Regioselective synthesis and in vitro anticancer activity of 4-aza-podophyllotoxin derivatives catalyzed by l -proline

Article abstract of DOI:10.1021/cc100003c

A series of 4-aza-podophyllotoxin derivatives have been synthesized regioselectively via the three-component reaction of aldehydes, aromatic amines, and tetronic acid catalyzed by l-proline. This method has the advantages of high yield, high regioselectiv

Full text of DOI:10.1021/cc100003c

430
J. Comb. Chem. 2010, 12, 430–434
Regioselective Synthesis and in Vitro Anticancer Activity of
4-Aza-podophyllotoxin Derivatives Catalyzed by L-Proline
Chunling Shi,† Juxian Wang,‡ Hui Chen,† and Daqing Shi*,†
Key Laboratory of Organic Synthesis of Jiangsu ProVince, College of Chemistry, Chemical Engineering
and Materials Science, Soochow UniVersity, Suzhou 215123, P. R. China, and Institute of Medicinal
Biotechnology, Chinese Academy of Medical Sciences and Perking Union Medical College,
Beijing 100050, P. R. China
ReceiVed January 11, 2010
A series of 4-aza-podophyllotoxin derivatives have been synthesized regioselectively via the three-component
reaction of aldehydes, aromatic amines, and tetronic acid catalyzed by L-proline. This method has the
advantages of high yield, high regioselectivity, extensive adaptability, easy operation, and environmental
friendliness. These compounds were also investigated in vitro, and some were found to have good anticancer
activity.
Introduction
Podophyllotoxin (Figure 1) is an antitumor lignan that
inhibits microtubule assembly.1 Because of mostly unsuc-
cessful attempts to use it for the treatment of human
neoplasia, extensive structural modifications have been
performed in order to obtain more potent and less toxic
anticancer agents.2 Among them, derivatives of 4-aza-
podophyllotoxin (Figure 1), reported as powerful DNA
topoisomerase II inhibitors, have recently attracted consider-
able interest. Takeya et al.3 reported the synthesis of 4-aza-
podophyllotoxin derivatives via three steps of condensation,
cyclization, and reduction. Tratrat et al.4 reported the one-
pot synthesis from an aldehyde, aniline, and tetronic acid in
refluxing ethanol. Tu et al.5 also reported a three-component
reaction for one-pot synthesis of 4-aza-podophyllotoxin
derivatives in water at higher temperature under microwave
irradiation conditions. However, these methods still suffer
from drawbacks such as needing rigorous conditions and a
special reaction instrument, and are limited to anilines.
Therefore, there is a need for milder and more efficient
methods for the one-pot synthesis of this type of compound.
Figure 1. Structure of podophyllotoxin and 4-aza-podophyllotoxin.
Scheme 1. Model Reaction
access to the construction of 4-aza-podophyllotoxin deriva-
tives using L-proline as catalyst.
Recently, organic reactions catalyzed by small organic
molecules have drawn attention. In particular, cinchona
alkaloids or L-proline and its derivatives have been used as
catalysts in various reactions with excellent yields.6–12
Recently, L-proline and its derivatives have been used in
multicomponent dissymmetric Biginelli13 and Hantzsch
reactions.14 We have previously reported the synthesis of
heterocycles using L-proline as catalyst.15 In continuing with
our earlier work, we would like to report herein efficient
Results and Discussion
In our initial study, the reaction of p-toluidine 1a,
4-bromobenzaldehyde 2a, and tetronic acid 3 (Scheme 1)
was chosen as a model reaction to optimize the reaction
conditions. The results are summarized in Table 1. The
reaction was first carried out in ethanol in the absence and
presence of several additives. It was found that only 35% of
the target compound 4a was obtained in the absence of
additive (Table 1, entry 1). Some proton acid (Table 1, entries
2-4), Lewis acid (Table 1, entries 5-10), and bases (Table
1, entries 11-12) can catalyze this reaction with moderate
yields. The best result was obtained when L-proline was used
according to the yield and the reaction time (Table 1, entry
13). So L-proline was chosen as the catalyst for this reaction.
* To whom correspondence should be addressed. Phone: +86-512-
65880049. Fax: +86-512-65880305. E-mail: dqshi@suda.edu.cn.
† Soochow University.
‡ Chinese Academy of Medical Sciences and Perking Union Medical
College.
10.1021/cc100003c  2010 American Chemical Society
Published on Web 05/26/2010

4-Aza-podophyllotoxin Derivatives
Journal of Combinatorial Chemistry, 2010 Vol. 12, No. 4 431
Table 1. Optimization of Catalysts, Solvents and Temperature
in the Synthesis of 4a
time
(h)
yielda
(%)
entry
additive (mol %)
solvent
T (°C)
1
2
3
4
5
6
7
8
none
HCl (10%)
H2SO4 (10%)
p-TsOH (10%)
CaCl2 (10%)
ethanol
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
b
40
60
65
100
150
120
70
4
35
51
49
47
49
65
46
60
76
70
35
43
93
90
92
93
c
58
68
92
36
83
90
79
ethanol
ethanol
ethanol
ethanol
ethanol
ethanol
ethanol
ethanol
ethanol
ethanol
ethanol
ethanol
ethanol
ethanol
ethanol
ethanol
ethanol
ethanol
methanol
water
1.5
1.5
2
2
FeCl3 · 6H2O (10%)
CoCl2 · 6H2O (10%)
AlCl3 (10%)
SnCl4 · 5H2O (10%)
SnCl2 · 2H2O (10%)
Et3N (10%)
pyridine (10%)
L-proline (10%)
L-proline (5%)
L-proline (15%)
L-proline (20%)
L-proline (10%)
L-proline (10%)
L-proline (10%)
L-proline (10%)
L-proline (10%)
L-proline (10%)
L-proline (10%)
L-proline (10%)
1.5
1.5
2
2.5
2.5
2
2
0.5
2
0.5
0.5
4
4
2
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
Figure 2. Possible structures of products 4t and 4v.
1.5
3
2.5
2.5
3
DMF
HOAc
acetonitrile
a Isolted yield. b Room temperature. c Trace.
Table 2. Synthesis of 4-Aza-podophyllotoxin Derivatives 4
Catalyzed by L-Proline
time yield
entry
Ar (1)
R2 (2)
4-BrC6H4
4-CH3C6H4
4-ClC6H4
4-NO2C6H4
3,4-Cl2C6H3
4-CH3C6H4
product
(h)
(%)
1
2
3
4
5
6
7
8
4-CH3C6H4
4-CH3C6H4
4-CH3C6H4
4-CH3C6H4
4-CH3C6H4
3-CH3C6H4
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
4q
4r
4s
0.5
0.5
0.5
0.5
1
0.5
0.15
0.2
0.15
0.2
0.2
0.5
0.5
1
0.5
0.5
1
0.5
0.5
1
93
92
89
91
93
91
95
92
94
93
92
93
91
92
94
92
91
93
91
93
92
94
92
91
Figure 3. The molecular structure of product 4t.
temperature, resulting in the isolation of 4a in a trace amount,
58%, 68%, and 93% yields (Table 1, entries 17-19 and 13),
respectively. Thus, 10 mol % L-proline and a reaction
temperature at reflux were optimal conditions. In addition,
methanol, water, DMF, HOAc, and CH3CN (Table 1, entries
20-24) were also tested as solvents. In these cases, product
4a was formed in slightly lower yields (Table 1, entries
20-24).
Under these appropriate reaction conditions (2 mL of
ethanol, 80 °C, 10 mol % L-proline), a series of 4-aza-
podophyllotoxin derivatives (4) were synthesized. The results
are summarized in Table 2.
3,4-OCH2OC6H3 3,4,5-(CH3O)3C6H2
3,4-OCH2OC6H3 4-FC6H4
9
3,4-OCH2OC6H3 4-CH3OC6H4
3,4-OCH2OC6H3 4-BrC6H4
3,4-OCH2OC6H3 4-ClC6H4
naphthalene-1-yl 4-CH3OC6H4
naphthalene-1-yl 4-BrC6H4
naphthalene-1-yl 4-ClC6H4
naphthalene-1-yl 3,4-OCH2OC6H3
naphthalene-1-yl 4-CH3C6H4
naphthalene-1-yl 2,4-Cl2C6H3
naphthalene-1-yl thiophen-2-yl
quinolin-6-yl
quinolin-6-yl
3-Cl-4-CH3C6H3 4-ClC6H4
3-Cl-4-CH3C6H3 4-BrC6H4
3-Cl-4-CH3C6H3 3,4-Cl2C6H3
3-Cl-4-CH3C6H3 4-NO2C6H4
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
4-CH3C6H4
4-ClC6H4
4t
4u
4v
4w
4x
2
2
3
2
As shown in Table 2, this protocol can be applied not only
to the aromatic aldehydes with either electron-withdrawing
groups (such as nitro and halide groups) or electron-donating
groups (such as hydroxyl and alkoxyl groups) but also to
heterocyclic aldehydes under the same conditions. Impor-
tantly, a wide range of aromatic amines including 3,4-
(methylenedioxy)aniline, p-toluidine, m-toluidine, 1-naph-
thylamine, quinolin-6-amine, and 3-chloro-4-methylaniline
were employed successfully in this reaction with excellent
results.
We also evaluated the amount of L-proline required for this
reaction. The results from Table 1 (entries 13-16) show that
10 mol % L-proline at reflux in ethanol is sufficient to initiate
the reaction. Higher loading of the catalyst had no significant
influence on the reaction yield. To find the optimum reaction
temperature, the reaction was carried out with 10 mol %
L-proline at room temperature, 40 °C, 60 °C, and reflux

432 Journal of Combinatorial Chemistry, 2010 Vol. 12, No. 4
Shi et al.
drance plays a critical role. It also can be explained from
the thermodynamic effect; the structures of 4t-1 and 4v-1
are more stable than 4t-2 and 4v-2, respectively.
1
All the products 4 were characterized by mp, IR, and H
NMR spectra, as well as HRMS.
Apart from the mild conditions of the process and its
excellent results, the simplicity of product isolation and the
possibility to recover and recycle the L-proline as catalyst
offer a significant advantage. Because L-proline is soluble
in the reaction medium (ethanol) and the desired products
are less soluble in ethanol, the products can be directly
separated by cooling to room temperature and filtering after
the reaction is completed. The filtrate containing L-proline
can directly be recovered and recycled. Studies using 1a,
2a, and 3 as model substrates showed that the recovered
reaction solution could be successively recycled in subse-
quent reactions without any decrease of yields (Table 3). It
is shown that after the reaction solution has been recycled
seven times, catalytic efficiency of L-proline did not change.
Based on the literature,4,17 the proposed mechanism for
the synthesis of 4-aza-podophllotoxin derivatives 4 is
described in Figure 5. We suggest that L-proline catalyzes
the formation of iminium ion 8 in a reversible reaction with
tetronic acid 3. The higher reactivity of the iminium ion
compared with the carbonyl species could facilitate the
addition of aniline 1, via intermediate 9, and after the
elimination of L-proline, 10 might be produced as intermedi-
ate. The product 4 can be produced by tautomerization of
intermediate 10.
Figure 4. The molecular structure of product 4v.
Table 3. Studies on the Reuse of L-Proline in the Preparation of
4a
round
yield(%)
1
2
3
4
5
6
7
8
92
93
92
91
93
92
90
90
When unsymmetrical aromatic amines are used in the
reaction, there may be two different structures of products.
For example, possible structures of product 4t and 4v are
shown in Figure 2. But only one product was obtained in
this reaction. The structures of 4t and 4v were established
by X-ray crystallographic analysis. The molecular structures
of 4t and 4v are shown in Figure 3 and Figure 4, respectively.
According to the results of X-ray crystallographic analysis,
the structures of 4t and 4v are 4t-1 and 4v-1, respectively.
The regioselectivity in the formation of 4t and 4v are
explained from the kinetic espect: for quinolin-6-amine, the
reactivity of its 5-position is higher than that of its 7-position,
and for 3-chloro-4-methylaniline, the effect of steric hin-
It is noted that the compounds obtained are recemic ones.
L-Proline plays a key role in this reaction. According to above
proposed mechanism, L-proline not takes part in the genera-
tion of the chiral center. So stereoselection is not achieved.
Finally, selected compounds were screened for anticancer
activity by the sulforhodamine B (SRB) method. The cells
used in this study were human liver cancer cell line HepG2.16
The results of the prescreening are listed in Table 4. It could
be seen that some products (e.g., 4b, 4e, 4f, 4i, 4j, 4k, 4p,
4v, and 4w) had the stronger inhibitory effects than 4-aza-
podophyllotoxin 4g, and to our happiness, the inhibition rates
Figure 5. Possible mechanism in the synthesis of product 4.

4-Aza-podophyllotoxin Derivatives
Journal of Combinatorial Chemistry, 2010 Vol. 12, No. 4 433
Table 4. Inhibition Rates of Compounds 4 in Certain
Concentration on HepG2 Cells
9-(4-Bromophenyl)-7-methyl-4,9-dihydrofuro[3,4-b]quin-
olin-1(3H)-one (4a). Mp > 300 °C (lit.5 mp > 300 °C). IR
(KBr) ν: 3232, 1711, 1637, 1545, 1500, 1349, 1205, 1036,
product
inhibition rate (%)
1
810 cm-1. H NMR (300 MHz, DMSO-d6) (δ, ppm): 9.92
4a
4b
4c
4d
4e
4f
4g
4h
4i
74.63
80.22
72.39
77.61
79.97
82.64
78.73
76.49
80.97
80.60
80.22
67.06
73.74
78.63
76.41
82.19
76.41
71.51
65.30
74.63
85.76
87.98
(s, 1H, NH), 7.43 (d, J ) 8.4 Hz, 2H, ArH), 7.13 (d, J )
8.7 Hz, 2H, ArH), 6.94 (d, J ) 7.8 Hz, 1H, ArH), 6.82 (d,
J ) 8.1 Hz, 2H, ArH), 4.97 (s, 1H, CH), 4.92 (d, J ) 15.6
Hz, 1H, CH2), 4.83 (d, J ) 15.6 Hz, 1H, CH2), 2.11 (s, 3H,
CH3). HRMS: found, m/z 355.0209 (M+), calcd for
C18H1479BrNO2, M, 355.0208.
4j
4k
4l
Acknowledgment. We gratefully acknowledge financial
support from the Foundation of Key Laboratory of Organic
Synthesis of Jiangsu Province.
4m
4n
4o
4p
4q
4r
4t
4u
4v
4w
Supporting Information Available. Experimental details,
spectroscopic characterization for compound 4, and crystal
data of 4t and 4v. This material is available free of charge
via the Internet at http://pubs.acs.org.
References and Notes
(1) (a) Eycken, J. V.; Bosmans, J. P.; Haver, D. V.; Vandewalle,
M.; Hulkenberg, A.; Veerman, W.; Nieuwenhuizeu, R. Tet-
rahedron Lett. 1989, 30, 3873–3876. (b) Tomioka, K.; Kubota,
Y.; Koga, K. Tetrahedron Lett. 1989, 30, 2953–2954. (c)
Bosmans, J. P.; Eycken, J. V.; Vandewalle, M.; Hulkenberg,
A.; Hes, R. V.; Veerman, W. Tetrahedron Lett. 1989, 30,
3877–3880.
of new compounds 4v and 4w were the highest among them.
Further studies about the activities of these products will be
carried out.
Conclusion
(2) (a) Tomioka, K.; Kubota, Y.; Koga, K. Tetrahedron 1993,
49, 1891–1900. (b) Lienard, P.; Royer, J.; Quirion, J. C.;
Husson, H. P. Tetrahedron Lett. 1991, 32, 2489–2492. (c)
Poli, G.; Giambastiani, G. J. Org. Chem. 2002, 67, 9456–
9459.
(3) (a) Hitosuyanagi, Y.; Kobayashi, M.; Fukuyo, M.; Takeya,
K.; Itokawa, H. Tetrahedron Lett. 1997, 38, 8295–8296. (b)
Hitosuyanagi, Y.; Kobayashi, M.; Morita, H.; Itokawa, H.;
Takeya, K. Tetrahedron Lett. 1999, 40, 9107–9110.
(4) Tratrat, C.; Renault, S. G.; Husson, H. P. Org. Lett. 2002, 4,
3187–3189.
(5) Tu, S. J.; Zhang, Y.; Zhang, J. Y.; Jiang, B.; Jia, R. H.; Zhang,
J. P.; Ji, S. J. Synlett 2006, 2785–2790.
(6) (a) Chandrasekhar, S.; Reddy, N. S.; Sultana, S. S.; Narsih-
mulu, C.; Reddy, K. V. Tetrahedron 2006, 62, 338–345. (b)
Li, H.; Wang, B.; Deng, L. J. Am. Chem. Soc. 2006, 128,
732–733.
In conclusion, we have developed an efficient and con-
venient method for the preparation of 4-aza-podophyllotoxin
derivatives by the three-component reaction of aldehydes,
aromatic amines, and tetronic acid catalyzed by L-proline.
A variety of substrates can participate in the process with
good yields. The procedure used commercially available
starting materials and is suitable for library synthesis and
drug discovery efforts. The short reaction time and easily
available materials render this method particularly attractive
for the efficient preparation of biologically and medicinally
interesting molecules. Importantly, almost all of the com-
pounds exhibited good anticancer activity.
Experimental Section
(7) Alcaide, B.; Almendros, P.; Luna, A.; Torres, M. R. J. Org.
Chem. 2006, 71, 4818–4822.
General Information. Melting points are uncorrected.
Infrared spectra were recorded on a Tensor 27 spectrometer
(8) (a) List, B.; Pojarliey, P.; Biller, W. T.; Martin, H. J. J. Am.
Chem. Soc. 2002, 124, 827–833. (b) Janey, J. M.; Hsiao, Y.;
Armstrong, J. D., III J. Org. Chem. 2006, 71, 390–392.
(9) (a) Rasalkar, M. S.; Potdar, M. K.; Mohile, S. S.; Salunkhe,
M. M. J. Mol. Catal. A: Chem. 2005, 235, 267–270. (b)
Kotrusz, P.; Toma, S. Molecules 2006, 11, 197–205. (c)
Kotrusz, P.; Toma, S. ArkiVoc 2006, 100–109.
(10) Ramachary, D. B.; Chowdari, N. S.; Barbas, C. F., III Angew.
Chem. 2003, 115, 4365–4369.
(11) Bogevig, A.; Juhl, K.; Kumaragurubaran, N.; Zhuang, W.;
Jorgensen, K. A. Angew. Chem., Int. Ed. 2002, 41, 1790–
1793.
1
in KBr with absorption in cm-1. H NMR spectra were
recorded on Varian 300-MHz and Bruker DPX 400-MHz
spectrometer as DMSO-d6 solution. J values are in hertz.
Chemical shifts are expressed in δ downfield from internal
tetramethylsilane. HRMS were obtained using TOF-MS
instrument. X-ray crystallographic analysis was performed
with a Smart-1000 CCD diffractometer.
General Procedure for the Synthesis of 4-Aza-podo-
phyllotoxin Derivatives 4. A mixture of aromatic amine 1
(1 mmol), aldehyde 2 (1 mmol), tetronic acid 3 (1 mmol),
L-proline (10 mol %) and ethanol (2 mL) in a 10 mL round-
bottom flask was stirred at 80 °C for given times. After
completion of the reaction, the reaction mixture was cooled
to room temperature. The precipitate was collected by
filtration and purified by recrystallization from EtOH and
DMF to give products 4.
(12) Hossein, A. O.; Elham, R.; Majid, M. H. J. Chem. Res. 2006,
246–247.
(13) (a) Yadav, J. S.; Kumar, S. P.; Kondaji, G.; Rao, R. S.;
Nagaiah, K. Chem. Lett. 2004, 33, 1168–1169. (b) Mabry, J.;
Ganem, B. Tetrahedron Lett. 2006, 47, 55–56.
(14) Kumar, A.; Maurya, R. A. Tetrahedron 2007, 63, 1946–1952.
(15) (a) Shi, C. L.; Shi, D. Q.; Kim, S. H.; Huang, Z. B.; Ji, S. J.;
Ji, M. Tetrahedron 2008, 64, 2425–2432. (b) Shi, C. L.; Shi,

434 Journal of Combinatorial Chemistry, 2010 Vol. 12, No. 4
Shi et al.
D. Q.; Kim, S. H.; Huang, Z. B.; Ji, M. Aust. J. Chem. 2008,
61, 547–551. (c) Li, Y. L.; Chen, H.; Shi, C. L.; Shi, D. Q.;
Ji, S. J. J. Comb. Chem. 2010, 12, 231–237.
Then certain concentrations of products (100, 10, or 20 mg/mL
in DMSO) were diluted 1000 times and added to the culture
using DMSO as control solution. After 24 h, the absorbencies
were read at 592 and 570 nm with SRB assay.
(16) The compounds were dissolved in DMSO as 100, 10, or 20 mg/
mL stock solutions before use and stored at-20 °C. HepG2 cells
were incubated in MEM culture medium containing 10% blood
serum. The cells were plated in flat-bottomed 96-well plates
(5000 cells/well) and cultured for 24 h in controlled atmosphere.
(17) Indumathi, S.; Perumal, S.; Menendez, J. C. J. Org. Chem.
2010, 75, 472–475.
CC100003C