Synthesis and anti-HIV-1 activities of novel podophyllotoxin derivatives

Article abstract of DOI:10.1016/j.bmcl.2006.11.070

In order to explore the range of biological activities of the podophyllotoxin compound class, a novel series of derivatives of podophyllotoxin, which were conjugates containing stavudine and different structural podophyllotoxin analogues, were designed, synthesized, and evaluated for their anti-HIV-1 activities in vitro. Among these compounds, 19d and 19c showed the highest anti-HIV-1 activities with EC₅₀ values of 0.17 and 0.29 μM and TI values of 466.9 and 354.5, respectively.

Full text of DOI:10.1016/j.bmcl.2006.11.070

Bioorganic & Medicinal Chemistry Letters 17 (2007) 2091–2095
Synthesis and anti-HIV-1 activities of novel
podophyllotoxin derivatives
Shi-Wu Chen,a,c Yun-Hua Wang,b,d Yan Jin,a Xuan Tian,a,* Yong-Tang Zheng,b,*
Du-Qiang Luob and Yong-Qiang Tua,*
aState Key Laboratory of Applied Organic Chemistry, Lanzhou University, Lanzhou 730000, PR China
bLaboratory of Molecular Immunopharmacology, Kunming Institute of Zoology, Chinese Academy of Sciences,
Kunming 650223, PR China
cSchool of Pharmacy, Lanzhou University, Lanzhou 730000, PR China
dGraduate School of Chinese Academy of Sciences, Beijing 100039, PR China
Received 2 September 2006; revised 13 October 2006; accepted 7 November 2006
Available online 1 December 2006
Abstract—In order to explore the range of biological activities of the podophyllotoxin compound class, a novel series of derivatives
of podophyllotoxin, which were conjugates containing stavudine and different structural podophyllotoxin analogues, were designed,
synthesized, and evaluated for their anti-HIV-1 activities in vitro. Among these compounds, 19d and 19c showed the highest anti-
HIV-1 activities with EC50 values of 0.17 and 0.29 lM and TI values of 466.9 and 354.5, respectively.
Ó 2007 Published by Elsevier Ltd.
In the past two decades, a worldwide search has been
made for new chemotherapeutic agents targeting the hu-
man immunodeficiency virus (HIV), the causative agent
of acquired immune deficiency syndrome (AIDS). Nine-
teen drugs, including nucleoside/nucleotide viral reverse
transcriptase (RT) inhibitors (NRTIs), nonnucleoside
RT inhibitors (NNRTIs), protease inhibitors (PIs),
and fusion (or entry) inhibitors (FIs), are now approved
for clinical use in the world.1 However, these drugs have
only limited or transient clinical benefit due to their side
effects and the development of drug-resistant viral
strains.2 Therefore, current searches for new anti-HIV
agents are focused on discovering compounds with nov-
el structures and different mechanisms of action.3
testicular carcinoma, lymphoma, and Kaposi’s sarcoma
(Fig. 1).5 In addition, there are GL-331,6 NK-611,7
TOP538 and Fl 17829 in clinical trail for treatment kinds
of tumors. As to mechanism, podophyllotoxin inhibits
the assembly of tubulin into microtubules through inter-
action with protein at the colchicine binding site, pre-
venting the formation of the spindle. While etoposide
and congeners induce a premitotic blockade in late S
stage of the cell cycle because of the inhibition of
DNA topoisomerase II (Top II), an enzyme required
for the unwinding of DNA during replication.4,5 Other
than their anti-cancer activity, podophyllotoxin was
used as antiviral agent in treatment of herpes simplex
type I and condyloma acuminatum caused by human
papilloma virus and other venereal and perianal warts.10
Lee’s group recently reported their initial anti-HIV
results for modified podophyllotoxin derivatives.3,11
Podophyllotoxin (1) as well as its congeners and deriva-
tives exhibit pronounced biological activity mainly as
strong antiviral agents and as antineoplastic drugs.4
The podophyllotoxin derivatives etoposide (VP-16, 2),
teniposide (VM-26, 3) and Etopophos (etoposide phos-
phate, 4) are thus successfully utilized in treatment of
a variety of cancers, including small-cell lung cancer,
Stavudine (20,30-didehydro-20,30-dideoxy-thymidine, d4T,
5) is an anti-HIV treatment in the class of drugs called
nucleoside reverse inhibitors. However, stavudine may
cause some serious side effects, including peripheral neu-
ropathy, tingling, burning, numbness, and pain in the
hands or feet.12
Keywords: Podophyllotoxin; d4T; Anti-HIV; Totoxicity.
*
To explore the range of biological activities of the pod-
ophyllotoxin compound class, we have synthesized a
Corresponding authors. Tel.: +86 931 8912410; fax: +86 931
8912582; e-mail: xuant@lzu.edu.cn
0960-894X/$ - see front matter Ó 2007 Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2006.11.070

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S.-W. Chen et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2091–2095
R'
A
O
A
O
B
O
OH
O
6
7
9
8
O
1
2
5
4
OH
O
N
R'
R''
H
O
O
B
C
D
OH
H3C
Me
2
3
9'
O
NH
8'
7'
3
O
O
O
2'
H
O
6'
E
S
O
HO
O
O
OH
OH
MeO
3' OMe
5'
4'
OMe
Me
P
4
MeO
OMe
Podophyllotoxin
d4T
1
5
OR''
Figure 1. Podophyllotoxin, Etoposide, Teniposide, Etopophos, and Stavudine.
novel series of derivatives of podophyllotoxin, which
were conjugates containing stavudine and different
structural podophyllotoxin analogues, and evaluated
their anti-HIV-1 activities in vitro. The preliminary re-
sults showed anti-HIV-1 activities of some synthesized
compounds are more distinct compared to those of
podophyllotoxin.
Preparation of 7-azido-compounds 11–14 and 17 was
according to the literature.17 In the presence of Lewis
acid, such as boron trifluoride etherate (BF3ÆEt2O), tri-
fluoroacetic acid (TFA), the reaction of the SN1 nucleo-
philic substitutions occurred, 7b-substituents were
major products. However, the main products resulting
from the reaction of 7-bromo-podophyllotoxin and its
40-O-demethyl compounds with sodium azide (NaN3)
were 7a-azido derivatives.
Compound 9 was synthesized as shown in Scheme 1, the
thymidine 6 was dimesylated and treated with aqueous
sodium hydroxide to afford 8 in 70% yield. Upon treat-
ment with sodium hydrogenate (NaH) in DMF, this yield-
ed 5 in good yield.13 The latter was converted into its
activated derivative 9 by treatment with p-nitrophenyl-
chloroformate in the presence of pyridine (Scheme 1).14
Further, the 7-amino compounds 18a–e were prepared
by reduction of the azides 11–14 and 17 using Pd as a
catalyst. Finally, introduction of the carbamate chains
was performed by reacting 9 with the appropriate
amines 18a–e in the presence of triethylamine, affording
compounds 19a–e (Scheme 3).14
Preparation of 7-azido-compounds 11–14 and 17 is
shown in Scheme 2. Podophyllotoxin 1 was first bromi-
nated and regioselectively 40-demethylated by treatment
with dried HBr and barium carbonate to afford 10 in
63% overall yield.15 The intermediate compound 16
was prepared from 10 in 83% yield by use of a modified
literature procedure.16 Boron trichloride has been used
in the selective cleavage of the methylenedioxy group
in the presence of aromatic methoxy group, followed
by mild basic hydrolysis with barium carbonate
(BaCO3). Methylation of 15 was prepared in 92% yield
using freshly prepared CH2N2.
In addition, 7b-cyano derivative 20 was prepared in 75%
yield by reaction of 16 with trimethylsilyl cyanide, in the
presence of BF3ÆEt2O at ꢀ15 °C. And 7b-carboxyl-de-
rivative 21 was obtained in 46% yield by hydrolyzing
20 in acetic acid (Scheme 4).18
All the target compounds were corroborated by
1H NMR, 13C NMR, IR spectroscopy, and high-resolu-
tion mass spectroscopy (HRMS).19 The assignment of
the configuration at C-7 position for target compounds
19a–e and 20–21 was based on the chemical shift for the
O
O
N
O
N
H3C
MsO
H3C
NaOH/MeOH
H3C
OH
NH
NH
NH
MsCl/Pyridine,rt
O
O
N
O
O
O
O
O
8
OMs
OH
NaH, DMF
H3C
7
6
O
N
O
N
H3C
NH
NH
p-NO2PhOCOCl
Pyridine/CH2Cl2
O
O
O
O
HO
O
O
O
O2N
9
5
Scheme 1. Synthesis compound 9 from thymidine 6.

S.-W. Chen et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2091–2095
2093
N3
N3
OH
O
O
O
O
O
O
O
O
O
PBr3/NaN3
HN3/BF3Et2O
O
O
O
MeO
OMe
MeO
OMe
MeO
OMe
OMe
OMe
OMe
12
1
11
HBr/CH2Cl2
BaCO3/H2O/Me2CO
OH
N3
N3
O
O
O
O
HBr/NaN3
HN3/BF3Et2O
O
O
O
O
O
O
O
O
MeO
OMe
MeO
OMe
MeO
OMe
OH
OH
OH
14
10
13
BCl3 /CH2Cl2
OH
OH
N3
MeO
OH
OH
MeO
MeO
CH2N2
TFA, NaN3
O
O
O
O
O
O
MeO
MeO
OMe
MeO
OMe
MeO
OMe
OMe
OH
OMe
15
16
17
Scheme 2. Preparation of 7-azido-compounds related to podophyllotoxin 11–14, 17.
O
NH
O
O
NH
NH2
N
O
O
RO
RO
Pd-C, H2
9
O
11~14, 17
RO
RO
Et3N, DMF
O
O
O
MeO
OMe
OR'
MeO
OMe
OR'
19a~e
18a~e
Componds
R
R'
CH3
CH3
H
H
CH3
7-configuration
11
12
13
14
17
18a
18b
18c
18d
18e
19a
19b
19c
19d
19e
R-R = -CH2-
R-R = -CH2-
R-R = -CH2-
R-R = -CH2-
CH3
7
7
7
7
7
Scheme 3. Synthesis of compounds 18a–e and 19a–e.
protons at C-6 and the J7,8 coupling constants. The pro-
tons at C-6, showing a singlet around 6.8, are always in
upfield in the C-7b substituted than in the C-7a substi-
tuted compounds (dH-6: 6.86 (19a, 7a) vs 6.78 (19b,
7b), 6.85 (19c, 7a) vs 6.77 (19d, 7b)). This difference is
due to the anisotropic effect of the aromatic ring at-
tached to the C-7a or C-7b substituted compounds. In
addition, the C-7b substituted compounds have

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S.-W. Chen et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2091–2095
CN
COOH
MeO
MeO
MeO
MeO
AcOH/HCl/H2O
TMSCN
O
O
16
BF3 . Et2O
O
O
MeO
OMe
MeO
OMe
OMe
OMe
20
21
Scheme 4. Synthesis of compounds 20 and 21.
J7,8 < 4.5 Hz due to a cis relationship between H-7 and
H-8. The C-7a substituted compounds, however, have
J7,8 > 8.5 Hz because H-7 is trans to H-8.20,21
In conclusion, modified podophyllotoxin derivatives
have demonstrated significant anti-HIV-1 activity.
7b-Amide or cyano group substitution and an opened
A-ring coupled with 40-demethylation resulted in com-
pounds with better anti-HIV-1 activity. These results
are encouraging and warrant further structural modifi-
cation to both decrease cytotoxicity and increase antivi-
ral inhibitory activity. Additional biological evaluation
is in progress to better define the anti- HIV-1 activity
of the podophyllotoxin compounds.
All target compounds were evaluated for inhibitory
activity against HIV-1 replication in acutely infected
C8166 cells following the previously described meth-
ods22 and the results are shown in Table 1. Among these
compounds, 19d and 19c showed the most anti-HIV-1
activity with EC50 values of 0.17 and 0.29 lM, and TI
values of 466.9 and 354.5, respectively.
As it can be seen in Table 1, the anti-HIV-1 activities of
the 40-methoxy compounds were much less than the
ones observed for the corresponding 40-hydroxy com-
pounds (19a to 19c, 19b to 19d). Simultaneously, the
C-7a and C-7b derivatives (19a to 19b, 19c to 19d) did
not show significant differences in their anti-HIV activi-
ties. As reported previously,3 the A-ring modified com-
pounds with 4,5-dimethoxy showed less cytotoxicity
and higher TI value (19e to 19b). These data indicate
that replacing the 4,5-methylenedioxy group with 4,5-di-
methoxy substitution increased their anti-HIV activities.
In addition, 7b-cyano substituted 4,5-dimethoxy com-
pound 20 showed promising anti-HIV activity (EC50
1.05 l M; TI 131.28). However, compound 21 obtained
by hydrolyzing 20 was significantly less active with EC50
value of 46.90 lM and TI value >9.31.
Acknowledgments
This work has been supported by the Interdisciplinary
Innovation Research Fund For Young Scholars, Lanz-
hou University (LZU200526); Natural Science Founda-
tion of Yunnan Province (2003C0022R); Key Scientific
and Technological Projects of China (2004BA719A14)
and Yunnan Province (2004NG12). We also would like
to acknowledge MRC (UK), AIDS Research Project for
providing cell lines and virus.
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.bmcl.
2006.11.070.
References and notes
Table 1. Anti-HIV-1 activities of Compounds 19a–e, 20, and 21
a
b
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IC50 (lM)
EC50 (lM)
TIc
19a
19b
19c
19d
19e
20
3.39
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D
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60-H), 5.98 (s, 2H, OCH2O), 5.91 (d, 1H, J = 5.7 Hz, 2000-
H), 5.37 (d, 1H, J = 6.9 Hz, NH), 5.03 (br, 1H, 4000-H), 4.97
(dd, 1H, J = 7.5, 3.6 Hz, 7-H), 4.56 (d, 1H, J = 3.9, 70-H),
4.36 (m, 2H, 11b-H, 5000b-H), 4.22 (dd, 1H, J = 11.7,
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