Synthesis of 4beta-amido and 4beta-sulphonamido analogues of podophyllotoxin as potential antitumour agents.

Article abstract of DOI:10.1016/j.bmc.2003.08.019

The new 4beta-amido analogues of podophyllotoxin or 4'-O-demethylepipodophyllotoxin have been prepared either by the coupling of 4beta-amino podophyllotoxin or 4beta-amino-4'-O-demethyl epipodophyllotoxin with the corresponding acids in presence of DCC in dichloromethane or by treating the appropriate acid chloride or sulphonyl chloride in presence of Et(3)N. These 4beta-amido and 4beta-sulphonamido derivatives of podophyllotoxin have been evaluated for their cytotoxicity against six human cancer cell lines. Some of these analogues have shown promising anticancer activity.

Full text of DOI:10.1016/j.bmc.2003.08.019

Bioorganic & Medicinal Chemistry 11 (2003) 5135–5142
Synthesis of 4ꢀ-amido and 4ꢀ-sulphonamido Analogues of
Podophyllotoxin as Potential Antitumour Agents
Ahmed Kamal,a,* B. Ashwini Kumar,a M. Arifuddina and Sunanda G. Dastidarb
aDivision of Organic Chemistry, Indian Institute of Chemical Technology, Hyderabad-500007, India
bRanbaxy Research Laboratories, Gurgaon-122001, Haryana, India
Received 9 May 2003; accepted 18 August 2003
Abstract—The new 4b-amido analogues of podophyllotoxin or 40-O-demethylepipodophyllotoxin have been prepared either by the
coupling of 4b-amino podophyllotoxin or 4b-amino-40-O-demethyl epipodophyllotoxin with the corresponding acids in presence of
DCC in dichloromethane or by treating the appropriate acid chloride or sulphonyl chloride in presence of Et3N. These 4b-amido
and 4b-sulphonamido derivatives of podophyllotoxin have been evaluated for their cytotoxicity against six human cancer cell lines.
Some of these analogues have shown promising anticancer activity.
# 2003 Elsevier Ltd. All rights reserved.
Introduction
congeners,9À11 for example NPF (5a) and GL-331 (5b).
Presently, GL-331 is undergoing phase II clinical trials
against gastric carcinoma, colon cancer, non-small cell
carcinoma, and etoposide-resistant malignancies.12 It
has also been indicated in the literature that bulky sub-
stitution at the C4 position usually enhances the cyto-
toxicity and DNA topoisomerase II inhibition
activity.11b,c It is observed that, amongst the C4-N-sub-
stituted congeners of podophyllotoxin that have been
synthesized and developed, C4b-N-amidopodophyllo-
toxin derivatives have received less attention. Further,
some of these compounds have shown improved biological
activity in comparison to etoposide.
Podophyllotoxin (1) and desoxypodophylltoxin (2) are
two well known naturally occurring aryltetralin lignans
and the former is the major constituent of a number of
plant species of the podophyllum family.1,2 Both these
compounds are cytotoxic and their derivatives are in use
as antitumour agents, for example etoposide3,4 (3) and
teniposide5 (4). Etoposide is the most widely used anti-
cancer agent for the treatment of leukemia, testicular
cancer and small cell lung cancer. Its clinical efficancy is
due to its ability to inhibit the enzyme DNA–topo-
isomerase II. These podophyllotoxin lignans block the
catalytic activity of DNA–toposiomerase II by stabiliz-
ing a cleavable enzyme DNA–complex in which the
DNA is cleaved and covalently linked to enzyme.6 This
lead has stimulated a renewed interest in the chemical
and biochemical studies of podophyllotoxin derived
antitumour agents.7 The replacement of the C-4 sugar
moiety of etoposide (VP-16) with a nonsugar substitu-
tion has proven to be significant in overcoming the drug
resistance of etoposide.8 The C-4 non-sugar substitu-
tions can be linked through O-, S- or N-linkage. In
general, the O-linked derivatives (ethers, esters) and the
S-linked derivatives (thioethers) are inactive or show
lower activity in comparison to the N-linked
Recently, C4b-amido analogue13 has been synthesized
and evaluated for its biological activity. This compound
(6) exhibits superior anticancer activity compared to
etoposide against some of the human cancer cell lines.
In earlier studies a series of 4b-amido aryl substituted
podophylltoxin derivatives (7)14 have been prepared and
evaluated for their biological activity. It is observed
from the results that some of these compounds exhibit
enhanced DNA–topoisomerase II inhibition in com-
parison to etoposide. In view of the available informa-
tion on structure–activity relationship studies and in
continuation of our ongoing project on the design and
development of structurally modified podophyllotoxin
congeners,15 it was considered of interest to prepare
C4b-amido and sulphonamido analogues of epipodo-
phylltoxin and 40-O-demethyl epipodophyllotoxin.16
*Corresponding author. Tel.: +91-40-2719-3157; fax: +91-40-2719-
3189; e-mail: ahmedkamal@iict.ap.nic.in
0968-0896/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2003.08.019

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A. Kamal et al. / Bioorg. Med. Chem. 11 (2003) 5135–5142
Hence, their preparation and biological evaluation,
particularly anticancer activity is reported herein.
(14–19). This reaction has also been performed by
employing 2-benzoyl substituted benzoic acid chlorides
to produce the corresponding amido analogues. It has
been observed that the work up is cleaner and simpler
and yields are improved when these coupling reactions
are carried out by acid chloride method. Similarly, aryl
substituted sulphonyl chlorides have been coupled to
the intermediates (9) and (13) to afford the correspond-
ing C4b-sulphonamido-podophyllotoxin analogues (20–
23). Furthermore, substituted nicotinic acid has been
coupled to the intermediates (9) and (13) in presence of
DCC to give the corresponding C4b-amino nicotinyl
derivatives of podophyllotoxin (24–27). Attempts to
prepare these derivatives from nicotinic acid chloride
did not produce the desired C4b-amidopodophyllotoxin
products (Scheme 2).
Chemistry
The synthesis of C4b-amido and sulphonamido analo-
gues of podophyllotoxin have been carried out from
podophyllotoxin as schemes in Schemes 1 and 2. The
key intermediate for the preparation of the new analo-
gues is C4b-aminopodophyllotoxin (9) C4b-amino-40-O-
demethylpodophyllotoxin (13). Podphyllotoxin is
mesylated followed by azidation to give C4b-azidopo-
dophyllotoxin (8). This upon reduction over Pd/C
provides a 7:3 mixture of C4b-aminopodophyllotoxin
and C4a-aminopodophyllotoxin. The required C4b-
aminopodophyllotoxin (9) has been purified by column
chromatography by employing chloroform–ethyl ace-
tate (2:1) as eluent, where as C4b-amino-40-O-deme-
thylpodophyllotoxin (13) has been prepared from C4b-
azido-40-O-demethylpodophyllotoxin. (12) The azido
compound (12) has been obtained from the azidation of
40-O-demethylepipodophyllotoxin (11) which in turn
has been obtained from the sequential in situ iodination
in presence of methane sulphonic acid/NaI reagent sys-
tem17 followed by hydrolysis in presence of barium car-
bonate (Scheme 1).
Biological Evaluation
These 4b-amido and 4b-sulphonamido analogues of
podophyllotoxin have been tested for their cytotoxic
activities against six human cancer cell lines that com-
prise of DU145, HT29, MCF7, MCF7ADR, NCIH460
and U251. The screening procedure is based on the
routine method adopted by the Developmental Ther-
apeutic Programme of National Cancer Institute (NCI),
Bethesda, MD, USA.
The intermediates, C4b-aminopodophyllotoxin (9) and
C4b-amino-40-O-demethyl podophyllotoxin (13) have
been coupled with 2-benzoyl substituted benzoic acids
in presence of N,N0-dicyclohexylcarbodiimide (DCC) to
afford the corresponding C4b-amido aryl substituted
podophyllotoxin and their 40-O-demethyl derivatives
4b-Amido-2-substituted benzophenone analogues of
podophyllotoxin (14–19) show moderate anticancer
activity. It is interesting to observe that one of the
compounds (20) in 4b-sulphonamido aryl substituted
podophylllotoxin derivatives (20–23) is highly potent

A. Kamal et al. / Bioorg. Med. Chem. 11 (2003) 5135–5142
5137
Scheme 1. Reagents: (i) MeSO2Cl/Et3N; (ii) NaN3/DMF; (iii) Pd/C/H2; (iv) MeSO3H/Nal, CH2Cl2, rt, 5 h; (v) CH3COCH3/H2O, BaCO3, rt, 30min;
(vi) NaN3/CF3COOH.
against all the six cancer cell lines. In the same series a
methyl substituent in the aryl ring at the 400-position
slightly reduces the activity. However, the 40-O-deme-
thyl analogue of compound (20) drastically reduces the
activity in almost all the cancer cell lines. The analogues
of 4b-nicotinylamido substitution (24–27) exhibit
promising cytotoxic activity. A number of these new
analogues possess comparable or superior in vitro anti-
cancer activity. In the literature mostly 40-O-demethyl
derivatives have been investigated for their anticancer
activity. Another interesting aspect observed in this
investigation is that a large number of analogues exhibit
improved cytotoxicity particularly in the presence of
40-O-methyl functionality (Table 1).
Conclusion
In conclusion, we can assert that the promising results
obtained for the new 4b-amido and 4b-sulphanamido
analogues of podophyllotoxin described in this study
make them potential candidates to take up further
synthesis and evaluation of such new analogues. This
investigation has also highlighted the importance of 40-
O-methyl functionality in such analogues for anticancer
activity. This will allow researcher in this area to pre-
pare podophyllotoxin related derivatives incorporating
such functionality. Some of the potential candidates are
undergoing detailed investigation particularly biochemical
studies.

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A. Kamal et al. / Bioorg. Med. Chem. 11 (2003) 5135–5142
Scheme 2. Reagents: (i) DCC, CH2Cl2, rt, 5 h; (ii) RCOCl, CH2Cl2, rt 5 h; (iii) CH2Cl2, rt, 5 h, Et3N.
Table 1. GI50 values of some of the 4b-amido and 4b-sulphonamido derivatives of podophyllotoxin
Compd
GI50 (mM)
DU145 (Prostate)
HT29 (Colon)
MCF7 (Breast)
MCF7ADR (Adr. res. breast)
NCIH460(Lung)
1.1
U251 (CNS)
6.4
Etoposidea
0.8
1.9
8.8
3.4
3.3
0.02
2.3
27.7
0.16
2.7
59
1.1
3.5
3.2
9.5
<0.004
1.7
28.9
0.03
1.8
4.3
5.4
4.3
8.2
10.0
0.03
5.01.3
46.2
0.05
3.5
116
2.64.03.0
1.3
2.05.2
15.5
0.01
4.3
11.2
0.59
1.9
14
16
18
19
20
21
22
24
25
26
3.8
2.8
0.3
0.02
2.7
4.7
3.9
0.01
30.8
0.08
3.7
25.0
0.02
3.3
0.05
<0.019
0.12
0.95
0.03
0.04
aValues from NCI database.
Experimental
spectra are recorded on CEC-21-100B, Finnigan Mat
1210or Micromass 7700spectrometers operating at
70ev using a direct inlet system. Optical rotations are
measured on Jasco Dip 360digital polarimeter. Melting
points are determined on an electro thermal melting
point apparatus and are uncorrected. TLC is performed
NMR spectra are recorded on Varian Gemini 200 MHz
spectrometer, using TMS as an internal reference. IR
spectra are recorded on Perkin-Elmer model 683 or
1310spectrometers with sodium chloride optics. Mass

A. Kamal et al. / Bioorg. Med. Chem. 11 (2003) 5135–5142
5139
with E. Merck precoated silica gel plates (60F-254) with
iodine as a developing agent. Acme, India silica gel,
100–200 mesh for column chromatography is used.
4ꢀ-(400-Methylbenzophenone-200-formyl)aminopodophyllo-
toxin (14). This compound was prepared according to
the methods described earlier employing 4-methylbenzo-
phenone - 2 - carboxylic acid (120mg, 0.5 mmol), DCC
(103 mg, 0.5 mmol) and 4b-aminopodophyllotoxin
(206 mg, 0.5 mmol) to give the compound 14. Yield:
4ꢀ-Aminopodophyllotoxin (9)
25
To a solution of 4b-azidopodophyllotoxin (1.54 g,
3.53 mmol) in 80mL of ethyl acetate was added 300mg,
of 10% palladium on activated carbon. The mixture was
stirred overnight under hydrogen, the reaction mixture
was filtered, and the filtrate was evaporated. The crude
product was purified by column chromatography by
employing chloroform/ethyl acetate (2:1) as an eluent to
85%; mp 173–175 ꢀC; ½aꢁD À34.5; IR (CHCl3) 3360,
3290, 2900, 1745, 1690, 1590 cmÀ1; 1H NMR (CDCl3) d
2.9 (s, 3H), 2.8 (m, 2H), 3.8 (s, 3H), 3.9 (s, 6H), 4.1–4.4
(m, 2H), 4.8 (m, 2H), 5.9 (s, 2H), 6.0(s, 1H), 6.4 (s, 1H),
6.5 (s, 2H), 6.7 (d, 1H, J=6.2 Hz), 7.1 (m, 2H), 7.3 (m,
2H), 7.4 (m, 1H), 7.5 (m, 2H), 7.7 (d, 2H), 7.8 (d, 1H,
J=3.8 Hz); MS 658 (M+sodium salt), 635 (M+), 604,
552, 524, 496, 468, 439, 397, 369, 339. Anal.
(C37H33NO9) calcd C: 69.91, H: 5.23, N: 2.20; found C:
69.73, H: 5.17, N: 2.11.
25
give compound 9. Yield 75%; mp 120–124 ꢀC; ½aꢁD
À62.0; IR (CHCl3) 3400, 2900, 1770, 1500, 1480,
1410cm À1; 1H NMR (CDCl3) d 2.8 (m, 1H), 3.2 (m, 1H),
3.8 (s, 9H), 4.3 (m, 2H), 4.6 (d, 1H, J=2.63 Hz), 4.8(d,
1H, J=7.8 Hz), 6.00 (d, 2H, J=5.2 Hz), 6.2 (s, 2H), 6.5
(s, 1H), 6.8 (s, 1H); MS 413 (M+), 396, 229, 185, 168.
4ꢀ-(400 -Methylbenzophenone-200 -formyl)amino-40 -O-de-
methylepipodophyllotoxin (15). This compound was pre-
pared according to the methods described earlier
employing 4-methylbenzophenone-2-carboxylic acid
(120 mg, 0.5 mmol), DCC (103 mg, 0.5 mmol) and 4b-
amino-40 -O-demethylepipodophyllotoxin (200 mg, 0.5
mmol) to give the compound 15. Yield 85%; mp 184–
4ꢀ-Amino-40-O-demthylepipodophyllotoxin (13). To a
solution of 4b-azido-40-O-demethyl-epipodophyllotoxin
(1.5 g, 3.53 mmol) in 80mL of ethyl acetate was added
300 mg, of 10% palladium on activated carbon. The
mixture was stirred overnight under hydrogen. The
reaction mixture was filtered and the filtrate was eva-
porated. The crude product was purified by column
chromatography by employing chloroform/ethyl acetate
(2:1) as an eluent to give compound 13. Yield 70%; mp
25
186 ꢀC; ½aꢁD À165.0; IR (CHCl3) 3360, 3190, 2920,
1
1750, 1590 cmÀ1; H NMR (CDCl3) d 2.4 (s, 3H), 3.2
(m, 2H), 3.8 (m, 2H), 3.9 (s, 6H), 4.1 (m, 2H), 4.4 (m,
2H), 6.0(d, 2H, J=3.5 Hz), 6.1 (s, 1H), 6.4 (s, 1H), 6.5
(s, 1H), 6.7 (m, 2H), 7.2 (m, 4H), 7.4 (m, 3H), 7.6 (m,
1H), 7.8 (d, 1H, J=5.1 Hz); MS 622 (M+1), 468, 383,
346, 325. Anal. (C36H31NO9) calcd C: 69.55, H: 5.02, N:
2.25; found C: 69.32, H: 5.05, N: 2.18.
25
134–138 ꢀC; ½aꢁD À63.0; IR (CHCl3) 3520, 3350, 2900,
1
1770, 1500, 1480, 1410 cmÀ1; H NMR (CDCl3) d 2.8
(m, 1H), 3.2 (m, 1H), 3.8 (s, 6H), 4.3 (m, 2H), 4.6 (d,
1H, J=8.0Hz), 4.8 (d, 1H, J=6.0Hz), 5.3 (s, 1H), 6.00
(d, 2H, J=6.1 Hz), 6.2 (s, 2H), 6.5 (s, 1H), 6.8 (s, 1H);
MS 400 (M+), 387, 229, 185, 168.
4ꢀ-(300-Chloro-400-methylbenzophenone-200-formyl)amino-
podophyllotoxin (16). This compound was prepared
according to the methods described earlier employing 3-
chloro-4-methylbenzophenone-2-carboxylic acid (137 mg,
0.5 mmol), DCC (103 mg, 0.5 mmol) and 4b-
aminopodophyllotoxin (206 mg, 0.5 mmol) to give the
General procedure for compounds 14–27
Method A. 4b-Aminopodophyllotoxin or 4b-amino-40-
O-demthylepipodophyllotoxin (0.5 m mol) was dis-
solved in 20mL of dried dichloromethane, followed by
addition of appropriate carboxylic acid (0.5 mmol) and
DCC (0.5 mmol). The reaction mixture was stirred at
room temperature for 5 h. Two drops of acetic acid was
added. The reaction mixture was filtered. The filtrate
was washed with saturated solution of NaHCO3, 10 %
hydrochloric acid and water respectively, dried over
anhydrous Na2SO4 and chromatographed through
silica gel using ethyl acetate/hexane (3:7) as an eluent to
obtain the pure products.
25
product 16. Yield 50%; mp 156–159 ꢀC; ½aꢁD À63.46; IR
1
(CHCl3): 3300, 3260, 2900, 1725, 1670, 1580 cmÀ1. H
NMR (CDCl3) d 2.4 (s, 3H), 2.8 (m, 2H), 3.8 (s, 9H),
3.9–4.2 (m, 2H), 4.5–4.6 (m, 2H), 5.8 (d, 1H, J=5.4 Hz),
5.9 (s, 2H), 6.2 (s, 2H), 6.5 (s, 1H), 6.7 (s, 1H), 7.0(m,
1H), 7.3 (m, 1H), 7.5 (m, 1H), 7.7 (s, 1H), 7.8 (s, 1H); MS
671 (M+1), 578, 551, 397. Anal. (C37H32ClNO9) calcd C:
66.31, H: 4.81, N: 2.09; found C: 66.24, H: 4.76, N: 2.06.
4ꢀ-(300-Chloro-400-methylbenzophenone-200-formyl)amino-
40-O-demethylepipodo-phyllotoxin (17). This compound
was prepared according to the methods described earlier
employing 3-chloro-4-methylbenzophenone-2-carboxylic
acid (137 mg, 0.5 mmol), DCC (103 mg, 0.5 m mol) and
Method B. To a solution containing 4b-aminopodo-
phyllotoxin or 4b-amino-40-O-demthylepipodophyllo-
toxin (0.5 mmol) triethylamine (1 mL) in 20 mL of
dichloromethane, appropriate acid chloride or sulpho-
nyl chloride (0.05 mmol) in 10 mL of dichloromethane
was added under nitrogen and stirred at room tem-
perature for about 5 h till the completion of the reaction
as monitored by TLC. The reaction mixture was washed
with water, extracted with ethyl acetate, dried over
anhydrous sodium sulphate and subjected to column
chromatography using ethyl acetate/hexane (3:7) as an
eluent to afford pure products.
4b-amino-40-O-demethylepipodophyllotoxin
(200 mg,
0.5 mmol) to give the compound 17. Yield in 80%; mp
25
177–179 ꢀC; ½aꢁD À26.25; IR (CHCl3) 3300, 3280, 2900,
1
1745, 1690, 1590 cmÀ1; H NMR (CDCl3) d 2.4 (s, 3H),
2.8 (m, 2H), 3.8 (s, 6H), 4.2 (m, 2H), 4.5 (m, 1H), 5.3 (br,
1H), 5.9 (d, 2H, J=2.5 Hz), 6.2 (s, 2H), 6.5 (s, 1H), 6.7
(s, 1H), 7.1 (d, 1H, J=5.6 Hz), 7.4 (m, 2H), 7.5 (m, 3H),
7.8 (m, 2H), 8.2 (d, 1H, J=4.2 Hz); MS 656 (M+1), 603,
577, 551, 523, 412, 397. Anal. (C36H30ClNO9) calcd C:
65.90, H: 4.60, N: 2.13; found C: 65.75, H: 4.62, N: 2.09.

5140
A. Kamal et al. / Bioorg. Med. Chem. 11 (2003) 5135–5142
4ꢀ-(400-Chlorobenzophenone-200-formyl)aminopodophyllo-
toxin (18). This compound was prepared according to
the methods described earlier employing 4-chloro-
benzophenone-2-carboxylic acid (130mg, 0.5 mmol),
DCC (103 mg, 0.5 mmol) and 4b-aminopodophyllotoxin
(206 mg, 0.5 mmol) to give the compound 18. Yield
demethylepipodophyllotoxin (200 mg, 0.5 mmol) to
give the compound 22. Yield 75%; mp 1763–165 ꢀC;
25
½aꢁD À207.5. IR (CHCl3) 3380, 3260, 2890, 1750,
1
1580cm À1; H NMR (CDCl3) d 2.8 (m, 1H), 3.2 (m,
1H), 3.7 (s, 6H), 4.0(m, 2H), 4.4 (m, 1H), 4.7 (m, 1H),
5.9 (d, 2H, J=5.2 Hz), 6.0(s, 1H), 6.2 (s, 2H), 6.3 (s,
1H), 6.4 (s, 1H), 7.6 (m, 3H), 7.9 (d, 2H, J=4.3 Hz), 8.1
(s, 1H); MS 539, 397. Anal. (C27H25NO9S) calcd C:
60.10, H: 4.67, N: 2.59; found C: 60.05, H: 4.64, N: 2.48.
25
75%; mp 153–156 ꢀC; ½aꢁD À19.25; IR (CHCl3) 330 0 ,
3250, 2890, 1750, 1680, 1590 cmÀ1; 1H NMR (CDCl3) d
3.3 (m, 3H), 3.5 (m, 1H), 3.7 (s, 3H), 3.8 (s, 6H), 4.1 (m,
2H), 4.5 (m, 1H), 5.6 (d, 1H), 5.9 (d, 2H, J=3.7 Hz), 6.
5 (s, 1H), 6.7 (s, 2H), 7.3 (t, 3H), 7.4 (m, 1H), 7.5 (m,
3H), 7.7 (m, 1H), 7.8 (d, 1H, J=5.2 Hz); MS 656, 603,
577,551, 523, 412, 397. Anal. (C36H30ClNO9) calcd C:
65.90, H: 4.60, N: 2.13; found C: 65.73, H: 4.55, N: 2.10.
4ꢀ-(p-Toluene sulphonyl)amino-40-O-demethylepipodo-
phyllotoxin (23). This compound was prepared accord-
ing to the method B described earlier employing
p-toulene sulphonyl chloride (95mg, 0.5 mmol) and
4b-amino-40-O-demethylepipodophyllotoxin (200mg, 0.5
mmol) to give the compound 23. Yield 80%; mp 154–
4ꢀ-(400-Chlorobenzophenone-200-formyl)amino-40-O-deme-
thylepipodophyllotoxin (19). This compound was pre-
pared according to the methods described earlier
employing 4-chlorobenzophenone-2-carboxylic acid
(130 mg, 0.5 mmol), DCC (103 mg, 0.5 mmol) and 4b-
amino-40 -O-demethylepipodophyllotoxin (200 mg, 0.5
mmol) to give the compound 19. Yield 85%; mp 182–
25
156 ꢀC; ½aꢁD À55.0; IR (CHCl3) 3400, 3290, 2860, 1750,
1
1580cm À1; H NMR (CDCl3) d 2.5 (s, 3H), 2.8–3.0(m,
2H), 3.8 (s, 6H), 4.3 (m, 2H), 4.4 (m, 2H), 5.6 (s, 1H),
5.9 (d, 2H, J=4.4 Hz), 6.2 (s, 2H), 6.4 (s, 1H), 7.3 (s,
1H), 7.4 (m, 2H), 7.8 (m, 3H), 8.1 (d, 1H, J=3.8 Hz).
MS 553, 397. Anal. (C28H27NO9S) calcd C: 60.75, H:
4.91, N: 2.53; found C: 60.64, H: 4.88, N: 2.51.
25
184 ꢀC; ½aꢁD À72.0; IR (CHCl3) 3380, 3250, 2900, 1735,
1
1690, 1580 cmÀ1; H NMR (CDCl3) d 2.8 (m, 1H), 3.4
(m, 1H), 3.8 (s, 6H), 4.2 (m, 1H), 4.6 (m, 1H), 5.2 (m,
1H), 5.3 (m, 1H), 5.8 (m, 1H), 6.0(d, 2H, J=2 Hz), 6.3
(s, 2H), 6.5 (s, 1H), 6.7 (s, 1H), 7.1 (m, 1H), 7.3 (m, 1H),
7.4 (m, 2H), 7.5 (m, 1H), 7.6 (m, 2H), 7.7 (m, 1H), 7.8
(d, 1H, J=5.2 Hz); MS 642, 484, 396, 382, 369, 337.
Anal. (C35H28ClNO9) calcd C: 65.47, H: 4.39, N: 2.18;
found C: 65.35, H: 4.32, N: 2.15.
4ꢀ-(200 -Chloropyridine-300 -formyl)aminopodophyllotoxin
(24). This compound was prepared according to
method A described earlier employing 2-chloronicotinic
acid (78 mg, 0.5 mmol), DCC (103 mg, 0.5 mmol) and
4b-aminopodophyllotoxin (206 mg, 0.5 mmol) to give the
25
compound 24. Yield 50%; mp 166–168 ꢀC; ½aꢁD À136.53;
IR (CHCl3) 3300, 3250, 2870, 1735, 1590 cmÀ1; 1H NMR
(CDCl3) d 2.8–3.0(m, 2H), 3.7 (s, 9H), 4.0–4.2 (t, 1H),
4.4–4.6 (m, 2H), 5.9 (d, 2H, J=5.5 Hz), 6.3 (s, 2H), 6.5 (s,
1H), 6.9 (s, 1H), 7.3 (m, 1H), 8.2 (m, 1H), 8.2 (d, 1H,
J=11.1 Hz), 8.4 (m, 1H); MS 552, 523, 496, 467, 397.
Anal. (C28H25ClN2O8) calcd C: 60.81, H: 4.55, N: 5.06;
found C: 60.74, H: 4.41, N: 5.01.
4ꢀ-(Benzene sulphonyl)aminopodophyllotoxin (20). This
compound was prepared according to method B descri-
bed earlier employing benzene sulphonyl chloride
(0.105 mL, 0.5 m mol) and 4b-aminopodophyllotoxin
(206 mg, 0.5 mmol) to give the compound 20. Yield
25
80%; mp 233–235 ꢀC; ½aꢁD À76.0; IR (CHCl3) 3350 ,
3200, 2860, 1745, 1560 cmÀ1; H NMR (CDCl3) d 2.9
4ꢀ-(200-Chloropyridine-300-formyl)amino-40-O-demethyle-
pipodophyllotoxin (25). This compound was prepared
according to method A described earlier employing
2-chloronicotinic acid (78 mg, 0.5 mmol), DCC (103 mg,
0.5 mmol) and 4b-amino-40-O-demethylepipodophyllo-
toxin (200 mg, 0.5 m mol) to give the compound 25. Yield
1
(m, 2H), 3.8 (s, 9H), 4.3–4.4 (m, 1H), 4.5 (m, 2H), 5.6 (s,
1H), 5.9 (d, 2H, J=2.5 Hz), 6.1 (s, 2H), 6.2 (s, 2H), 6.1
(s, 1H), 7.7 (m, 3H), 8.0(d, 2H, J=10.0 Hz); MS 553,
397. Anal. (C28H27NO9S) calcd C: 60.75, H: 4.91, N:
2.53; found C: 60.62, H: 4.90, N: 2.47.
25
45%; mp 180–182 ꢀC; ½aꢁD À23.5; IR (CHCl3) 3360,
1
4ꢀ-(p-Toulene sulphonyl)aminopodophyllotoxin (21).
This compound was prepared according to method B
described earlier employing p-toulene sulphonyl chlo-
ride (95 mg, 0.5 mmol) and 4b-aminopodophyllotoxin
(206 mg, 0.5 mmol) to give the compound 21. Yield
3280, 2890, 1745, 1580 cmÀ1; H NMR (CDCl3) d 2.9
(m, 1H), 3.1 (m, 1H), 3.8 (s, 6H), 4.0(m, 1H), 4.5–4.6
(m, 2H), 5.2 (br, 1H), 6.0(d, 2H, J=7.1 Hz), 6.3 (s, 2H),
6.6 (s, 1H), 6.8 (s, 1H), 7.0(m, 1H), 7.1 (m, 1H), 8.2
(m,1H), 8.5 (d,1H, J=4.7 Hz); MS 538, 503, 397, 367.
Anal. (C27H23ClN2O8) calcd C: 60.17, H: 4.30, N: 5.19;
found C: 60.12, H: 4.21, N: 5.09.
25
85%; mp 209–212 ꢀC; ½aꢁD À14.5; IR (CHCl3) 3390 ,
3280, 2890, 1735, 1580 cmÀ1; 1H NMR (CDCl3) d 2.5 (s,
3H), 2.8 (m, 1H), 3.0(m, 1H), 3.7 (s, 6H), 3.8 (s, 3H),
4.3 (m, 2H), 4.4 (m, 1H), 4.5 (m, 1H), 4.9 (d, 1H,
J=5.7 Hz), 5.7 (s, 1H), 5.9 (s, 2H), 6.2 (s, 2H), 6.4 (s,
1H), 7.4 (d, 2H, J=4.5 Hz), 7.8 (d, 2H, J=4.0Hz); MS
567(M+), 397, 229. Anal. (C29H29NO9S) calcd C: 61.36,
H: 5.14, N: 2.46; found C: 61.29, H: 5.09, N: 2.40.
4ꢀ-(600 -Chloropyridine-300 -formyl)aminopodophyllotoxin
(26). This compound was prepared according to the
methods described earlier employing 2-chloronicotinic
acid (78 mg, 0.5 mmol), DCC (103 mg, 0.5 mmol) and
4b-aminopodophyllotoxin (206 mg, 0.5 mmol) to give the
25
compound 26. Yield 45%; mp 173–174 ꢀC; ½aꢁD À163.2;
4ꢀ-(Benzene sulphonyl)amino-40-O-demethylepipodophyl-
lotoxin (22). This compound was prepared according to
method B described earlier employing benzene sulpho-
nyl chloride (0.105 mL, 0.5 mmol) and 4b-amino-40-O-
IR (CHCl3) 3360, 3290, 2890, 1750, 1570 cmÀ1 1H
;
NMR (CDCl3) d 2.8–3.2 (m, 1H), 3.8 (s, 9H), 4.0–4.2
(m, 2H), 4.4–4.4 (m, 2H), 5.9 (d, 2H, J=5.2 Hz), 6.3 (s,
1H), 6.5 (s, 2H), 6.9 (s, 1H), 7.0(m, 1H), 7.4 (m, 1H),

A. Kamal et al. / Bioorg. Med. Chem. 11 (2003) 5135–5142
5141
8.3 (m,1H), 8.8 (m, 1H), 8.9 (m, 1H); MS 552, 523, 496,
467, 397. Anal. (C28H25ClN2O8) calcd C: 60.81, H: 4.55,
N: 5.06; found C: 60.69, H: 4.53, N: 5.01.
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4ꢀ-(600-Chloropyridine-300-formyl)amino-40-O-demethyle-
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6-chloronicotinic acid (78 mg, 0.5 mmol), DCC (103 mg,
0.5 mmol) and 4b-amino-40-O-demethylepipodophyllo-
toxin (200 mg, 0.5 mmol) to give the compound 27.
25
Yield: 40%; mp 185–187 ꢀC; ½aꢁD : À25.25; IR (CHCl3):
3360, 3280, 2910, 1750, 1590 cmÀ1; 1H NMR (CDCl3) d
2.9–3.1 (m, 1H), 3.2–3.4 (m, 1H), 3.8 (s, 6H), 3.9–4.0(m,
2H), 4.3–4.6 (m, 2H), 5.4 (br, 1H), 5.9 (d, 2H,
J=5.4 Hz), 6.2 (d, 2H, J=2.7 Hz), 6.5 (s, 1H), 6.8 (s,
1H), 7.1 (m, 1H), 7.4 (d, 1H, J=10.8 Hz), 8.2 (d, 1H,
J=16.2 Hz), 8.8 (d, 1H, J=3.2 Hz); MS 540(M +2), 538
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Biological evaluation
In vitro evaluation of cytotoxic activity. In routine
screening, each agent is tested over a broad concen-
tration range (10-fold dilutions starting from >100 mM
to ꢂ10nM) against six human cancer cell lines com-
prised of different tumour types. Standard compound
doxorubicin is tested in each assay as a positive control.
The cells are maintained in growing condition in RPMI
1640medium containing 1%0 fetal calf serum and
incubated at 37 ꢀC under 5% CO2 atmosphere. All cell
lines are inoculated onto a series of standard 96-well
microtitre plate on day zero, followed by 24-h incu-
bation in the absence of test compound. The inoculation
densities used in this screen are as per the procedure of
Monks et al.18 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 incubated for 48 h. After incubation the assay is
terminated by adding 50 mL of trichloro acetic acid
(TCA) and incubating at 4 ꢀC for 30min. The pre-
cipitated cells are washed and stained with sulphorho-
damine B dye for 30min and the excess dye is washed
off with acetic acid. Adsorbed dye is solublised in Tris
base (alkaline pH) and quantitated by measuring the
OD at 490nm in an ELISA reader. GI 50 (concentration
that inhibits the cell growth by 50%) is calculated
according to the method of Boyd.19
Acknowledgements
13. Pan, J. L.; Wang, Y. G.; Chen, Y. Z. Curr. Sci. 1997, 72,
268.
14. Zhou, X. M.; Wang, Z.; Chen, H. X.; Cheng, Y. C.; Lee,
K. H. Pharm. Res. 1993, 10, 214.
15. (a) Kamal, A.; Gayatri, N. L. Tetrahedron Lett. 1996, 37,
3359. (b) Kamal, A.; Laxminarayana, B.; Gayatri, N. L. Tet-
rahedron Lett. 1997, 38, 6871. (c) Kamal, A.; Gayatri, N. L.;
Rao, N. V. Bioorg. Med. Chem. Lett. 1998, 8, 3097. (d)
Kamal, A.; Damyanthi, Y. Bioorg. Med. Chem. Lett. 1997, 7,
657. (e) Gayatri, N. L. PhD Thesis, Osmania University, 1999.
(f) Laxman, E. L. PhD Thesis, Osmania University, 2000. (g)
Kamal, A.; Laxman, E.; Arifuddin, M.; Khanna, G. B. R. US
This work was financially supported by Department of
Science and Technology, India under the drugs and
pharmaceutical research programme. One of the
author’s, B.A.K., is thankful to CSIR, New Delhi for
the award of research fellowship.
References and Notes
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