Design, synthesis, and biological evaluation of novel pyridine acid esters of podophyllotoxin and esters of 4′-demethylepipodophyllotoxin

Article abstract of DOI:10.1007/s00044-007-9010-2

Podophyllotoxin and related analogs present numerous challenges associated with optimal antitumor activity and severe unpredictable toxicity. In the course of our ongoing investigation of quantitative structure-activity relationships to find biorational antitumor drugs, two series of pyridine acid ester derivatives of podophyllotoxin and 4′-demethylepipodophyllotoxin have been prepared by reacting the corresponding pyridine acids with the hydroxyl group of podophyllotoxin and 4′-demethyl epipodophyllotoxin in the presence of dimethylaminopyridine (DMAP) and N,N-dicyclohexylcarbodiimde (DCC). The structures of the compounds were extensively characterized by using ¹H-nuclear magnetic resonance, mass spectroscopy, infrared, and elemental analysis and evaluated for their in vitro cytotoxicity on two neoplastic cell lines (P-388 murine leukemia, A-549 human lung carcinoma) using a MTT-based assay, the results indicated significantly higher efficacy of these compounds in comparison with the prototypical inhibitor etoposide. On the basis of the preliminary biological testing results, it suggested that the cytotoxic activity of OCH₃ at C-4′ is more potent than that of its demethylated analogs and the podophyllotoxin substitution at C-4 may be optimal for synthesizing more potent cytotoxic compounds.

Full text of DOI:10.1007/s00044-007-9010-2

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2007) 16:319–330
DOI 10.1007/s00044-007-9010-2
ORIGINAL RESEARCH
Design, synthesis, and biological evaluation of novel
pyridine acid esters of podophyllotoxin and esters of
40-demethylepipodophyllotoxin
Ying-Qian Liu Æ Liu Yang Æ Xuan Tian
Received: 9 July 2006 / Accepted: 9 January 2007 / Published online: 8 January 2008
¨
Ó Birkhauser Boston 2008
Abstract Podophyllotoxin and related analogs present numerous challenges
associated with optimal antitumor activity and severe unpredictable toxicity. In the
course of our ongoing investigation of quantitative structure–activity relationships
to find biorational antitumor drugs, two series of pyridine acid ester derivatives of
podophyllotoxin and 40-demethylepipodophyllotoxin have been prepared by react-
ing the corresponding pyridine acids with the hydroxyl group of podophyllotoxin
and 40-demethyl epipodophyllotoxin in the presence of dimethylaminopyridine
(DMAP) and N,N-dicyclohexylcarbodiimde (DCC). The structures of the com-
1
pounds were extensively characterized by using H-nuclear magnetic resonance,
mass spectroscopy, infrared, and elemental analysis and evaluated for their in vitro
cytotoxicity on two neoplastic cell lines (P-388 murine leukemia, A-549 human
lung carcinoma) using a MTT-based assay, the results indicated significantly higher
efficacy of these compounds in comparison with the prototypical inhibitor etopo-
side. On the basis of the preliminary biological testing results, it suggested that the
cytotoxic activity of OCH3 at C-40 is more potent than that of its demethylated
analogs and the podophyllotoxin substitution at C-4 may be optimal for synthesizing
more potent cytotoxic compounds.
Keywords Antitumor activity ꢀ 4’-Demethylepipodophyllotoxin ꢀ
Podophyllotoxin ꢀ Pyridine acid ꢀ Structure ꢀ activity relationship
Y.-Q. Liu (&) ꢀ X. Tian
State Key Laboratory of Applied Organic Chemistry, Lanzhou Universitry, Gansu Lanzhou 730000,
P. R. China
e-mail: liuyq02@st.lzu.edu.cn
L. Yang
Analytic Center of Environment Engineering, Environmental and Municipal Engineering School,
Lanzhou Jiaotong Universtitry, Gansu Lanzhou 730000, P.R.China

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Med Chem Res (2007) 16:319–330
Introduction
Podophyllotoxin (1) is a natural bioactive plant-based lignan known to have
significant antineoplastic and antiviral properties (Jardine, 1980; Bedows and
Hatfield, 1982; Ayres and Loike, 1990; Bohlin and Rosen, 1996; Charlton, 1998;
Canel et al., 2000; Gordaliza et al., 2001; Castro et al., 2003; Zhu et al., 2004). Its
powerful cytotoxic properties have been attributed to its binding to tubulin during
mitosis and thus inhibiting microtubule assembly (King and Sullivan, 1946;
`
Desbene and Giorgi-Renault, 2001). Extensive attempts to chemically modify
podophyllotoxin to obtain more potent and less toxic antitumor agents have resulted
in the clinical introduction of etoposide (2) and teniposide (3), and more recently,
etopophos (4), which possesses a disodium phosphate salt at the 40-phenolic oxygen
of 2 and is used in the clinic as a prodrug of etoposide (as shown in Fig. 1).
Interestingly, these semisynthetic derivatives and the parent compound, podo-
phyllotoxin, show different mechanisms of action. Podophyllotoxin inhibits tubulin
polymerization through its interaction at the colchicines binding site, while
etoposide and congeners involve the inhibition of topoisomerase II-mediated DNA
ligation by reversible cleavable complex stabilization, resulting in tumor cell death
and its corresponding ortho-quinone via oxidative demethylation (Berger et al.,
1996; Burden and Osheroff, 1998; Hande, 1998; Imbert, 1998; Botta et al., 2001;
Gordaliza et al., 2004; Islam and Iskander, 2004). However, a series of
disadvantages of podophyllotoxin and its congeners as chemotherapeutic agents
derive not only from their undesirable side effects (such as anemia, alopecia,
carcinogenicity, gastrointestinal disturbances, etc.), but also from their lack of
selectivity against tumoral cells and their insolubility Gordaliza et al., 1996; Lopez-
Perez et al., 2004). In this regard, it is necessary to continuously research and
synthesize podophyllotoxin and etoposide analogs that encompass a broad spectrum
of antineoplastic activities and overcome these clinical limitations; if possible, this
type of drug should work via tubulin polymerization inhibition or, much better, by
inhibition of both tubulin and DNA topoisomerase II.
A number of investigations have demonstrated the importance of an ester group
in the cytotoxicity and antileukemic activity of compounds in certain classes (Lee
O
O
R
O
O
O
CH3
O
O
O
OH
HO
HO
HO
HO
5
O
11
10
O
6
7
4
3
2
O
A
O
O
O
O
O
C
D O
12
O
B
8
O
O
9
1
1'
6'
2'
E
3'
OCH3
5'
H3CO
OCH3
H3CO
H3CO
OCH3
4'
OCH3
O
ONa
ONa
OH
P
O
Etoposide 2 R= CH3
Teniposide 3 R=
Podophyllotoxin 1
Etopophos 4
S
Fig. 1 Structures of podophyllotoxin 1, etoposide 2, teniposide 3, and etopophos 4

Med Chem Res (2007) 16:319–330
321
et al., 1973, 1982; Levy et al., 1983; Nagao et al., 1991). The specific role of the
bioactivity of pyridine ring has been recognized and the introduction of some
pyridine analogs into the molecules of drugs could result in new compounds that
have significant biological activity compared with the parent compounds Straub
et al., 1997; Bolognese et al., 2002; Tiwari et al., 2002; Kuo et al., 2005). It was also
reported that a diverse array of naturally occurring alkaloids in sponges exhibit
potent bioactivities because of the presence of a pyridine moiety (Vincent et al.,
1997; Gordon et al., 2005).
Based on the preceding, we designed 12 novel ester derivatives with the hope that
the C-4-hydroxyl group esterified by a pyridine moiety would simultaneously
circumvent the various limitations and result in superior pharmacological profiles.
We examined them for their in vitro antitumor activity against P-388 murine
leukemia cell growth and against A-549 human lung carcinoma cell growth.
In view of the available information on structure–activity relationship studies and
in continuation of our ongoing project on the design and development structurally
modified podophyllotoxin congeners we describe herein synthesis and structure–
activity relationships of a series of pyridine acyl compounds derived from
podophyllotoxin and 40-O-demethylepipodophyllotoxin.
Results and Discussion
Chemistry
Podophyllotoxin (1) was isolated from a Chinese medicinal herb, Podophyllum
emodi Wall var Chinesis Sprague, and served as the starting material for the
preparations of all the derivatives. The reaction of podophyllotoxin with hydrogen
bromide simultaneously O-demethylated at the 40-position and generated the
reactive 4b-bromo-deoxy-demethylpodophyllotoxin as the major product, the
reaction of 4b-bromo-deoxy-demethylpodophyllotoxin with H2O used to obtain
40-O-demethylepipodophyllotoxin (7) (Kuhn et al., 1969). The desired compounds
5a–5f and 8a–8f were prepared in almost quantitative yields by the straightforward
acylation of podophyllotoxin or 40-demethylepipodophyllotoxin with the corre-
sponding pyridine acids in the presence of the coupling agent N,N-dic-
yclohexylcarbodiimde (DCC) and a catalytic amount of 4-dimethylaminopyridine
(DMAP) to form an ester linkage under an atmosphere of nitrogen at room
temperature (Figs. 2 and 3). DCC can preserve the configuration of the chiral
reactants to a greater extent in the esterifying reaction. The average yield of
esterification was greater than 80% and the structures of the desired compounds
were extensively characterized by using 1H-nuclear magnetic resonance (1H-NMR),
mass spectroscopy (MS), infrared (IR), and elemental analysis.
The reaction of the hydroxyl group at C-4 of podophyllotoxin and 40-
demethylepipodophyllotoxin with the various pyridine acids (viz. isonicotinic acid,
nicotinic acid, picolinic acid, 2-chloronicotinic acid, 6-chloronicotinic acid, 5-
bromonicotinic acid) gave the corresponding podophyllotoxin and 40-demethyle-
pipodophyllotoxin ester derivatives in almost quantitative yields. To simplify the

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Med Chem Res (2007) 16:319–330
O
C
OH
O
R
O
O
O
O
O
O
i
N
5b
Cl
N
5a
N
5c
N
5d
O
O
Br
OCH3
OCH3
H3CO
H3CO
N
Cl
OCH3
OCH3
N
5f
1
5e
5a-5f
Reagents and conditions: i: RCOOH, DCC/DMAP, stir, 2hr.
Fig. 2 The synthetic methodology for the preparation of compounds 5a–5f Reagents and conditions: i:
RCOOH, DCC/ DMAP, stir, 2 h
OH
OH
Br
O
O
O
O
O
O
O
O
O
i
ii
O
O
O
OCH3
OCH3
OCH3
H3CO
H3CO
H3CO
OH
7
OCH3
1
OH
6
O
C
R
O
O
O
O
iii
N
8b
Cl
N
8a
N
8c
N
8d
O
Br
OCH3
H3CO
OH
N
8e
N
8f
Cl
8a-8e
; iii: RCOOH, DCC/DMAP, stir, 2hr.
Reagents and conditions: i: hydrogen bromide; ii: water-acetone, BaCO3
Fig. 3 The synthetic methodology for the preparation of compounds 7 and 8a–8f Reagents and
conditions: i: hydrogen bromide; ii: water–acetone, BaCO3; iii: RCOOH, DCC/ DMAP, stir, 2 h
naming of the target compounds 5a–5f and 8a–8f, podophyllotoxin and 40-
demethylepipodophyllotoxin are considered as the alcohol moiety in these
reactions, thus we have coined the prefixes for these ester derivatives as
‘‘podophyllotoxinyl’’ and ‘‘40-demethylepipodophyllotoxinyl.’’ All of these ester
derivatives were white solids and required an 8:1 (vol:vol) mixture of dichlorom-
ethane–acetone as eluent for thin-layer chromatography (TLC) analysis. The
chemical shifts of various protons of pyridine acid and podophyllotoxin or 40-
demethylepipodophyllotoxin moieties of these derivatives were successfully
assigned with the help of 1H-NMR. The configuration at the C-4 position for
compounds 5a–5f and 8a–8f was assigned on the basis of their J3, 4 coupling
constants. The C-4a-substituted compounds have a J3, 4 ꢁ8.5 Hz as H-3 is trans to
H-4, whereas C-4b-substituted compounds have a J3, 4 <4.5Hz, owing to the cis
relationship between H-3 and H-4 (Fred Brewer, 1979; Hansen et al., 1993).
Moreover, the absence of C-4b isomers for compounds 5a–5f may be explained
through the reaction mechanism involving the assistance of DCC in the presence of

Med Chem Res (2007) 16:319–330
323
a catalytic amount of DMAP to form the ester linkages (Smith et al., 1958;
Doleachall and Lempert, 1963), as the a-stereoselectivity of C-4 is retained as the a-
bond between C-4 and the hydroxyl group is not involved in the chemical reaction,
the absence of C-4a isomers for compounds 8a–8f may have the same explanation
as the above reaction mechanism, the previous spectral studies have further
supported stereospecific in these molecules (Nagao et al., 1991; Greenwald et al.,
1999; Lie Ken Jie et al., 1999). On the basis of these facts, we were able to elucidate
the configuration at the C-4 position in the series. We could thus verify that coupling
reactions between various pyridine acids and podophyllotoxin or 40-demethylepip-
odophyllotoxin are stereospecific in nature.
Biological evaluation
Cytotoxicities of the target compounds 5a–5f and 8a–8f were tested in vitro against
two cell lines (P-388 murine leukaemia and A-549 human lung carcinoma cells)
using the MTT assay as previously described (Minko et al., 2002). The prototypical
inhibitor etoposide was included as a reference standard. The results of these assays
were used to obtain the corresponding inhibition rates, as shown in Table 1.
As can be seen, the introduction of a number of the pyridine acid moieties at the
C-4 hydroxyl of podophyllotoxin and 40-demethylepipodophyllotoxin gave in vitro
cytotoxicity against the two tested neoplastic cell lines comparable or superior to
that of the prototypical inhibitor etoposide, which represented an improvement in
their potency with respect to the latter. Another interesting aspect observed in this
investigation is that a large of number of analogs exhibit improved cytotoxicity
particularly in the presence of 40-O-methyl functionality. The inhibition rate values
of all of esters of podophyllotoxin were, in general, better than those of 40-
demethylepipodophyllotoxin, which demonstrated that this activity may mainly be
due to different biological mechanisms. Compounds 5a–5f, which belong to the
podophyllotoxin series (4 a-substitution and 40-OMe), may exhibit an inhibition of
microtubule assembly of the same magnitude as podophyllotoxin itself, while they
were almost devoid of topoisomerase II activity. The cytotoxicity for compounds
8a–8f may mainly result from DNA topoisomerase II inhibition (Gupta and
Chenchaiah, 1987; Lee et al., 1989; Damayanthi and Low, 1998; Canel et al., 2000;
Gordaliza et al., 2000).
Conclusions
This is the first report of the application of the DCC coupling chemistry procedure to
introduce pyridine moieties into the molecule of podophyllotoxin analogs. The
structures of the compounds were extensively characterized by using 1H-NMR, MS,
IR, and elemental analysis. The preliminary biological testing has shown that the
introduction of the pyridine acyl moieties at C-4-OH of podophyllotoxin and 40-
demethylepipodophyllotoxin results in improved biological activity in comparison
to etoposide. This investigation has also highlighted the importance of the 40-O-
methyl functionality in such analogs for antitumor activity. The present data provide

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Med Chem Res (2007) 16:319–330
Table 1 In vitro cytotoxicity of 5a–5f and 8a–8f against P-388 murine leukemia and A-549 human lung
carcinoma
Compound
C (mol/L)
Inhibition rate (%)
Compound
8a
C (mol/L)
Inhibition rate (%)
P-388a
A-549a
P-388a
A-549a
5a
0ꢂ4
89.0
85.1
84.8
84.8
83.8
87.5
84.8
84.1
83.8
83.4
87.7
87.0
86.0
85.5
85.4
87.3
85.8
85.0
84.7
80.6
92.1
88.3
85.0
84.3
80.1
97.7
87.4
85.0
82.8
80.6
99.2
70.6
25.5
6.0
89.2
84. 4
82.6
81.8
81.4
87.5
84.9
82.8
82.2
81.0
89.2
84.0
82.3
81.8
81.4
86.6
82.5
81.3
80.4
79.5
87.9
87.0
85.9
83.0
81.5
91.1
84.7
82.7
80.9
77.3
97.5
50.7
45.0
0
10ꢂ4
10ꢂ5
10ꢂ6
10ꢂ7
10ꢂ8
10ꢂ4
10ꢂ5
10ꢂ6
10ꢂ7
10ꢂ8
10ꢂ4
10ꢂ5
10ꢂ6
10ꢂ7
10ꢂ8
10ꢂ4
10ꢂ5
10ꢂ6
10ꢂ7
10ꢂ8
10ꢂ4
10ꢂ5
10ꢂ6
10ꢂ7
10ꢂ8
10ꢂ4
10ꢂ5
10ꢂ6
10ꢂ7
10ꢂ8
10ꢂ4
10ꢂ5
10ꢂ6
10ꢂ7
10ꢂ8
100
86.9
76.5
53.5
19.8
100
87.9
72.1
71.4
4.4
95.1
67.8
66.4
66.1
54.0
91.4
79.9
74.0
72.6
71.9
91.6
78.8
73.7
56.0
53.7
92.5
78.1
69.8
62.5
56.6
85.4
93.9
72.5
70.7
59.3
87.3
82.0
78.4
74.6
71.0
97.5
50.7
45.0
0
10ꢂ5
10ꢂ6
10ꢂ7
10ꢂ8
10ꢂ4
10ꢂ5
10ꢂ6
10ꢂ7
10ꢂ8
10ꢂ4
10ꢂ5
10ꢂ6
10ꢂ7
10ꢂ8
10ꢂ4
10ꢂ5
10ꢂ6
10ꢂ7
10ꢂ8
10ꢂ4
10ꢂ5
10ꢂ6
10ꢂ7
10ꢂ8
10ꢂ4
10ꢂ5
10ꢂ6
10ꢂ7
10ꢂ8
10ꢂ4
10ꢂ5
10ꢂ6
10ꢂ7
10ꢂ8
5b
5c
5d
5e
5f
2
8b
8c
8d
8e
8f
2
100
88.6
73.5
68.0
39.7
100
84.7
72.3
12.8
9.1
100
81.1
73.4
28.4
20.2
100
87.4
66.1
63.5
2.0
99.2
70.6
25.5
6.0
1.8
0
1.8
0
a
MTT method, drug exposure was for 72 h
a promising outlook for creating novel antitumor agents on the basis of pyridine
ester derivatives of podophyllotoxin analogs, and this will allow researchers in this
area to prepare podophyllotoxin related analogs incorporating such functionalities.

Med Chem Res (2007) 16:319–330
325
Some of the potential candidates are undergoing detailed investigation, particularly
biochemical studies.
Experimental
General
All melting points were taken on a Kofler melting point apparatus and are
1
uncorrected, IR spectra were obtained on NIC-5DX spectra photometer, H-NMR
spectra were obtained via a Bruker AM–400, and all chemical shifts were reported
in ppm downfield from trimethylsilane (TMS). Mass spectral analysis was done on a
ZAB-HS and Bruker Daltonics APEXII49e instrument. Optical rotations were
determined on Perkin Elmer Model 341 spectropolarimeter. Elemental analysis was
taken on a YANACO-CHN-CODERMT-3 instrument. Anhydrous dichloromethane
˚
was obtained by distillation from phosphorus pentoxide and stored over 4 A
molecular sieves. All reactions that required anhydrous conditions were performed
under a positive nitrogen flow, and all glassware was oven-dried and/or flame-dried.
Natural product source
Podophyllotoxin (1) was isolated from the ethanol extract of the Chinese medicinal
herb Podophyllum emodi Wall var Chinesis Sprague via column chromatography
and elution with a CHCl3-MeOH (20:1) mobile phase. Fractions containing the
product were evaporated in vacuo and the podophyllotoxin was recrystallized from
C6H6. The resulting material was of greater than 97% purity, which was confirmed
by direct comparison with an authentic sample and spectral data reported previously
(Fred Brewer et al., 1979).
40-Demethyl-epipodophyllotoxin
Podophyllotoxin (5g) was dissolved in dry CH2Cl2 (75 ml) and dry HBr gas was
bubbled through the solution to saturation. The reaction mixture was capped and
allowed to stand at room temperature for 48 h. Removal of the solvent yielded 40-
demethyl-4-bromo-4-desoxypodophyllotoxin, which was treated with H2O (25 ml),
acetone (50 ml), and calcium carbonate (5g) and heated at reflux for 1 h. The
reaction mixture was extracted with chloroform and chromatographed on a Si gel
column. The product was obtained by elution with CHCl3-CH3OH (30:1) to give 2.5
g (52%) of 7. Spectral data, specific rotation and m.p. agreed with those in the
literature (Kuhn et al., 1969).
General procedure for synthesis of target compounds 5a–5f and 8a–8f
A mixture of acid (0.001 mol), podophyllotoxin (0.414 g, 0.001 mol) or 40-demethyl
epipodophyllotoxin (0.400 g, 0.001 mol), dimethylaminopyridine (DMAP, 0.1 g)
was stirred in anhydrous dichloromethane (10 ml) for 5 min at room temperature

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Med Chem Res (2007) 16:319–330
under nitrogen. N,N-dicyclohexylcarbodiimide (DCC, 0.21 g, 0.001 mol) was added
and the reaction mixture was stirred for 2 h. The reaction mixture was filtered and
the filtrate was evaporated. The residue was separated by flash-column chromatog-
raphy (gradient elution with mixtures of dichloromethane–acetone) on silica gel and
monitored by TLC. Synthesized target compounds 5a–5f and 8a–8f were
1
characterized by m.p., H-NMR, IR, MS spectral analyses, and elemental analysis.
4-O-Podophyllotoxinyl-isonicotinate (5a): Yield: 82%; m.p. 172–1748C;
[a]20D= –89o (c = 0.1, CH3COCH3); IR (KBr) (cmꢂ1): 1779 (lactone), 1728
(CO), 1588, 1505, 1484 (aromatic C = C), 928 (OCH2O), 787, 759, 708 (pyridinyl
1
ring); H-NMR: (CD3COCD3) d ppm: 3.08 (1H, m, 3-H), 3.38 (1H, dd, 2-H), 3.72
(6H, s, 30,50-OCH3), 3.78 (3H, s, 40-OCH3), 4.53-4.68 (2H, t, 11a,11b-H), 4.70 (1H,
d, 1-H), 6.04 (2H, OCH2O), 6.29 (1H, d, J = 9.6 Hz, 4-H), 6.49 (2H, d, 20,60-H), 6.65
(1H, s, 8-H), 7.10 (1H, s, 5-H), 7.94 (2H, pyridine ring-200,600-H), 8.82 (2H, pyridine
ring-300,500-H); MS (FAB) m/z (int.%): 520 (M++1, 38), 397 (100); Anal.
(C28H25O9N) calcd. C: 64.74, H: 4.82, N: 2.70; found C: 64.44, H: 4.92, N: 2.52.
4-O-Podophyllotoxinyl-nicotinate (5b): Yield: 80%; m.p. 177–1788C; [a]20
=
D
–798 (c = 0.1, CH3COCH3); IR (KBr) (cmꢂ1): 1766 (lactone), 1725 (CO), 1589,
1505, 1482 (aromatic C = C), 931 (OCH2O), 790, 767, 743 (pyridinyl ring); H-
1
NMR: (CD3COCD3) d ppm: 2.82 (1H, m, 3-H), 3.35 (1H, dd, 2-H), 3.70 (6H, s,
30,50-OCH3), 3.78 (3H, s, 40-OCH3), 4.15 (2H, t, 11a, 11b-H), 4.78 (1H, d, 1-H),
5.99 (2H, -OCH2O), 6.30 (1H, d, J = 9.2Hz, 4-H), 6.50 (2H, d, 20,60-H), 6.65 (1H, s,
8-H), 7.11 (1H, s, 5-H), 7.60 (1H, pyridine ring-500-H), 8.40 (1H, pyridine ring-600-
H), 8.83 (1H, pyridine ring-400-H), 9.20 (1H, pyridine ring-200-H); MS (FAB) m/z
(int.%): 520 (M++1, 42), 397 (100); Anal. (C28H25O9N) calcd. C: 64.74, H: 4.82, N:
2.70; found C: 64.81, H: 4.67, N: 2.71.
4-O-Podophyllotoxinyl-picolinate (5c): Yield: 80%; m.p. 172–1748C; [a]20D= –
838 (c = 0.1, CH3COCH3); IR (KBr) (cmꢂ1): 1776 (lactone), 1726 (CO), 1587,
1
1505, 1482 (aromatic C = C), 930 (OCH2O), 789,766,747 (pyridinyl ring); H-
NMR: (CD3COCD3)dppm: 2.78(1H, m, 3-H), 3.05(1H, dd, 2-H), 3.67(6H, s, 30,50-
OCH3), 3.78 (3H, s, 40- OCH3), 4.15(2H, t, 11a,11b-H), 4.57 (1H, d, 1-H), 6.04 (2H,
-OCH2O), 6.30 (1H, d, J = 9.6 Hz, 4-H), 6.48 (2H, d, 20,60-H), 7.07(1H, s, 8-H),
7.19(1H, s, 5-H), 7.66 (1H, pyridine ring-400-H), 8.03 (1H, pyridine ring-500-H), 8.18
(1H, pyridine ring-300-H), 8.74 (1H, pyridine ring-600-H). MS (FAB) m/z (int.%): 520
(M++1, 40), 397 (100). Anal. (C28H25O9N) calcd C: 64.74, H: 4.82, N: 2.70; found
C: 64.62, H: 4.75, N: 2.47.
4-O-Podophyllotoxinyl-200-chloronicotinate (5d): Yield: 85%; m.p. 131–
1338C; [a]20D= –1048 (c = 0.1, CH3COCH3); IR (KBr) (cmꢂ1): 1778 (lactone),
1735 (CO), 1584, 1505, 1483 (aromatic C = C), 928 (OCH2O), 795,765,745
(pyridinyl ring); 1H-NMR: (CD3COCD3)dppm: 3.13 (1H, m, 3-H), 3.36 (1H, dd, 2-
H), 3.68 (6H, s, 30,50 -OCH3), 3.70 (3H, s, 40-OCH3),4.39 (1H, t, 11), 4.54 (1H, t,
11b-H), 4.68 (1H, d, 1-H), 6.02 (2H, OCH2O), 6.32 (1H, d, J = 9.2Hz, 4-H), 6.46
(2H, d, 20,60-H), 6.64 (1H, s, 8-H), 7.11 (1H, s, 5-H), 7.60 (1H, pyridine ring-600-H),
8.43 (1H, pyridine ring-500-H), 8.60 (1H, pyridine ring-400-H). MS (FAB) m/z
(int.%): 553 (M+, 38), 397 (100). Anal. (C28H24O9NCl) calcd C: 60.76, H: 4.34, N:
2.53; found C: 60.01, H: 4.27, N: 2.47.

Med Chem Res (2007) 16:319–330
327
4-O-Podophyllotoxinyl-6’’-chloronicotinate (5e): Yield: 83%; m.p. 137–
1398C; [a]20D= –778 (c = 0.1, CH3COCH3); IR (KBr) (cmꢂ1): 1779 (lactone),
1725 (CO), 1587, 1506, 1484 (aromatic C = C), 928 (OCH2O), 795,766,746
(pyridinyl ring ); 1H-NMR: (CD3COCD3)dppm: 3.11(1H, m, 3-H), 3.14 (1H, dd, 2-
H), 3.68 (6H, s, 30, 50- OCH3), 3.70 (3H, s, 40-OCH3), 4.37 (1H, t, 11), 4.50 (1H, t,
11b-H), 4.68 (1H, d, 1-H), 6.04 (2H, OCH2O), 6.28 (1H, d, J = 9.2Hz, 4-H), 6.56
(2H, d, 20,60-H), 6.65 (1H, s, 8-H), 7.11 (1H, s, 5-H), 7.66 (1H, pyridine ring-500-H),
8.42 (1H, pyridine ring-600-H), 9.00 (1H, pyridine ring-200-H). MS (FAB) m/z
(int.%): 553 (M+, 40), 397 (100). Anal. (C28H24O9 NCl) calcd C: 60.76, H: 4.34, N:
2.53; found C: 60.72, H: 4.18, N: 2.67.
4-O-Podophyllotoxinyl-500-bromonicotinate (5f): Yield: 85%; m.p. 178–1798C;
[a]20D= –808 (c = 0.1, CH3COCH3); IR (KBr) (cmꢂ1): 1779 (lactone), 1725 (CO),
1586, 1505, 1484 (aromatic C = C), 928 (OCH2O), 798, 764,742 (pyridinyl ring),
1H-NMR: (CD3COCD3)dppm: 3.13 (1H, m, 3-H), 3.14 (1H, dd, 2-H), 3.68 (6H, s,
30,50-OCH3), 3.70 (3H, s, 40-OCH3), 4.49 (1H, t, 11a), 4.51 (1H, t, 11b-H), 4.69 (1H,
d, 1-H), 6.04 (2H, OCH2O), 6.32 (1H, d, J = 9.2 Hz, 4-H), 6.52 (2H, d, 20,60-H), 6.65
(1H, s, 8-H), 7.12 (1H, s, 5-H), 8.52 (1H, pyridine ring-400-H), 8.93 (1H, pyridine
ring-600-H), 9.14 (1H, pyridine ring-200-H). MS (FAB) m/z (int.%): 597 (M+, 37),
397 (100). Anal. (C28H24O9NBr) calcd C: 56.28, H: 4.02, N: 2.34; found C: 56.19,
H: 4.13, N: 2.27.
4-O-Demethylepipodophyllotoxinyl-isonicotinate (8a): Yield: 83%; m.p. 139–
1418C; [a]20D= –438 (c = 0.1, CH3COCH3); IR (KBr) (cmꢂ1): 3418 (aromatic ring
OH), 1774 (lactone), 1754 (CO), 1601, 1506, 1483 (aromatic C = C), 932 (OCH2O),
1
803,758,703 (pyridinyl ring); H-NMR: (CD3COCD3)dppm: 3.00 (1H, m, 3-H),
3.43 (1H, dd, 2-H), 3.68 (6H, s, 30,50-OCH3), 4.40 (2H, dd, 11a, 11b-H), 4.70 (1H, d,
1-H), 4.95 (1H, d, J = 3.6 Hz, 4-H), 6.03 (2H, OCH2O), 6.40 (2H, s, 20,60-H), 6.57
(1H, s, 8-H), 7.10 (1H, s, 5-H), 8.00 (2H, pyridine ring-200,600-H), 8.87 (2H, pyridine
ring-300,500-H). MS (FAB) m/z (int.%): 506 (M++1, 41), 397 (100). Anal.
(C27H23O9N) calcd C: 64.16, H: 4.55, N: 2.77; found C: 64.02, H: 4.61, N: 2.67.
4-O-Demethylepipodophyllotoxinyl-nicotinate (8b): Yield: 89%; m.p. 174–
1768C; [a]20D= –428 (c = 0.1, CH3COCH3); IR (KBr) (cmꢂ1): 3417 (aromatic ring
OH), 1773 (lactone), 1700 (CO), 1600, 1507, 1483 (aromatic C = C), 940 (OCH2O),
1
802,765,706 (pyridinyl ring); H-NMR: (CD3COCD3)dppm: 2.87 (1H, m, 3-H),
3.41 (1H, dd, 2-H), 3.70 (6H, s, 30,50-OCH3), 4.42 (2H, dd, 11a, 11b-H), 4.72 (1H, d,
1-H), 4.92 (1H, d, J = 3.8 Hz, 4-H),5.99 (2H, OCH2O), 6.39 (2H, s, 20, 60-H), 6.45
(1H, s, 8-H), 7.08 (1H, s, 5-H), 7.40 (1H, pyridine ring-500-H), 8.44 (1H, pyridine
ring-600-H), 8.70 (1H, pyridine ring-400-H), 9.25 (1H, pyridine ring-200-H). MS
(FAB) m/z (int.%): 506(M++1, 34), 397 (100). Anal. (C27H23O9N) calcd C: 64.16,
H: 4.55, N: 2.77; found C: 64.10, H: 4.70, N: 2.58.
4-O-Demethylepipodophyllotoxinyl-picolinate (8c): Yield: 89%; m.p. 178–
1798C; [a]20D= –39o (c = 0.1, CH3COCH3); IR (KBr) (cmꢂ1): 3417 (aromatic ring
OH), 1771 (lactone), 1770 (CO), 1602, 1507, 1483 (aromatic C = C), 931 (OCH2O),
1
802,764,745 (pyridinyl ring ); H-NMR: (CD3COCD3)dppm: 2.91 (1H, m, 3-H),
3.37 (1H, dd, 2-H), 3.71 (6H, s, 30,50-OCH3), 4.33 (2H, dd, 11a,11b-H), 4.70 (1H, d,
1-H), 4.95 (1H, d, J = 3.2Hz, 4-H), 6.02 (2H, OCH2O), 6.41 (2H, s, 20,60-H), 6.59
(1H, s, 8-H), 6.95 (1H, s, 5-H), 7.68 (1H, pyridine ring-400-H), 8.05 (1H, pyridine

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Med Chem Res (2007) 16:319–330
ring-500-H), 8.22 (1H, pyridine ring-300-H), 8.76 (1H, pyridine ring-6’’-H). MS
(FAB) m/z (int.%): 506(M++1, 44), 397 (100). Anal. (C27H23O9N) calcd C: 64.16,
H: 4.55, N: 2.77; found C: 63.95, H: 4.58, N: 2.70.
4-O-Demethylepipodophyllotoxinyl-200-chloronicotinate (8d): Yield: 87%;
m.p. 179–1808C; [a]20D= –368 (c = 0.1, CH3COCH3); IR (KBr) (cmꢂ1): 3415
(aromatic ring OH),1764 (lactone), 1764 (CO), 1602, 1506, 1483 (aromatic C = C),
1
939 (OCH2O), 802,761,713 (pyridinyl ring); H-NMR: (CD3COCD3)dppm: 2.95
(1H, m, 3-H), 3.34 (1H, dd, 2-H), 3.74 (6H, s, 30,50-OCH3), 4.35 (2H, dd, 11a, 11b-
H), 4.70 (1H, d, 1-H), 4.95 (1H, d, J = 3.6 Hz, 4-H), 6.00 (2H, OCH2O), 6.54 (2H, s,
20,60-H), 6.62 (1H, s, 8-H), 7.11 (1H, s, 5-H), 7.61 (1H, pyridine ring-600-H), 8.38
(1H, pyridine ring-500-H), 8.57 (1H, pyridine ring-4’’-H). MS (FAB) m/z (int.%):
540 (M++1, 40), 397 (100). Anal. (C27H22O9NCl) calcd C: 60.11, H: 4.08, N: 2.60;
found C: 60.04, H: 4.12, N: 2.42.
4-O-Demethylepipodophyllotoxinyl-600-chloronicotinate (8e): Yield: 83%;
m.p. 173–1758C; [a]20D= –308 (c = 0.1, CH3COCH3); IR (KBr) (cmꢂ1): 3415
(aromatic ring OH), 1774 (lactone), 1748 (CO), 1600, 1506, 1483 (aromatic C = C),
1
940 (OCH2O), 803,760,711 (pyridinyl ring); H-NMR: (CD3COCD3)dppm: 2.98
(1H, m, 3-H), 3.39 (1H, dd, 2-H), 3.70 (6H, s, 30,50-OCH3),4.37 (2H, dd, 11a, 11b-
H), 4.71 (1H, d, 1-H), 4.95 (1H, d, J = 3.2 Hz, 4-H), 6.00 (2H, OCH2O), 6.51 (2H, s,
20,60-H), 6.62 (1H, s, 8-H), 6.99 (1H, s, 5-H), 7.70 (1H, pyridine ring-500-H), 8.47
(1H, pyridine ring-600-H), 9.06 (1H, pyridine ring-200-H). MS (FAB) m/z (int.%): 540
(M++1, 45), 397 (100). Anal. (C27H22O9NCl) calcd C: 60.11, H: 4.08, N: 2.60;
found C: 59.93, H: 4.24, N: 2.59.
4-O-Demethylepipodophyllotoxinyl-5’’-bromonicotinate (8f): Yield: 87%;
m.p. 193–1958C; [a]20D= –328 (c = 0.1, CH3COCH3); IR (KBr) (cmꢂ1): 3415
(aromatic ring OH), 1787 (lactone), 1746 (CO), 1598, 1508, 1484 (aromatic C = C),
1
929 (OCH2O), 803,753,713 (pyridinyl ring); H-NMR: (CD3COCD3)dppm: 2.96
(1H, m, 3-H), 3.38 (1H, dd, 2-H), 3.72 (6H, s, 30,50-OCH3), 4.35 (2H, dd, 11a, 11b-
H), 4.70 (1H, d, 1-H), 4.94 (1H, d, J = 3.2 Hz, 4-H), 6.02 (2H, OCH2O), 6.52 (2H, s,
2’,6’-H), 6.62 (1H, s, 8-H), 6.96 (1H, s, 5-H), 8.56 (1H, pyridine ring -400-H), 8.97
(1H, pyridine ring-600-H), 9.19 (1H, pyridine ring-200-H). MS (FAB) m/z (int.%): 584
(M++1, 45), 397 (100). Anal. (C27H22O9NBr) calcd C: 55.57, H: 3.77, N: 2.40;
found C: 55.32, H: 3.86, N: 2.32.
MTT assay
A total of 1200 cells per well were plated in 96-well plates. After culturing for 24 h,
test compounds were added onto triplicate wells with different concentration, and
0.1% dimethyl sulfoxide (DMSO) for control. After 4 days of incubation, 10 ml of
MTT (3-[4,5-dimethyl thiazol-2-yl]-2,5-diphenyltetrazolium bromide) solution
(5 mg mlꢂ1) was added to each well, and after shaking for 1 min the plate was
incubated further for 4 h. Formazan crystals were dissolved with 100 ml of DMSO.
The absorbance (OD) was quantitated with microplate spectrophotometer at 570
nm. Wells containing no drugs were used as blanks for the spectrophotometer. The
survival of cells was expressed as percentage of untreated control wells. The

Med Chem Res (2007) 16:319–330
329
prototypical inhibitor etoposide was included as reference standard, the results of
these assays were used to obtain the corresponding inhibition rates. Further
biological evaluation is in progress to better define the antitumor activity of these
compounds and to clarify whether the types of podophylltoxin analogs exhibit their
cytotoxicity activity through different mechanism of action.
References
Ayres DC, Loike JD (1990) Lignans: Chemical, Biological and Clinical Properties, Vols. 3 and 4.
Cambridge, UK: Cambridge University Press
Bedows E, Hatfield GM (1982) An investigation of the antiviral activity of podophyllum peltatum. J Nat
Prod 45:725–729
Berger JM, Gambin SJ, Harrison SC, Wang JC (1996) Structure and mechanism of DNA top oisomerase
II. Nature 379:225–232
Bohlin L, Rosen B (1996) Podophyllotoxin derivatives: drug discovery and development. Drug Discov
Today 1:343–351
Bolognese A, Correale G, Manfra M, Lavecchia A, Mazzoni O, Novellino E, Barone V, Colla PL, Lodda
R (2002) Antitumor agents. 2. Synthesis, structure-activity relationships, and biological evaluation
of substituted 5H-pyridophenoxazin-5-ones with potent antiproliferative activity. J Med Chem
45:5217–5223
Botta B, Monache GD, Misiti D, Vitali A, Zappia G (2001) Aryltetralin lignans: chemistry, pharmacology
and biotransformations. Curr Med Chem 8:1363–1381
Burden DA, Osheroff N (1998) Mechanism of action of eukaryotic. topoisomerase II and drugs targeted
to the enzyme. Biochim Biophys Acta 1400:139–154
Canel C, Moraes RM, Dayan FE, Ferreira D (2000) Podophyllotoxin. Phytochemistry 54:115–120
´
Castro MA, Miguel del Corral JM, Gordaliza M, Gomez-Zurita MA, Luz de la Puente M, Betancur-
Galvis LA, Sierra J, Feliciano AS (2003) Synthesis, cytotoxicity and antiviral activity of
podophyllotoxin analogues modified in the E-ring. Eur J Med Chem 38:899–911
Charlton JL (1998) Antiviral activity of lignans. J Nat Prod 61:1447–1451
Damayanthi Y, Low JW (1998) Podophyllotoxins: current status and recent developments. Curr Med
Chem 5:205–252
`
Desbene S, Giorgi-Renault S (2001) Drugs that inhibit tubulinpolymerization: the particular case of
podophyllotoxin andanalogues. Curr Med Chem Anti-Cancer Agents 1:71–90
Doleachall G, Lempert K (1963) On the mechanism of carboxyl condensations by carbodiimides.
Tetrahedron Lett 18:1195–1199
Fred Brewer C, Loike JD, Horwitz SB (1979) Conformational analysis of podophyllotoxin and its
congeners. Structure-activity relationship in microtubule assembly. J Med Chem 22:215–221
Gordaliza M, Castro MA, Miguel del Corral JM, San Feliciano A (2000) Antitumor properties of
podophyllotoxin and related compounds. Curr Pharmaceut Des 6:1811–1839
Gordaliza M, Faircloth GT, Castro MA, San Feliciano A (1996) Immunosuppressive cyclolignans. J Med
Chem 39:2865–2868
´
´
Gordaliza M, Garcıa PA, Miguel del Corral JM, Castro MA, Gomez-Zurita MA (2004) Podophyllotoxin:
distribution, sources, applications and new cytotoxic derivatives. Toxicon 44:441–459
´
´
Gordaliza M, Miguel del Corral JM, Castro MA, Garcıa-Garcıa PA, Feliciano AS (2001) Cytotoxic
cyclolignans related to podophyllotoxin. Il Farmaco 56:297–304
Gordon M, Cragg D, Newman J (2005) Plants as a source of anti-cancer agents. J Ethnopharmacol
100:72–79
Greenwald RB, Conover CD, Pendri A, Choe YH, Martinez A, Wu D, Guan SY, Yao ZL, Shum KL
(1999) Drug delivery of anticancer agents: water soluble 4-poly (ethylene glycol) derivatives of the
lignan, podophyllotoxin. J Control Release 61:281–294
Gupta RS, Chenchaiah PC (1987) Synthesis and structure activity relationship among glycosidic
derivatives of 4’-demethylepipodophyllotoxin and epipodophyllotoxin, showing VM26- and VP16-
213-like activities. Anti-Cancer Drug Des 2:13–23

330
Med Chem Res (2007) 16:319–330
Hande KR (1998) Etoposide: four decades of development of a topoisomerase II inhibitor. Eur J Cancer
34:1514–1521
Hansen HF, Jensen RB, Willumsen AM, Norskov-Lauritsen N, Ebbesen P, Nielsen PE, Buchardt O
(1993) New compounds related to podophyllotoxin and congeners: synthesis, structure elucidation
and biological testing. Acta Chem Scand 47:1190–1200
Imbert TF (1998) Discovery of podophyllotoxins. Biochimie 80:207–222
Islam MN, Iskander MN (2004) Microtubulin binding sites as target for developing anticancer agents.
Mini-Rev Med Chem 4:1077–1104
Jardine I (1980) Podophyllotoxin in Anticancer Agents Based on Natural Products Models. New York:
Academic Press, pp. 319–351
King JL, Sullivan M (1946) The similarity of the effects of podophyllin and colchicines and their use in
the treatment of codylomata acuminata. Science 104:244–245
Kuhn VM, Keller-Juslen C, Von Wartburg A (1969) Partial synthese von 40-demethylepipodophyllotoxin.
Helv Chim Acta 52:944–947
Kuo GH, Prouty C, Wang AH, Emanuel S, Angelis AD, Zhang Y, Song FB, Beall L, Connolly PJ, Jolliffe
L, Murray W (2005) Synthesis and structure-activity relationships of pyrazine-pyridine biheteroaryls
as novel, potent, and selective vascular endothelial growth factor receptor-2 inhibitors. J Med Chem
48:4892–4909
Lee KH, Imakuura Y, Haruna M, Beers SA, Thurston LS, Dai HJ, Chen CH (1989) Antitumor agents,
107. New cytotoxic 4-alkylamino analogues of 40-demethyl- epipodophyllotoxin as inhibitors of
human DNA topoisomerase II. J Nat Prod 52:606–613
Lee KH, Meck R, Piantadosi C, Huang ES (1973) Antitumor agents. 4. Cytotoxicity and in vivo activity
of helenalin esters and related derivatives. J Med Chem 16:299–301
Lee KH, Okam M, Hall IH, Broni DA, Soltmann B (1982) Antitumor agents XLV: Bisbrusatolyl and
brusatolyl esters and related compounds as novel potent antileukemic agents. J Pharm Sci 71:338–
343
Levy RK, Hall IH, Lee KH (1983) Synthesis and biological evaluation of podophyllotoxin esters and
related derivatives. J Pharm Sci 72:1158–1161
Lie Ken Jie MSF, Mustafa J, Pasha MK (1999) Synthesisand spectral characteristics of some unusual
fatty esters of podophyllotoxin. Chem Phys Lipid 100:165–170
Lopez-Perez JL, del Olmo E, de Pascual-Teresa B, Abad A, San Feliciano A (2004) Synthesis and
cytotoxicity of hydrophobic esters of podophyllotoxins. Bioorg Med Chem Lett 14:1283–1286
Minko T, Paranjpe PV, Qiu B, Lalloo A, Won R, Stein S, Sinko PJ (2002) Enhancing the anticancer
efficacy of camptothecin using biotinylated poly(ethyleneglycol) conjugates in sensitive and
multidrug-resistant human ovarian carcinoma cells. Cancer Chemother Pharmacol 50:143–150
Nagao Y, Musta J, Sano S, Ochiiai M (1991) Different mechanism of action of long chain fatty acid esters
of podophyllotoxin and esters of epipodophyllotoxin against P388 lymphocytic leukemia in mice.
Med Chem Res 1:295–299
Simth M, Moffat JG, Khorana HG (1958) Carbodiimides. VIII. Observations on the reactions of
carbodiimides with acids and some new applications in the synthesis of phosphoric acid esters. J Am
Chem Soc 80:6204
Straub T, Boesenberg C, Gekeler V, Boege F (1997) The dihydropyridine dexniguld ipine hydrochloride
inhibits cleavage and religation reactions of eukaryotic DNA topoisomerase I. Biochemistry
36:10777–10783
Tiwari A, Riordan JM, Waud WR, Struck RF (2002) Synthesis and antitumor activity of several new
analogues of penclomedine and its metabolites. J Med Chem 45:1079–1085
Vincent RM, Lopez-Meyer M, Mcknight TD, Nessler CL (1997) Sustained harvest of camptothecin from
the leaves of Camptotheca acuminata. J Nat Prod 60:618–619
Zhu XK, Guan J, Xiao ZY, Cosentino LM, Lee KH (2004) Anti-AIDS agents. Part 61: Anti-HIV activity
of new podophyllotoxin derivatives. Bioorg Med Chem 12:4267–4273