Studies on novel 4β-[(4-substituted)-1,2,3-triazol-1-yl] podophyllotoxins as potential anticancer agents

Article abstract of DOI:10.1016/j.ejmech.2007.09.015

A series of 4β-[(4-substituted)-1,2,3-triazol-1-yl] podophyllotoxin congeners have been designed and synthesized with significant regioselectivity by employing Cu(I) catalyzed 1,3-dipolar cycloaddition reaction of C4β-azido podophyllotoxin and C4β-azido-4′-O-demethyl podophyllotoxin with N-prop-2-yn-1-ylanilines. These compounds were evaluated for anticancer activity against a panel of seven human cancer cell lines. It was interesting to note that all the compounds exhibited promising activity especially against SF-295 (CNS), HCT-15 (colon) and 502713 (colon) cell lines. Compound 11e was found to be the most promising in this study.

Full text of DOI:10.1016/j.ejmech.2007.09.015

Available online at www.sciencedirect.com
European Journal of Medicinal Chemistry 43 (2008) 2067e2072
http://www.elsevier.com/locate/ejmech
Original article
Studies on novel 4b-[(4-substituted)-1,2,3-triazol-1-yl]
podophyllotoxins as potential anticancer agents*
Bilal A. Bhat, P. Bhaskar Reddy, Satyam Kumar Agrawal, A.K. Saxena,
*
H.M. Sampath Kumar , G.N. Qazi
Synthetic Chemistry Division, Pharmacology Division, Indian Institute of Integrative Medicine (CSIR), Canal Road, Jammu-Tawi 180001, India
Received 4 April 2007; received in revised form 14 September 2007; accepted 18 September 2007
Available online 29 September 2007
Abstract
A series of 4b-[(4-substituted)-1,2,3-triazol-1-yl] podophyllotoxin congeners have been designed and synthesized with significant regiose-
lectivity by employing Cu(I) catalyzed 1,3-dipolar cycloaddition reaction of C4b-azido podophyllotoxin and C4b-azido-40-O-demethyl podo-
phyllotoxin with N-prop-2-yn-1-ylanilines. These compounds were evaluated for anticancer activity against a panel of seven human cancer
cell lines. It was interesting to note that all the compounds exhibited promising activity especially against SF-295 (CNS), HCT-15 (colon)
and 502713 (colon) cell lines. Compound 11e was found to be the most promising in this study.
Ó 2007 Published by Elsevier Masson SAS.
Keywords: Podophyllotoxin; Regioselectivity; N-prop-2-yn-1-ylanilines; Cancer cell lines
1. Introduction
etoposide and teniposide are clinically useful against various
cancers, including small-cell lung cancer, testicular carci-
noma, lymphoma and Kaposi’s sarcoma [1,5]; however,
their therapeutic uses are often hindered by acquired drug-
resistance. In another podophyllotoxin glycoside derivative,
NK611, 4, the 200-hydroxyl group in the glucose ring of 2
has been replaced with an N,N-dimethylamino moiety. It
is currently undergoing phase I clinical evaluation [6]. In or-
der to obtain better therapeutic agents, extensive synthetic
efforts have been devoted to overcome drug-resistance and
improve topoisomerase II inhibition [7,8].
The recent synthetic studies on podophyllotoxin have been
focused on the synthesis of the C-4 non-sugar substituted an-
alogs, which exhibit improved topoisomerase II inhibition.
The replacement of the C-4 sugar moiety of etoposide and te-
niposide with a non-sugar substituent has improved the thera-
peutic value of etoposide [9]. The C-4 non-sugar substituent
can be linked through O-, S- or N-linkage. In general, the
O-linked (ethers, esters) and S-linked (thioethers) compounds
are less active in comparison to the N-linked congeners
[3,10e12] like GL-331 and NPF (Fig. 1).
Podophyllum peltatum L. (and Podophyllum emodi L.,
found in China and India) also known as American man-
drake or May apple is the rich source of podophyllotoxin.
Podophyllotoxin has been known to inhibit the tubulin as-
sembly into microtubules through tubulin binding, but its
high toxicity has limited its application as a drug in cancer
chemotherapy [1]. Several podophyllotoxin derivatives that
are potent inhibitors of mitosis have been synthesized and
examined as antitumor agents. Etoposide, 2 [2] and tenipo-
side, 3 [3], the two semi-synthetic epipodophyllotoxin deriv-
atives are in clinical use as antineoplastic agents. Their
clinical efficacy is due to their ability to inhibit the enzyme
DNA-topoisomerase II. These podophyllotoxin lignans block
the catalytic activity of DNA-topoisomerase II by stabilizing
a cleavable enzymeeDNA complex in which the DNA is
cleaved and covalently linked to the enzyme [4]. Both
*
IIIM communication SCL07/10.
* Corresponding author. Tel.: þ91 (0191) 2579117; fax: þ91 (0191) 2548607.
E-mail address: hmskumar@yahoo.com (H.M.S. Kumar).
0223-5234/$ - see front matter Ó 2007 Published by Elsevier Masson SAS.
doi:10.1016/j.ejmech.2007.09.015

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B.A. Bhat et al. / European Journal of Medicinal Chemistry 43 (2008) 2067e2072
O
O
R1
O
HO
OH
OR2
O
O
O
O
O
O
O
O
O
MeO
OMe
OMe
MeO
OH
OMe
R1 = CH3,R2 = OH; Etoposide
2.
3.
1. podophyllotoxin
R1 =
,R2 = OH; Teniposide
S
R1 = CH3; R2 = NMe2; NK-611
4.
R
N
N
HN
O
O
O
O
O
O
O
O
MeO
OMe
MeO
OMe
OH
OH
7. TOP-553
5. R = NO2; GL-331.
6. R = F; NPF
Fig. 1. Structure of some podophyllotoxin derivatives.
GL-331 and TOP-53 [13] have been found to have poten-
tial activity, especially selective in resistant malignancies [14]
and nonsmall-cell lung cancers [15], respectively. Both of
them showed topoisomerase II inhibitory activity, antitumor
spectra, and drug-resistance profiles significantly different
from those of 2, which suggested an important role of various
C-4 substitutions on the activity profiles of 2-related analogs
and the feasibility of optimizing this compound class through
rational C-4 modification. This postulation coincides with the
composite pharmacophore model proposed by MacDonald
et al. [4] and the comparative molecular field analysis
(CoMFA) model generated by Cho et al. [16]. Both models
indicated that the C-4 molecular area could accommodate
considerable structural diversity. Wang and co-workers have
reported a brief synthesis of 4b-[(5-substituted)-1,2,3-tria-
zol-1-yl] podophyllotoxin derivatives as a new class of antitu-
mor compounds [17]. This prompted us to synthesize
a library of 4b-[(4-substituted)-1,2,3-triazol-1-yl] podophyllo-
toxin derivatives as new anticancer compounds using click-
chemistry protocols. Click chemistry enables a modular
approach to generate these novel pharmacophores utilizing
a collection of reliable chemical reactions [18]. Of particular
interest is the Huisgen [3 þ 2] cycloaddition between a termi-
nal alkyne and an azide to generate substituted 1,2,3-triazoles
[19]. Using this click reaction a series of novel regioselective
4b-[(4-substituted)-1,2,3-triazol-1-yl] podophyllotoxin deriva-
tives have been synthesized and screened for anticancer
activity against a panel of seven human cancer cell lines.
From the IC50 values it is found that all the compounds are
having promising anticancer activity, better than the standard
compounds like podophyllotoxin and etoposide.
2. Results and discussion
2.1. Chemistry
As illustrated in Scheme 1, 4b-[(4-substituted)-1,2,3-
triazol-1-yl] podophyllotoxin derivatives were synthesized by
the cycloaddition reaction of C4b-azido podophyllotoxin, 8
and C4b-azido-40-O-demethyl podophyllotoxin, 9 with N-
prop-2-yn-1-ylanilines, obtained by the reaction of anilines
with propargyl bromide in the presence of potassium carbon-
ate in acetone under reflux conditions. C4b-Azido-40-O-
demethyl podophyllotoxin was synthesized according to the
literature procedure in which podophyllotoxin was first con-
verted to 40-O-demethyl epipodophyllotoxin by the inversion
of 4-hydroxyl and simultaneous demethylation of 40-OCH3 us-
ing CH3SO3H and NaI in dichloromethane to the correspond-
ing iodo-derivative followed by hydrolysis with H2O/Me2CO/
barium carbonate to 40-O-demethyl epipodophyllotoxin [20].
The key intermediate, 4b-azido-40-O-demethyl podophyllo-
toxin was obtained in excellent yield by treating the latter
with TFA and NaN3. On the other hand, C4b-azido-podophyl-
lotoxin was obtained by the reaction of podophyllotoxin with

B.A. Bhat et al. / European Journal of Medicinal Chemistry 43 (2008) 2067e2072
2069
R2
N
N
H
N3
N
N
O
O
O
O
CuSO4 / Sodium ascorbate
t-BuOH:H2O (1:2)
O
+
HN
O
O
O
R2
MeO
OMe
MeO
OMe
OR1
OR1
11
8. R
9. R
1 = CH3
1 = H
10
Scheme 1. Click-chemistry strategy for the synthesis of novel 4b-[(4-substituted)-1,2,3-triazol-1-yl] podophyllotoxin derivatives.
CH3SO3H and NaI in acetonitrile followed by hydrolysis with
H2O/Me2CO/barium carbonate and reaction with NaN3 in
TFA. The azides 8 and 9 obtained were allowed to react with
the above terminal alkynes, in the presence of CuSO4$5H2O
and sodium ascorbate, in t-butyl alcohol and water (1:2) at
room temperature to yield selectively 4b-[(4-substituted)-
1,2,3-triazol-1-yl] derivatives in excellent yields (84e90%).
Using this click-chemistry protocol a series of 10 compounds
differing in substitution at R1 and R2 have been synthesized
(Table 1).
All the products were characterized by 1H NMR, 13C NMR,
IR, ESI-MS and elemental analyses, which were found to be
within the range of ꢀ0.4 from theoretical values. In 1H
NMR, cyclization of azides to triazoles was confirmed by res-
onance of H-5 of triazole ring in aromatic region and that of
C4a-H around d 6.
In conclusion, a series of 4b-[(4-substituted)-1,2,3-triazol-
1-yl] podophyllotoxin derivatives were synthesized and
screened for anticancer activity against a panel of seven hu-
man cancer cell lines. From the data it was found that all
the compounds are having promising anticancer activity and
compound 11e was found to be the most active in this study.
3. Experimental
Melting points were recorded on Buchi Melting point appa-
ratus D-545 and IR spectra (KBr) on Bruker Vector 22 instru-
ment. NMR spectra were recorded on Bruker DPX200
instrument in CDCl3 with TMS as an internal standard. Chem-
ical shift values are reported in d (ppm) and coupling constants
in hertz. Mass spectra were recorded on ESI-esquire 3000
Bruker Daltonics instrument. The progress of all reactions
was monitored by TLC on 2 ꢁ 5 cm pre-coated silica gel 60
F254 plates of thickness 0.25 mm (Merck). The chromatograms
were visualized under UV 254e366 nm and iodine.
2.2. Biology
3.1. Click chemistrydgeneral procedure
All the compounds were assayed for in vitro cytotoxicity
against a panel of seven human cancer cell lines including
DU-145, PC-3 (prostrate), SF-295 (CNS), HCT-15, 502713
(Colon), HEP-2 (liver) and A-549 (lung) using sulforhodamine
B [21,22]. Podophyllotoxin and etoposide were taken as refer-
ence compounds and the results are reported in terms of IC50
values (Table 2).
From the IC50 values, it is clear that all the compounds have
significant cytotoxic activity against prostrate, CNS, colon and
liver derived cancer cell lines. However, it may be noted that
these compounds showed comparatively lesser activity against
A-549, a lung derived cancer cell line. Compound 11e, with
m-nitro-substitution on aniline part with trimethoxy moiety
on ring E of lignan, showed significant cytotoxicity against
DU-145, PC-3, SF-295, HCT-15 and 502713 cell lines, how-
ever, its activity was found to be maximum against HCT-15
cell line with IC50 value 0.04 mM. Compounds 11ae11c,
11g and 11h were also found to have promising activity
against the above mentioned cell lines. From the IC50 values,
it is clear that the compounds with trimethoxy moiety in ring E
are having more cytotoxic activity than those with dimethoxy
moiety.
To a solution of 9 (1 mmol) in t-butyl alcohol and water
(1:2, 8 mL) was added CuSO4$5H2O (1 mmol), sodium ascor-
bate (2 mmol) followed by 4b-azido-podophyllotoxin (44 mg,
0.1 mmol). The reaction mixture was stirred at room tempera-
ture for 8 h. After completion, the reaction mixture was diluted
with 80 mL of water and extracted with ethylacetate
(2 ꢁ 20 mL). The combined extracts were washed with brine,
dried over Na2SO4 and evaporated in vacuo. The crude prod-
uct obtained was precipitated in diethyl ether to yield the pure
product 11.
3.1.1. 9-[4-(o-Tolylamino-methyl)-[1,2,3]-triazol-1-yl]-
5-(3,4,5-trimethoxy-phenyl)-5,8,8a,9-tetrahydro-5aH-
furo[30,40:6,7]naphtho[2,3-d][1,3]dioxol-6-one (11a)
White solid; mp: 128e130 ꢂC; [a]D25 ꢃ41.3 (c 0.5, CHCl3);
1H NMR (CDCl3): d 2.19 (s, 3H), 3.06 (m, 1H), 3.23 (m, 2H),
3.64 (s, 6H), 3.89 (s, 3H), 4.03 (d, 1H, J ¼ 2.29 Hz), 4.40e4.51
(m, 3H), 4.75 (d, 1H, J ¼ 4.76 Hz), 6.08 (m, 3H), 6.33 (s, 2H),
6.59e6.76 (m, 4H), 7.13 (m, 3H); 13C NMR (CDCl3): d 17.4,
33.7, 37.1, 41.6, 43.5, 56.3, 60.7, 67.4, 71.4, 101.9, 108.3,

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B.A. Bhat et al. / European Journal of Medicinal Chemistry 43 (2008) 2067e2072
Table 1
Various podophyllotoxin 4b-[(4-substituted)-1,2,3-triazol-1-yl] derivatives
Table 2
IC50 values (mM) of 4b-[(4-substituted)-1,2,3-triazol-1-yl] podophyllotoxin
against a panel of human cancer cell lines
R2
Entry Prostate
DU-145 PC-3
CNS
Colon
Liver
Lung
O
A
B
C
O
D
SF-295 HCT-15 502713 HEP-2 A-549
O
11a
11b
11c
11d
11e
11f
11g
11h
11i
11j
1
1.06
1.19
0.93
4.16
0.65
4.37
1.12
1.09
3.57
2.75
2.97
3.85
1.09
1.53
1.18
1.37
0.74
6.63
0.88
1.03
3.45
4.49
1.94
0.96
0.84
3.11
0.68
4.20
0.78
0.70
1.00
6.03
5.69
0.36
0.14
0.34
4.22
0.04
1.55
0.14
0.40
1.43
1.69
1.00
1.48
0.78
0.62
0.71
0.98
0.56
1.19
0.61
0.68
1.88
1.34
3.88
2.42
9.14
1.50
1.37
1.22
3.65
4.10
4.64
2.96
1.26
3.27
1.42
2.15
7.88
5.17
4.99
8.63
5.78
7.43
6.10
7.46
9.24
7.41
7.63
5.62
O
E
MeO
OMe
OR1
Entry
R1
R2
Yielda
88
N
N
N
N
N
N
N
N
N
N
11a
CH3
N
N
H
18.9
17.4
2
13.3
N
N
11b
11c
11d
11e
11f
H
86
87
88
84
88
87
83
N
H
3.79 (s, 6H), 3.99 (d, 1H, J ¼ 2.28 Hz), 4.35 (m, 1H), 4.47 (s,
2H), 4.71 (d, 1H, J ¼ 4.82 Hz), 6.02 (m, 3H), 6.32 (s, 2H),
6.59e6.73 (m, 4H), 7.11 (m, 3H); 13C NMR (CDCl3):
d 18.7, 33.7, 37.6, 40.1, 42.9, 56.99, 60.3, 67.8, 103.5, 108.4,
109.5, 112.2, 117.1, 122.1, 127.2, 129.1, 130.4, 134.7,
135.09, 144.8, 145.4, 146.8, 172.4; IR (KBr): 3418.1, 1778.2,
1605.1, 1485.07, 1231.9, 1115.4, 762.8 cmꢃ1; ESI-MS: 593.1
(M þ Na).
N
N
Cl
Cl
CH3
H
N
H
N
N
N
H
N
NO2
CH3
H
N
N
H
3.1.3. 9-{4-[(3-Chloro-phenylamino)-methyl]-
[1,2,3]triazol-1-yl}-5-(3,4,5-trimethoxy-phenyl)-
5,8,8a,9-tetrahydro-5aH-furo[30,40:6,7]naphtho
[2,3-d][1,3]dioxol-6-one (11c)
N
NO2
N
N
H
White solid; mp: 136e138 ꢂC; [a]D25 ꢃ24.1 (c 0.47,
N
11g
11h
CH3
H
N
N
H
1
CHCl3); H NMR (CDCl3): d 3.01 (m, 1H), 3.21 (m, 2H),
3.76 (s, 6H), 3.81 (s, 3H), 4.12e4.21 (m, 1H), 4.40e4.53
(m, 3H), 4.73 (d, 1H, J ¼ 4.84 Hz), 5.99e6.07 (m, 3H), 6.51
(s, 2H), 6.51e6.72 (m, 5H), 7.07 (m, 2H); 13C NMR
(CDCl3): d 37.1, 39.7, 41.6, 43.6, 56.3, 58.7, 60.7, 67.3,
102.0, 108.3, 108.7, 110.5, 111.7, 113.0, 113.2, 118.2,
118.5, 124.5, 130.2, 133.2, 134.2, 135.0, 137.7, 148.0,
148.5, 149.5, 152.8, 173.1; IR (KBr): 3389.2, 1778.6,
1598.5, 1505.3, 1485.2, 1238.0, 1002.6, 766.7 cmꢃ1; ESI-
MS: 627 (M þ Na).
N
N
N
H
N
N
N
H
11i
CH3
CH3
88
90
Cl
N
N
11j
N
N
H
a
3.1.4. 9-{4-[(3-Chloro-phenylamino)-methyl]-[1,2,3]
triazol-1-yl}-5-(4-hydroxy-3,5-dimethoxy-phenyl)-
5,8,8a,9-tetrahydro-5aH-furo[30,40:6,7]naphtho
[2,3-d][1,3]-dioxol-6-one (11d)
Isolated yields.
108.8, 110.5, 118.2, 124.7, 127.1, 130.2, 133.3, 134.3, 137.7,
148.09, 149.45, 152.8, 173.1; IR (KBr): 3425, 1778.6,
1588.5, 1506.3, 1237.8, 1126.4, 751.0 cmꢃ1; ESI-MS: 607
(M þ Na).
White solid; mp: 144e147 ꢂC; [a]D25 ꢃ29.5 (c 0.47,
1
CHCl3); H NMR (CDCl3): d 3.18 (m, 1H), 3.22 (m, 2H),
3.79 (s, 6H), 3.92 (d, 1H, J ¼ 2.27 Hz), 4.38 (m, 3H), 4.72
(d, 1H, J ¼ 4.66 Hz), 5.99e6.05 (m, 3H), 6.32 (s, 2H),
6.47e6.76 (m, 5H), 7.08 (m, 2H); 13C NMR (CDCl3):
d 37.1, 39.7, 41.6, 56.3, 58.7, 67.3, 72.8, 102.0, 108.3,
108.7, 110.5, 111.7, 113.0, 118.2, 124.5, 130.2, 134.2,
135.0, 137.7, 148.0, 149.5, 152.8, 173.1; IR (KBr): 3405.5,
1777.5, 1598.3, 1505.3, 1232.2, 1114.5, 1036.9, 765.9 cmꢃ1
;
ESI-MS: 590 (M þ Na).
3.1.2. 5-(4-Hydroxy-3,5-dimethoxy-phenyl)-
9-[4-(o-tolylamino-methyl)-[1,2,3]triazol-1-yl]-5,8,8a,9-
tetrahydro-5aH-furo[30,40:6,7]naphtho[2,3-d][1,3]dioxol-
6-one (11b)
White solid; mp: 145e148 ꢂC; [a]D25 ꢃ25.3 (c 0.47, CHCl3);
1H NMR (CDCl3): d 2.16 (s, 3H), 3.02 (m, 1H), 3.18 (m, 2H),

B.A. Bhat et al. / European Journal of Medicinal Chemistry 43 (2008) 2067e2072
2071
3.1.5. 9-{4-[(3-Nitro-phenylamino)-methyl]-[1,2,3]triazol-
1-yl}-5-(3,4,5-trimethoxy-phenyl)-5,8,8a,9-tetrahydro-5aH-
furo[30,40:6,7]naphtho[2,3-d][1,3]dioxol-6-one (11e)
1602.4, 1504.2, 1485.1, 1237.5, 1125.7, 752.2 cmꢃ1; ESI-
MS: 579 (M þ Na).
White solid; mp: 208e211 ꢂC; [a]D25 ꢃ42.2 (c 0.40, CHCl3);
1H NMR (CDCl3): d 3.08 (m, 1H), 3.29 (m, 2H), 3.82 (s, 6H),
3.87 (s, 3H), 4.07e4.21 (m, 1H), 4.40e4.52 (m, 3H), 4.80 (d,
1H, J ¼ 4.82 Hz), 6.10 (m, 3H), 6.37 (s, 2H), 6.61 (s, 1H),
6.70 (s, 1H), 6.98 (d, 1H, J ¼ 8.04 Hz), 7.24e7.45 (m, 3H),
7.62 (d, 1H, J ¼ 9.12 Hz); 13C NMR (CDCl3): d 34.9, 37.7,
40.1, 42.7, 56.4, 58.6, 60.0, 70.3, 105.5, 107.6, 108.3, 108.6,
110.1, 112.1, 120.3, 124.1, 125.2, 130.2, 133.3, 134.1, 137.1,
148.3, 149.5, 152.2, 173.2; IR (KBr): 3389.5, 1778.2, 1588.7,
1530.1, 1348.8, 1237.7, 1125.6, 1092.95, 1002.35, 736.4,
675.3 cmꢃ1; ESI-MS: 638 (M þ Na).
3.1.9. 9-{4-[(2-Chloro-phenylamino)-methyl]-
[1,2,3]triazol-1-yl}-5-(3,4,5-trimethoxy-phenyl)-5,8,8a,9-
tetrahydro-5aH-furo[30,40:6,7]naphtho[2,3-d][1,3]dioxol-
6-one (11i)
White solid; mp: 167e170 ꢂC; [a]D25 ꢃ25.7 (c 0.46, CHCl3);
1H NMR (200 MHz, CDCl3): d 2.96 (m, 1H), 3.20 (m, 2H),
3.77 (s, 6H), 3.82 (s, 3H), 3.93 (d, 1H, J ¼ 2.35 Hz), 4.41 (br
s, 3H), 4.74 (d, 1H, J ¼ 4.71 Hz), 6.01e6.09 (m, 3H), 6.32
(s, 2H), 6.48e6.73 (m, 5H), 7.09 (m, 2H); 13C NMR
(125 MHz, CDCl3): d 36.8, 37.7, 40.4, 42.2, 56.9, 59.3, 61.3,
67.9, 102.5, 105.1, 108.8, 110.5, 111.7, 113.0, 113.2, 118.2,
118.5, 124.5, 130.2, 133.2, 134.2, 135.0, 137.7, 148.0, 148.5,
149.5, 153.8, 173.6; IR (KBr): 3389.2, 1778.6, 1598.5,
1505.3, 1485.2, 1238.0, 1002.6, 766.7 cmꢃ1; ESI-MS: 627
(M þ Na).
3.1.6. 5-(4-Hydroxy-3,5-dimethoxy-phenyl)-
9-{4-[(3-nitro-phenylamino)-methyl]-[1,2,3]triazol-1-yl}-
5,8,8a,9-tetrahydro-5aH-furo[30,40:6,7]naphtho
[2,3-d][1,3]dioxol-6-one (11f)
White solid; mp: 221e223 ꢂC; [a]D25 ꢃ44.1 (c 0.50,
3.1.10. 9-[4-( p-Tolylamino-methyl)-[1,2,3]triazol-1-yl]-
5-(3,4,5-trimethoxy-phenyl)-5,8,8a,9-tetrahydro-5aH-
furo[30,40:6,7]naphtho[2,3-d][1,3]dioxol-6-one (11j)
White solid; mp: 146e148 ꢂC; [a]D25 ꢃ33.4 (c 0.75, CHCl3);
1H NMR (CDCl3): d 2.19 (s, 3H), 3.06 (m, 1H), 3.23 (m, 2H),
3.64 (s, 6H), 3.89 (s, 3H), 4.03 (d, 1H, J ¼ 2.29 Hz), 4.40e4.51
(m, 3H), 4.75 (d, 1H, J ¼ 4.76 Hz), 6.08 (m, 3H), 6.32 (s, 2H),
6.63 (s, 1H), 6.66 (s, 1H), 6.79 (m, 4H); 13C NMR (125 MHz,
CDCl3): 18.1, 34.9, 41.5, 42.8, 56.5, 58.7, 67.3, 101.9, 107, 8,
108.5, 110.4, 114.7, 124.5, 129.6, 130.2, 130.8, 133.63, 134.4,
134.4, 146.6, 147.7, 155.8, 173.1; IR (KBr): 3437, 2905, 1768,
1505.3, 1486, 1238.0, 1095, 1002.6, 766.7 cmꢃ1; ESI-MS: 607
(M þ Na).
1
CHCl3); H NMR (CDCl3): d 3.08 (m, 1H), 3.29 (m, 2H),
3.82 (s, 6H), 4.07e4.23 (m, 1H), 4.40e4.52 (m, 3H), 4.80
(d, 1H, J ¼ 4.82 Hz), 6.10 (m, 3H), 6.37 (s, 2H), 6.59 (s,
1H), 6.68 (s, 1H), 6.98 (d, 1H, J ¼ 7.88 Hz), 7.24e7.45 (m,
3H), 7.62 (d, 1H, J ¼ 9.12 Hz); 13C NMR (CDCl3): d 34.9,
37.7, 40.1, 42.7, 56.4, 61.7, 70.3, 105.5, 107.6, 108.3, 108.6,
110.1, 112.1, 120.3, 124.1, 125.2, 130.2, 133.3, 134.1,
137.1, 148.3, 149.5, 152.2, 173.2; IR (KBr): 3389.5, 1778.2,
1588.7, 1530.1, 1348.8, 1237.7, 1125.6, 1092.95, 1002.35,
736.4, 675.3 cmꢃ1; ESI-MS: 624 (M þ Na).
3.1.7. 9-(4-Phenylaminomethyl-[1,2,3]triazol-1-yl)-
5-(3,4,5-trimethoxy-phenyl)-5,8,8a,9-tetrahydro-5aH-
furo[30,40:6,7]naphtho[2,3-d][1,3]dioxol-6-one (11g)
White solid; mp: 168e171 ꢂC; [a]D25 ꢃ33.4 (c 0.44,
3.2. Biological assay
1
CHCl3); H NMR (CDCl3): d 2.99 (m, 1H), 3.16 (m, 2H),
The effect of 4b-[(4-substituted)-1,2,3-triazol-1-yl] podo-
phyllotoxin derivatives on the growth of cancer cell lines
was evaluated according to the procedure adopted by the Na-
tional Cancer Institute for in vitro anticancer drug screening
that uses the protein-binding dye sulforhodamine B to estimate
cell growth. Briefly, cells in their log phase of growth were
harvested, counted and seeded (104 cells/well in 100 mL me-
dium) in 96-well microtitre plates. After 24 h of incubation
at 37 ꢂC and 5% CO2 to allow cell attachment, cultures
were treated with varying concentrations (0.1e100 mM) of
test samples made with 1:10 serial dilutions. Four replicate
wells were set up for each experimental condition. Test sam-
ples were left in contact with the cells for 48 h under same
conditions. Thereafter cells were fixed with 50% chilled
TCA and kept at 4 ꢂC for 1 h, washed and air-dried. Cells
were stained with sulforhodamine B dye. The adsorbed dye
was dissolved in Tris-buffer and the plates were gently shaken
for 10 min on a mechanical shaker. The optical density (OD)
was recorded on ELISA reader at 540 nm. The cell growth
was calculated by subtracting mean OD value of the respective
blank from the mean OD value of experimental set. Percentage
of growth in the presence of test material was calculated
3.76 (s, 6H), 3.94 (s, 3H), 3.94 (d, 1H, J ¼ 2.28 Hz), 4.36e
4.43 (m, 3H), 4.70 (d, 1H, J ¼ 4.67 Hz), 6.00 (m, 3H), 6.31
(s, 2H), 6.58e6.79 (m, 5H), 7.12e7.22 (m, 3H); 13C NMR
(CDCl3): d 38.1, 41.1, 42.3, 47.1, 57.3, 59.6, 61.7, 103.6,
108.9, 110.1, 112.7, 115.8, 119.4, 123.5, 125.4, 132.2,
133.7, 134.9, 145.4, 149.5, 150.4, 157.2, 173.3; IR (KBr):
3405.4, 1777.6, 1602.4, 1504.2, 1485.1, 1237.5, 1125.7,
752.2 cmꢃ1; ESI-MS: 593 (M þ Na).
3.1.8. 5-(4-Hydroxy-3,5-dimethoxy-phenyl)-
9-(4-phenylaminomethyl-[1,2,3]triazol-1-yl)-5,8,8a,9-
tetrahydro-5aH-furo[30,40:6,7]naphtho[2,3-d][1,3]dioxol-
6-one (11h)
White solid; mp: 81e182 ꢂC; [a]D25 ꢃ29.9 (c 0.61, CHCl3);
1H NMR (200 MHz, CDCl3): d 2.98 (m, 1H), 3.19 (m, 2H),
3.80 (s, 6H), 3.95 (d, 1H, J ¼ 2.28 Hz), 4.36e4.44 (m, 3H),
4.72 (d, 1H, J ¼ 4.71 Hz), 5.99e6.06 (m, 3H), 6.32 (s, 2H),
6.59e6.85 (m, 5H), 7.12e7.27 (m, 3H); 13C NMR (CDCl3):
d 38.1, 41.1, 42.3, 47.1, 57.3, 59.6, 103.6, 108.9, 110.1,
112.7, 115.8, 119.4, 123.5, 125.4, 132.2, 133.7, 134.9,
145.4, 149.5, 150.4, 157.2, 173.3; IR (KBr): 3405.4, 1777.6,

2072
B.A. Bhat et al. / European Journal of Medicinal Chemistry 43 (2008) 2067e2072
[9] S.Y. Liu, B.D. Hwang, M. Haruna, Y. Imakura, K.H. Lee, Y.C. Cheng,
Mol. Pharmacol. 36 (1989) 78e82.
considering the growth in the absence of any test material as
100% and the results are reported in terms of IC50 values. Po-
dophyllotoxin and etoposide were taken as positive controls. A
comparison of IC50 values for the tested compounds with the
standards is provided in Table 2.
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Acknowledgement
Authors, B.A.B., P.B.R. and S.K.A., are grateful to CSIR
(India) for Senior Research Fellowships.
[14] W. Choy, Genelabs Technologies, personal communication.
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