Anti-AIDS agents. Part 61: Anti-HIV activity of new podophyllotoxin derivatives

Article abstract of DOI:10.1016/j.bmc.2004.04.048

A series of novel podophyllotoxin derivatives containing structural modifications at C-4 (7-14), C-4′ (16-17), and the methylenedioxy A-ring (23-28) was synthesized and tested for inhibition of HIV replication. Four of these compounds (25-28) were previou

Full text of DOI:10.1016/j.bmc.2004.04.048

Bioorganic & Medicinal Chemistry 12 (2004) 4267–4273
Anti-AIDS agents. Part 61: Anti-HIV activity of
new podophyllotoxin derivativesq
Xiao-Kang Zhu,a Jian Guan,a Zhiyan Xiao,c L. Mark Cosentinob and Kuo-Hsiung Leea,*
aNatural Products Laboratory, School of Pharmacy, University of North Carolina, Chapel Hill, NC 27599-7360, USA
bBiotech Research Laboratories, 3 Taft Court, Rockville, MD 20850, USA
cDepartment of Synthetic Medicinal Chemistry, Chinese Academy of Medicinal Sciences and Peking Union Medical College,
Beijing 100050, China
Received 23 February 2004; accepted 12 April 2004
Abstract—A series of novel podophyllotoxin derivatives containing structural modifications at C-4 (7–14), C-40 (16–17), and the
methylenedioxy A-ring (23–28) was synthesized and tested for inhibition of HIV replication. Four of these compounds (25–28) were
previously reported to show EC50 values of <0.001 lg/mL and therapeutic index (TI) values >120. Three of the newly tested
compounds (8, 12, and 20) showed good activity with EC50 values of 0.012, <0.001, and 0.389 lg/mL and TI values of 19.1, >16, and
19.4, respectively. A comparison of the anti-HIV activity of these derivatives suggested that an opened A-ring with 6,7-dimethoxy
substitution and a 40-demethylated E ring enhanced anti-HIV activity.
Ó 2004 Elsevier Ltd. All rights reserved.
1. Introduction
now approved for clinical use in the US. However, these
drugs have only limited or transient clinical benefit due
to their adverse side effects and the development of
drug-resistant viral strains.6–9 Therefore, current sear-
ches for new anti-HIV agents are focused on discovering
compounds with novel structures and different mecha-
nisms of action.
In the past two decades, a worldwide search has been
made for new chemotherapeutic agents targeting the
human immunodeficiency virus (HIV), the causative
agent of acquired immune deficiency syndrome (AIDS).
Nineteen drugs, including nucleoside/nucleotide viral
reverse transcriptase (RT) inhibitors (NRTIs),2;3 non-
nucleoside RT inhibitors (NNRTIs),4 protease inhibi-
tors (PIs),4 and fusion (or entry) inhibitors (FIs),5 are
Podophyllotoxin (1) is a lignan isolated from the roots
of the North American Podophyllum peltatum Linnaeus,
q For Part 60, see Ref. 1.
* Corresponding author. Tel.: +1-919-962-0066; fax: +1-919-966-3893; e-mail: khlee@unc.edu
0968-0896/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.04.048

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the Tibetan P. emodi Wall, or the Taiwanese species
Podophyllum pleinthum. Hence10 extensive structural
modification and anti-tumor studies of 1 have resulted
in clinically useful anti-cancer drugs, such as etoposide
(2) and teniposide (3). In our continuing studies on the
synthesis and biological evaluation of podophyllotoxin-
and etoposide-related derivatives, we have prepared
several series of C-4 modified podophyllotoxins bearing
various substituents including alkyl- and aryl-amino
groups [e.g., 4 (GL331)], oxy and thio ethers, and ke-
tones.11–16 Additional structural changes have been
made in the methylenedioxy A-ring, C-ring, and the
lactone D-ring.15;17;18 To explore the range of biological
activities of the podophyllotoxin compound class, we
recently reported our initial anti-HIV results19 for
modified podophyllotoxin derivatives. Based on these
promising results, we have extended our studies and
herein report results of analogues containing structural
modifications at C-4 (7–14), C-40 (16–17), and the
methylenedioxy A-ring (23–24).
erential formation of C-4b-oriented products. The 40-
hydroxy group of 5 was selectively reacted with benzyl
or vinyl chloroformate to give the carbonate derivatives
16 and 17.
6,7-O-Demethylene-40-O-demethyl-podophyllotoxin (18)
and 6,7-O-demethylene-podophyllotoxin (19) were pre-
pared by treating podophyllotoxin (1) with BCl3 fol-
lowed by weak basic hydrolysis with BaCO3.15
Methylation of both 18 and 19 was achieved using
methyl iodide with K2CO3 and Et4NF as catalysts.
40-Demethyl-6,7-O-demethylene-6,7-O-dimethyl-4b-sub-
stituted-amino-4-desoxypodophyllotoxin
derivatives
(25–28) were prepared by an efficient four-step synthetic
sequence starting from podophyllotoxin (Scheme 2). In a
one-pot synthesis, intermediate 20 underwent 4-haloge-
nation and -epimerization coupled with 40-demethylation
using trimethylsilyl iodide (TMSI) to give 22. Then
without purification, amination of the 4-position of 22
with the appropriate p-substituted aniline yielded the
target compounds 25–28 as the major products. This
protocol proceeded effectively to afford 25–28 without
any detectable 6,7-demethylation (Scheme 2). Com-
pounds with an intact 40-methoxy group (e.g., 23 and 24)
were minor products.
2. Chemistry
40-Demethyl-epipodophyllotoxin (5) was prepared from
podophyllotoxin according to a previously published
method12 and intermediate 6 was synthesized from 5 via
a modified Kuhn’s method.16 As shown in Scheme 1,
compounds 7–14 were prepared by treating 6 with
appropriately substituted arylamines in the presence of
barium carbonate. Although this reaction occurs via an
SN1 mechanism,15 the bulky C-1a pendent aromatic ring
directs a stereoselective substitution, resulting in pref-
3. Biological results and discussion
All three compound types were evaluated for inhibitory
activity against HIV-1 replication in acutely infected H9
cells. Table 1 shows the data for Type-1 compounds,
which have different substituents at the 4-position of
40-demethyl-desoxypodophyllotoxin (7–15, 2, and 4).
Scheme 1.

X.-K. Zhu et al. / Bioorg. Med. Chem. 12 (2004) 4267–4273
4269
Scheme 2.
Among these compounds, 8 and 12 showed the highest
anti-HIV activity with EC50 values of 0.012 and
<0.001 lg/mL and TI values of 19.1 and >16, respec-
tively.
compounds were the phenol 5 and its corresponding
vinyl carbamate 17 with EC50 values of 0.057 and
0.034 lg/mL, respectively. The benzyl carbamate (16)
was devoid of anti-HIV activity.
As shown in Table 1, addition of an o-methoxy group to 4
increased anti-HIV activity and therapeutic index (see
compound 8). Among the three disubstituted analogues,
the 2-methoxy-4-nitroanilino analogue (8) showed the
highest anti-HIV potency, the 2-methoxy-5-nitroanilino
derivative (9) was less active, and the 2-nitro-4-methoxy-
anilino compound (7) was inactive. Both trisubstituted
compounds (10 and 11) were inactive. Replacing the p-
nitro group of 4 with bulky substituents (benzamidizole,
12, or camphanoylamido, 13) led to significantly increased
anti-HIV activity, as well as cytotoxicity. However, plac-
ing a benzimidazole directly at the 4-position of podo-
phyllotoxin resulted in total loss of anti-HIV activity (14).
Data for the Type-3 compounds are shown in Table 3.
As reported previously,19 the 4b-substituted anilino-6,7-
dimethoxy compounds 25–28 showed promising activity
(EC50 < 0.001 lg/mL; TI > 120 ꢀ> 166). However, com-
pounds 4 and 15 (both with an intact methylenedioxy
ring A) were significantly less active with EC50 values of
0.055 and 0.1 lg/mL and TI values of 2.43 and 10,
respectively (Table 1). A similar order of activity was
found with podophyllotoxin and two derivatives (19 and
20) with a 4a-OH and various 6,7-substitution patterns.
Compound 20 (6,7-dimethoxy) was active (EC50
0.389 lg/mL, TI 19.4), 19 (6,7-dihydroxy) was less active
(EC50 4.30 lg/mL, TI 2.23), and 1 (6,7-methylenedioxy)
was inactive (Table 2). These data indicate that replac-
ing the 6,7-methylenedioxy group with 6,7-dimethoxy
substitution increased anti-HIV activity. In a final
comparison, the 40-methoxy compounds 23 and 24 were
The Type-2 compounds (podophyllotoxin and epipod-
ophyllotoxins) in Table 2 (1, 5, 16, and 17) were less
active than the Type-1 compounds. The most active

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X.-K. Zhu et al. / Bioorg. Med. Chem. 12 (2004) 4267–4273
Table 1. Anti-HIV activity of podophyllotoxin derivatives-Type 1
R
O
O
O
O
H3CO
OCH3
OH
Compd
R
EC50 (lg/mL)a
IC50 (lg/mL)b
TIc
HO
O
OH
O
O
2
4
0.030
0.055
1.27
42.7
O
0.132
2.43
NO2
HN
O2N
7
8
9
Not active
0.012
1.69
0.23
0.229
––
OCH3
NO2
HN
H3CO
HN
19.1
H CO
3
0.29
7.92
HN
NO
2
H3CO
HN
10
Not active
1.76
––
NO2
OCH3
H3C
HN
11
12
Not active
<0.001
38.7
––
NO2
OCH3
N
0.016
>16
HN
N
H
O
13
0.0023
0.018
7.82
O
NH
HN
O
N
14
Not active
0.1
0.206
1.0
––
HN
N
H
15d
10
F
HN
a EC50 refers to the concentration of drug that causes 50% reduction in total virus replication.
b IC50 refers to the concentration of drug that causes 50% reduction in total cell number. Drugs that have IC50 values >100 lg/mL cannot be tested at
higher concentrations for a more exact IC50 value due to the effect of the solvent, DMSO.
c Therapeutic Index is a ratio of the IC50 value/EC50 value. Therefore, when the IC50 value is >100 lg/mL (refer to footnote a, above), the TI value
must also be reported as greater than.
d From Ref. 19.
much less active than the corresponding 40-hydroxy
compounds 25 and 26.
compounds with better anti-HIV activity. These results
are encouraging and warrant further structural modifi-
cation to both decrease cytotoxicity and increase anti-
viral inhibitory activity. Although the precise biological
target is not known, studies by Hara et al.20 on the anti-
HIV activity of numerous aryltetralin lignan analogues
provide some insight and suggestions. Their work showed
In summary, modified podophyllotoxin derivatives have
demonstrated significant anti-HIV activity. Bulky, para-
substitution on the 4b-aniline (12 and 13) and an opened
A-ring (25–28) coupled with 40-demethylation resulted in

X.-K. Zhu et al. / Bioorg. Med. Chem. 12 (2004) 4267–4273
Table 2. Anti-HIV activity of podophyllotoxin derivatives-Type 2
4271
R1
O
O
O
O
H3CO
OCH3
OR2
Compd
R1
R2
EC50 (lg/mL)a
IC50 (lg/mL)a
TIa
––
1
CH3
Not active
0.0025
OH
OH
5
H
0.057
0.171
0.201
3.01
––
OH
16
Not active
COH2C
O
17
0.034
0.136
4.02
COCH=CH2
O
OH
a See Table 1.
that lignan analogues bind to reverse transcriptase at the
same site as many other RT inhibitors. Additional bio-
logical evaluation is in progress to better define the anti-
HIV activity of the podophyllotoxin compounds.
4.2. Synthesis of 40-O-demethyl-40-acyl-epipodophyllo-
toxins (16 and 17)
To a solution of 40-O-demethyl-epipodophyllotoxin (5)
(2 mmol) in 10 mL of anhydrous dichloroethane and
1 mL of pyridine was added the appropriate chlorofor-
mate in 2 mL of dichloroethane dropwise under nitrogen
at room temperature. The reaction mixture was then
stirred for 1–3 days, concentrated in vacuo to remove
solvent. The residue was diluted with CH2Cl2, washed
with water, dried over anhydrous magnesium sulfate,
and purified via silica gel column chromatography using
CH2Cl2–acetone–EtOAc (100:5:5) as eluent.
4. Experimental
All melting points were taken on Fisher–Johns and Mel-
Temp II melting point instruments and are uncorrected.
IR spectra were recorded on a Perkin–Elmer 1320
spectrophotometer. 1H NMR spectra were obtained
using Bruker AC-300 and WM 250 NMR spectrometers
with TMS as the internal standard. All chemical shifts
are reported in ppm. FABMS and HRFABMS spectral
analyses were determined on a JOEL HX-110 instru-
ment. Analytical thin-layer chromatography (TLC) was
carried out on Merck precoated aluminum silica gel
sheets (Kieselgel 60 F-254). Optical rotations were
measured with a JASCO DIP-1000 polarimeter. All
target compounds were characterized by 1H and IR
spectral analyses and MS analyses.
4.3. 40-O-Demethyl-40-carbobenzoxy-epipodophyllotoxin
(16)
21:4
D
Yield 64%; crystals from MeOH; mp 116–118 °C; ½aꢁ
)29.4 (c 0.17, CHCl3); IR (KBr) 3495 (OH), 2900 (ali-
phatic C–H), 1755 (lactone), 1595, 1500, 1475, 1450,
1420 (aromatic C@C) cmꢂ1; MS m=e: 534 [M]þ; 1H
NMR (DMSO, D2O exchange): d 6.95 (s, 1 H, 5-H),
6.54 (s, 1H, 8-H), 6.33 (s, 2H, 20, 60-H), 6.01 (s, 2H, 12-
H), 5.48 (d, J ¼ 5:8 Hz, 1H, exchangeable, 4-OH), 4.74
(m, 1H, 4-H), 4.60 (d, J ¼ 5:8 Hz, 1H, 1-H), 4.36 (t,
J ¼ 7:9 Hz, 1H, 11-H), 4.19 (t, J ¼ 7:9 Hz, 1H, 11-H),
3.62 (s, 6H, 30, 50-OCH3), 3.32 (m, 1H, 2-H), 2.82 (m,
1H, 3-H); Anal. (C29H26O10) C, H.
4.1. Synthesis of 4b-poly-substituted-anilino-40-demethyl-
desoxypodophyllotoxins (7–14)
A solution containing 40-O-demethyl-4b-bromo-4-des-
oxypodophyllotoxin (6) (695 g, 1.5 mmol), anhydrous
BaCO3 (590 g, 3.0 mmol), and the appropriate arylamine
(1.65 mmol) in 15 mL of dry dichloroethane under
nitrogen was stirred overnight at room temperature. The
reaction mixture was filtered, diluted with EtOAc,
washed with water, dried over anhydrous MgSO4, and
purified via silica gel column chromatography using
CH2Cl2–acetone–EtOAc (100:5:5) as eluent. The physi-
cal and spectral data of all compounds agreed with those
reported by us previously.21
4.4. 40-O-Demethyl-40-vinyloxy-carbonyl-epipodophyllo-
toxin (17)
Yield 54.3%; crystals from acetone; mp 246–249 °C;
23:5
D
½aꢁ
)59.1 (c 0.18, CHCl3); IR (KBr) 3530 (OH), 2900
(aliphatic C–H), 1765 (lactone), 1580, 1475, 1450 (aro-
matic C@C) cmꢂ1; MS m=e: 470 [M]þ; 1H NMR
(CDCl3, D2O exchange): d 7.13 (dd, J ¼ 6:2 Hz, 13.8 Hz,

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X.-K. Zhu et al. / Bioorg. Med. Chem. 12 (2004) 4267–4273
Table 3. Anti-HIV activity of podophyllotoxin derivatives-Type 3
R1
R3
R3
O
O
O
O
H3CO
OCH3
OR2
Compd
R1
R2
R3
EC50 (g/mL)a
IC50 (lg/mL)a
TIa
19
CH3
H
4.30
9.57
2.23
19.4
OH
OH
HN
20
23
CH3
CH3
CH3
CH3
0.389
2.45
7.53
5.29
2.16
9.50
NO
2
24
CH3
H
CH3
CH3
CH3
0.151
<0.001
<0.001
1.43
F
HN
HN
HN
HN
25a
26a
0.158
0.166
>158
NO
2
H
>166
>141
F
27a
28a
H
H
CH3
CH3
<0.001
0.141
CN
<0.001
0.0239
0.120
500
>120
CO C H
2 5
HN
2
AZT
22,650
a See Table 1.
1H, 100-H), 6.90 (s, 1H, 5-H), 6.57 (s, 1H, 8-H), 6.34 (s,
2H, 20, 60-H), 6.00 (d, J ¼ 7:0 Hz, 2 H, 12-H), 5.03 (dd,
J ¼ 2:1, 13.9 Hz, 1H, 4-H), 4.89 (t, J ¼ 3:7 Hz, 1H, 1-
H), 4.66 (m, 2H, 200-H), 4.41 (m, 2H, 11-H), 3.73 (s, 6H,
30, 50-OCH3), 3.32 (dd, J ¼ 5:1, 14.1 Hz, 1H, 2-H), 2.84
(m, 1H, 3-H); Anal. (C24H22O10) C, H.
(25–28) as major product (52% yield for 25) and the
undemethylated compound as minor product (6% yield
for 23). Spectral and physical data of all compounds
agreed with those reported in our previous literature.15
4.6. Anti-HIV assay
The T cell line, H9, was maintained in continued culture
with complete medium (RPMI 1640 with 10% fetal calf
serum supplemented with L-glutamine at 5% CO2 and
4.5. Synthesis of 40-O-demethyl-6,7-O-demethylene-6,7-
dimethyl-4-substituted-aniline-4-desoxypodophyllotoxins
(25–28) and 6,7-O-demethylene-6,7-dimethyl-4-substi-
tuted-aniline-4-desoxypodophyllotoxin (23, 24)
37 C). Aliquots of this cell line were only used in exper-
iments when in log-phase growth. Test samples were first
dissolved in dimethyl sulfoxide. The following final drug
concentrations were routinely used for screening: 100,
20, 4, and 0.8 lg/mL. For active agents, additional
dilutions were prepared for subsequent testing so that an
accurate EC50 value (defined below) could be achieved.
As the test samples were being prepared, an aliquot of
the H9 cell line was infected with HIV-1 (IIIB isolate)
while another aliquot was mock-infected with complete
medium. The stock virus used for these studies typically
had a TCID50 value of 104 infectious units/milliliter. The
appropriate amount of virus for a multiplicity of infec-
tion (m.o.i.) between 0.1 and 0.01 infectious units/cell
was added to the first aliquot of H9 cells. The other
aliquot only received culture medium, and these mock-
infected cells were used for toxicity determinations (IC50,
defined below). After a 4 h incubation at 37 °C and 5%
To a solution of 6,7-O-demethylene-6,7-O-dimethyl-
podophyllotoxin 20 (86 g, 0.2 mmol), prepared as pre-
viously described in Ref. 15, in dry 1,2-dichloroethane
(10 mL) was added a solution of TMSI (140 mg, 0.1 mL,
0.7 mmol) at )20 °C. The reaction mixture was stirred
for 6 h at )20 to )10 °C then concentrated in vacuo at
room temperature to give a brown residue. This crude
product was dissolved in dry 1,2-dichloroethane (5 mL),
and then BaCO3 (80 mg, 0.4 mmol) and the appropriate
substituted aniline (0.22 mmol) were added successively.
The mixture was stirred for 12 h at room temperature,
filtered, and concentrated under reduced pressure. The
crude product was purified on a silica gel preparative
TLC plate with hexane–MeOH–acetone (90:5:5) as sol-
vent system to give the 40-O-demethylated compound

X.-K. Zhu et al. / Bioorg. Med. Chem. 12 (2004) 4267–4273
4273
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This investigation was supported by grant AI-33066
from the National Institute of Allergy and Infectious
Diseases awarded to K.H.L.
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