH3).