Inhibition of IGF-1R and lipoxygenase by nordihydroguaiaretic acid (NDGA) analogs

Article abstract of DOI:10.1016/j.bmcl.2007.04.092

Herein, we pursue the hypothesis that the structure of nordihydroguaiaretic acid (NDGA) can be refined for selective potency against the insulin-like growth factor 1 receptor (IGF-1R) as a potential therapeutic target for breast cancer while diminishing its action against other cellular targets. Thus, a set of NDGA analogs (7a-7h) was prepared and examined for inhibitory potency against IGF-1R kinase and an alternative target, 15-lipoxygenase (15 LOX). The anti-cancer effects of these compounds were determined by their ability to inhibit IGF-1 mediated cell growth of MCF-7 breast cancer cells. The design of the analogs was based upon a cursory Topliss approach in which one of NDGA's aromatic rings was modified with various substituents. Structural modification of one of the two catechol rings of NDGA was found to have little effect upon the inhibitory potency against both kinase activity of the IGF-1R and IGF-1 mediated cell growth of MCF-7 cells. 15-LOX was found to be most sensitive to structural modifications of NDGA. From the limited series of NDGA analogs examined, the compound that exhibited the greatest selectivity for IGF-1 mediated growth compared to 15-LOX inhibition was a cyclic analog 7h with a framework similar to a natural product isolated from Larrea divaricata. The results for 7h are significant because while NDGA displays biological promiscuity, 7h exhibits greater specificity toward the breast cancer target IGF-1R with that added benefit of possessing a 10-fold weaker potency against 15-LOX, an enzyme which has a purported tumor suppressing role in breast cancer. With increased specificity and potency, 7h may serve as a new lead in developing novel therapeutic agents for breast cancer.

Full text of DOI:10.1016/j.bmcl.2007.04.092

Bioorganic & Medicinal Chemistry Letters 17 (2007) 4026–4029
Inhibition of IGF-1R and lipoxygenase by
nordihydroguaiaretic acid (NDGA) analogs
Joseph E. Blecha,a Marc O. Anderson,a Jennifer M. Chow,a Christle C. Guevarra,a
Celia Pender,b Cristina Penaranda,b Marianna Zavodovskaya,b
Jack F. Youngrenb,* and Clifford E. Berkmana,*
aDepartment of Chemistry & Biochemistry, San Francisco State University, San Francisco, CA 94132-4163, USA
bDiabetes and Endocrine Research, University of California, San Francisco/Mt. Zion Medical Center, Box 1616, San Francisco,
CA 94143-1616, USA
Received 15 February 2007; revised 21 April 2007; accepted 25 April 2007
Available online 29 April 2007
Abstract—Herein, we pursue the hypothesis that the structure of nordihydroguaiaretic acid (NDGA) can be refined for selective
potency against the insulin-like growth factor 1 receptor (IGF-1R) as a potential therapeutic target for breast cancer while dimin-
ishing its action against other cellular targets. Thus, a set of NDGA analogs (7a–7h) was prepared and examined for inhibitory
potency against IGF-1R kinase and an alternative target, 15-lipoxygenase (15 LOX). The anti-cancer effects of these compounds
were determined by their ability to inhibit IGF-1 mediated cell growth of MCF-7 breast cancer cells. The design of the analogs
was based upon a cursory Topliss approach in which one of NDGA’s aromatic rings was modified with various substituents. Struc-
tural modification of one of the two catechol rings of NDGA was found to have little effect upon the inhibitory potency against both
kinase activity of the IGF-1R and IGF-1 mediated cell growth of MCF-7 cells. 15-LOX was found to be most sensitive to structural
modifications of NDGA. From the limited series of NDGA analogs examined, the compound that exhibited the greatest selectivity
for IGF-1 mediated growth compared to 15-LOX inhibition was a cyclic analog 7h with a framework similar to a natural product
isolated from Larrea divaricata. The results for 7h are significant because while NDGA displays biological promiscuity, 7h exhibits
greater specificity toward the breast cancer target IGF-1R with that added benefit of possessing a 10-fold weaker potency against 15-
LOX, an enzyme which has a purported tumor suppressing role in breast cancer. With increased specificity and potency, 7h may
serve as a new lead in developing novel therapeutic agents for breast cancer.
Ó 2007 Elsevier Ltd. All rights reserved.
Nordihydroguaiaretic acid (NDGA, Fig. 1) is isolated
from the resinous extract of the creosote bush Larrea
divaricata.1 It has also been reported that NDGA inhib-
its the growth of tumors, both in vitro and in vivo2 but
relatively high concentrations are required to achieve
efficacy in most cases. Preliminary in vivo studies have
Figure 1. Structure of nordihydroguaiaretic acid (NDGA).
revealed that NDGA inhibits tumor growth by inhibit-
ing receptor tyrosine kinase (RTK) phosphorylation.3
quent signaling through the anti-apoptotic pathway in
NDGA has been shown to inhibit several RTKs overex-
pressed in certain cancer cells. In a previous study, we
breast cancer cell.3,4 These and other RTKs such as
the epidermal growth factor receptor (EGFR) are over-
noted that NDGA inhibits both the IGF-1R and
expressed in breast and other cancers and contribute to
HER2/neu with moderate potency and impairs subse-
a poor prognosis.5–7
However, NDGA is known to directly interact with
multiple non-RTK targets, several of which could con-
Keywords: Nordihydroguaiaretic acid; NDGA; Insulin-like growth
factor 1 receptor; IGF-1R; 15-Lipoxygenase; 15-LOX.
tribute to its anti-cancer effects. NDGA directly inhibits
the interaction of fatty acid synthase (FAS) with the
substrate malonyl CoA.8 NDGA can also directly inhi-
*
Corresponding authors. Tel.: +1 415 885 7552; fax: +1 415 885 7727
(J.F.Y.); tel.: +1 415 338 1288; fax: +1 415 338 2384 (C.E.B); e-mail
addresses: jack.youngren@ucsf.edu; cberkman@sfsu.edu
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.04.092

J. E. Blecha et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4026–4029
4027
bit 5-a reductase,9 thereby blocking the conversion of
testosterone into the biologically active dihydrotestos-
terone. Inhibitors of both FAS and 5-a reductase can in-
hibit growth or induce apoptosis in certain cancer cell
lines.9,10 NDGA’s reported antioxidant properties are
attributed to its activity as a non-selective inhibitor of
lipoxygenases (LOX).11 The role of LOX isozymes in
cancer has been questionable, as the anti-cancer effects
of NDGA were originally attributed to reduction of cel-
lular levels of leukotrienes generated from arachidonic
acid by 5- and 12-LOX,12–14 but recent studies suggest
that in breast cancer, 15-LOX has a tumor suppressing
role.15. The multiplicity of NDGA’s effects may, in some
cases, enhance its anti-tumor actions, but may also
antagonize its therapeutic effects or increase the undesir-
able side effects in non-tumor sites. Therefore, in design-
ing potential therapeutics for breast cancer, compounds
capable of inhibiting RTKs such as IGF-1R and HER2/
neu but lacking potency against 15-LOX or other targets
could prove promising. The development of NDGA
analogs with more specific potencies for various cellular
targets is expected to result in an enhancement of its
anti-cancer properties.
chain and one of the hydroxyls of a catechol ring were
absent.
The synthesis of NDGA analogs 7a–7g is outlined in
Scheme 1. Carboxylic acids 1a and 1b were initially re-
19
duced to the corresponding alcohols with LiAlH4
and subsequently oxidized to the intermediate aldehydes
2a and 2b with pyridinium chlorochromate (PCC).20
Next, treatment with substituted-aryl Grignard reagents
(3a–3f) yielded alcohols21 4a–4g, which were oxidized
with PCC20 to yield ketones 5a–5g prior to their com-
plete reduction.
As halogenated aryl rings can undergo dehalogenation
under the simple conditions of catalytic hydrogenation
to reduce benzylic ketones,22 the halogen-containing
aryl ketones 5d and 5e were subjected to adapted
Wolff–Kishner conditions employing hydrazine hy-
drate23 to generate an initial hydrazone intermediate.
Hydrazones were subsequently reduced with KOH23,24
to yield alkanes 6d and 6e. The remaining non-halogen
substituted aryl ketones (5a–5c, 5f, and 5g) were reduced
by simple catalytic hydrogenation. Next, precursors 6a–
25
6e were demethylated with BBr3 to yield the NDGA
analogs 7a–7e. 3,4-Methylenedioxy-protected catechol
The focus of the present study was to demonstrate that
the structure of NDGA could be refined for selective po-
tency against the IGF-1R as a therapeutic target for
breast cancer16 while diminishing its action against
15-LOX. Primarily based upon a cursory Topliss
approach,17,18 our objective was to prepare a small set
of NDGA analogs in which the substituents on one of
NDGA’s aromatic rings were adjusted to explore a
range of hydrophobicity, electronics, and steric proper-
ties. We also pursued two simplified analogs of NDGA
in which the methyl substituents on the bridging butyl
26
6f was selectively deprotected using PCl5 to yield 7f.
Similar conditions were applied to 6g, followed by sub-
sequent demethylation with BBr3 to generate 7g, the
desmethyl analog of NDGA. Both 7a and 7g have been
reported previously.27,28
A cyclic NDGA analog 7h was prepared as outlined in
Scheme 2. Ketone 5a was reduced with Et3SiH29 and
TFA, and was expected to proceed through two steps
of carbocation formation followed by reduction through
Scheme 1. Reagents and conditions: (a) LiAlH4, CH2Cl2, rt, 3.5 h; (b) PCC, CH2Cl2, rt, 1 h; (c) Et2O or THF, À78 °C, then rt, 30 min; (d) PCC,
CH2Cl2, rt, 1 h; (e) H2, 10% Pd/C, THF, rt, 1.5 h; (f) H2NNH2.H2O and KOH, 165 °C, 4 h; (g) BBr3, CH2Cl2, À78 °C, then rt, 30 min; (h) PCl5,
PhMe, 160 °C, then H2O.

4028
J. E. Blecha et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4026–4029
Scheme 2. Reagents and conditions: (a) TFA, Et3SiH, 0 °C then rt, 2.5 h; (b) BBr3, CH2Cl2, À78 °C, then rt, 30 min.
hydrogen transfer by the silane. Applied to this system,
these conditions generated product 9 which may have
occurred through cyclization of the benzylic carbocation
intermediate 8. Subsequent demethylation provided cyc-
lic NDGA analog 7h. Incidentally, this cyclic framework
has also been isolated as a plant-derived natural product
in Larrea spp.30
NDGA. These results suggest that with respect to the
structure of NDGA, a single catechol ring is sufficient
to affect inhibition of IGF-1R. The inhibition of IGF-1
mediated growth of MCF-7 cells by NDGA and the
analogs presented an approximate 3-fold range in po-
tency. The slight variation of potency compared to the
results of IGF-1R inhibition may be due to variances
in passive membrane permeability, metabolism, or inter-
actions with other cellular targets. In contrast, NDGA
and its analogs displayed larger differences in their
inhibitory potency against 15-LOX. The desmethyl ana-
log (7g) exhibited similar anti-lipoxygenase activity as
NDGA and both these compounds were the most po-
tent in the series that were examined. Given the symme-
try of NDGA and 7g, the greater accessibility and
probability of catechol rings binding in the enzyme
may be responsible for their enhanced potency. Analogs
possessing a single catechol ring and bearing sterically
demanding substituents on the remote non-catechol ring
exhibited weaker anti-lipoxygenase activity. The most
dramatic loss of activity was observed for the cyclic ana-
log 7h, especially when directly compared to its linear
Compounds 7a–7h were screened along with NDGA in
three separate assays for activity. The first assay tested
direct inhibition of IGF-1R phosphorylation of a syn-
thetic, non-specific, protein tyrosine kinase substrate.
The second examined the inhibitory effect of the NDGA
analogs on IGF-1 mediated cell growth of MCF-7
breast cancer cells. The third assay screened the analogs
for the inhibition of 15-LOX, a non-target enzyme and
one for which NDGA is a known inhibitor.31 The results
from the three assays for NDGA and its synthetic ana-
logs are summarized in Table 1.
In general, the analogs did not possess inhibitory po-
tency against IGF-1R kinase activity distinct from
Table 1. Inhibitory potency and selectivity of NDGA analogs 6a–6ha
Inhibitor
IGF-1R inhibition IC50 (lM)
Inhibition of IGF-1 mediated growth IC50 (lM)
15-Lipoxygenase inhibition IC50 (lM)
NDGA
7a
7b
0.9 (0.3)
0.8 (0.1)
0.8 (0.1)
1.0 (0.1)
0.8 (0.4)
1.0 (0.2)
1.1 (0.1)
0.6 (0.1)
1.0 (0.4)
24.6 (10.7)
11. 2 (1.8)
15.1 (4.1)
36.2 (6.4)
13.4 (3.3)
21.5 (3.2)
15.7 (4.0)
25.4 (6.6)
12.3 (2.4)
3.8 (0.8)
7.3 (0.9)
21.6 (0.5)
9.4 (0.3)
15.0 (5.5)
33.6 (1.6)
15.0 (1.4)
3.9 (1.1)
38.6 (1.7)
7c
7d
7e
7f
7g
7h
a IC50 values represent the means of a minimum of three determinations with standard deviation in parentheses.

J. E. Blecha et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4026–4029
4029
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Acknowledgments
This work was supported by a grant from the National
Cancer Institute, U-56 CA92616. The authors would
also like to extend their gratitude to W. Tam and the
NMR facility at SFSU for their expert assistance.
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