Facile and efficient one-pot synthesis of 4β-arylamino-podophyllotoxins: Synthesis of DNA topoisomerase II inhibitors (NPF and W-68)

Article abstract of DOI:10.1016/S0960-894X(00)00407-8

A series of 4β-arylamino-4'-O-demethylepipodophyllotoxins and 4β-arylaminoepipodophyllotoxins have been synthesized with significant stereoselectivity and improved yields by employing the methanesulphonic acid/sodium iodide reagent system. Compounds NPF, W-68 and other DNA topoisomerase II inhibitors are prepared in good to excellent yields by this method and these are active or more active than etoposide in their inhibition of the human DNA topoisomerase II. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0960-894X(00)00407-8

Bioorganic & Medicinal Chemistry Letters 10 (2000) 2059±2062
Facile and Ecient One-Pot Synthesis of 4ꢀ-Arylamino-
podophyllotoxins: Synthesis of DNA Topoisomerase II
Inhibitors (NPF and W-68)y
Ahmed Kamal,* N. Laxman and G. Ramesh
Division of Organic Chemistry, Indian Institute of Chemical Technology, Hyderabad-500 007, India
Received 6 March 2000; accepted 27 June 2000
AbstractÐA series of 4b-arylamino-40-O-demethylepipodophyllotoxins and 4b-arylaminoepipodophyllotoxins have been synthe-
sized with signi®cant stereoselectivity and improved yields by employing the methanesulphonic acid/sodium iodide reagent system.
Compounds NPF, W-68 and other DNA topoisomerase II inhibitors are prepared in good to excellent yields by this method and
these are active or more active than etoposide in their inhibition of the human DNA topoisomerase II. # 2000 Elsevier Science Ltd.
All rights reserved.
Etoposide and teniposide are semisynthetic drugs
derived from podophyllotoxin and are in clinical use for
the treatment of many cancers, particularly small cell
lung cancer and testicular cancer.1 These compounds
block the catalytic activity of DNA topoisomerase II
and concurrent enzyme-mediated production of lethal
DNA strands leading to DNA damage and cytotoxi-
city.2 Etoposide is widely used in clinical cancer therapy
and this has stimulated a renewed interest by the research-
ers in the chemical and biological studies of podophyllo-
toxin derived antitumour agents.3 7 The recent synthetic
studies on podophyllotoxin have been focussed on the
synthesis of the C-4 non-sugar substituted analogues
which show improved topoisomerase II inhibition and
cytotoxicity. In this endeavour, Lee and co-workers8
have synthesized a large number of 4b-anilino derivatives
of podophyllotoxin, in particular, they have identi®ed
40-O-demethyl-4b-(400-¯uoroanilino)-4-desoxypodophyl±
lotoxin (NPF) and 40-O-demethyl-4b-(400-nitroanilino)-4-
desoxypodophyllotoxin (W-68) as potential derivatives.
This new anticancer agent NPF has been found to
exhibit 10-fold more potent topoisomerase II inhibition
and 100-fold more cytotoxic activity against various
human tumour cells and etoposide-KB resistant cells.
The synthetic route for the 4b-anilino substituted 40-O-
demethylepipodophyllotoxin compounds reported8 in
the literature comprises of three steps, i.e. C-4 epimeriza-
tion, C-4 bromination (modi®ed Kuhn's method) and
nucleophilic displacement with anilino compounds giving
the overall yields ranging from 14 to 30%. In our recent
studies,9 we have improved the selectivity and yields in
yIICT Communication No. 4517.
*Corresponding author. Tel.: +91-40-717-3874 ext. 2638; fax: +91-40-717-3387; e-mail: ahmedkamal@iict.ap.nic.in
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00407-8

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A. Kamal et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2059±2062
the last step of this nucleophilic displacement by the
addition of tetrabutylammonium iodide (Bu4N+I )
through the dynamic kinetic resolution process. Recently,
a one-pot synthesis of NPF has been reported10
employing trimethylsilyl iodide (TMSI), wherein 40-O-
demethylation and 4b-iodo displacement with p-¯uoro-
aniline takes place with improved yield. There are some
synthetic routes reported11 in the literature and as well
as developed by us12 14 for the preparation of 4b-
aminopodophyllotoxin and 4b-amino-40-O-demethyl-
epipodophyllotoxin as important precursors towards
the synthesis of newer podophyllotoxin congeners.15,16
iodide as a reagent system for the conversion of 1 to 2 is
a practical and useful procedure.
Interestingly, when this reaction has been carried out in
MeCN as a solvent, 40-O-demethylation (3) product is
not observed for 12 h even under re¯ux conditions by
using 3 mmol of the above reagent system, whereas this
reaction in chlorinated solvents like CH2Cl2 or (CH2)2Cl2
in 5 h at room temperature and in 2 h at re¯ux condi-
tions proceeds to 40-O-demethylation (2). Therefore, by
using these two di€erent type of solvents, the selectivity
of 40-O-demethylation could be manoeuvred by
employing this methodology.
In this paper, we wish to report the synthesis of 4b-
arylaminopodophyllotoxin derivatives employing sul-
phonic acid/sodium iodide as an ecient reagent system.
This reagent has not been explored in the literature
except for the deoxygenation of sulphoxides to the cor-
responding sulphides.17 During a project directed
towards the synthesis of new podophyllotoxin con-
geners, we investigated the methanesulphonic acid and
p-toluenesulphonic acid/sodium iodide system for iodi-
nation of benzylic alcohol and simultaneously attacking
the hindered methoxy group in a regiospeci®c fashion in
the podophyllotoxin ring system. This observation
clearly indicates that hydrogen iodide is formed in situ
from methanesulphonic acid/sodium iodide. With these
considerations in mind, this reagent system has been
considered for its potential application for both the
aspects of 40-O-demethylation and C-4 epimerization in
the podophyllotoxin system. Thus, treatment of podo-
phyllotoxin 1 by the above reagent system in CH2Cl2 fol-
lowed by weak basic hydrolysis (water:acetone, BaCO3)
gave 40-O-demethylepipodophyllotoxin 2 (yield 65%).
In comparison to theꢀliterature methods, i.e., reaction of
1 with HBr gas at 0 C followed by hydrolysis18,19 and
treatment with trimethylsilyl iodide (TMSI),10 the present
route employing the methanesulphonic acid/sodium
Further, reaction of podophyllotoxin with this reagent
system followed by the addition of arylamines in pres-
ence of bases like BaCO3 or K2CO3 or CsCO3 gave the
4b-arylaminopodophyllotoxin analogues. As a result,
employing CH2Cl2 as solvent produced 40-O-demethyl-
ation and C-4 epimerization to give 4b-arylamino-40-O-
demethylpodophyllotoxin20 4a±4f (40±55% yields),
whereas the use of MeCN as a solvent produced C-4
epimerization alone without 40-O-demethylation to
a€ord the corresponding 4b-arylaminopodophyllotoxin
analogues21 4g±4i (80±85% yields). This may be attrib-
uted to the facile generation of hydrogen iodide in
chlorinated solvents as in the case of TMSI, although its
role is not clear.22 However, the use of p-toluenesul-
phonic acid instead of methanesulphonic acid and also
the use of other bases like K2CO3 or CsCO3 in place of
BaCO3 for the preparation of some representative com-
pounds has not shown any noticeable e€ect on the
yields as well as on the rate of the reaction process.
In conclusion, we have developed a highly practical one-
pot procedure for the preparation of 4b-arylaminopodo-
phyllotoxin congeners. This methodology also demon-
strates the selectivity in 40-O-demethylation by
Scheme 1. Reagents and conditions: (i) MeSO3H/NaI, CH2Cl2, rt, 5 h; (ii) H2O/Me2CO, BaCO3, rt, 30 min; (iii) MeSO3H/NaI, MeCN, rt, 15 min.

A. Kamal et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2059±2062
2061
Scheme 2. Reagents and conditions: (i) MeSO3H/NaI, CH2Cl2, rt, 5 h; (ii) THF, RNH2, BaCO3, rt, 8 h; (iii) MeSO3H/NaI, MeCN, rt, 15 min.
employing the desired type of solvents. This manipula-
tion is useful to prepare selectively 40-O-demethyl or
epipodophyllotoxin analogues in good yields. Further-
more, this new methodology has been demonstrated for
the facile synthesis of DNA topoisomerase II inhibitors
NPF and W-68, and it is extremely cost e€ective, and
this one-pot process could assist other researchers in the
preparation of important analogues of this class of
podophyllotoxin congeners.
15. Gayatri, N. L. PhD Thesis, 1999, submitted to Osmania
University.
16. Yu, Y.-P.; Chen, S.-Y.; Wang, Y. G.; Chen, Y.-Z. Tetra-
hedron Lett. 1999, 40, 1967.
17. Drabowicz, J.; Dudzinski, B.; Mikolajczyk, M. Synlett.
1992, 252.
18. Kuhn, M.; von Wartburg, A. Helv. Chim. Acta 1969, 52,
948.
19. Thurston, L. S.; Imakura, Y.; Haruna, M.; Li, D.-H.; Liu,
Z.-C.; Liu, S.-Y.; Cheng, Y.-C.; Lee, K.-H. J. Med. Chem.
1989, 32, 604.
20. General procedure for the synthesis of compounds (4a±f).
To a solution of podophyllotoxin 1 (414 mg, 1 mmol) in dry
CH2Cl2 (10 mL), NaI (447 mg, 3 mmol) was added and stirred
for 5 min. To this stirred suspension, MeSO3H (288 mg,
3 mmol) was added dropwise with syringe at 0 ꢀC and the
stirring was continued for another 5 h at room temperature.
Nitrogen was bubbled through the solution to drive o€ the
excess hydrogen iodide. This solution was then evaporated in
vacuo and used for the next reaction without further puri®ca-
tion. To the above crude product, anhydrous BaCO3 (395 mg,
2 mmol) and the appropriate arylamine (1.2 mmol) in 10 mL of
dry THF under nitrogen were added and stirred for 8 h at
room temperature. The reaction mixture was ®ltered, diluted
with ethylacetate and washed with water, 10% Na2S2O4 solu-
tion, dried, and puri®ed via column chromatography (40 g of
silica gel with ethyl acetate/hexane as eluent).
Acknowledgements
We are thankful to DST, New Delhi for ®nancial sup-
port under the Drugs and Pharmaceutical Research
Programme and one of the authors (NL) is thankful to
CSIR, New Delhi for the award of a Senior Research
Fellowship.
References and Notes
1. Stahelin, H.; von Wartburg, A. Cancer Res. 1991, 51, 5.
2. Macdonald, T. L.; Lehnert, E. K.; Loper, J. T.; Chow, K.
C.; Ross, W. E. In DNA Topoisomerase in Cancer; Potmesil,
M., Kohn, K. W., Eds.; Oxford University Press: New York,
1991; pp 119 and references cited therein.
3. Leteurte, F.; Madalengoita, J.; Orr, A.; Cuzi, T. J.; Leh-
nert, E.; Macdonald, T.; Pommier, Y. Cancer Res. 1992, 52,
4478.
4. Ward, R. S. Synthesis 1992, 719 and references therein.
5. Sauliner, M. G.; LeBoulluec, K. L.; McGee, D. P. C.;
Long, B. H.; Crosswell, A. R.; Vyas, D. M. Bioorg. Med.
Chem. Lett. 1993, 3, 1619.
6. Watt, P. M.; Hickson, I. D. Biochem. J. 1994, 303, 681 and
references therein.
40 - Demethyl - 4ꢀ - (40 - ¯uoroanilino) - 4 - desoxypodophyllotoxin
(NPF) (4a). Yield 55%; mp 175±177 ꢀC; [a]D30= 102ꢀ (c=1,
CHCl3); IR (KBr) 3504 (OH), 3388 (NH), 1772 (lactone),
1619, 1513 and 1473 (aromatic CˆC) cm 1; 1H NMR (CDCl3)
d 6.90 (t, J=6.90 Hz, 2H, 300, 500-H), 6.75 (s, 1H, 5-H), 6.53 (s,
1 H, 8-H), 6.49 (q, J=2.5, 6.7 Hz, 2H, 200, 600-H), 6.29 (s, 2H,
20, 60-H), 5.95 (d, J=4.6 Hz, 2H, OCH2O), 5.36 (s, 1H,
exchangeable, 40-OH), 4.60 (d, 2H, 4-H and 1-H), 4.36 (t,
J=8.0 Hz, 1H, 11-H), 3.99 (t, J=8.0 Hz, 1H, 11-H), 3.88 (br,
1H, exchangeable NH), 3.80 (s, 6H, 30, 50-OCH3), 3.15 (dd,
J=4.8, 13.7Hz, 1H, 2-H), 3.05 (m, 1H, 3-H); FAB MS: 493
(M+H)+; FABHRMS calcd for [C27H24NO7F+H]+ 493.4846,
found 493.1536.
21. General procedure for the synthesis of compounds (4g±i).
To a solution of podophyllotoxin 1 (414 mg, 1 mmol) in dry
MeCN (10 mL), NaI (298 mg, 2 mmol) was added and stirred
for 5 min. To this stirred suspension, MeSO3H (192 mg,
2 mmol) was added dropwise with a syringe at 0 ꢀC and the
stirring was continued for another 15 min at room tempera-
ture. Nitrogen was bubbled through the solution to drive o€
the excess hydrogen iodide. This solution was then evaporated
in vacuo and used for the next reaction without further puri®ca-
tion. To the above crude product, anhydrous BaCO3 (395 mg,
2 mmol), and the appropriate arylamine (1.2mmol) in 10 mL of
dry THF under nitrogen were added and stirred for 8 h at room
temperature. The reaction mixture was ®ltered, diluted with
7. Hitosuyanagi, Y.; Kobayashi, M.; Takeya, K.; Itokawa, H.
J. Chem. Soc., Perkin Trans. 1 1995, 1387.
8. Tawa, R.; Takami, M. J.; Imakura, Y.-J.; Lee, K.-H.;
Sakuri, H. Bioorg. Med. Chem. Lett. 1997, 7, 489.
9. Kamal, A.; Gayatri, N. L. Tetrahedron Lett. 1996, 37, 3359.
10. Daley, L.; Meresse, P.; Bertounesque, E.; Monneret, C.
Tetrahedron Lett. 1997, 38, 2673.
11. Lee, K.-H.; Imakura, Y.; Haruna, M.; Beers, S. A.;
Thurston, L. S.; Dai, H. J.; Chen, C.-H.; Liu, S.-Y.; Cheng,
Y.-C. J. Nat. Prod. 1989, 52, 606.
12. Kamal, A.; Damayanthi, Y. Bioorg. Med. Chem. Lett.
1997, 7, 657.
13. Kamal, A.; Laxminarayana, B.; Gayatri, N. L. Tetra-
hedron Lett. 1997, 38, 6871.
14. Kamal, A.; Gayatri, N. L.; Venugopal Rao, N. Bioorg.
Med. Chem. Lett. 1998, 8, 3097.

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A. Kamal et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2059±2062
ethyl acetate and washed with water, 10% Na2S2O4 solution,
dried, and puri®ed via column chromatography (40 g of silica
gel with ethyl acetate/hexane as eluent).
6.75 (s, 1H, 5-H), 6.53 (s, 1H, 8-H), 6.49 (q, J=2.5, 6.7 Hz, 2H,
200, 600-H), 6.29 (s, 2H, 20, 60-H), 5.95 (d, J=4.7 Hz, 2H, OCH2O),
4.60 (d, 2H, 4-H and 1-H), 4.36 (t, J=8.0 Hz, 1H, 11-H), 3.99 (t,
J=8.0 Hz, 1H, 11-H), 3.88 (br, 1H, exchangeable NH), 3.80 (s,
9H, 30, 40, 50-OCH3), 3.15 (dd, J=4.8, 13.7>Hz, 1H, 2-H), 3.05
(m, 1H, 3-H); FAB MS: 507 (M+H)+; FABHRMS calcd for
[C28H26NO7F+H]+ 507.4814, found 507.1693.
4ꢀ-(400-¯uoroanilino)-4-desoxypodophyllotoxin (4g). Yield 85%;
mp 128±130 ꢀC; [a]D30= 78ꢀ (c=1, CHCl3); IR (KBr) 3388
(NH), 1772 (lactone), 1619, 1513 and 1473 (aromatic CˆC)
cm 1; H NMR (CDCl3) d 6.90 (t, J=6.90 Hz, 2H, 300, 500-H),
22. Kanai, O.; Irifune, S.; Ogawa, M. Synthesis 1989, 283.
1