Design and synthesis of novel cytotoxic podophyllotoxin derivatives

Article abstract of DOI:10.1016/j.cclet.2010.03.040

In order to investigate the effect of different C4 linkage moieties on the cytotoxicity of podophyllotoxin derivatives, novel 4-N- and 4-C-substituted 4'-O-demethylepipodophyllotoxin derivatives were designed and synthesized. All the compounds were tested against A549 and MCF-7 tumor cells in vitro, and six compounds showed significant cytotoxicity. The most active compound 9f was superior to GL-331, and exhibited potent cytotoxicity with IC₅₀ value at 10^-7mol/L level.

Full text of DOI:10.1016/j.cclet.2010.03.040

Available online at www.sciencedirect.com
Chinese Chemical Letters 21 (2010) 1153–1156
www.elsevier.com/locate/cclet
Design and synthesis of novel cytotoxic podophyllotoxin derivatives
Wen Li Xi a, Qian Cai a, Yan Bo Tang b, Hua Sun b, Zhi Yan Xiao b,
*
a School of Pharmacy, Liaoning University of Traditional Chinese Medicine, Dalian 116600, China
b Key Laboratory of Bioactive Substances and Resources Utilization of Chinese Herbal Medicine, Ministry of Education & Institute of Materia
Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, China
Received 25 January 2010
Abstract
In order to investigate the effect of different C4 linkage moieties on the cytotoxicity of podophyllotoxin derivatives, novel 4-N-
and 4-C-substituted 40-O-demethylepipodophyllotoxin derivatives were designed and synthesized. All the compounds were tested
against A549 and MCF-7 tumor cells in vitro, and six compounds showed significant cytotoxicity. The most active compound 9f was
superior to GL-331, and exhibited potent cytotoxicity with IC50 value at 10ꢀ7 mol/L level.
# 2010 Zhi Yan Xiao. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.
Keywords: Podophyllotoxin; C4-derivation; Cytotoxicity
Podophyllotoxin 1 is a bioactive lignan isolated from the roots of Podophyllum peltatum L (Fig. 1). Although the
therapeutic application of podophyllotoxin is limited to topical use due to its high toxicity, its semisynthetic
derivatives Etoposide 2 and Teniposide 3 have been widely used as important anticancer drugs in clinic, which has
prompted extensive structural modification, particularly at the C4 position of podophyllotoxin. As highlights, C4
derivatives, GL-331 4 and TOP-53 5, have displayed unique antitumor spectra and reached clinical trials [1].
Previous structure–activity relationship (SAR) indicated that bulky groups are well-tolerated at C4 and the moiety
immediate to C4 would affect the antitumor spectra of podophyllotoxin derivatives significantly [1]. Although former
modification has provided potent derivatives with both 4-N- (e.g. GL-331) and 4-C- (e.g. TOP-53) substitution, the
effect of different C4 linkages on the pharmacological profiles of 1-derivatives is still to be clarified. Accordingly, we
designed two series of novel derivatives with C4 linkage units of either p-aminobenzamido or amido, which were
extended with bulky tails to optimize the antitumor activity and incorporated with tertiary amino groups to improve the
bioavailability profile of the target compounds.
40-Demethylepipodophyllotoxin (DMEP, 6) was synthesized from 1 stereoselectively through successive 40-
demethylation, 4-iodination, and nucleophilic substitution [2]. The intermediates 7 and 8 were prepared from DMEP
as previously reported [3,4]. Briefly, DMEP was subjected to nucleophilic displacement by p-aminobenzoic acid and
trimethylsilyl cyanide to provide 7 and 11 respectively, and 11 was then readily hydrolyzed to the carboxylic acid 8. 7
and 8 were subsequently condensed with appropriate amines in the presence of 2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-
tetramethyluronium hexafluorophosphate (HATU) and N,N-diisopropylethylamine (DIEA) to yield target compounds
* Corresponding author.
E-mail address: xiaoz@imm.ac.cn (Z.Y. Xiao).
1001-8417/$ – see front matter # 2010 Zhi Yan Xiao. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.
doi:10.1016/j.cclet.2010.03.040

1[(ig._1)TD$FIG] 154
W.L. Xi et al. / Chinese Chemical Letters 21 (2010) 1153–1156
Fig. 1. Structures of Podophyllotoxin 1, Etoposide 2, Teniposide 3, GL-331 4, and TOP-53 5.
[(Schme_1)TD$FIG]
Scheme 1. Conditions and reagents: (a) MeSO3H/NaI; CH2Cl2, then H2O/BaCO3; acetone, 57%; (b) MeSO3H/NaI; CH2Cl2, then p-aminobenzoic
acid, BaCO3, THF, 30%; (c) BF3ꢁEt2O/Me3SiCN/CHCl3, 57%; (d) AcOH/HCl/H2O, 78%; and (e) HATU, DIEA, CH2Cl2, RNH2.
Table 1
Data of compounds 9a–f and 10a–h.
20
½aꢂD (CHCl3)
No.
Yield%
Mp (8C)
9a
9b
35.9
87.3
76.4
72.7
35.6
74.8
62.6
79.3
96.7
59.3
44.1
64.5
10.3
53.8
186–189
168–169
157–159
165–168
193–195
241–243
165–167
124–127
148–152
182–185
177–179
190–191
161–163
228–231
ꢀ119 (c, 0.05)
ꢀ232 (c, 0.05)
ꢀ127 (c, 0.05)
ꢀ67 (c, 0.05)
ꢀ70 (c, 0.05)
ꢀ196 (c, 0.05)
ꢀ17 (c, 0.05)
ꢀ67 (c, 0.05)
ꢀ119 (c, 0.1)
ꢀ70 (c, 0.1)
9c
9d
9e
9f
10a
10b
10c
10d
10e
10f
10g
10h
ꢀ132 (c, 0.1)
ꢀ104 (c, 0.1)
ꢀ96 (c, 0.1)
ꢀ104 (c, 0.05)

W.L. Xi et al. / Chinese Chemical Letters 21 (2010) 1153–1156
1155
9 and 10 (Scheme 1). All the 14 compounds are novel structures and their physical and spectral data are elaborated in
Tables 1 and 2. In particular, the C4-configuration of 6, 7 and 11 was deduced from the reaction mechanism as well as
evidences from NMR data. It is generally accepted that the nucleophilic substitution occurred at C4 follows an SN1
mechanism, and due to the steric effect posed by the bulky C1-substituent, C4b-configuration is usually preferred
regardless of the reacting configuration in C4a or C4b [5]. It was also previously observed that C4a- and C4b-
derivatives present different J3,4 values. The C4b-substituted compounds show a J3,4 around 4.0 Hz, whereas the
Table 2
Spectral date of compounds 9a–f and 10a–h.
No.
1H NMR (300 MHz, d) and HR-ESI-MS (m/z)
9a
CDCl3: 7.76 (d, 2H, J = 8.7 Hz), 7.62 (s, 1H), 7.53 (d, 2H, J = 8.7 Hz), 6.94 (d, 2H, J = 7.8 Hz), 6.77 (s, 1H),
6.60 (d, 2H, J = 8.4 Hz), 6.55 (s, 1H), 6.33 (s, 2H), 5.98 (d, 2H, J = 6.0 Hz), 5.43 (s, 1H), 4.76–4.84 (m, 1H),
4.61 (d, 1H, J = 4.2 Hz), 4.40 (t, 1H, J = 7.8 Hz), 4.26 (d, 1H, J = 6.0 Hz), 3.88–3.96 (m, 5H), 3.82 (s, 6H),
3.09–3.15 (m, 6H); 680.2620 [M+H]+ (calcd. 680.2608)
9b
9c
CDCl3: 7.67 (d, 2H, J = 8.4 Hz), 6.75 (s, 1H), 6.66 (s, 1H), 6.56 (d, 2H, J = 8.7 Hz), 6.53 (s, 1H), 6.32 (s, 2H),
5.97 (d, 2H, J = 4.2 Hz), 4.74–4.77 (m, 1H), 4.60 (d, 1H, J = 4.5 Hz), 4.39 (t, 1H, J = 7.2 Hz), 4.25 (d, 1H, J = 6.3 Hz),
3.94 (t, 1H, J = 10.2 Hz), 3.80 (s, 6H), 3.72 (m, 4H), 3.51–3.57 (m, 2H), 3.00–3.14 (m, 2H), 2.59–2.63 (m, 2H),
2.52 (m, 4H); 632.2612 [M+H]+ (calcd. 632.2603)
CDCl3: 7.68 (d, 2H, J = 8.1 Hz), 6.87 (s, 1H), 6.76 (s, 1H), 6.52–6.57 (m, 3H), 6.32 (s, 2H), 5.96 (d, 2H, J = 3.0 Hz),
4.72–4.78 (m, 1H), 4.59 (d, 1H, J = 4.2 Hz), 4.38 (t, 1H, J = 8.1 Hz), 4.23 (d, 1H, J = 5.7 Hz), 3.96 (t, 1H, J = 9.6 Hz),
3.78 (s, 6H), 3.44–3.52 (m, 2H), 2.92–3.14 (m, 2H), 2.54–2.58 (t, 2H, J = 5.4 Hz), 2.45 (s, 4H), 1.38–1.66
(m, 6H); 630.2801 [M+H]+ (calcd. 630.2810)
9d
9e
CDCl3: 7.64 (d, 2H, J = 8.1 Hz), 7.30–7.33 (m, 5H), 6.75 (s, 1H), 6.53–6.56 (m, 3H), 6.33 (s, 2H), 5.97 (d, 2H, J = 6.0 Hz),
5.87 (d, 1H, J = 7.5 Hz), 5.30 (s, 1H), 4.75 (m, 1H), 4.60 (d, 1H, J = 3.0), 4.36 (m, 1H), 4.22 (d, 1H, J = 5.7 Hz),
3.79 (s, 6H), 3.56 (s, 2H), 3.52 (m, 1H), 2.90–3.08 (m, 2H), 1.96–2.90 (m, 8H); 692.2978 [M+H]+ (calcd. 692.2966)
CDCl3: 7.76 (d, 2H, J = 8.1 Hz), 7.64 (s, 1H), 7.47 (d, 2H, J = 8.4 Hz), 6.78 (s, 1H), 6.76 (d, 2H, J = 9.0),
6.60 (d, 2H, J = 8.1 Hz), 6.55 (s, 1H), 6.34 (s, 2H), 5.99 (d, 2H, J = 7.8 Hz), 5.31 (s, 1H), 4.79 (s, 1H),
4.60 (d, 1H, J = 3.6 Hz), 4.32–4.41 (m, 2H), 3.95 (t, 1H, J = 9.6 Hz), 3.82 (s, 6H), 3.10–3.16 (m, 2H),
2.95 (s, 6H); 638.2488 [M+H]+ (calcd. 638.2497)
9f
DMSO-d6: 8.31 (s, 1H), 7.99 (t, 1H, J = 5.4 Hz), 7.63 (d, 2H, J = 8.7 Hz), 6.77 (s, 1H), 6.70 (d, 2H, J = 8.7 Hz),
6.56 (s, 1H), 6.25 (s, 2H), 5.98 (d, 2H, J = 9.9 Hz), 4.92–4.96 (m, 1H), 4.51 (d, 1H, J = 4.8 Hz), 4.36 (t, 1H, J = 8.1 Hz),
3.63–3.67 (m, 7H), 3.23–3.28 (m, 3H), 3.00–3.10 (m, 1H), 2.37 (t, 2H, J = 7.2 Hz), 2.16 (s, 6H); 590.2490 [M+H]+
(calcd. 590.2497)
10a
10b
10c
10d
10e
10f
DMSO-d6: 10.04 (s, 1H), 8.17 (s, 1H), 7.41 (d, 2H, J = 8.1 Hz), 7.01 (s, 1H), 6.57 (s, 1H), 6.89 (d, 2H, J = 8.1 Hz),
5.99 (s, 2H), 5.98 (s, 2H), 4.44–4.46 (m, 2H), 4.22 (d, 1H, J = 7.5 Hz), 3.94 (d, 1H, J = 9.9 Hz), 3.72 (m, 4H),
3.41 (s, 6H), 2.86 (m, 4H), 2.86–2.90 (m, 2H); 589.2191 [M+H]+ (calcd. 589.2181)
CDCl3: 7.11 (s, 1H), 6.47 (s, 1H), 6.16 (s, 1H), 6.08 (s, 2H), 5.95 (d, 2H, J = 4.5 Hz), 4.39–4.45 (m, 1H),
4.33 (d, 1H, J = 5.1 Hz), 4.14 (d, 1H, J = 9.6 Hz), 3.87 (d, 1H, J = 7.8 Hz), 3.76 (s, 6H), 3.73 (s, 2H), 3.70 (m, 2H),
3.11–3.45 (m, 3H), 2.85 (dd, 1H, J = 4.8, 12.6 Hz), 2.45–2.51 (m, 6H); 541.2196 [M+H]+ (calcd. 541.2181)
CDCl3: 7.08 (s, 1H), 6.60 (s, 1H), 6.48 (s, 1H), 6.11 (s, 2H), 5.93 (d, 2H, J = 2.1 Hz), 5.30 (s, 1H), 4.39–4.45 (m, 1H),
4.28 (d, 1H, J = 5.1 Hz), 4.13 (d, 1H, J = 9.9 Hz), 3.88 (d, 1H, J = 7.8 Hz), 3.76 (s, 6H), 3.15–3.36 (m, 3H),
2.88 (dd, 1H, J = 5.1, 12.6 Hz), 2.38–2.70 (m, 6H), 1.40–1.64 (m, 6H); 539.2390 [M+H]+ (calcd. 539.2393)
CDCl3: 7.32 (m, 5H), 7.07 (s, 1H), 6.39 (s, 1H), 6.06 (s, 2H), 5.93 (s, 3H), 4.32–4.35 (m, 1H), 4.14 (d, 1H, J = 4.2 Hz),
4.04 (d, 1H, J = 10.2 Hz), 3.82 (d, 1H, J = 8.1 Hz), 3.73 (s, 6H), 3.52 (s, 2H), 3.17–3.23 (m, 1H), 2.79–2.85 (m, 2H),
2.70 (dd, 1H, J = 4.2, 12.0 Hz),1.42–2.12 (m, 7H); 601.2547 [M+H]+ (calcd. 601.2544)
CDCl3: 7.71 (s, 1H), 7.35 (d, 2H, J = 8.4 Hz), 7.10 (s, 1H), 6.75 (d, 2H, J = 8.1 Hz), 6.43 (s, 1H), 6.08 (s, 2H), 5.96 (s, 2H),
5.38 (s, 1H), 4.42–4.47 (m, 1H), 4.35 (d, 1H, J = 4.5 Hz), 4.19 (d, 1H, J = 10.2 Hz), 3.88 (d, 1H, J = 7.8 Hz), 3.60 (s, 6H),
3.21–3.30 (m, 1H), 2.84–2.93 (m, 7H); 547.2077 [M+H]+ (calcd. 547.2075)
CDCl3: 7.10 (s, 1H), 6.47 (s, 1H), 6.44 (s, 1H), 6.08 (s, 2H), 5.94 (d, 2H, J = 4.8 Hz), 4.40–4.45 (m, 1H),
4.27 (d, 1H, J = 4.5 Hz), 4.12 (d, 1H, J = 9.9 Hz), 3.85 (d, 1H, J = 7.8 Hz), 3.74 (s, 6H), 3.35–3.37 (m, 1H),
3.19–3.27 (m, 2H), 2.86 (dd, 1H, J = 5.1, 12.9 Hz), 2.38–2.46 (m, 2H), 2.28 (s, 6H); 499.2083 [M+H]+ (calcd. 499.2075)
CDCl3: 8.45 (s, 1H), 7.41 (d, 2H, J = 7.8 Hz), 7.14 (d, 2H, J = 8.1 Hz), 7.06 (s, 1H), 6.33 (s, 1H), 6.02 (s, 2H),
5.96 (d, 2H, J = 10.2 Hz), 5.37 (s, 1H), 4.40–4.43 (m, 1H), 4.27 (d, 1H, J = 4.5 Hz), 4.14 (d, 1H, J = 9.9 Hz),
3.87 (d, 1H, J = 7.8 Hz), 3.53 (s, 6H), 3.20–3.30 (m, 1H), 2.76 (dd, 1H, J = 4.5, 12.0 Hz), 1.30–2.52 (m, 11H); 586.2449 [M+H]+
(calcd. 586.2435)
10g
10h
CDCl3: 7.16–7.35 (m, 5H), 7.08 (s, 1H), 6.40 (s, 1H), 6.03 (s, 2H), 5.93 (d, 2H, J = 4.5 Hz), 5.70 (t, 1H, J = 4.5 Hz),
5.39 (s, 1H), 4.31–4.36 (m, 1H), 4.14 (d, 1H, J = 4.2 Hz), 3.98 (d, 1H, J = 9.9 Hz), 3.81 (d, 1H, J = 7.8 Hz),
3.74 (s, 6H), 3.42–3.66 (m, 2H), 3.08–3.12 (m, 1H), 2.83 (t, 2H, J = 5.4 Hz), 2.70 (dd, 1H, J = 5.1, 12.6 Hz); 532.1968 [M+H]+
(calcd. 532.1966)

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W.L. Xi et al. / Chinese Chemical Letters 21 (2010) 1153–1156
Table 3
Cytotoxicity of selected compounds.
IC50 (10ꢀ6 mol/L)
9b
9c
9d
9e
9f
10g
4
a
A549
2.27
NAb
1.11
3.23
2.73
NAb
6.35
NAb
0.71
0.92
NAb
8.15
3.54
2.13
MCF-7
a
IC50: concentration that causes a 50% reduction of cell growth.
NA: not active.
b
C4a-compounds display a J3,4 above 10.0 Hz [6]. The J3,4 values for 6, 7 and 11 are 3.3, 4.2 and 5.7 Hz, respectively.
Therefore, the C4-configuration of 6, 7 and 11 is assigned as C4b.
All compounds were tested in parallel with GL-331 4 against A549 and MCF-7 tumor cells with the MTT method
in vitro. Preliminary results indicated that compound series 9 were generally more active than series 10, and six
compounds with considerable cytotoxicity are recorded in Table 3. The most active compounds 9c and 9f were
comparable or superior to GL-331 in cytotoxicity.
In summary, based on previous SAR, two series of novel podophyllotoxin derivatives with different C4 linkage
were designed and synthesized to enhance antitumor activity and improve bioavailability. It appears that the 4b-p-
aminobenzamido series are more active than the 4b-amido series. Further analogue synthesis following this molecular
design is ongoing and will be reported in due course.
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