Synthesis of 4β-triazole-podophyllotoxin derivatives by azide-alkyne cycloaddition and biological evaluation as potential antitumor agents

Article abstract of DOI:10.1016/j.ejmech.2011.07.024

A representative synthetic process of derivatizing the natural product podophyllotoxin utilizing the copper-catalyzed azide-alkyne cycloaddition (CuAAC) is described including molecular design, reaction optimization and X-ray structure confirmation. Evalu

Full text of DOI:10.1016/j.ejmech.2011.07.024

European Journal of Medicinal Chemistry 46 (2011) 4709e4714
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Synthesis of 4
b-triazole-podophyllotoxin derivatives by azideealkyne
cycloaddition and biological evaluation as potential antitumor agents
,
d
,
a
,
b
,
c
a
a
a
a
Hong Chen a , Song Zuo
, Xiaochen Wang , Xiaowei Tang , Ming Zhao , Yanling Lu , Liting Chen ,
*
**
Jing Liu a, Yongfeng Liu a, Dailin Liu a, Shi Zhang a, Tan Li a
a Pharmacognosy Division, Medical College of Chinese People’s Armed Police Force, Tianjin 300162, China
b Chengdu Branch of General Unit of Chinese People’s Armed Police Force Hospital in Sichuan, Chengdu 600041, China
c Department of Chemistry and Biochemistry, University of Maryland, College Park, MD 20742, USA
d Tianjin Key Laboratory for Biomarkers of Occupational and Environmental Hazard, Tianjin 300162, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A representative synthetic process of derivatizing the natural product podophyllotoxin utilizing the
copper-catalyzed azideealkyne cycloaddition (CuAAC) is described including molecular design, reaction
optimization and X-ray structure confirmation. Evaluation of cytotoxicity against human cancer cell lines
(Hela, K562 and K562/A02) using MTT assay proves that these triazole derivatives have good antitumor
activities. High activities toward the drug resistant K562/A02 cell line reveal promising future for these
derivatives. The rarely prepared 1,5-disubstituted triazole isomers, which would be omitted by the “click
chemistry”, were found to have superior cytotoxicities to that of the 1,4-disubstituted isomers.
Ó 2011 Elsevier Masson SAS. All rights reserved.
Received 24 January 2011
Received in revised form
4 July 2011
Accepted 14 July 2011
Available online 23 July 2011
Keywords:
Podophyllotoxin
Triazole
1,3-Dipolar cycloaddition
Antitumor
1. Introduction
disubstituted-1,2,3-triazoles are commonly formed, whereas 1,5-
disubstituted isomers can be selectively prepared via ruthenium
Podophyllotoxin, an aryl tetralin lignan, is able to inhibit tubulin
by binding with its colchicine domain [1]. Several podophyllotoxin
derivatives such as etoposide, teniposide are in clinical use as
antineoplastic agents. They block the DNA topoisomerase II by
stabilizing enzymeeDNA complex [2e5]. However, their high
toxicity, low water solubility, acquired drug-resistance and
gastrointestinal disturbances have limited their application in
cancer chemotherapy. Numerous structure modifications had been
performed since the 1950s. Among them, NK611, NPF, GL-311 and
TOP53 are presently under clinical trials [6e8]. Since 1,2,3-triazole
ring is a widespread functional group in drugs [9,10], it is intriguing
to attach 1,2,3-triazoles to podophyllotoxin parent nucleus.
catalysis [16,17] and the reactions without metal catalysis result into
mixtures of 1,4- and 1,5-disubstituted isomers. Previously, 4 -[(4-
b
substituted)-1,2,3-triazol-1-yl]podophyllotoxins reported by Kumar
[18] showed promising antitumor activity which revealed significant
outlook for further in-depth research. In this paper, a classic structure
modificationonpodophyllotoxin isdescribed including methodology
research, structure confirmation of isomers, further chemical
synthesis, and biological evaluation.
2. Results and discussion
2.1. Chemistry
The click chemistry of copper-catalyzed Huisgen 1,3-dipolar azi-
deealkyne cycloaddition (CuAAC) forming 1,2,3-triazole has exhibi-
ted significant advantages with respect to high chemoselectivities
and mild reaction conditions [11e15]. During the process, 1,4-
To find out the most compatible reaction conditions for
synthesizing this series of natural product derivatives, a wide range
of reaction parameters were tested by altering the catalyst and the
additive as well as the temperature and the solvent in a test reac-
* Corresponding author. Pharmacognosy Division, Medical College of Chinese
People’s Armed Police Force, Tianjin 300162, China. Tel./fax: þ86 22 60578193.
** Corresponding author. Chengdu Branch of General Unit of Chinese People’s Armed
Police Force Hospital in Sichuan, Chengdu 600041, China. Tel./fax: þ86 28 85425419.
E-mail addresses: chenhongtian06@yahoo.com.cn (H. Chen), zuosong203@
yahoo.com.cn (S. Zuo).
tion of 4b-azido-podophyllotoxin and methyl propiolate (Table 1).
As can be clearly seen in Table 1, the CuAAC reactions resulted in
regioselective products. Single 1,4-disubstituted-1,2,3-triazole was
formed in entries 1e7. 2,6-Lutidine was a beneficial additive for CuI
compared to other basic additives while
L-ascorbic acid was
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.07.024

4710
H. Chen et al. / European Journal of Medicinal Chemistry 46 (2011) 4709e4714
Table 1
Screening of the reaction conditions for the cycloaddition reaction between 4
b-azido-podophyllotoxin and methyl propiolate.
5
CO2Me
OH
4
N
O
N
N
1
N
O
3
N
2
N
O
CuAAC
1, 4- Isomers
+
CO2Me
O
CO2Me
5
4
N
H3CO
OCH3
1
N
N
OCH3
3
2
1, 5- Isomers
β
4
-Azido-podophyllotoxin, 1
Triazole-substituted
Podophyllotoxin
Podophyllotoxin
Entry
Catalyst
Additive
Solvent
t (h)
T (ꢁC)
Yielda (%)
1,4-Isomers
1,5-Isomers
1
2
3
4
5
6
7
8
CuSO4$5H2O
CuSO4$5H2O
CuSO4$5H2O
CuSO4$5H2O
CuSO4$5H2O
CuSO4$5H2O
CuI
CuI
CuI
CuI
CuSO4$5H2O
CuI
L
L
L
L
L
L
-Ascorbic acid
-Ascorbic acid
-Ascorbic acid
-Ascorbic acid
-Ascorbic acid
-Ascorbic acid
t-BuOH/H2O (1:1)
t-BuOH/H2O (1:1)
t-BuOH/H2O (1:1)
THF/H2O (1:1)
DMF/H2O (1:1)
DMSO/H2O (1:1)
CHCl3
CHCl3
CHCl3
t-BuOH/H2O (1:1)
t-BuOH/H2O (1:1)
PEG400/H2O (1:1)
CH2Cl2
2
2
2
2
2
25
50
75
25
25
25
0
0
0
25
25
25
25
70b
95b
65b
0
0
0
0
0
0
0
0
0
17
0
80b
83b
90b
30b
20
2
2,6-Lutidine
Et3N
Pyridine
12
12
12
12
2
9
10
11
12
13
L-Ascorbic acid
0
15
95
15
2,6-Lutidine
None
None
10
0
12
2
6
None
a
Isolated yield (%) after column chromatography.
Multiple side products were detected without 1,5-disubstituted products.
b
necessary for CuSO4 to generate the active Cu(I). Later, the solvent
PEG400 (entry 12) was found to greatly enhance the reaction rate in
CuI catalyzed reactionwithout anyadditive [19]. Entries 2, 7,12 were
picked out as candidate conditions for further synthesis (Table 2).
1,5-Disubstituted triazole podophyllotoxins obtainedbyaccident
was found to have comparative or even superior anticancer activity.
Therefore, the traditional thermal condition without any catalyst
was applied and the obtained mixture of the 1,4-disubstitued and
the 1,5-disubstituted triazole podophyllotoxins were subjected to
column separation. In most cases, ratio of two regioisomers formed
were approximately 1:1 (Table 3). X-ray crystallography confirmed
that the product formed through the CuAAC reaction was the
1,4-disubstituted triazole podophyllotoxins (Fig. 1) [20].
Although Wang et al. has previously published a short report on the
synthesis and antitumor activities of a few 1,5-disubstituted triazole
podophyllotoxins [21], to the best of our knowledge, this is the first
time that a systematic comparison on antitumor activities of two
regioisomers is being made. Previous studies indicate that VP-16
and VM-26, which have a free hydroxyl group at E-ring, are inhibi-
tors of topoisomerase II [2e5], whereas podophyllotoxin targets
microtubule [22]. Same as podophyllotoxin, the derivatives we
prepared do not have free hydroxyl groups at E-ring, and in our
preliminary studies they also target microtubule [23].
3. Conclusion
In conclusion, CuAAC reactions showed a broad and profound
perspective for structural modification on natural products. In this
2.2. Pharmacology
communication, nine new
4
b-[(4-substituted)-1,2,3-triazol-1-yl]
podophyllotoxinswerepreparedandevaluatedforcytotoxicityagainst
human cancer cell lines. Some of these derivatives exhibit excellent
activities, even toward the multidrug-resistant cell line. Meanwhile,
the superior activities of 1,5-disubstitued triazole podophyllotoxins
prove the importance of developing methodology leading to the less
accessible regioisomers. In order to get 1,5-disubstituted triazoles, the
traditional method without any catalyst are worth trying. Further
synthesis of 1,4- and 1,5-disubstituted triazole podophyllotoxins and
in vivo tests will be reported soon.
These derivatives were evaluated for cytotoxicity against human
cancer cell lines HeLa, K562, K562/A02, using the MTT assay in vitro
with comparison to the parent compound VP-16, podophyllotoxin
and ADM. Most of 1,4-disubstituted triazole podophyllotoxins were
demonstrated to have significant antitumor activity (Table 2, IC50
values were found at the range of 10ꢀ6e10ꢀ8 mol/L while the IC50
values of VP-16 is in 10ꢀ6 magnitude). Some derivatives exhibited
high cytotoxicities toward the drug resistant K562/A02 leukemic
cell line, whereas VP-16 was not active. Among them, compound 3g
showed excellent cytotoxicities, more than 40 times more cytotoxic
than VP-16 in HeLa, K562 and K562/A02. In the structure-activity
analysis, free carboxylic acids in R greatly reduce the cytotoxicity
(compounds 3b and 3c) while esters and amides are well tolerated
(compounds 3a and 3g). Secondary amine attached to an aromatic
ring in R (compounds 3d and 3f) is another promising functionality
for future development of analogs. Meanwhile, the 1,5-disubstituted
products had comparative or even superior anticancer activity
in vitro (Table 3). Up to 20 folds of enhancement on cytotoxicity was
observed compared to their 1,4-disubstituted counterparts.
4. Experimental
4.1. Materials and methods
All materials and reagents were obtained from commercial
sources and used without further purification unless stated. Podo-
phyllotoxin (98% purity) was purchased from Qingze Corporation of
Nanjing in China. CH2Cl2 was redistilled over P2O5. Melting points
were determined on an electric X-4 digital visual melting point

H. Chen et al. / European Journal of Medicinal Chemistry 46 (2011) 4709e4714
4711
Table 2
Table 3
Synthesis of triazole derivatives.a
Synthesis of 4b
-triazole-podophyllotoxin derivatives with no catalysta and
comparison on cytotoxicities of two regioisomers against human cancer cell lines.
R
N
N
N
N
N
N
N
5
R
N
N
4
O
N
O
O
O
O
N
N
1
O
CuAAC
O
3
R
+
O
2
O
Entry 2, 7 or 12
+
R
3X
1
1, 4- Isomers,
R
in Table 1
O
O
O
5
2
Alkynes,
2
Alkyne,
N
4
H3CO
OCH3
1
H3CO
OCH3
N
N
3
H3CO
OCH3
OCH3
OCH3
2
1, 5- Isomers,3X2
OCH3
β
1,4-Disubstituted Triazole
4
-Azido-podophyllotoxin, 1
Triazole-substituted
Podophyllotoxin, 3
β
1
4
-Azido-podophyllotoxin,
Podophyllotoxin, 3
X=a, b, e, j, k
Comp. 3
R
Yield %b IC50
Hela
(m
mol/L)
K562
Entry Alkynes
R
Yield %b
IC50 (mmol/L)
K562/
A02
Hela K562 K562/
A02
O
3a
3b
3c
3d
95
65
60
95
1.12
4.15
2.069
O
3a1 30% 1.12
3a2 25% 0.42
4.15
0.40
2.069
0.60
OCH3
O
1
2
3
4
2a
2b
2e
2j
OCH3
O
>10
5.4
8.87
3b1 25% >10
3b2 20% n.d.
5.4
n.d.
8.87
n.d.
OH
OH
N
OH
3e1 30% 35.3
3e2 27% 1.46
>10
0.39
>10
1.23
>10
>10
>10
O
O
3j1 18% >100 >100 >100
3j2 15% >10
2.36
5.73
5.96
5.1
19.8
N
H
HN
CH3
O
3k1 34% 3.53
3k2 17% 0.92
4.25
0.85
2.21
0.69
N
5
2k
3e
3f
93
80
35.3
>10
>10
OEt
a
Reaction condition: ethanol, 80 ꢁC.
CH3
b
0.845
0.082
0.47
0.52
Total isolated yields (%) were calculated after column chromatography; The
N
H
ratio of isomers were determined by HPLC.
O Cl
4 mol% CuSO4$5H2O (1.0 mg) or 1 equiv of 2,6-lutidine (11 mg,
0.1 mmol) and 5 mol% CuI (1.0 mg) were added into a reaction vial
charged with a magnetic stir bar. t-BuOH/H2O (5.0 ml, 1:1) were
used to dissolve the reactants. After stirring for 2 h at corre-
sponding temperature, the reaction mixture was quenched with
water (15 ml), and extracted with ethyl acetate (2 ꢂ 20 ml). The
combined organic layers were washed with water (2 ꢂ 10 ml), and
then the solvent was removed under reduced pressure. The residue
was dissolved in acetone and purified on silica gel to afford the
product.
3g
60
0.053
0.059
HN
H3C
3h
3i
85
57
>10
>10
>10
N
CH3
O
>10
>10
>10
NEt2
VP-16
6.27
2.11 151.69
Podophyllotoxin
ADM
0.10
0.43
0.03
0.36
0.17
19.21
4.3. General procedure for the traditional 1,3-dipolar cycloaddition
a
Reaction conditions from Table 1 (entries 2, 7 or 12) were applied.
Isolated yield.
1
equiv of 4b-azido-podophyllotoxin (44 mg, 0.1 mmol),
b
1.2 equiv of alkynes (0.12 mmol) were added into ethanol (5.0 ml)
and stirred for 8 h at 80 ꢁC. The workup was similar to the proce-
dure described for the CuAAC Reactions.
apparatus. The 1H NMR spectra were obtained using a BRUKER ARX-
300 instrument (300 MHz) with tetramethylsilane (TMS) as the
internal standard, and the multiplicity were marked as s ¼ singlet,
d ¼ doublet, t ¼ triplet, q ¼ quartet, m ¼ multiplet. Mass spectra data
were obtained on Agilent 6210 TOP-MS and are reported as m/z.
4.4. Product characterization
4.4.1. Synthesis of 4b-(4-methoxy-carbonyl-1,2,3-triazole)-podo-
4.2. General procedure for the CuAAC reactions
phyllotoxin (3a1) by CuAAC
Yield: 95%. White solid. Mp 212 ꢁC. ½a D25
ꢀ85 (c 0.35 CHCl3). IR
ꢃ
1
equiv of
4b
-azido-podophyllotoxin (44 mg, 0.1 mmol),
-ascorbic acid (2 mg) and
(KBr)
(CDCl3) d/ppm 7.85 (s, 1H, triazoleeH), 6.64 (s, 1H), 6.33 (s, 2H),
n
1772, 1762 (C]O), 1603 (N]N), 1555, 1484 (C]C). 1H NMR
10 equiv of alkynes (1.0 mmol), 10 mol%
L

4712
H. Chen et al. / European Journal of Medicinal Chemistry 46 (2011) 4709e4714
HR-ESI-MS calculated for C25H23N3NaO9 [M þ Na]þ 532.1327, found
532.1354.
4.4.4. Synthesis of 4b-(4-(2-carboxyl)-ethyl-1,2,3-triazole)-
podophyllotoxin(3c) by CuAAC
Yield: 60%. Yellow solid. Mp 151e153 ꢁC. ½a D25
ꢀ79 (c 0.36
ꢃ
CHCl3). IR (KBr) n 3455 (OH), 1773, 1752 (C]O), 1605 (N]N), 1556,
1446 (C]C). 1H NMR (CDCl3)
d/ppm 7.09 (s, 1H, triazoleeH), 6.62
(s, 1H, AreH), 6.59 (s, 1H, AreH), 6.30 (s, 2H, 20,60-H), 6.03e6.04 (d,
1H, J ¼ 3.2 Hz, 4-H), 6.00 (d, H, J ¼ 1.1 Hz, OCH2O), 5.98 (d, H,
J ¼ 1.1 Hz, OCH2O), 4.72e4.74 (d, 1H, J ¼ 4.7 Hz, 1-H), 4.32e4.39 (m,
1H, 1-H-a), 4.08e4.15 (m, 1H, 1-H-b), 3.80 (s, 3H, 40-OCH3), 3.75 (s,
6H, 30,50-OCH3), 3.13e3.21 (m, 2H, 2,3-H), 3.98e3.03 (t, 2H,
J ¼ 7.1 Hz), 2.75e2.80 (t, 2H, J ¼ 6.8Hz). 13C NMR (CDCl3)
d 30.8,
33.1, 37.1, 41.5, 43.6, 56.2, 58.6, 60.5, 67.4, 101.9, 108.2, 108.8, 110.3,
122.5, 124.8, 133.2, 134.5, 137.4, 146.3, 148.0, 149.3, 152.7, 173.6,
176.1. HR-ESI-MS calculated for C27H27N3NaO9 [M þ Na]þ 560.1640,
found 560.1641.
4.4.5. Synthesis of 4b-(4-anilino-methylene-1,2,3-triazole)-
podophyllotoxin (3d) by CuAAC
a 25
ꢁ
Yield: 95%. Mp 165e166 C. ½ ꢃD ꢀ82 (c 0.35 CHCl3). IR (KBr)
n
3320 (NeH), 1766 (C]O), 1601 (N]N), 1553, 1480 (C]C), 1475. 1H
NMR (CDCl3) /ppm 8.02 (1H, s, NeH), 7.40 (1H, s, triazoleeH), 7.00
d
(d, 1H, J ¼ 10.5 Hz, AreH), 6.88 (d, 2H, J ¼ 7.9 Hz, AreH), 6.70 (s, 1H,
5-H), 6.65 (d, 2H, J ¼ 8.6 Hz, AreH), 6.38 (s, 2H, 20,60-H), 6.18 (s, 1H,
8-H), 5.98 (d, 1H, J ¼ 1.3 Hz, OCH2O), 5.96 (d, 1H, J ¼ 1.3, OCH2O),
5.78 (s, 1H, 4-H), 4.38e4.60 (m, 4H, 1-H, 11-H-a, eCH2e), 3.85e3.91
(m, 1H, 11-H-b), 3.78 (s, 6H, 30,50-OCH3), 3.60 (m, 2H, 2,3-H). This
compound has been characterized before [18].
Fig. 1. X-ray structure of
[20]. (Hydrogen atoms are omitted for clarity).
4b-(4-methoxy-carbonyl-1,2,3-triazole)-podophyllotoxin
6.61 (s, 1H), 6.18e6.19 (d, 1H, J ¼ 3.9 Hz), 6.04 (d, 1H, J ¼ 1.1 Hz,
OCH2O), 6.01 (d, 1H, J ¼ 1.1 Hz, OCH2O), 4.75e4.77 (d, 1H,
J ¼ 5.0 Hz), 4.37e4.46 (m, 1H), 4.04e4.16 (m, 1H), 3.92 (s, 3H), 3.81
4.4.6. Synthesis of 4b-(4-pyridyl-1,2,3-triazole)-podophyllotoxin
(3e1) by CuAAC
(s, 3H), 3.77 (s, 6H), 3.02e3.36 (m, 2H). 13C NMR (CDCl3)
d
30.9,
Yield: 93%. Yellow solid. Mp 139e141 ꢁC. ½a D25
ꢀ85 (c 0.41
ꢃ
37.0, 41.3, 43.6, 56.4, 59.2, 60.8, 67.3, 102.1, 108.3, 108.7, 110.7,
123.9128.0, 133.5, 134.0, 137.7, 139.9, 148.3, 149.7, 152.8, 160.8, 172.7.
HR-ESI-MS calculated for C26H25N3NaO9 [M þ Na]þ 546.1483,
found 546.1446.
CHCl3). IR (KBr) n 1768 (C]O), 1601 (N]N), 1588, 1528 (C]C), 1472.
1H NMR (CDCl3)
d
/ppm 8.75 (d, 1H, J ¼ 7.3 Hz), 8.07 (s, 1H, tri-
azoleeH), 7.93 (dd, 1H, J ¼ 2.2, 7.8 Hz), 7.77 (d, 1H, J ¼ 7.8 Hz), 7.43
(m, 1H), 6.98 (d, 1H, J ¼ 8.2 Hz), 6.63 (s, 1H), 6.51 (s, 1H), 6.42 (s, 2H),
5.96 (d, 1H, J ¼ 1.3 Hz, OCH2O), 5.90 (d, 1H, J ¼ 1.3 Hz, OCH2O), 4.65
(d, 1H, J ¼ 3.4 Hz), 4.42 (m, 1H), 4.09 (m, 1H), 3.82 (s, 3H), 3.78 (s,
4.4.2. Synthesis of 4b-(5-methoxy-carbonyl-1,2,3-triazole)-podo-
phyllotoxin (3a2) by the traditional 1,3-dipolar cycloaddition
6H), 3.16e3.25 (m, 2H). 13C NMR (CDCl3)
d 30.9, 41.6, 43.7, 56.4,
Yield: 25%. White solid. Mp 164e165 ꢁC. ½a D25
ꢃ
ꢀ87 (c 0.41 CHCl3).
58.9, 60.8, 67.5, 102.0, 108.2, 108.9, 110.5, 120.5, 120.3, 123.3, 124.4,
133.3, 134.2, 137.3, 137.6, 148.2, 148.3, 149.3, 149.6, 149.6, 152.8,
173.0, HR-ESI-MS calculated for C29H27N4O7 [M þ H]þ 543.1874,
found 543.1890.
IR (KBr)
NMR (CDCl
n
1775, 1760 (C]O), 1605 (N]N), 1559, 1481 (C]C). 1H
) d/ppm 8.23 (s, 1H, triazoleeH), 6.76e6.78 (d, 1H,
3
J ¼ 5.3 Hz), 6.63 (s, 1H), 6.47 (s, 1H), 6.36 (s, 2H), 5.99 (d, 1H,
J ¼ 1.2 Hz, OCH2O), 5.93 (d, 1H, J ¼ 1.2 Hz, OCH2O), 4.81e4.83 (d, 1H,
J ¼ 5.1 Hz), 4.30e4.36 (m, 1H), 4.02 (s, 3H), 3.80e3.85 (m, 1H), 3.82
4.4.7. Synthesis of 4b-(5-pyridyl-1,2,3-triazole)-podophyllotoxin
(3e2) by the traditional 1,3-dipolar cycloaddition
(s, 3H), 3.78 (s, 6H), 3.00e3.35 (m, 2H). 13C NMR (CDCl3)
d
30.9, 37.0,
41.3, 43.6, 56.4, 59.2, 60.8, 67.3, 102.2, 108.3, 108.7, 110.7, 123.8,
128.0, 133.5, 134.0, 137.8, 140.1, 148.4, 149.8, 152.9, 160.8, 172.6. HR-
ESI-MS calculated for C26H25N3NaO9 [M þ Na]þ 546.1483, found
546.1460.
Yield: 27%. Yellow solid. Mp 145e147 ꢁC. ½a D25
ꢃ
ꢀ81 (c 0.37 CHCl3).
IR (KBr)
(CDCl3)
n
d
1766 (C]O),1605 (N]N),1585,1550 (C]C),1481.1H NMR
/ppm 8.56 (d, 1H), 8.20 (dd, 1H, J ¼ 1.7, 7.6 Hz), 7.85 (s, 1H,
triazoleeH), 7.85 (dd, 1H, J ¼ 1.2, 7.6 Hz), 7.27 (m, 1H), 6.69 (s, 2H),
6.34 (s, 2H), 6.19 (d, 1H, J ¼ 8.3 Hz), 6.04 (d, 1H, J ¼ 1.2 Hz, OCH2O),
5.98 (d, 1H, J ¼ 1.2 Hz, OCH2O), 4.77 (d, 1H, J ¼ 5.0 Hz), 4.46 (m, 1H),
4.10 (m, 1H), 3.85 (s, 3H), 3.78 (s, 6H), 3.20e3.38 (m, 2H). 13C NMR
4.4.3. Synthesis of 4b-(4-carboxyl-1,2,3-triazole)-podophyllotoxin
(3b1) by CuAAC
Yield: 65%. White solid. Mp 177e179 ꢁC. ½a D25
ꢃ
ꢀ81 (c 0.31
(CDCl3) d 30.9, 41.8, 43.8, 56.4, 58.9, 61.5, 67.5, 103.2, 108.6, 108.9,
CHCl3). IR (KBr)
n
3510 (OH), 1768, 1752 (C]O), 1611 (N]N), 1559,
110.5, 120.7, 120.4, 123.4, 124.8, 134.4, 134.6, 137.8, 138.7, 148.6,
148.9, 149.3, 149.8, 149.3, 152.9, 173.4. HR-ESI-MS calculated for
C29H27N4O7 [M þ H]þ 543.1874, found 543.1882.
1443 (C]C). 1H NMR (CDCl 3 /ppm 8.30 (s, 1H, 50-H), 8.05 (s, 1H,
) d
triazoleeH), 6.80 (d, 1H, J ¼ 5.3 Hz, 4-H), 6.66 (s, 1H, 5-H), 6.60 (s,
1H, 8-H), 6.37 (s, 2H, 20,60-H), 6.02 (d, 1H, J ¼ 1.3 Hz, OCH2O), 5.99
(d, 1H, J ¼ 1.3 Hz, OCH2O), 4.82 (d, 1H, J ¼ 5.1 Hz, 1-H), 4.35e4.45
(m, 1H, 11-H-a), 4.04e4.16 (m, 1H, 11-H-b), 3.92 (s, 3H, eOCH3),
3.77 (s, 6H, 30,50-OCH3), 3.02e3.36 (m, 2H, 2,3-H). 13C NMR (CDCl3)
4.4.8. Synthesis of 4b-(4-(4-methylbenzenamino)-methylene-
1,2,3-triazole)-podophyllotoxin (3f) by CuAAC
Yield: 80%. White solid. Mp 153e154 ꢁC. ½a D25
ꢀ85 (c 0.42 CHCl3).
ꢃ
d
36.8, 40.2, 43.5, 56.3, 56.5, 60.4, 67.6, 102.1, 108.6, 109.2, 110.3,
IR (KBr) n 3365 (NeH), 1767 (C]O), 1611 (N]N), 1558, 1550 (C]C),
123.7, 126.1, 130.0, 133.7, 135.7, 137.0, 147.5, 148.1, 152.6, 162.1, 174.0.
1475.1H NMR (CDCl3)
d/ppm 7.18 (s,1H, 5-H), 7.02e6.94 (m, 2H), 6.61

H. Chen et al. / European Journal of Medicinal Chemistry 46 (2011) 4709e4714
4713
(s,1H, 8-H), 6.58(d, 2H, J ¼ 10.6 Hz, AreH), 6.31 (s, 2H, 20,60-H), 6.08(s,
1H, 4-H), 5.97(d,1H, J¼ 1.3Hz, OCH2O), 5.93(d,1H, J¼1.3Hz, OCH2O),
4.76 (s, 1H,1-H) 4.43 (s, 2H, eCH2e), 4.38 (m, 1H, 11-H-a), 3.85e3.91
(m,1H,11-H-b), 3.82 (s, 3H, 40-OCH3), 3.78 (s, 6H, 30,50-OCH3), 3.18 (m,
2H, 2,3-H). This compound has been characterized before [18].
4.4.13. Synthesis of 4b-(4-ethoxy-carbonyl-1,2,3-triazole)-podo-
phyllotoxin (3k1) by CuAAC
Yield: 73%. White solid. Mp 177e178 ꢁC. ½a D25
ꢃ
ꢀ86 (c 0.33 CHCl3).
IR (KBr)
NMR
n
1772, 1760 (C]O), 1608 (N]N), 1561, 1466 (C]C). 1H
d/ppm 8.24 (s, 1H, triazoleeH), 6.75 (s, 1H), 6.54e6.55 (d, 1H,
J ¼ 3.8 Hz), 6.44 (s, 2H), 6.99 (d, 1H, J ¼ 1.3 Hz, OCH2O), 5.80 (d, 1H,
J ¼ 1.3 Hz, OCH2O), 4.71e4.77 (d, 1H, J ¼ 2.0 Hz), 4.69 (m, 1H),
4.25e4.33 (m, 2H, OCH2), 4.19e4.24 (m, 1H), 3.85 (s, 6H, OCH3),
3.83 (s, 3H, OCH3), 3.63e3.79 (m, 2H), 1.34 (t, 3H, J ¼ 7.3 Hz, CH3).
4.4.9. Synthesis of 4b-[4-(2-chloro-phenyl-amino-carbonyl)-1,2,3-
triazole]-podophyllotoxin (3g) by CuAAC
Yield: 60%. White solid. Mp 167e168 ꢁC. ½a D25
ꢀ82 (c 0.37 CHCl3).
ꢃ
IR (KBr)
n
3360 (NeH), 1770, 1650 (C]O), 1605 (N]N), 1557, 1550
13C NMR (CDCl3)
d14.3, 24.2, 37.0, 41.3, 43.6, 56.4, 59.2, 60.4, 67.3,
(C]C),1452. 1H NMR (CDCl3)
d
/ppm 8.32 (s,1H, triazoleeH), 7.82 (s,
102.1, 108.7, 109.7, 110.7, 123.9, 127.9, 133.4, 133.5, 137.7, 140.4, 148.3,
149.8, 152.6, 160.4, 172.7. HR-ESI-MS calculated for C27H27N3NaO9
[M þ Na]þ 560.1640, found 560.1619.
1H, NeH), 7.74 (dd, 1H, J ¼ 3.3, 7.0 Hz), 7.77 (d, 2H, J ¼ 3.3 Hz), 7.34
(m, 1H), 6.82 (d,1H, J ¼ 8.4 Hz), 6.63 (s, 1H), 6.54 (s, 1H), 6.38 (s, 2H),
5.99 (d, 1H, J ¼ 1.4 Hz, OCH2O), 5.96 (d, 1H, J ¼ 1.4 Hz, OCH2O), 4.83
(d,1H, J ¼ 3.4 Hz), 4.45 (m,1H), 4.09 (m,1H), 3.82 (s, 3H), 3.83 (s, 6H),
4.4.14. Synthesis of 4b-(5-ethoxy-carbonyl-1,2,3-triazole)-podo-
3.11e3.20 (m, 2H). 13C NMR (CDCl3)
d
30.6, 37.0, 41.2, 42.7, 56.4,
phyllotoxin (3k2) by the traditional 1,3-dipolar cycloaddition
60.8, 67.2, 102.2, 108.3, 108.5, 110.5, 117.9, 120.0, 124.9, 126.9, 128.8,
130.3, 130.9, 133.9, 134.5, 135.0, 138.4, 143.0, 148.0, 148.5, 152.9,
157.6, 172.6. HR-ESI-MS calculated for C31H28ClN4O8 [M þ H]þ
619.1590, found 619.1574.
Yield: 17%. White solid. Mp 135e136 ꢁC. ½a D25
ꢀ82 (c 0.37
ꢃ
CHCl3). IR (KBr)
n 1769, 1761 (C]O), 1601 (N]N), 1555, 1484 (C]
C), 1456. 1H NMR
d/ppm 7.91 (s, 1H, triazoleeH), 6.69 (s, 1H), 6.38
(s, 2H), 6.44 (s, 2H), 6.25 (s, 1H), 6.00 (d, 1H, J ¼ 1.2 Hz, OCH2O),
5.97 (d, 1H, J ¼ 1.2 Hz, OCH2O), 5.92 (d, 1H, J ¼ 4.8 Hz), 4.57 (d,
1H, J ¼ 2.5 Hz,), 4.43e4.54 (m, 2H, OCH2), 4.36e4.43 (m, 2H,
OCH2), 3.83 (s, 3H, OCH3), 3.80 (s, 6H, OCH3), 3.50e3.67 (m, 2H),
4.4.10. Synthesis of 4b-(4-(N,N-dimethylamino)methylene-1,2,3-
triazole)-podophyllotoxin (3h) by CuAAC
Yield: 85%. White solid. Mp 172e174 ꢁC. ½a D25
ꢃ
ꢀ79 (c 0.35 CHCl3).
1.45 (t, 3H, J ¼ 7.1 Hz, CH3). 13C NMR (CDCl3)
d 14.4, 24.1, 36.7,
IR (KBr)
(CDCl 3)
n
1769 (C]O), 1603 (N]N), 1562, 1495 (C]C). 1H NMR
40.5, 42.3, 56.8, 59.3, 60.4, 67.2, 101.4, 108.6, 109.8, 110.8, 124.0,
127.5, 131.6, 133.4, 137.7, 140.5, 147.9, 150.2, 152.2, 162.4, 173.9;
HR-ESI-MS calculated for C27H27N3NaO9 [M þ Na]þ 560.1640,
found 560.1614.
d/ppm 7.19 (1H, s, triazoleeH), 6.62 (1H, s, AreH), 6.60 (1H,
s, AreH), 6.31 (2H, s, 20,60-H), 6.07e6.09 (1H, d, J ¼ 4.1Hz, 4-H), 6.01
(d, 1H, d, J ¼ 1.2 Hz, OCH2O), 5.98 (d, H, J ¼ 1.2 Hz, OCH2O),
4.72e4.72 (d, 1H, J ¼ 4.9 Hz, 1-H), 4.38e4.41 (m, 1H, 1-H-a),
3.79e3.83 (m, 1H, 11-H-b), 3.81 (s, 1H, 40-OCH3), 3.76 (s, 6H,
30,50-OCH3), 3.57 (s, 2H, eCH2eNe), 2.26 (s, 6H, eNeCH3). 13C NMR
4.5. Cell culture and cytotoxic assay
(CDCl3)
d 32.2, 37.2, 41.7, 43.7, 56.3, 58.4, 60.7, 62.2, 67.5, 102.0,
All the cell lines were supplied by the Institute of Materia
Medica, Chinese Academy of Medical Sciences & Peking Union
Medical College. Tumor cells were maintained in RPM11640
medium containing 10% heat-inactivated fetal bovine serum,
108.2, 108.9, 110.4, 121.1, 125.0, 133.1, 134.4, 137.5, 148.1, 148.3, 149.3,
152.8, 173.3. HR-ESI-MS calculated for C27H31N4O7 [M þ H]þ
523.2187, found 523.2195.
penicillin (100 units/ml), streptomycin (100 mg/ml) under humidi-
fied air with 5% CO2 at 37 ꢁC. Exponentially growing cells were
seeded into 96-well tissue culture-treated plates and precultured
for 1 day. The test compounds at various concentrations were
added, and the cells were incubated for additional 2 days. The
cytotoxic activity was measured by MTT assay [24], and the IC50
values were obtained from doseeresponse curves. VP-16, Podo-
phyllotoxin and ADM were used as the positive control.
4.4.11. Synthesis of 4b-(4-(N,N-diethylamino)carbonyl-1,2,3-triaz-
ole)-podophyllotoxin (3i) by CuAAC
Yield: 57%. White solid. Mp 180e181 ꢁC. ½a D25
ꢀ87 (c 0.38 CHCl3).
ꢃ
IR (KBr)
NMR (CDCl3)
n
1775, 1765 (C]O), 1601 (N]N), 1560, 1483 (C]C). 1H
d
/ppm 7.83 (s, 1H, triazoleeH), 6.64 (s, 1H, 5-H), 6.59
(s, 1H, 8-H), 6.32 (s, 2H, 20,60-H), 6.03 (d, 1H, J ¼ 1.1 Hz, OCH2O), 6.03
(d, 1H, J ¼ 1.1 Hz, OCH2O), 4.83 (d, 1H, J ¼ 4.9 Hz), 4.45 (t, 1H,
J ¼ 7.2 Hz), 4.09 (m, 1H), 4.00 (q, 2H, J ¼ 7.1 Hz, NCH2), 3.83 (s, 6H,
OCH3), 3.82 (s, 3H, OCH3), 3.50 (q, 2H, J ¼ 7.1 Hz, NCH2), 3.11e3.20
Acknowledgment
(m, 2H), 1.38 (t, 3H, J ¼ 7.1 Hz, CH3), 1.22 (t, 3H, J ¼ 7.1 Hz, CH3). 13
C
NMR (CDCl3) d 19.1, 36.4, 37.0, 43.6, 44.9, 45.0, 56.3, 60.7, 67.6, 102.1,
This work was supported by the National Natural Science
Foundation of China (No. 30873363), the Great Program of Science
Foundation of Tianjin (09ZCKFNC01200) and Program of Science
Foundation of Tianjin (08JCYBJC070000).
108.2, 108.8, 110.6, 124.1, 126.0, 133.3, 134.2, 137.5, 143.3, 148.2,
149.6, 152.8, 159.8, 172.9. HR-ESI-MS calculated for C29H33N4O8
[M þ H]þ 565.2293, found 565.2279.
4.4.12. Synthesis of 4b-[5-(4-methylphenylamino-carbonyl)-1,2,3-
triazole]-podophyllotoxin (3j2) by the traditional 1,3-dipolar
References
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