New nordihydroguaiaretic acid derivatives as anti-HIV agents

Article abstract of DOI:10.1016/j.bmcl.2008.02.018

Reaction of nordihydroguaiaretic acid with various alkyl chloride, 1-piperidinecarbonyl chloride, methyl chloroformate, or 1,1′-carbonyldiimidazole under alkaline conditions produced the corresponding phenol ethers, carbamates and carbonates, respectively

Full text of DOI:10.1016/j.bmcl.2008.02.018

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 1884–1888
New nordihydroguaiaretic acid derivatives as anti-HIV agents
Jih Ru Hwu,a,b, Ming-Hua Hsu and Ru Chih C. Huangc,*
a,c
*
aDepartment of Chemistry, National Tsing Hua University, 101, Section 2, Kuang-fu Road, Hsinchu 30013, Taiwan, ROC
bDepartment of Chemistry, National Central University, Jhongli 32001, Taiwan, ROC
cDepartment of Biology, The Johns Hopkins University, Baltimore, MD 21218, USA
Received 13 November 2007; revised 1 February 2008; accepted 7 February 2008
Available online 10 February 2008
Abstract—Reaction of nordihydroguaiaretic acid with various alkyl chloride, 1-piperidinecarbonyl chloride, methyl chloroformate,
or 1,10-carbonyldiimidazole under alkaline conditions produced the corresponding phenol ethers, carbamates and carbonates,
respectively, in 67–83% yields. Among these derivatives, the nitrogen-containing compounds were converted to the corresponding
hydrochloride salts. Having good solubility, these NDGA derivatives were found stable in aqueous solution. These new compounds
exerted appealing activity against HIV Tat-regulated transactivation in human epithelial cells. The most potent compound meso-2,3-
dimethyl-1,4-bis(3,4-[2-(piperdino)ethoxyphenyl])butane tetrakishydrochloride salt (5b) showed IC50 value of 0.88 lM.
Ó 2008 Elsevier Ltd. All rights reserved.
Nordihydroguaiaretic acid (NDGA, 1 in Fig. 1) is a
lignan found in the leaves and twigs of the shrub Larrea
tridentata. Being a lipoxygenases inhibitor, NDGA can
induce cystic nephropathy in the rat.1 In addition, it
HO
OH
MeO
OMe
shows various bioactivities, including inhibition of pro-
tein kinase C,2 induction of apoptosis,3 alterations of
membrane,4 elevation of cellular Ca2+ level5 and activa-
tion of Ca2+ channels in smooth muscle cells,6 break-
down of pre-formed Alzheimer’s b-amyloid fibrils
in vitro,7 anti-oxidation,8 etc. Although having been
banned in some countries (e.g., USA), this natural prod-
uct is used commercially as a food additive to preserve
fats and butter. Recently, the derivatives of the plant lig-
nan NDGA have been used for block of viral replication
through the inhibition of viral transcription.9–16 These
compounds can inhibit production of HIV,9–13 herpes
simplex virus,14,15 and HPV transcripts16 by deactiva-
tion of their Sp1-dependent promoters. Moreover, (tet-
ra-O-methyl)nordihydroguaiaretic acid (M4N, 2) can
function as an anti-HIV proviral transcription inhibitor;
this NDGA derivative also causes growth arrest of a
variety of transformed human and mouse cells in culture
and in mice.17,18
OH
OH
OMe
OMe
2 (M4N)
1 (NDGA)
O
O
Me2N
NMe2
O
O
O
O
O
O
HO
OMe
OH
OH
.
4 HCl
Me2N
NMe2
4 (G4N)
3 (Mal.4)
Figure 1. The structures of NDGA derivatives.
We have been focusing on design of NDGA derivatives
that can selectively arrest growth and induce apoptosis
of cancer cells with minimal toxicity to normal tissues.
Huang et al.12 reported that (30-O-methyl)nord-
ihydroguaiaretic acid (Mal.4, 3) can directly and specif-
ically interfere wit21h the binding of Sp1 protein to the
Sp1 sites of the HIV long terminal repeat. The M4N (2)
appears to induce cell cycle arrest in mammalian cell
lines18 and thus inhibits tumor cell growth. Nevertheless,
M4N and other methylated NDGA show poor water
solubility, which limits their applicability. To circum-
Keywords: Anti-HIV; NDGA; Nordihydroguaiaretic acid; DNA; HIV
Tat-regulated transactivation.
*
Corresponding authors. Tel.: +1 886 35 725813; fax: +1 886 35
721594; e-mail: jrhwu@mx.nthu.edu.tw
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.02.018

J. R. Hwu et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1884–1888
1885
vent this problem, we designed and synthesized (tetra-O-
glycyl)nordihydroguaiaretic acid (G4N, 4), a water
soluble derivative of NDGA.11 It can act as mutation-
insensitive transcription inhibitor to HSV-1.10,15 The
high water solubility associated with G4N allows it to
work efficiently in inhibition of HSV despite its short
half-life in aqueous solution. The instability comes from
the ester bonds therein, which connect the glycine moie-
ties onto the NDGA main skeleton. Accordingly, we
planned to synthesize a new series of NDGA derivatives
with appealing anti-HIV activity as well as good water
solubility and stability.
transactivation in a great extent and represents a new
lead of anti-HIV drugs.
We first treated NDGA (1) with an alkyl chloride bear-
ing a hydrocarbon spacer and a nitrogen—containing
five- or six-membered ring in the presence of sodium car-
bonate and acetone (see Scheme 1). These intermediates
were then allowed to react with HCl(g) in situ to give tet-
ra-O-alkylated NDGAs 5a–d in 67–82% overall yields.
Their solubility in aqueous solution was found 379–
541 mg/mL.
Furthermore, we prepared NDGA derivatives in the
families of carbamate and carbonate as shown in
Scheme 2. Treatment of NDGA with 1-piperidinecar-
bonyl chloride in the presence of pyridine at 0 °C
produced the NDGA carbamate 6a in 72% yield. Under
the same conditions, NDGA reacted with methyl
chloroformate or 1,10-carbonyldiimidazole afforded
carbonates 6b and 7, respectively. The latter product
was generated through an intramolecular cyclization
process.
Being a potent transcription activator encoded by the
human immunodeficiency virus-1 (HIV-1), HIV Tat is
required for replication of the deadly virus.19 Tat-regu-
lated transactivation has since become an attractive
strategy for the development of anti-HIV drugs.20
On the other hand, Berger et al.21 reported a novel
eukaryotic reporter gene, the secreted alkaline phospha-
tase (SEAP). By adopting these methods, we screened
our newly synthesized NDGA derivatives against HIV
by using HIV long terminal repeat (LTR) promoter,
SEAP, and CMV promoter driven Tat. We found that
compound 5b was able to inhibit of HIV Tat-regulated
These new NDGA derivatives were found stable in
aqueous solution; >99% of these compounds remained
intact after 28 days (see Fig. 2(b)). In a sharp contrast,
>96% G4N (4) decomposed in aqueous solution within
24 h (see Fig. 2(a)).
We used the transactivation assay involving transfection
of plasmid constructs21 to test the effect of synthesized
NDGA derivatives on Tat-regulated secreted alkaline
phosphatase (SEAP). This protein was produced in
COS cells as described previously.12,13,21 The plasmid
constructs included a cytomegalovirus (CMV) promoter
driven tat gene and an HIV LTR promoter driven repor-
ter gene (i.e., SEAP). A standard SEAP assay in the ab-
sence of drug was initially run to find out efficiency of
transfection. Our results shown in Figure 3 indicate a
nearly 5 times increment in Tat-induced SEAP expres-
sion after 60 min in comparison with the control (i.e.,
1. RCl, K2CO3(s)
Acetone
RO
OR
2. HCl(g)
HO
OH
O
OR
R
OH
OH
.
4 HCl
1
5
R =
c. (CH2)2
N
N
O
(CH2)2
(CH2)2
N
a.
d.
N
(CH2)3
O
b.
Scheme 1. Synthesis of new NDGA derivatives.
RCl
pyridine, 0 oC
O
RO
OR
OR
RO
a.
N
R =
6
b.
COOMe
HO
OH
OH
OH
O
N
1
N
N
N
THF, reflux
O
O
O
O
O
O
7
Scheme 2. Synthesis of new NDGA derivatives.

1886
J. R. Hwu et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1884–1888
NDGA derivatives inhibited the Tat-regulated SEAP
production to the level >90%; at 20 lM, the inhibition
was still >50%. All of these newly synthesized deriva-
tives exhibited a greater inhibitory activity than the par-
ent NDGA (1, IC50 = 20 lM) and Mal.4 (3,
IC50 = 25 lM).12 Except 5a (IC50 = 17.2 lM) and 5c
(IC50 = 17.3 lM), all other NDGA derivatives showed
greater potency than M4N (2, IC50 = 11.1 lM).9 Com-
pound 5b (IC50 = 0.88 lM) was found to be the stron-
gest inhibitor among all of the new NDGA derivatives
for the HIV Tat-regulated transactivation.
For determining cell cytotoxicity in vitro, we adopted
the MTT assay22 by growing cultured cancer cells in
the presence of the NDGA derivatives. Remaining via-
ble cells were then measured by a spectrophotometric
method involving the reduction of the yellow 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(i.e., MTT) to a purple–blue formazan by mitochon-
drial dehydrogenases of metabolically active cancer
cells. The IC50 value indicates the concentration that
resulted in a 50% decrease in cell growth relative to
an untreated control.22 Our results are shown in
Table 2, which includes the cell growth inhibition by
the NDGA derivatives to COS cells without transfec-
ted by HIV/SEAP.
In comparison with the NDGA derivatives 6a, b and 7,
the ether linkage in compounds 5a–d enabled them
much more stable in aqueous medium. On the other
hand, our results from their inhibition of the HIV
Tat-regulated transactivation indicate that the piperi-
dine derivative 5b possessed greater activity than the
pyrrolidine derivative 5a. The angle strain in five-mem-
bered rings is bigger than that in six-membered ring,
which may limit the bioactivity. On the other hand,
placement of an oxygen atom at the 4-position in the
six-membered ring decreased the anti-cancer activity;
the morpholine derivative 5c (IC50 = 17.34 lM) was
Figure 2. UV–visible spectra for indication of stability of NDGA
derivatives (a) G4N (4) degradation in Milli-Q Water. (b) P4N (5b)
present in Milli-Q Water after 28 days.
2.0
control (no DNA)
1.8
HIV/Seap
HIV/Seap + Tat
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
less potent than piperidine derivative 5b (IC50
=
0.88 lM). In the morpholine series (cf. 5c and 5d), we
found that the activity was increased by elongating
the spacer from two methylene units to three units.
The longer spacer in 5d than that in 5c could allow
the morpholine to have a greater flexibility for reaching
the blocking side. Among all of these NDGA deriva-
tives, compound 5b showed most potent activity in
HIV Tat-regulated transactivation. It could be re-
garded as a lead in the development of new anti-HIV
agents.
0
10
20
30
40
50
60
Johansen et al.23 reported the binding of Sp1 family
proteins with DNA at a GC box through the major
groove. The NDGA derivative 5b could also block
the GC box of DNA through the major groove to
form a complex as shown in Figure 5. We performed
this graphic molecular modeling by using the programs
Builder and Biopolymer for the construction of struc-
tures. The energies for conformations were minimized
by use of the program Discover with the consistent va-
lence forcefield (CVFF). The guest 5b, however, was
expelled from the minor groove of the host DNA be-
cause of steric congestion.
Time (minutes)
Figure 3. Induction of SEAP expression in SEAP standard assay.
without DNA) levels of the enzyme and the non-selected
gene (HIV/SEAP) alone. Both of them were close to the
baseline.
We treated the NDGA derivatives 5a–d, 6a, b, and 7
with the SEAP assay, of which the results displayed a
dose-dependent inhibitory activity of Tat transactiva-
tion (see Table 1 and Fig. 4). At 80 lM, all of these

J. R. Hwu et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1884–1888
1887
Table 1. Inhibition (%) of HIV Tat-regulated transactivation in COS cells by NDAG derivatives
b
Compound
Concentrationa (lM)
IC50 (lM)
0.10
0.25
0.50
1.0
2.5
5.0
10
20
59.1
100
58.9
40
94.8
100
86.0
80
97.1
100
96.8
100
90.1
100
100
5a
5b
5c
5d
6a
6b
7
18.1
8.7
5.0
15.0
0
15.4
4.3
27.2
29.0
9.9
19.6
56.8
8.1
31.0
90.6
4.6
14.1
97.0
13.3
89.5
78.6
38.0
47.1
26.3
99.8
25.4
96.0
84.0
51.6
51.2
17.23
0.88
17.34
2.41
2.79
9.41
8.54
97.7
100
89.5
16.5
15.9
3.5
10.6
5.8
9.5
52.7
46.2
15.1
23.7
99.9
86.1
58.4
68.8
99.8
83.8
70.8
85.7
11.7
13.1
5.9
89.8
88.9
91.6
9.8
7.5
12.3
18.1
16.0
4.2
93.4
96.3
a All data represent the average of three experiments.
b Concentrations exhibiting 50% inhibitory (IC50) represented the mean of the triplicate determinations with standard deviations.
SEAP Assay
MTT Assay
1
10
100
Figure 5. Possible complexation between NDGA derivative 5b and a
segment of DNA at its GC box through the major groove. The
graphical results were obtained by CVFF calculations through local
energy minimization.
Drug Concentration [μM]
Figure 4. Inhibition of HIV Tat-regulated transactivation in COS cells
by compound 5b. The COS cells were co-transfected with a Tat-
producing plasmid. A plasmid containing the HIV LTR linked to the
SEAP reporter gene was treated with various concentrations of 5b.
After 48 h, aliquots of media were removed and analysis of SEAP
activity was performed. The percent inhibition of SEAP activity by 5b
was calculated in comparison with that presented in the medium of
untreated cells. In a separate experiment, COS-7 cells were exposed for
the same 48 h time period to media containing increasing concentra-
tions of compound 5b and assayed for viability by use of the MTT
colorimetric method. The data are presented as percent inhibition of
cell viability, which was calculated relative to the value for untreated
cells.
the seven new NDGA derivatives, heterocycle-contain-
ing ether NDGAs were found stable in aqueous medium
and possessed inhibitory activity toward HIV Tat-regu-
lated transactivation in human epithelial cells. The
piperidine containing NDGA 5b with IC50 0.88 lM
exhibited the greatest potency for inhibiting Tat-induced
transactivation. This compound could be regarded as an
ideal lead for anti-HIV drug.
a,b
Table 2. Comparative potency (TC50 and IC50
)
and relative safety
(selective indexes,cSI) in COS cells by NDAG derivatives
Acknowledgment
Compound
TC50 (lM)
IC50 (lM)
SI
This work was supported by the National Science Coun-
cil of Republic of China.
5a
5b
5c
5d
6a
6b
7
27.32
3.83
17.23
0.88
17.34
2.41
2.79
9.41
8.54
1.59
4.35
1.14
0.74
1.98
3.69
1.15
19.71
1.79
5.53
Supplementary data
34.75
9.84
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.bmcl.2008.
02.018.
a Toxicity concentration (TC50) and inhibition concentration (IC50
obtained as the average of triplicate determination.
)
b TC50 and IC50 values were determined after a 48 h exposure to the
test compound.
c The selective index was defined as the ratio: TC50/IC50
.
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