Synthesis, characterization, and anti-melanoma activity of tetra-O-substituted analogs of nordihydroguaiaretic acid

Article abstract of DOI:10.1016/j.bmcl.2009.06.063

Synthesis of seven semi-synthetic analogs of NDGA is described. An approach to NDGA derivatization is described in which the ortho-phenolic groups are tethered together by one atom, forming a 5-membered heterocyclic ring. The analogs were evaluated for cy

Full text of DOI:10.1016/j.bmcl.2009.06.063

Bioorganic & Medicinal Chemistry Letters 19 (2009) 4752–4755
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis, characterization, and anti-melanoma activity of tetra-O-substituted
analogs of nordihydroguaiaretic acid
b
c
a
d
a
Ross O. Meyers a, , Joshua D. Lambert , Nicole Hajicek , Alan Pourpak , John A. Kalaitzis , Robert T. Dorr
*
a University of Arizona Cancer Center, 1515 N. Campbell Ave., Tucson, AZ 85724, USA
b Department of Food Science, The Pennsylvania State University, University Park, PA 16802, USA
c Department of Pharmacology, University of Illinois, College of Medicine, Chicago, IL 60612, USA
d Department of Pharmacy, University of Arizona, Tucson, AZ 85724, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Synthesis of seven semi-synthetic analogs of NDGA is described. An approach to NDGA derivatization is
described in which the ortho-phenolic groups are tethered together by one atom, forming a 5-membered
heterocyclic ring. The analogs were evaluated for cytotoxicity in four cancer cell lines and compared to
NDGA and tetra-O-methyl-NDGA (M4N) (1a). NDGA bis-cyclic sulfate (2a), NDGA bis-cyclic carbonate
(2b), and methylenedioxyphenyl-NDGA (2d) and NDGA tetra acetate (1b) showed anti-cancer activity
in vitro. Two compounds, (1b) and (2b), were evaluated for anticancer activity in a mouse xenograft
model of human melanoma and showed dose-dependent activity.
Received 20 April 2009
Revised 8 June 2009
Accepted 15 June 2009
Available online 21 June 2009
Keywords:
Nordihydroguaiaretic acid
NDGA
Ó 2009 Elsevier Ltd. All rights reserved.
Analogs
Melanoma
Cancer
Nordihydroguaiaretic acid (NDGA) is a lignan that is found at up
to 10–15% by dry weight in the leaves and twigs of the creosote
bush (Larrea tridentata (Sesse and Moc.) Coville, Zygophyllaceae).1
NDGA is a 5-lipoxygenase inhibitor with a well-defined mecha-
nism of action requiring ‘free’ phenolic groups for activity.2
Tetra-substituted, ‘masked’ phenolic group NDGA analogs were
found to be devoid of inhibitory activity against soybean, human
12, and human 15- lipoxygenase.3 NDGA has shown many biolog-
ical activities including anticancer activity. In vitro, NDGA inhib-
ited DNA synthesis in K562 chronic myelogenous leukemia blast
NDGA analogs were shown to have a similar antiviral mechanism.
Recently, NDGA analogs have shown anti-HIV activity in an in vitro
Tat-regulated secreted alkaline phosphatase assay.16
Heller et al. have reported that M4N arrested C3 cells, a HPV-16/
ras-transformed, tumorigenic mouse embryo cell line, in the G2
phase of the cell cycle. The growth inhibitory activity of M4N was
associated with a decrease in the expression of cyclin-dependent ki-
nase (Cdc2), an Sp-1-promoter dependent gene which progresses
the cell through G2/M. In vivo, intratumoral injections of M4N
caused a decrease in C3 xenograft tumor size in mice which was cor-
related with an observed decrease in protein levels of CDC2.17 M4N
inhibition of Sp-1 binding was further investigated as a mechanism
of anticancer activity. Sp-1 promoter binding is responsible for Cdc2
and survivin gene expression. Survivin, an inhibitor of apoptosis, is
overexpressed in most cancer cells. Its expression is G2/M specific
and Sp-1 dependent. M4N-induced apoptosis in the C3 cell line
was correlated to inhibition of survivin, suggesting that inhibition
of survivin expression may be an underlying mechanism of action
of M4N. M4N-treated C3 cells showed a decrease in CDC2 and survi-
vin at the mRNA and protein level. A non-Sp-1 dependent promoter
showed resistance to M4N-induced cytotoxic activity.18 M4N sup-
pressed tumor growth in Hep3B hepatocellular carcinoma, LnCaP
prostate carcinoma, HT-29 colorectal carcinoma, MCF-7 breast car-
cinoma, and K-562 erythroleukemia in a nude mouse xenograft
model. A decrease in Cdc2 and survivin gene expression was corre-
lated to tumor growth inhibition.19
cells with an IC50 of 230 l
M.4 NDGA inhibited the proliferation of
human small cell lung,5 non-small cell (NSCLC)6 lung, human pan-
creatic, and cervical cancer cells in vitro.7 In vivo, NDGA inhibited
tumor growth in esophageal adenocarcinoma8 and NSCLC xeno-
grafts in mice.6
Natural9,10 and semi-synthetic, O-methylated11,12 analogs of
NDGA have been studied for their antiviral activities. The mecha-
nism of antiviral activity was reported to be inhibition of host
Sp-1 transcription factor binding with subsequent inhibition of
Sp-1-dependent viral gene expression.12 The degree of inhibition
was directly proportional to the degree of phenolic group methyl-
ation with the tetra-O-methylated NDGA (M4N) showing the
strongest inhibition.11 Tetra-acetyl13 and tetra-glycinated14,15
* Corresponding author. Tel.: +1 520 626 7892.
E-mail address: rom9765@hotmail.com (R.O. Meyers).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.06.063

R. O. Meyers et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4752–4755
4753
M4N inhibited growth in MCF-7 human breast, A549 human
lung, SW480 human colon cancer cell lines and was especially po-
tent against A375 and ACC375 human melanoma cells with IC50
a method described by Grazia et al.23 Analogs 1a, 2a, 2d were all
purified to >95% by semi-preparative HPLC and purity was as-
sessed by three-dimensional UV scan by this method. Analogs1b,
1c, 2b, 2c were all purified to >95% (as assessed by three-dimen-
sional UV scan) using recrystallization from isopropanol.
The MTT assay was used to assess in vitro cytotoxicity of the
test compounds. All IC50 determinations are for 5-day treatment
times. Compounds 1a, 1b, and NDGA showed the highest potency
in the MTT assay against the panel of cell lines with IC50 values
values of 2.5 and 5.0
M4N was non-schedule dependent and induced apoptosis. At
50 M, M4N inhibited DNA synthesis by 66% after 1 h and by
lM, respectively. In the ACC375 cell line,
l
95% of control after 24 h demonstrating an early event in growth
inhibition. M4N arrested melanoma cells in G1/G0 and G2/M phases
of the cell cycle, suggesting it may affect cell cycle checkpoint pro-
teins. In vivo, M4N inhibited tumor growth in the B16 murine mel-
anoma model, as well as in SW480 human colon cancer and A375
human melanoma xenografts in SCID mice.20
Tetra-O-substituted NDGA analogs show decreased in vivo tox-
icity, for example, the lethal dose 50% (LD50) of NDGA was found to
be 75 mg/kg ip21 M4N was well-tolerated at 1000 mg/kg ip,18
approximately 11 times the LD50 of NDGA.
The purpose of this project was to generate new tetra-O-substi-
tuted NDGA analogs for the continued investigation of in vivo,
anti-melanoma therapeutic activity demonstrated in our prior stud-
ies.20 This work compares the in vitro cytotoxicity of NDGA and M4N
to NDGA tetra-acetate and NDGA tetra-methanesulfonate (Scheme
1, R1). In addition, we investigate an approach to tetra-O-substitu-
tion of NDGA by the tethering together of the ortho phenolic groups
by a single atom forminga 5-membered heterocyclic ring (Scheme1,
R2). Analogs from this group were evaluated for their cytotoxicity in
A375, human malignant melanoma cells, HT-29, human colorectal
adenocarcinoma cells, MCF-7, human mammary gland adenocarci-
noma cells, and HepG2 human hepatoma cancer cell lines. Cytotox-
icity was evaluated by 5-day 3-(4,5-dimethylthiazol-2-yl)-2,
5-diphenyltetrazolium bromide (MTT) assays and is summarized
in Table 1. One analog from each group was evaluated in vivo using
the A375 xenograft model in SCID mice.
ranging from 8.5 to 79.4 lM (Table 1). Compounds 2a and 2b
had moderate inhibitory activity against MCF-7 cells. Compounds
1c and 2d were 2.2–116-fold less potent than NDGA against the
panel. Compound 2c showed no significant cytotoxicity against
any of the cell lines tested.
The cytotoxicity of the analogs against A375 cells was further
evaluated by determining the inhibitory effects of the analogs
against DNA, RNA, and protein synthesis by a method described
by Mayr et al.24 The results are shown in Table 1. There was rela-
tively good agreement between cytotoxicity and inhibition of mac-
romolecular synthesis inhibition with the exception of 1c. This
compound was inactive below 1000
lM in the MTT assay but
showed strong inhibitory activity against RNA and DNA synthesis
at 85 and 100
are needed.
lM, respectively. Further studies of this discrepancy
Effects of the test compounds on tumor growth were deter-
mined in two studies. In study 1, 1b, 2a, and 2b were suspended
in 100% Tween 80 (Sigma–Aldrich, St. Louis, MO.). Mice were im-
planted sc with 10 ꢀ 106 A375 human melanoma cells in 100
ll
phosphate buffer saline in the rear right flank. The mice were im-
planted on day 0 and were dosed with test compounds on days 1,
5, and 9. Controls (n = 4) received 100% Tween 80. The method for
study 2 was identical to study 1, except there were eight mice per
group and the analogs were suspended in 100% PEG 300. Controls
(n = 4) received 100% PEG 300. Tumor volumes were measured 3
The analogs 2a, 2b, and 2c were synthesized by a modification
of the procedure of Tickner et al.22 Analog 2d was synthesized by
NDGA
HO
HO
OH
OH
R1O
R1O
O
R2
OR1
OR1
O
O
O
R2
R2
R1
2a: SO2
2b: C=O
2c: C=S
2d: CH2
1a: -CH3
1b: -COCH3
1c: -SO2CH3
Scheme 1. Reagents and conditions 1a: potassium carbonated, acetone, dimethylsulfate, reflux 1b: acetyl chloride, pyridine, methylene chloride 1c: methanesulfonyl
chloride, pyridine 2a: 1,10-sulfuryldiimidazole, N,N0-dimethylacetamide, KF 2b: N,N0-carbonyldiimidazole, N,N0-dimethylacetamide, KF 2c: 1,10-thiocarbonyldiimidazole,
pyridine, paradioxane 2d: bromochloromethane, DMF, cesium carbonate.

4754
R. O. Meyers et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4752–4755
Table 1
Cytotoxicity and macromolecular synthesis inhibition by NDGA analogs in cancer cell lines
Compound ID
5-day MTT IC50
MCF-7
(
l
M) SD
Macromolecular synthesis inhibition (% of control SD)
HT-29
HepG2
A375
DNA
RNA
Protein
NDGA
1a
1b
1c
2a
2b
2c
2d
51.3 0.6
61.5 0.9
25.6 0.6
343 0.6
105.4 0.7
72.3 0.7
>1000 NA
89.9 0.4
8.5 0.2
54.1 1.0
44.5 0.7
51.3 1.0
542.2 0.8
98.5 0.6
164.5 0.6
54.3 1.0
16.6 0.8
79.4 0.8
>1,000 NA
124.5 1.0
150.3 0.8
>1000 NA
235.5 1.0
75 18
72
31
68 16
23
75 11
115 11
100 12
98 25
6
3
74
70
81
81
55
76
6
6
7
6
3
3
42.4 0.3
24.0 0.2
103.4 0.2
51.4 0.3
41.1 0.3
>1000 NA
102.7 0.3
23
8
77 18
43 12
102 31
184 54
5
85
89 33
34
6
98 28
84
175.3 1.0
2
aDoxorubicin
bActinamycin D
cCyclohexamide
7
0.7 02
3
0.2
Doses of positive controls: a10
NA = not applicable.
l l lg/mL.
g/ml, b50 g/mL, c10
Cytotoxicity was determined by the MTT assay. Inhibition of DNA, RNA and protein synthesis was determined in A375 human melanoma cells at a concentration of 50
each test compound to compare to the IC50 of NDGA and to maintain analog solubility.
lM for
times/week and were determined based on the following formula:
V(mm3) = length ꢀ (width)2/2. The in vivo tumor growth inhibition
model data was analyzed by determining % T/C as described by
Bissery et al.25 and are summarized in Table 2. A percent T/C be-
tween 42% and 10% is considered moderate tumor growth inhibi-
tion. Both 1b and 2b showed moderate growth inhibitory effects
against xenograft tumors in the first experiment, however in the
second study, tumor growth inhibition was observed only for 1b
at the 300 mg/kg dose. This may be the result of lower solubility
of 2b in a PEG 300 formulation compared to the Tween 80 formu-
lation in study 1. In study 2, PEG 300 was used as the vehicle be-
cause Tween 80 displayed some tumor growth inhibition
compared to untreated controls in study 1 (data not shown). Com-
pounds 1b and 2b were well-tolerated at doses of 100–300 mg/kg
and no sign of general toxicity was observed compared to control
mice. Compound 2a displayed significant toxicity after a single
injection at the doses tested and studies of this compound were
discontinued.
in vivo and were well-tolerated at doses 2.6 and 3.4 times the
LD50 of NDGA. The fused-ring heterocyclic derivatives of NDGA,
such as 2b, represent a new class of derivatives that should be fur-
ther investigated for their potential antitumor activity and the pos-
sible underlying mechanisms.
Acknowledgments
The authors would like to acknowledge Bhashyam S. Iyengar
and William A. Remers for their assistance in the naming and char-
acterization of the compounds described in this Letter. Sponsor-
ship: The study was funded by grant CA017094 from the
National Institutes of Health, Bethesda, Maryland, USA.
Supplementary data
Supplementary data (materials, methods and results of synthe-
sis, purification characterization and bioassay) associated with this
article can be found, in the online version, at doi:10.1016/
j.bmcl.2009.06.063.
In conclusion, among the tetra-substituted analogs, 1a and 1b
were clearly more potent in vitro than 1c, although 1c was rather
insoluble in vitro above 100 l
M. The compounds in Scheme 1, R2,
showed markedly reduced cytotoxicity compared to Scheme 1, R1,
with the exception of analogs 2a and 2b which showed IC50 values
similar to Scheme 1, R1 in the MCF-7 cell line. Moreover, com-
pounds 1b and 2b demonstrated significant antitumor activity
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Table 2
A375 tumor growth inhibition parameters
Analog
Dose (mg/kg)
N
%T/Ca
T–Cb
Tdc
TCKd
Study 1
1b
1b
2b
2b
100
200
100
200
4
4
4
4
34
16
21
18
2.5
3.5
3.5
3.5
7.4
7.4
7.4
7.4
0.1
0.1
0.1
0.1
Study 2
1b
2b
300
300
8
8
44
68
1
1
2.6
2.6
0.1
0.1
a
Percent T/C value = median tumor volume of the treated/median tumor volume
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1100 mm3.
b
Tumor growth delay (T–C value) was calculated by comparing the median time
in days for the treated and control group tumors to reach a predetermined volume.
c
Tumor doubling time (Td) was calculated from the line of best fit from a log(y or
tumor volume) versus linear (x or time) plot when the control tumors were in
exponential growth between 100 and 1000 mm3.
d
Tumor cell kill (TCK) were approximated by the following formula: Log10 cell
kill = T–C value in days/3.32 ꢀ Td.
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