Synthesis and evaluation of novel podophyllotoxin analogs

Article abstract of DOI:10.1016/j.bmcl.2012.05.033

Because prior studies have shown inconsistency between structure-activity relationships for podophyllotoxin derivatives as topoisomerase II inhibitors and cytotoxic agents, eight novel podophyllotoxin analogs were synthesized to further explore the effects of structural variations on both A and D rings on activity. The new compounds contain a 4,5-dimethoxy substituted A ring and opened D-ring variants and were prepared by appropriate functional and stereochemical operations at the methylenedioxy group, C7, C8, and C8′. Four compounds (15, 18, 21 and 22) demonstrated noticeable inhibitory activity against A549, DU145, KB and KBvin tumor cells, and the most active compound 18 showed IC₅₀ values less than 10 μg/mL.

Full text of DOI:10.1016/j.bmcl.2012.05.033

Bioorganic & Medicinal Chemistry Letters 22 (2012) 4293–4295
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and evaluation of novel podophyllotoxin analogs
Jie Li a,b, Hui Ming Hua b, Yan Bo Tang a, Shipeng Zhang a, Emika Ohkoshi c, Kuo-Hsiung Lee c,d, Zhi yan Xiao a,
⇑
a Beijing Key Laboratory of Active Substance Discovery and Druggability Evaluation, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union
Medical College, Beijing 100050, China
b Key Laboratory of Structure-Based Design and Discovery, Ministry of Education, Shenyang Pharmaceutical University, Shenyang 110016, China
c Natural Products Research Laboratories, UNC Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, NC 27599-7568, USA
d Chinese Medicine Research and Development Center, China Medical University and Hospital, Taichung, Taiwan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 23 February 2012
Revised 2 May 2012
Accepted 8 May 2012
Available online 15 May 2012
Because prior studies have shown inconsistency between structure–activity relationships for podophyl-
lotoxin derivatives as topoisomerase II inhibitors and cytotoxic agents, eight novel podophyllotoxin ana-
logs were synthesized to further explore the effects of structural variations on both A and D rings on
activity. The new compounds contain a 4,5-dimethoxy substituted A ring and opened D-ring variants
and were prepared by appropriate functional and stereochemical operations at the methylenedioxy
group, C7, C8, and C80. Four compounds (15, 18, 21 and 22) demonstrated noticeable inhibitory activity
against A549, DU145, KB and KBvin tumor cells, and the most active compound 18 showed IC50 values
Keywords:
Podophyllotoxin
Structural modification
Cytotoxicity
less than 10 lg/mL.
Ó 2012 Elsevier Ltd. All rights reserved.
The natural lignan podophyllotoxin (1) has been the focus of
extensive chemical modification and biological investigation in re-
cent decades. In particular, the discovery of the semi-synthetic
anticancer drugs etoposide and teniposide has stimulated
prolonged research interest in this structural phenotype.
believed to be essential for Topo II inhibition.1 However, podophyl-
lotoxin derivatives 2 and 3, which lack the trans-lactone D ring,
showed significant cytotoxicity against various tumor cell lines.2,3
Furthermore, although the A-ring modified derivatives 4 and 5
were only weak inhibitors of Topo II catalytic activity, they
NO2
NO2
O
O
R
OH
5
8
COOCH3
HN
HN
O
7
A
O
D
N
N
HO
HO
O
4
8'
O
O
O
OCH3
O
H3CO
OCH3
OCH3
O
H3CO
OCH3
OCH3
4'
2
3
R = CHO
R =
H3CO
OCH3
H3CO
OH
OH
CH2CH3
N
1
4
5
Two alternative molecular mechanisms are generally involved
in the antineoplastic activity of podophyllotoxin analogs: prevent-
ing the assembly of tubulin into microtubules and inhibiting the
catalytic activity of DNA topoisomerase II (Topo II). As the primary
mechanism for therapeutically useful podophyllotoxin analogs,
Topo II inhibition has been the major focus for previous struc-
ture-activity relationship (SAR) studies, and intact A/D rings are
inhibited KB cell growth at sub-micromolar concentrations.4 These
results implied conflicting SAR for Topo II inhibition and
cytotoxicity, and supported further SAR exploration on various
molecular areas of the structural phenotype, particularly the A
and D rings.
Accordingly, we synthesized a series of novel podophyllotoxin
analogs with structural variations on both A and D rings (15–22).
These new analogs feature 4,5-dimethoxy substitution as well as
structural alterations at C7, C8, and C80. To investigate the effects
of C80 stereochemistry on cytotoxicity, D-ring variants with
opposite chirality at C80 were deliberately incorporated. We report
⇑
Corresponding author.
E-mail address: xiaoz@imm.ac.cn (Z.y. Xiao).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.05.033

4294
J. Li et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4293–4295
herein the synthesis,5 structural characterization, and preliminary
biological evaluation of these novel podophyllotoxin analogs.
The key intermediate 8 was synthesized from 40-demethylepip-
odophyllotoxin (DMEP, 6) in two steps with a protocol modified
from the literature method (Scheme 1).6
provide analogs 15–19. The trans-D9,10 stereochemistry in com-
pounds 15–19 was confirmed by the measured J9,10 values (around
15.0 Hz).
Another three analogs 20–22 with b-configuration at C80 were
also prepared from 8 (Scheme 3). Aminolysis of 8 under reflux with
pyrrolidine as both reactant and solvent provided the dihydroxya-
mide 13 in good yield. Swern oxidation of 13 afforded the alde-
hyde–amide 14, and subsequent aldol condensation of 14 with
the corresponding acetophenones produced compounds 20–22.
The C80b-configuration in analogs 20–22 was achieved with a basic
reaction milieu and supported by the 1H NMR data. It has been well
recognized that C80 epimerization occurs readily under even mildly
basic conditions. In fact, C80 epimerization was previously ob-
served in the presence of 0.1 M piperidine.8 Furthermore, the
chemical shifts of H-70 in 20–22 were upfield from those in
15–19 (ꢀ4.34 vs ꢀ4.56), which is consistent with the previous
observation for etoposide and its C80b-isomer.9
As illustrated in Scheme 2, five analogs 15–19 with a-configu-
ration at C80 were prepared from 8. Oxidization with pyridinium
dichromate (PDC) and subsequent acid-catalyzed methanolysis
gave the D-ring opened variant 10. Stereoselective reduction of
the 7-carboxyl in 10 afforded compound 11. The stereoselectivity
in the reduction of 10 should be attributed to the asymmetric
environment of the Re and Si faces of the 7-carbonyl. The bulky
aromatic group and carboxylate substitution preclude hydrogen
addition from the rear face (i.e., the Re face), therefore, reduction
from the front face (i.e., the Si face) would be dominant. Under
Swern conditions, oxidation and dehydration of 11 occurred
simultaneously to yield the unsaturated aldehyde 12.2,7 Com-
pound 12 was reacted with the appropriate acetophenones in
the presence of p-toluenesulfonic acid (p-TsOH) as catalyst to
Analogs 15–22 were evaluated for their inhibitory activity
against the growth of tumor cell lines with an SRB assay. Four
OH
OH
OH
HO
HO
H3CO
O
O
O
O
O
H3CO
a
b
O
O
O
H3CO
OCH3
H3CO
OCH3
H3CO
OCH3
OH
OH
OCH3
7
8
DMEP 6
Scheme 1. Reagents conditions: (a): (i) BCl3/CH2Cl2, 0 oC, 6 h; (ii) acetone–water–CaCO3, reflux, 3 h; (b): CH3I, K2CO3, Et4NF, acetone, rt, 24 h; (yield 82% for two steps).
OH
O
O
OH
OH OH
O
O
H3CO
H3CO
H3CO
H3CO
H3CO
H3CO
H3CO
H3CO
H3CO
H3CO
H
OCH3
O
O
OCH3
OCH3
d
a
c
b
O
O
O
O
H3CO
OCH3
OCH3
8
H3CO
OCH3
OCH3
9
H3CO
OCH3
OCH3
H3CO
OCH3
OCH3
11
H3CO
OCH3
OCH3
12
10
O
R
NO2
15 R =
H3CO
H3CO
OH
18 R =
19 R =
e
OCH3
16 R =
17 R =
O
H3CO
OCH3
OCH3
15-19
Scheme 2. Reagents and conditions: (a): PDC/CH2Cl2, rt, 2 h, 51%; (b): H2SO4/CH3OH, reflux, 2 h, 55%; (c): NaBH4/CH3OH, rt, 0.5 h, 90%; (d): (COCl)2, DMSO, Et3N, CH2Cl2, ꢁ65
to ꢁ70 oC, 74%; (e): corresponding acetophenones, p-TsOH, DCM, reflux, 2–5 d.
O
O
OH
OH OH
O
O
R
H3CO
H3CO
H3CO
H3CO
H3CO
H3CO
H3CO
H3CO
O
20 R =
21 R =
H
N
O
N
N
b
c
a
O
O
O
H3CO
OCH3
OCH3
H3CO
OCH3
OCH3
13
H3CO
OCH3
OCH3
14
H3CO
OCH3
OCH3
20-22
22 R =
8
Scheme 3. Reagents and conditions: (a): pyrrolidine, reflux, 2 h; (b): (COCl)2, DMSO, Et3N, CH2Cl2, ꢁ65 to ꢁ70 oC, 66%; (c): corresponding acetophenones, p-TsOH, DCM,
reflux, 2–5 d, 20–50%.

J. Li et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4293–4295
4295
Table 1
corresponding target molecules. Compound 15: Yield: 16.9%; mp: 100ꢁ102 °C;
½ ꢂ
a 2D0 +322.4 (c, 0.55, CHCl3); HR-ESI-MS: m/z 590.2011 [M+H]+ (calcd 590.2021);
Inhibitory activity of selected analogs against A549, DU145, KB and KBvin tumor cell
lines
1H NMR (300 MHz, CDCl3): d 8.31 (d, 2H, J = 8.4 Hz, –ArH), 8.08 (d, 2H, J = 8.4 Hz,
–ArH), 7.68 (d, 1H, J = 15.6 Hz, 9-H), 7.10 (d, 1H, J = 15.6 Hz, 10-H), 7.07 (s, 1H, 7-
H), 6.85 (s, 1H, 6-H), 6.62 (s, 1H, 3-H), 6.59 (s, 2H, 20, 60-H), 4.57 (d, 1H, J = 6.9 Hz,
70-H), 3.73ꢁ3.93 (16H, 5ꢃOCH3, 80-H), 3.50 (s, 3H, –COOCH3). Compound 16:
Compound
IC50
(
l
g/mL)
A549
DU145
KB
KBvin
Yield: 55.1%; mp: 107ꢁ109 °C; ½a D20
ꢂ
+265.2 (c, 0.7, CHCl3); HR-ESI-MS: m/z
627.2928 [M+H]+ (calcd 627.2952); 1H NMR (300 MHz, CDCl3): d 7.90 (d, 2H,
J = 8.1 Hz, ꢁArH), 7.62 (d, 1H, J = 15.6 Hz, 9-H), 7.29 (d, 2H, J = 8.1 Hz, ꢁArH),
7.17 (d, 1H, J = 15.3 Hz, 10-H), 6.99 (s, 1H, 7-H), 6.83 (s, 1H, 6-H), 6.60 (s, 3H, 20,
60-H, 3-H), 4.56 (d, 1H, J = 7.5 Hz, 70-H), 3.72ꢁ3.93 (16H, 5ꢃOCH3, 80-H), 3.49 (s,
3H, –COOCH3), 2.56 (m, 1H, –CH–), 1.26ꢁ1.86 (m, 10H, –(CH2)5–). Compound 17:
15
18
21
16.8 1.32
6.79 0.76
12.0 0.92
24.6 2.02
0.113 0.014
20.3 0.61
5.84 0.91
12.8 2.15
15.46 1.19
16.0 1.66
23.0 0.35
5.17 0.96
12.29 2.36
16.9 0.71
2.10 0.378
5.90 1.13
12.28 1.84
16.6 3.87
0.819 0.162
22
GL-331*
0.800 0.056
Yield: 9.2%; mp: 95ꢁ97 oC; ½a D20
ꢂ
+260 (c, 0.5, CHCl3); HR-ESI-MS: m/z 601.2780
7.91 (d, 2H,
[M+H]+ (calcd 601.2796); 1H NMR (300 MHz, acetone-d6):
d
*
GL-331 is a podophyllotoxin analog that previously reached clinical trials.1
J = 8.1 Hz, –ArH), 7.60 (d, 1H, J = 15.3 Hz, 9-H), 7.33 (d, 2H, J = 8.1 Hz, –ArH), 7.24
(d, 1H, J = 15.3 Hz, 10-H), 7.16 (s, 1H, 7-H), 7.02 (s, 1H, 6-H), 6.76 (s, 2H, 20, 60-H),
6.67 (s, 1H, 3-H), 4.56 (d, 1H, J = 7.1 Hz, 70-H), 3.99 (d, 1H, J = 7.1 Hz, 80-H),
3.66ꢁ3.85 (15H, 5ꢃOCH3), 3.32 (s, 3H, –COOCH3), 2.56 (d, 1H, J = 7.1 Hz, –CH–),
1.91 (m, 1H, –CH–), 0.99 (s, 3H, CH3), 0.96 (s, 3H, CH3). Compound 18: Yield:
compounds (15, 18, 21, and 22) demonstrated noticeable inhibi-
tory activity against A549, DU145, KB and KBvin tumor cells, and
the most active compound 18 exhibited IC50 values less than
41.7%; mp: 126ꢁ129 °C; ½a D20
ꢂ
+260.9 (c, 0.7, CHCl3); HR-ESI-MS: m/z 561.2132
[M+H]+ (calcd. 561.2119); 1H NMR (300 MHz, CDCl3): d 7.93 (d, 2H, J = 8.4 Hz, –
ArH), 7.62 (d, 1H, J = 15.3 Hz, 9-H), 7.16 (d, 1H, J = 15.6 Hz, 10-H), 6.99 (s, 1H, 7-
H), 6.88 (d, 2H, J = 9.0 Hz, –ArH), 6.83 (s, 1H, 6-H), 6.60 (s, 3H, 20, 60-H, 3-H), 4.56
(d, J=7.2 Hz, 1H, 70-H), 3.87 (s, 1H, 80-H), 3.72ꢁ3.92 (15H, 5ꢃOCH3), 3.49 (s, 3H, -
10
lg/mL (Table 1).
In summary, a series of novel podophyllotoxin analogs featuring
COOCH3). Compound 19: Yield: 13.7%; mp: 124ꢁ126 °C; ½a D20
ꢂ
+311.1 (c, 0.3,
4,5-dimethoxy substitution and an opened D ring were synthe-
sized and evaluated for cytotoxic activity. In contrast to previous
SAR deduced from Topo II inhibition, which requires intact A and
D rings for retention of activity, analogs with modified A and D
rings, such as 18, exhibited evident in vitro anticancer activity.
CHCl3); HR-ESI-MS: m/z 621.2471 [M+H]+ (calcd 621.2483); 1H NMR (300 MHz,
CDCl3): d 8.05 (d, 2H, J = 8.4 Hz, –ArH), 7.45ꢁ7.69 (m, 5H, –ArH), 7.63 (d, 2H,
J = 8.7 Hz, –ArH), 7.47 (d, 1H, J = 14.4 Hz, 9-H), 7.22 (d, 1H, J = 15.0 Hz, 10-H),
7.03 (s, 1H, 7-H), 6.85 (s, 1H, 6-H), 6.61 (s, 3H, 20, 60-H, 3-H), 4.57 (d, 1H,
J = 6.9 Hz, 70-H), 3.73ꢁ3.93 (16H, 5ꢃOCH3, 80-H), 3.51 (s, 3H, -COOCH3).
20
Compound 20: Yield: 10.4%; mp: 110ꢁ113 °C ; [
a
]
D
-244.4 (c, 0.6, CHCl3);
HR-ESI-MS: m/z 628.2564 [M+H]+ (calcd. 649.2541); 1H NMR (300 MHz, CDCl3):
d 7.55 (d, 1H, J = 15.6 Hz, 9-H), 7.43 (dd, 1H, J = 8.4; 1.5 Hz, –ArH), 7.36 (d, 1H,
J = 1.5 Hz, –ArH), 7.08 (s, 1H, 7-H), 6.82 (d, 1H, J = 9.0 Hz, –ArH), 6.81 (s, 1H, 6-H),
6.74 (d, 1H, J = 15.6 Hz, 10-H), 6.55 (s, 1H, 3-H), 6.46 (s, 2H, 20, 60-H), 6.04 (s, 2H,
-OCH2O-), 4.34 (d, 1H, J = 6.3 Hz, 70-H), 3.96 (d, 1H, J = 6.3 Hz, 80-H), 3.78ꢁ3.91
(15H, 5ꢃOCH3), 3.32ꢁ3.56 (m, 4H, –N(CH2)2–); 1.87ꢁ2.04 (m, 4H, –(CH2)2–).
Acknowledgments
This investigation was supported by the National S&T Major
Project (No. 2009ZX09301-003) and also in part by the Taiwan
Department of Health, China Medical University Hospital Cancer
Research Center of Excellence (DOH100-TD-C-111-005).
Compound 21: Yield: 10.0%; mp: 113ꢁ115 °C; ½a D20
ꢁ488.9 (c, 0.3, CHCl3); HR-
ꢂ
ESI-MS: m/z 666.3406 [M+H]+ (calcd 666.3425); 1H NMR (300 MHz, CDCl3): d
7.77 (d, 2H, J = 8.4 Hz, –ArH), 7.56 (d, 1H, J = 15.9 Hz, 9-H), 7.27 (d, 2H, J = 8.1 Hz,
–ArH), 7.08 (s, 1H, 7-H), 6.81 (s, 1H, 6-H), 6.78 (d, 1H, J = 16.2 Hz, 10-H), 6.56 (s,
1H, 3-H), 6.46 (s, 2H, 20, 60-H), 4.33 (d, 1H, J = 6.0 Hz, 70-H), 3.97 (d, 1H, J = 6.0 Hz,
80-H), 3.78ꢁ3.91 (15H, 5ꢃOCH3), 3.31ꢁ3.59 (m, 4H, –N(CH2)2–), 2.56 (m, 1H, –
CH(CH2)2–), 1.79ꢁ1.99 (m, 8H, –(CH2)2–; –CH(CH2)2–), 1.33ꢁ1.41 (m, 6H, –
References and notes
1. Cragg, G. M.; Kingston, D. G. I.; Newman, D. J. Anticancer Agents from Natural
Products; CRC Press Taylor & Francis Group: London UK, 2005. 1st ed., pp. 71.
2. Gordaliza, M.; Castro, M. A.; Miguel del Corral, J. M.; López-Vázquez, M. L.;
García, P. A.; Feliciano, A. S.; García-Grávalos, M. D.; Broughton, H. Tetrahedron
1997, 53, 15743.
3. Castro, M. A.; MigueldelCorral, J. M.; Gordaliza, M.; García, P.; Gómez-Zurita, M.
A.; García-Grávalos, M. D.; de la Iglesia-Vincente, J.; Gajate, C.; An, F.; Mollinedo,
F.; Feliciano, A. S. J. Med. Chem. 2004, 47, 1214.
(CH2)3–). Compound 22: Yield: 21.2%; mp: 115ꢁ117 °C; ½a D20
ꢁ358.8 (c, 0.55,
ꢂ
CHCl3); HR-ESI-MS: m/z 660.2937 [M+H]+ (calcd 660.2956); 1H NMR (300 MHz,
CDCl3): d 7.92 (d, 2H, J = 8.4 Hz, –ArH), 7.66 (d, 2H, J = 8.1 Hz, –ArH), 7.42ꢁ7.64
(m, 5H, –ArH), 7.47 (d, 1H, J = 14.4 Hz, 9-H), 7.11 (s, 1H, 7-H), 6.82 (d, 1H,
J = 15.9 Hz, 10-H), 6.82 (s, 1H, 6-H), 6.56 (s, 1H, 3-H), 6.47 (s, 2H, 20, 60-H), 4.35
(d, 1H, J = 6.0 Hz, 70-H), 3.99 (d, 1H, J = 6.3 Hz, 80-H), 3.79ꢁ3.91 (15H, 5ꢃOCH3),
3.34ꢁ3.60 (m, 4H, –N(CH2)2–); 1.84ꢁ2.00 (m, 4H, –(CH2)2–).
6. Wang, Z. Q.; Hu, H.; Chen, H. X.; Cheng, Y. C.; Lee, K. H. J. Med. Chem. 1992, 35,
871.
4. Cho, S. J.; Kashiwada, Y.; Bastow, K. F.; Cheng, Y. C.; Lee, K. H. J. Med. Chem. 1996,
39, 1396.
5. General Preparation of Compounds 15ꢁ22: To a solution of 12 or 14 (0.25 mmol)
in 15 mL of anhydrous CH2Cl2 were added p-TsOH (0.15 mmol) and the
corresponding acetophenones (0.5 mmol). The reaction mixture was stirred at
room temperature for 2 to 5 days and then washed with 5% NaHCO3 and
saturated aqueous NaCl. The organic layer was dried over Na2SO4 and
concentrated. The residue was chromatographed on silica gel and afforded the
7. Mancuso, A. J.; Swern, D. Synthesis 1981, 165.
8. Gensler, W. J.; Gatsons, C. D. J. Org. Chem. 1966, 31, 3224.
9. Aso, Y.; Hayashi, Y.; Yoshioka, S.; Takeda, Y.; Kita, Y.; Nishimura, Y.; Arata, Y.
Chem. Pharm. Bull. 1989, 37, 422.