Synthesis and biological evaluation of new 4β-anilino- and 4β-imido-substituted podophyllotoxin congeners

Article abstract of DOI:10.1016/j.bmc.2005.06.032

A series of C-4-anilino- and C-4-imido-substituted new podophyllotoxin congeners have been designed, synthesized, and evaluated for their cytotoxicity and DNA topoisomerase-II (topo-II) inhibition potential. Some of these compounds have exhibited promisin

Full text of DOI:10.1016/j.bmc.2005.06.032

Bioorganic & Medicinal Chemistry 13 (2005) 6218–6225
Synthesis and biological evaluation of new 4b-anilino-
and 4b-imido-substituted podophyllotoxin congeners
Ahmed Kamal,a,* N. Lakshmi Gayatri,a D. Rajasekhar Reddy,a
P. S. Murali Mohan Reddy,a M. Arifuddin,a Sunanda G. Dastidar,b
Anand K. Kondapic and M. Rajkumarc
aDivision of Organic Chemistry, Indian Institute of Chemical Technology, Hyderabad 500007, Andhra Pradesh, India
bRanbaxy Research Laboratories, Gurgaon 122001, Haryana, India
cDepartment of Biochemistry, School of Life Sciences, University of Hyderabad, Hyderabad 500046, Andhra Pradesh, India
Received 6 June 2005; revised 17 June 2005; accepted 21 June 2005
Available online 2 August 2005
Abstract—A series of C-4-anilino- and C-4-imido-substituted new podophyllotoxin congeners have been designed, synthesized, and
evaluated for their cytotoxicity and DNA topoisomerase-II (topo-II) inhibition potential. Some of these compounds have exhibited
promising in vitro anticancer and topo-II inhibition activity.
Ó 2005 Elsevier Ltd. All rights reserved.
1. Introduction
During the course of preparing new C-4-nonsugar deriv-
atives of podophyllotoxin, it has been observed that the
N-linked congeners like GL 331 (6) and NPF (7) have
exhibited improved cytotoxicity and topo-II inhibition
activity (Fig. 1).4a,5,8 A recent study on molecular-area-
oriented chemical modifications of podophyllotoxin has
revealed certain structural features that are critical for
the topo-II inhibition.9 The comparative molecular field
analysis 8b,9 model further demonstrated that bulky sub-
stituents at C-4 might be favorable for topo-II inhibition
(Fig. 2). Recently, we have been involved in the develop-
ment of new synthetic procedures10 for the podophyllo-
toxin-based compounds and also in the design and
synthesis of new analogs of podophyllotoxin as potential
anticancer agents.11 In conjunction with these efforts, it
was of interest to prepare 4b-(200-benzoyl) anilino- and
4b-imido-substituted podophyllotoxin analogs12 with
the objective to enhance their topo-II inhibition as well
as in vitro anticancer activity particularly by increasing
the bulkiness at the C-4 position.
Podophyllotoxin (1) is a naturally occurring aryltetralin
lignan obtained from a number of plant species of the
Podophyllum family.1,2 It has cytotoxic properties and
is known as an antimicrotubule agent acting at the colchi-
cine-binding site on tubulin. However, the semisynthetic
derivatives of podophyllotoxin, namely etoposide3,4 (2)
and teniposide5 (3), inhibit DNA topoisomerase-II
(topo-II) by stabilizing the covalent topo-II DNA cleav-
able complex.6 Both these compounds are in clinical
usage for the treatment of various cancers including small
cell lung cancer, testicular carcinoma, and lymphoma.3,7
Their therapeutic use has encountered certain limitations
such as acquired drug resistance and poor water solubili-
ty. To overcome such problems, extensive synthetic ef-
forts have been carried out by a number of researchers.
This led to the development of etopophos (4), NK 611
(5), and GL 331 (6). Etopophos is a water-soluble pro-
drug which readily converts into the active drug and
exhibits similar biological profile to that of etoposide.7b,7c
The other analogs of podophyllotoxin like NK 611 (5)
and GL 331 (6) are in different stages of clinical studies.
2. Chemistry
The synthesis of C-4b-substituted analogs of podophyl-
lotoxin has been carried out from podophyllotoxin (1).
The key intermediate for the preparation of the new
analogs is 4b-bromopodophyllotoxin/4b-bromo-40-
demethylpodophyllotoxin (8) that was obtained by a
Keywords: Podophyllotoxin; DNA topoisomerase-II; Etoposide;
Substituted 2-aminobenzophenone.
*
Corresponding author. Tel.: +91 40 27193157; fax: +91 40
27193189; e-mail: ahmedkamal@iict.res.in
0968-0896/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.06.032

A. Kamal et al. / Bioorg. Med. Chem. 13 (2005) 6218–6225
6219
Figure 1. Structures of podophyllotoxin (1), etoposide (2), teniposide (3), etopophos (4), NK 611 (5), GL 331 (6), and NPF (7).
Figure 2. Pharmacophore model of podophyllotoxin analogs.
previously reported method13 employing HBr in dichlo-
romethane. The intermediate (8) thus obtained has been
utilized for the synthesis of the final target molecules
10a–j and 11a–e. Compounds 10a–j are obtained by
coupling of 8 with substituted 2-aminobenzophenones
employing Bu4NI, and NEt3 in tetrahydrofuran (THF)
at room temperature in good yields (40–70%), whereas
coupling of 8 with various imides like succinimide,
phthalimide, and naphthalimide using Bu4NI, and
NEt3 in THF at room temperature gives the correspond-
ing 4b-imido-substituted podophyllotoxin analogs
(11a–e) in 30% yield (Scheme 1).
U251. The screening procedure is based on the routine
method adopted by NCI as described in the previous re-
port.11a These analogs of podophyllotoxin (10a–j and
11a–d) have exhibited an interesting profile of in vitro
anticancer activity. The compound 10j having C1 and F
substituents in 4b-(200-benzoyl) anilino-substituted series
is highly potent against all the cancer cell lines and similar-
ly 11d has exhibited very high in vitro anticancer activity
(Tables 1 and 2). However, the cytotoxicity (IC50 in KB
cell lines) of compound 6 has been reported5 as 1.3 lM.
On the basis of the in vitro anticancer activity, 10j has
been investigated for its in vivo activity but unfortunately
this compound has problems of bioavailability.
3. Biological evaluation
Some representative compounds in the 4b-anilino series
(10a, 10c, 10e, and 10g) have been evaluated in the 60
cancer cell line assay of NCI (Table 3). Interestingly,
both the 40-O-methyl and the 40-O-demethyl analogs
exhibited good in vitro anticancer activity. As demon-
strated by the mean graph pattern for compound 10c
These 4b-anilino- and 4b-imido-substituted podophyllo-
toxin analogs have been tested for their cytotoxic activi-
ties against six human cancer cell lines that comprise of
DU145, HT29, MCF7, MCF7ADR, NCIH460, and

6220
A. Kamal et al. / Bioorg. Med. Chem. 13 (2005) 6218–6225
Scheme 1. Reagents and conditions: (i) HBr, CH2Cl2, 0 °C, 45 min (8a); HBr, CH2Cl2, 0 °C, rt, 48 h (8b); (ii) Bu4NI, Et3N, THF, rt; (iii) succinimide,
phthalimide, naphthalimide, Bu4NI, Et3N, THF, rt.
(Fig. 3), it is seen that it exhibits not only significant
activity but also more sensitivity for certain cancer cell
lines. Therefore, from the structure–activity relationship
point of view, it is not clear about the role played by this
functionality. In literature, it has been observed that
demethylation of the 40-methoxy moiety eliminates anti-
mitotic activity but there is no clarity about its relation-
ship for the topo-II inhibition activity. For the
podophyllotoxin lignans, DNA topo-II is the pharmaco-
logical target of clinical relevance; therefore, some repre-
sentative compounds have been evaluated for its
inhibition. Most of the compounds (e.g., 10b, 10g, 10j,
and 11d) exhibited comparable in vitro inhibition of
topo-II catalytic activity to m-AMSA and the results
are illustrated in Figure 4. The topo-II-mediated relaxa-
tion assay performed is according to the previous
procedure.14
cell lines. Two of the compounds from this series exhib-
ited interesting DNA topo-II inhibition activity, sug-
gesting that these new molecules also exhibit biological
activity based on this mechanism similar to the etopo-
side prototypes. Further, these results agree with the
hypothesis that the bulky substitution at C-4 position
can enhance the activity profile for this class of com-
pounds. This investigation suggests that these podophyl-
lotoxin congeners irrespective of their interaction with
DNA possess potential in vitro anticancer activity.
Moreover, efforts are in progress to improve the bio-
availability of these compounds.
5. Experimental
The NMR spectra are recorded on Varian Gemini
200 MHz spectrometer, using TMS as an internal refer-
ence. IR spectra are recorded on Perkin-Elmer model
683 or 1310 spectrometer with sodium chloride optics.
Mass spectra are recorded on CEC-21-100B, Finnigan
Mat 1210, or Micromass 7070 spectrometer operating
at 70 eV using a direct inlet system. Optical rotations
are measured on Jasco Dip 360 digital polarimeter.
4. Conclusion
In summary, the newly designed and synthesized C-4-
modified podophyllotoxin congeners exhibit promising
in vitro cytotoxic activities in a number of human cancer

A. Kamal et al. / Bioorg. Med. Chem. 13 (2005) 6218–6225
6221
Table 1. Average GI50 values of the 4b-anilino-substituted analogs of
podophyllotoxin in six cell lines
Table 3. In vitro cytotoxicity of compounds 10a, 10c, 10e, and 10g in
selected human cancer cell lines
Cancer panel/cell line
GI50 (lM)
10c 10e
10a
10g
Leukemia
CCRF-CEM
SR
0.36
0.34
0.04
0.03
—
0.15
0.41
0.27
Nonsmall cell lung
NCI-H522
0.25
0.25
0.09
0.03
0.14
0.25
0.15
0.20
Colon
KM 12
CNS
SF-295
SF-539
0.34
0.25
0.05
0.02
0.27
0.18
0.36
0.26
Renal
ACHN
0.35
0.34
0.02
0.02
0.21
0.51
0.48
—
Breast
Compound
R
R1
R2
GI50 (lM)
HS-578T
Etoposidea (2)
—
—
H
—
H
0.80–116
0.04–0.5
15–382
10a
10b
10c
10d
10e
10f
10g
10h
10i
CH3
H
5.1. Chemistry
H
H
CH3
H
4-Cl
4-Cl
4-NO2
4-NO2
4-Cl
4-Cl
4-Cl
4-Cl
2-Cl
2-Cl
H
0.059–0.876
0.10–0.24
<10 nm–0.28
0.01–0.24
0.07–1.10
14–270
5.1.1. 4b-Bromopodophyllotoxin (8a). To a stirred solu-
tion of podophyllotoxin (1) (1.1 g, 2.65 mmol) in dichlo-
˚
romethane and 1 mL of ether, molecular sieves (4A)
CH3
H
H
powder was added. Dry HBr gas was bubbled through
the solution to saturation (about 45 min) at 0 °C. Later,
the solution was filtered (to remove molecular sieves
powder) and the solvent was removed under reduced
pressure. The product obtained in 60% yield and utilized
for next step without purification.
CH3
H
H
H
CH3
H
2-F
2-F
0.14–0.30
0.004–0.10
10j
a Values from NCI database.
5.1.2. 40-O-Demethyl-4b-bromo-4-desoxypodophyllotoxin
(8b). To a stirred solution of podophyllotoxin (1) (1.1 g,
2.65 mmol) in dichloromethane and 1 mL of ether,
Table 2. Average GI50 values of the 4b-imido-substituted analogs of
podophyllotoxin in six cell lines
˚
molecular sieves (4A) powder was added. Dry HBr gas
was bubbled through the solution to saturation (about
45 min) at 0 °C. The reaction mixture was allowed to stir
for 48 h at room temperature and filtered to remove the
molecular sieves powder. The filtrate was evaporated
under reduced pressure to leave the residue, which was
used for next step without further purification.
5.1.3. 4b-100-[200-(Benzoyl) anilino]-4-desoxypodophyllo-
toxin (10a). 4b-Bromo-4-desoxypodophyllotoxin (0.1 g,
0.21 mmol) was reacted with 2-aminobenzophenone
(0.045 g, 0.23 mmol) in the presence of Et3N (0.032 g,
0.32 mmol) and Bu4N+Iꢀ (0.015 g, 0.042 mmol) in dry
tetrahydrofuran at room temperature for 4 h. After com-
pletion of the reaction, the solvent was removed in vacuo.
The residue was subjected to silica gel column chromatog-
raphy using chloroform/methanol (9.8:0.2) as an eluent.
Compound
X
R
GI50 (lM)
Etoposidea (2)
—
—
H
0.80–116
0.16–0.50
0.03–0.40
0.10–0.24
0.004–0.02
—
11a
11b
11c
11d
11e
Succinimido
Succinimido
Phthalimido
Phthalimido
Naphthalimido
CH3
H
CH3
CH3
25
Yield 60%, mp 140–142 °C; ½aꢁ ꢀ112 (c 0.1, CHCl3);
a Values from NCI database.
D
IR (KBr) 3400, 2900, 1780, 1500, 1480, 1410, 1300,
1
1250 cmꢀ1; H NMR (CDCl3) d 8.82 (d, 1H), 7.50 (m,
Melting points are determined on an electrothermal
melting point apparatus and are uncorrected. TLC is
performed with E. Merck precoated silica gel plates
(60F-254) with iodine as a developing agent. Acme, In-
dia silica gel (100–200 mesh) is used for column
chromatography.
7H), 6.80 (s, 1H), 6.75 (d, 1H), 6.68 (d, 1H), 6.55 (s,
1H), 6.35 (s, 2H), 5.96 (d, 2H), 4.92 (m, 1H), 4.65 (d,
1H), 4.35 (t, 1H), 3.96 (t, 1H), 3.82 (d, 9H), 3.20 (q,
1H), 3.05 (m, 1H); MS 593 (M+Å). Anal. Calcd for
C35H31NO8: C, 70.82; H, 5.26; N, 2.36. Found: C,
70.85; H, 5.28; N, 2.39.

6222
A. Kamal et al. / Bioorg. Med. Chem. 13 (2005) 6218–6225
Figure 3. Log10GI50, log10TGI, and log10LC50 data from the NCI 60 cell line screen for the compound 10c.
25
Yield 50% mp 154–156 °C; ½aꢁ 111 (c 1.1, CHCl3);
D
IR (KBr) 3550, 3400, 2900, 1750, 1650, 1500, 1480,
1
1410, 1300, 1250 cmꢀ1; H NMR (CDCl3) d 8.85 (d,
1H), 7.50 (m, 7H), 6.80 (s, 1H), 6.75 (d, 1H), 6.68
(d, 1H), 6.55 (s, 1H), 6.35 (s, 2H), 5.96 (d, 2H),
5.38 (s, 1H), 4.92 (m, 1H), 4.65 (d, 1H), 4.35 (t,
1H), 3.96 (t, 1H), 3.82 (s, 6H), 3.20 (q, 1H), 3.05
(m, 1H); MS 579 (M+Å). Anal. Calcd for
C34H29NO8: C, 70.46; H, 5.04; N, 2.42. Found: C,
70.50; H, 5.08; N, 2.39.
5.1.5.
4b-100-[200-(2-Chlorobenzoyl)-400-chloroanilino]-4-
desoxypodophyllotoxin (10c). 4b-Bromo-4-desoxypodo-
phyllotoxin (0.10 g, 0.21 mmol) was reacted with 2-ami-
no-20,50-dichlorobenzophenone (0.06 g, 0.23 mmol) in
the presence of Et3N (0.032 g, 0.32 mmol) and Bu4N+Iꢀ
(0.015 g, 0.042 mmol) in dry tetrahydrofuran at room
temperature for 5 h. After completion of the reaction,
the solvent was removed in vacuo. The residue was
subjected to silica gel column chromatography using
chloroform/methanol (9.7:0.3) as an eluent.
Figure 4. Inhibition of DNA topoisomerase-II catalyzed relaxation of
supercoiled DNA by compounds 10b, 10g, 10j, and 11d. Lanes 1–5:
Compounds 10b, 10g, 10j, and 11d (200, 100, 50, 25, and 10 lM,
respectively); lane 6: m-AMSA (200 lM) postive control; lane 7: Topo-
II a negative control; lane 8: 0.6 lg PRYG plasmid DNA.
5.1.4. 40-O-Demethyl-4b-100-[200-(benzoyl) anilino]-4-des-
oxypodophyllotoxin (10b). 4b-Bromo-40-O-demethyl-4-
desoxypodophyllotoxin (0.1 g, 0.21 mmol) was reacted
with 2-aminobenzophenone (0.045 g, 0.23 mmol) in
the presence of Et3N (0.032 g, 0.32 mmol) and
Bu4N+Iꢀ (0.015 g, 0.042 mmol) in dry tetrahydrofu-
ran at room temperature for 4 h. After completion
of the reaction, the solvent was removed in vacuo.
The residue was subjected to silica gel column chro-
matography using chloroform/methanol (9.8:0.2) as
an eluent.
25
Yield 64% mp 142–145 °C; ½aꢁ ꢀ84 (c 0.87, CHCl3); IR
D
(KBr) 3350, 2900, 1760, 1640, 1550, 1480, 1250 cmꢀ1
;
1H NMR (CDCl3) d 9.10 (d, 1H), 7.40 (m, 5H), 7.20
(d, 1H), 6.78 (s, 1H), 6.75 (d, 1H), 6.52 (s, 1H), 6.35
(s, 2H), 5.96 (d, 2H), 4.97 (m, 1H), 4.65 (d, 1H), 4.35
(t, 1H), 3.90 (t, 1H), 3.77 (d, 9H), 3.20 (q, 1H), 3.10
(m, 1H); MS 663 (M+Å). Anal. Calcd for C35H29Cl2NO8:
C, 63.45; H, 4.41; N, 2.11. Found: C, 63.48; H, 4.44; N,
2.14.

A. Kamal et al. / Bioorg. Med. Chem. 13 (2005) 6218–6225
6223
5.1.6.
chloroanilino]-4-desoxypodophyllotoxin (10d). 4b-Bro-
mo-40-O-demethyl-4-desoxypodophyllotoxin
(0.10 g,
40-O-Demethyl-4b-100-[200-(2-chlorobenzoyl)-400-
5.1.9. 4b-100-[200-(Benzoyl)-400- chloroanilino]-4-desoxypod-
ophyllotoxin (10g). 4b-Bromo-4-desoxypodophyllotoxin
(0.10 g, 0.21 mmol) was reacted with 2-amino-5-chloro-
benzophenone (0.053 g, 0.23 mmol) in the presence of
Et3N (0.032 g, 0.32 mmol) and Bu4N+Iꢀ (0.015 g,
0.042 mmol) in dry tetrahydrofuran at room tempera-
ture for 6 h. After completion of the reaction, the sol-
vent was removed in vacuo. The residue was subjected
to silica gel column chromatography using chloroform/
methanol (9.7:0.3) as an eluent.
0.21 mmol) was reacted with 2-amino-20,50-dichloro-
benzophenone (0.06 g, 0.23 mmol) in the presence of
Et3N (0.032 g, 0.32 mmol) and Bu4N+Iꢀ (0.015 g,
0.042 mmol) in tetrahydrofuran at room temperature
for 5 h. After completion of the reaction, the solvent
was removed in vacuo. The residue was subjected to sil-
ica gel column chromatography using chloroform/meth-
anol (9.7:0.3) as an eluent.
25
Yield 56% mp 139–142 °C; ½aꢁ ꢀ103 (c 0.93, CHCl3);
D
IR (KBr) 3350, 2900, 1780, 1660, 1500, 1480, 1410,
25
Yield 70% mp 151–153 °C; ½aꢁ ꢀ91 (c 0.93, CHCl3); IR
D
(KBr) 3320, 2900, 1760, 1650, 1550, 1480, 1410,
1
1250 cmꢀ1; H NMR (CDCl3) d 8.72 (d, 1H), 7.60 (m,
1
1250 cmꢀ1; H NMR (CDCl3) d 9.10 (d, 1H), 7.40 (m,
7H), 6.80 (s, 1H), 6.75 (d, 1H), 6.55 (s, 1H), 6.35 (s,
2H), 5.98 (d, 2H), 4.95 (m, 1H), 4.65 (d, 1H), 4.40 (t,
1H), 3.95 (t, 1H), 3.80 (d, 9H), 3.20 (q, 1H), 3.10 (m,
1H); MS 628 (M+Å). Anal. Calcd for C35H30ClNO8: C,
66.93; H, 4.81; N, 2.23. Found: C, 66.94; H, 4.84; N,
2.26.
5H), 7.20 (d, 1H), 6.77 (s, 1H), 6.70 (d, 1H), 6.30 (s,
2H), 5.96 (d, 2H), 5.40 (s, 2H), 4.90 (m, 1H), 4.65 (d,
1H), 4.30 (t, 1H), 4.10 (t, 1H), 3.80 (s, 6H), 3.20 (q,
1H), 3.10 (m, 1H); MS 649 (M+Å). Anal. Calcd for
C34H27Cl2NO8: C, 62.97; H, 4.20; N, 2.16. Found: C,
62.94; H, 4.23; N, 2.14.
5.1.10. 40-O-Demethyl-4b-100-[200-(benzoyl)]-400-chloroani-
lino]-4-desoxypodophyllotoxin (10h). 4b-Bromo-40-O-
demethyl-4-desoxypodophyllotoxin (0.1 g, 0.21 mmol)
was reacted with 2-amino-5-chlorobenzophenone
(0.053 g, 0.23 mmol) in the presence of Et3N (0.032 g,
0.32 mmol) and Bu4N+Iꢀ (0.015 g, 0.042 mmol) in dry
tetrahydrofuran at room temperature for 6 h. After
completion of the reaction, the solvent was removed in
vacuo. The residue was subjected to silica gel column
chromatography using chloroform/methanol (9.7:0.3)
as an eluent.
5.1.7. 4b-100-[200-(Benzoyl)-400-nitroanilino]-4-desoxypodo-
phyllotoxin (10e). 4b-Bromo-4-desoxypodophyllotoxin
(0.1 g, 0.21 mmol) was reacted with 2-amino-5-nitro-
benzophenone (0.056 g, 0.23 mmol) in the presence of
Et3N (0.032 g, 0.32 mmol) and Bu4N+Iꢀ (0.015 g,
0.042 mmol) in dry tetrahydrofuran at room tempera-
ture for 8 h. After completion of the reaction, the sol-
vent was removed in vacuo. The residue was subjected
to silica gel column chromatography using chloroform/
methanol (9.5:0.5) as an eluent.
25
Yield 40%, mp 163–167 °C; ½aꢁ ꢀ85 (c 1.2, CHCl3); IR
25
Yield 50%, mp 146–149 °C; ½aꢁ ꢀ105 (c 0.97, CHCl3);
D
D
IR (KBr) 3500, 3360, 2900, 1750, 1640, 1500, 1480,
(KBr) 3450, 2950, 1740, 1650, 1550, 1480, 1250 cmꢀ1
;
1H NMR (CDCl3) d 9.50 (d, 1H), 8.57 (d, 1H), 8.32
(q, 1H), 7.60 (m, 4H), 6.75 (d, 1H), 6.70 (s, 1H) 6.60
(d, 1H), 6.40 (d, 1H), 6.30 (s, 2H), 6.00 (d, 2H), 5.05
(m, 1H), 4.70 (d, 1H), 4.40 (t, 1H), 3.90 (t, 1H), 3.80
(d, 9H), 3.15 (d, 1H), 2.95 (m, 1H); MS 638 (M+Å). Anal.
Calcd for C35H30N2O10: C, 65.83; H, 4.73; N, 4.39.
Found: C, 65.85; H, 4.76; N, 4.41.
1230 cmꢀ1; H NMR (CDCl3) d 8.68 (d, 1H), 7.52 (m,
1
7H), 6.72 (s, 1H), 6.65 (d, 1H), 6.50 (s, 1H) 6.30 (s,
2H), 5.96 (d, 2H), 5.35 (s, 1H), 4.85 (m, 1H), 4.60 (d,
1H), 4.30 (t, 1H), 3.85 (d, 1H), 3.80 (s, 6H), 3.10 (q,
1H), 3.00 (m, 1H); MS 614 (M+Å). Anal. Calcd for
C34H28ClNO8: C, 66.50; H, 4.60; N, 2.28. Found: C,
66.54; H, 4.57; N, 2.26.
5.1.8. 40-O-Demethyl-4b-100-[200-(benzoyl)]-400-nitroanili-
no]-4-desoxypodophyllotoxin (10f). 4b-Bromo-40-O-dem-
ethyl-4-desoxypodophyllotoxin (0.1 g, 0.21 mmol) was
reacted with 2-amino-5-nitro-benzophenone (0.056 g,
0.23 mmol) in the presence of Et3N (0.032 g, 0.32 mmol)
and Bu4N+Iꢀ (0.015 g, 0.042 mmol) in dry tetrahydrofu-
ran at room temperature for 8 h. After completion of the
reaction, the solvent was removed in vacuo. The residue
was subjected to silica gel column chromatography
using chloroform/methanol (9.5:0.5) as an eluent.
5.1.11. 4b-100-[200-(2-Fluorobenzoyl)-400-chloroanilino]-4-
desoxypodophyllotoxin (10i). 4b-Bromo-4-desoxypodo-
phyllotoxin
(0.10 g,
0.21 mmol)
was
reacted
with 2-amino-5-chloro-20-fluorobenzophenone (0.057 g,
0.23 mmol) in the presence of Et3N (0.032 g, 0.32 mmol)
and Bu4N+Iꢀ (0.015 g, 0.042 mmol) in dry tetrahydrofu-
ran at room temperature for 5 h. After completion of the
reaction, the solvent was removed in vacuo. The residue
was subjected to silica gel column chromatography
using chloroform/methanol (9.7:0.3) as an eluent.
25
Yield 38%, mp 169–171 °C; ½aꢁ ꢀ89 (c 1.0, CHCl3); IR
25
Yield 68% mp 123–128 °C; ½aꢁ ꢀ89 (c 1.0, CHCl3); IR
D
(KBr) 3560, 3400, 2900, 1740, 1650, 1500, 1480,
D
(KBr) 3400, 2950, 1760, 1650, 1500, 1480, 1300,
1
1
1250 cmꢀ1; H NMR (CDCl3) d 9.47 (d, 1H), 8.55 (d,
1250 cmꢀ1; H NMR (CDCl3) d 9.10 (d, 1H), 7.45 (m,
1H), 8.30 (q, 1H), 7.60 (m, 4H), 6.80 (d, 1H), 6.70 (s,
1H), 6.55 (s, 1H) 6.35 (d, 1H), 6.30 (s, 2H), 6.00 (d,
2H), 5.87 (s, 1H), 5.00 (m, 1H), 4.65 (d, 1H), 4.30 (m,
2H), 3.80 (d, 6H), 3.15 (d, 1H), 3.00 (m, 1H); MS 624
(M+Å). Anal. Calcd for C34H28N2O10: C, 65.38; H,
4.52; N, 4.49. Found: C, 65.35; H, 4.56; N, 4.51.
6H), 6.80 (s, 1H), 6.75 (d, 1H), 6.55 (s, 1H), 6.35 (s,
2H), 6.00 (d, 2H), 4.95. (m, 1H), 4.70 (d, 1H), 4.40 (t,
1H), 3.95 (t, 1H), 3.82 (d, 9H), 3.20 (q, 1H), 3.10 (m,
1H); MS 646 (M+Å). Anal. Calcd for C35H29ClFNO8:
C, 65.07; H, 4.52; N, 2.17. Found: C, 65.09; H, 4.55;
N, 2.19.

6224
A. Kamal et al. / Bioorg. Med. Chem. 13 (2005) 6218–6225
5.1.12.
40-O-Demethyl-4b-100-[200-(2-fluorobenzoyl)-400-
presence of Et3N (0.032 g, 0.32 mmol) and Bu4N+Iꢀ
(0.015 g, 0.042 mmol) in dry tetrahydrofuran at room
temperature for 5 h. After completion of the reaction,
the solvent was removed in vacuo. The residue was sub-
jected to silica gel column chromatography using ethyl
acetate/hexane (8:2) as an eluent.
chloroanilino]-4-desoxypodophyllotoxin (10j). 4b-Bro-
(0.10 g,
mo-40-O-demethyl-4-desoxypodophyllotoxin
0.21 mmol) was reacted with 2-amino-5-chloro-2-fluoro-
benzophenone (0.057 g, 0.23 mmol) in the presence of
Et3N (0.032 g, 0.32 mmol) and Bu4N+Iꢀ (0.015 g,
0.042 mmol) in dry tetrahydrofuran at room temperature
for 5 h. After completion of the reaction, the solvent was
removed in vacuo. The residue was subjected to silica gel
column chromatography using chloroform/methanol
(9.7:0.3) as an eluent.
25
Yield 30% mp 135–139 °C; ½aꢁ ꢀ69 (c 1.1, CHCl3); IR
D
(KBr) 2940, 1760, 1740, 1530, 1410, 1250 cmꢀ1
;
1H
NMR (CDCl3): d 7.85 (m, 2H), 7.75 (m, 2H), 6.75 (s,
1H), 6.55 (s, 1H), 6.20 (s, 2H), 5.97 (d, 2H), 4.57 (d,
1H), 4.33. (d, 1H), 4.27 (d, 2H), 3.75 (d, 9H), 3.35 (q,
1H), 2.80 (m, 1H); MS 544 (M+Å). Anal. Calcd for
C30H25NO9: C, 66.29; H, 4.64; N, 2.58. Found: C,
66.26; H, 4.62; N, 2.61.
25
Yield 60% mp 164–167 °C; ½aꢁ ꢀ85 (c 1.01, CHCl3); IR
D
(KBr) 3520, 3440, 2900, 1750, 1650, 1500, 1480, 1300,
1250 cmꢀ1; H NMR (CDCl3) d 9.05 (d, 1H), 7.48 (m,
1
6H), 6.80 (s, 1H), 6.75 (d, 1H), 6.52 (s, 1H), 6.35 (s, 2H),
6.00 (d, 2H), 5.10 (s, 1H), 4.98 (m, 1H), 4.70 (d, 1H), 4.40
(t, 1H), 3.95 (t, 1H), 3.82 (s, 6H), 3.20 (q, 1H), 3.10 (m,
1H); MS 632 (M+Å). Anal. Calcd for C34H27ClFNO8: C,
64.61; H, 4.31; N, 2.22. Found: C, 64.64; H, 4.35; N, 2.18.
5.1.16.
40-O-Demethyl-4b-phthalimido-4-desoxypodo-
phyllotoxin (11d). 4b-Bromo-40-O-demethyl-4-desoxyp-
odophyllotoxin (0.10 g, 0.21 mmol) was reacted with
phthalimide (0.034 g, 0.23 mmol) in the presence of
Et3N (0.032 g, 0.32 mmol) and Bu4N+Iꢀ (0.015 g,
0.042 mmol) in dry tetrahydrofuran at room tempera-
ture for 5 h. After completion of the reaction, the sol-
vent was removed in vacuo. The residue was subjected
to silica gel column chromatography using ethyl ace-
tate/hexane (8:2) as an eluent.
5.1.13.
40-O-Demethyl-4b-succinimido-4-desoxypodo-
phyllotoxin (11a). 4b-Bromo-40-O-demethyl-4-desoxyp-
odophyllotoxin (0.10 g, 0.21 mmol) was reacted with
succinimide (0.024 g, 0.23 mmol) in the presence of
Et3N (0.032 g, 0.32 mmol) and Bu4N+Iꢀ (0.015 g,
0.042 mmol) in dry tetrahydrofuran at room temperature
for 6 h. After completion of the reaction, the solvent was
removed in vacuo. The residue was subjected to silica gel
column chromatography using ethyl acetate/hexane (7:3)
as an eluent.
25
Yield 30% mp 156–160 °C; ½aꢁ ꢀ58 (c 1.0, CHCl3); IR
D
(KBr) 3300, 2940, 1760, 1740, 1530, 1410, 1250 cmꢀ1
;
1H NMR (CDCl3) d 7.85 (m, 2H), 7.75 (m, 2H), 6.78
(s, 1H), 6.55 (s, 1H), 6.22 (s, 2H), 5.95 (d, 2H), 5.32 (s,
1H), 4.55. (d, 1H), 4.35 (d, 1H), 4.29 (d, 2H), 3.80 (s,
6H), 3.35 (q, 1H), 2.85 (m, 1H); MS 530 (M+Å). Anal.
Calcd for C29H23NO9: C, 65.78; H, 4.38; N, 2.65.
Found: C, 65.74; H, 4.32; N, 2.61.
25
Yield 30% mp 197–201 °C; ½aꢁ ꢀ104 (c 0.05, EtOAc:-
D
MeOH); IR (KBr) 3500, 2880, 1800, 1630, 1530,
1
1250 cmꢀ1; H NMR (CDCl3) d 6.85 (s, 1H), 6.55 (s,
1H), 6.25 (s, 2H), 6.00 (d, 2H), 5.35 (s, 1H), 4.85. (d,
1H), 4.58 (d, 1H), 4.35 (d, 2H), 3.78 (s, 6H), 3.24 (q,
1H), 2.75 (s,4H), 2.30 (m, 1H); MS 481 (M+Å). Anal. Calcd
for C25H23NO9: C, 62.37; H, 4.82; N, 2.91. Found: C,
62.35; H, 4.80; N, 2.89.
5.1.17. 4b-Naphthalimido-4-desoxypodophyllotoxin (11e).
4b-Bromo-4-desoxypodophyllotoxin (0.10 g, 0.21 mmol)
was reacted with naphthalimide (0.056 g, 0.23 mmol) in
the presence of Et3N (0.032 g, 0.32 mmol) and Bu4N+Iꢀ
(0.015 g, 0.042 mmol) in dry tetrahydrofuran at room
temperature for 7 h. After completion of the reaction,
the solvent was removed in vacuo. The residue was sub-
jected to silica gel column chromatography using ethyl
acetate/hexane (8:2) as an eluent.
5.1.14. 4b-Succinimido-4-desoxypodophyllotoxin (11b).
4b-Bromo-4-desoxypodophyllotoxin (0.10 g, 0.21 mmol)
was reacted with succinimide (0.023 g, 0.23 mmol) in the
presence of Et3N (0.032 g, 0.32 mmol) and Bu4N+Iꢀ
(0.015 g, 0.042 mmol) in dry tetrahydrofuran at room
temperature for 6 h. After completion of the reaction,
the solvent was removed in vacuo. The residue was sub-
jected to silica gel column chromatography using ethyl
acetate/hexane (7:3) as an eluent.
25
D
1
Yield 30% mp 132–136 °C; ½aꢁ ꢀ64 (c 0.7, CHCl3); H
NMR (CDCl3) d 7.72 (m, 2H), 7.55 (m, 2H), 6.8 (s, 1H),
6.55 (t, 2H), 6.23 (s, 2H), 6.10 (s, 1H), 5.97 (d, 2H), 4.58.
(d, 1H), 4.35 (d, 1H), 4.28 (t, 2H), 3.76 (d, 9H), 3.35 (dd,
1H), 2.85 (m, 1H). MS 593 (M+Å). Anal. Calcd for
C34H27NO9: C, 68.80; H, 4.58; N, 2.36. Found: C,
68.84; H, 4.53; N, 2.40.
25
D
Yield 30% mp 168–172 °C; ½aꢁ ꢀ97 (c 0.6, EtOAc:-
MeOH, 1:1); IR (KBr) 2850, 1800, 1620, 1500, 1480,
1410, 1250 cmꢀ1; 1H NMR (CDCl3) d 6.85, (s, 1H), 6.52
(s, 1H), 6.25 (s, 2H), 5.90 (d, 2H), 4.85 (d, 1H), 4.55 (d,
1H), 4.35 (d, 2H), 3.75 (d, 9H), 3.25 (q, 1H), 2.75 (s,
4H), 2.40 (m, 1H); MS 495 (M+Å). Anal. Calcd for
C26H25NO9: C, 63.03; H, 5.09; N, 2.83. Found: C,
63.06; H, 5.12; N, 2.87.
5.2. Biological evaluation
5.2.1. In vitro evaluation of cytotoxic activity. In routine
screening, each agent is tested over a broad concentra-
tion range (10-fold dilutions starting from >100 lM to
ꢂ10 nM) against six human cancer cell lines comprised
of different tumor types. Standard compound doxorubi-
cin is tested in each assay as a positive control. The cells
are maintained in growing condition in RPMI 1640
5.1.15. 4b-Phthalimido-4-desoxypodophyllotoxin (11c).
4b-Bromo-4-desoxypodophyllotoxin (0.10 g, 0.21 mmol)
was reacted with phthalimide (0.033 g, 0.23 mmol) in the

A. Kamal et al. / Bioorg. Med. Chem. 13 (2005) 6218–6225
6225
2. MacRae, W. D.; Hudson, J. B.; Towers, B. H. N. Planta
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3. Jardine, I. In Anti-Cancer Agents Based on Natural
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medium containing 10% fetal calf serum and incubated
at 37 °C under 5% CO2 atmosphere. All cell lines are
inoculated onto a series of standard 96-well microtiter
plate on day 0, followed by 24 h incubation in the ab-
sence of test compound. The inoculation densities used
in this screen are as per the procedure of Monks
et al.15 All NCEs are dissolved in DMSO and diluted
further in culture medium. An aliquot of each dilution
is added to the growing cells in 96-well plates and incu-
bated for 48 h. After incubation, the assay is terminated
by adding 50 lL of trichloro acetic acid and incubating
at 4 °C for 30 min. The precipitated cells are washed and
stained with sulphorhodamine B dye for 30 min and the
excess dye is washed off with acetic acid. Adsorbed dye
is solublized in Tris base (alkaline pH) and quantitated
by measuring the OD at 490 nm in an ELISA reader.
GI50 (concentration that inhibits the cell growth by
50%) is calculated according to the method of Boyd.16
5.2.2. Immunoprecipitation of topoisomerase-II a. Brain
extracts (100 lg total protein) prepared from cerebellum
of embryos (E11, E18, and 1-day-old) and whole brain,
cerebellum, cerebral cortex, and midbrain regions of the
young, adult, and old age groups were taken in Eppendorf
tubes for immunoprecipitation and topo-II a or b anti-
body (1:1000 dilution in immunoprecipitation buffer con-
taining 100 mM Tris–HCl, pH 8, 750 mM NaCl, 2 mM
EDTA, 1 mM PMSF, and 0.75% Nonidet) was added
to each sample. The antigen–antibody mixture was incu-
bated at room temperature for 1 h and 25 ll of 6% protein
An agarose beads was added. The beads were incubated at
4 °C for 15 min, spun down and the supernatant was re-
moved. The protein An agarose beads were washed twice
with 0.5% Triton X-100 in PBS. The beads were directly
used for monitoring the relaxation activity of topo-II.17
9. Cho, S. J.; Tropsha, A.; Suffness, M.; Cheng, Y. C.; Lee,
K. H. J. Med. Chem. 1996, 39, 1383.
10. (a) Kamal, A.; Gayatri, N. L. Tetrahedron Lett. 1996, 37,
3359; (b) Kamal, A.; Laxminarayana, B.; Gayatri, N. L.
Tetrahedron Lett. 1997, 38, 6871; (c) Kamal, A.; Dama-
yanthi, Y. Bioorg. Med. Chem. Lett. 1997, 7, 657; (d)
Kamal, A.; Gayatri, N. L.; Rao, N. V. Bioorg. Med.
Chem. Lett. 1998, 8, 3097; (e) Kamal, A.; Laxman, N.;
Ramesh, G. Bioorg. Med. Chem. Lett. 2000, 10, 2059; (f)
Kamal, A.; Laxman, N.; Ramesh, G. Indian Patent Nos.
191366 and 192429; (g) Kamal, A.; Kumar, B. A.;
Arifuddin, M. Tetrahedron Lett. 2004, 44, 8457.
11. (a) Kamal, A.; Kumar, B. A.; Arifuddin, M.; Sunanda, G.
D. Bioorg. Med. Chem. 2003, 11, 5135; (b) Kamal, A.;
Laxman, E.; Khanna, G. B. R.; Reddy, P. S. M. M.;
Rehana, T.; Arifuddin, M.; Neelima, K.; Kondapi, A. K.;
Dastidar, S. G. Bioorg. Med. Chem. 2004, 12, 4197.
12. (a) Kamal, A.; Gayatri, N. L. Indian Patent No. 186131.;
(b) Kamal, A.; Reddy, P. S. M. M. PCT Appl. No. PCT/
IN02/00091, International Publication No. WO 03/082876
A1.
Acknowledgments
This work was financially supported by the Department
of Science and Technology, India, under the drugs and
pharmaceutical research program. We thank National
Cancer Institute, Maryland, USA, for the in vitro anti-
cancer assay in human cancer cell lines. The authors
D.R.S.R., P.S.M.M.R., and M.R. are grateful to CSIR,
New Delhi, for the award of research fellowships.
13. Khun, M.; Keller-Juslen, C.; von Wartburg, A. Helv.
Chem. Acta 1969, 52, 948.
14. Gopal, Y. N. V.; Jayaraju, D.; Kondapi, A. K. Biochem-
istry 1999, 38, 4382.
15. Monks, A.; Scudiero, D.; Skehan, P.; Shoemaker, R.;
Paull, K.; Vistica, D.; Hose, C.; Langley, J.; Cronise, P.;
Vaigro-Wolff, A.; Gray-Goodrich, M.; Campbell, H.;
Mayo, J.; Boyd, M. J. Natl. Cancer Inst. 1991, 83, 757.
16. Boyd, M. R.; Paull, K. D. Drug. Dev. Res. 1995, 34, 91.
17. Kondapi, A. K.; Mulpuri, N.; Mandraju, R. K.; Sasika-
ran, B.; Rao, K. S. Int. J. Dev. Neurosci. 2004, 22, 19.
References and notes
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