Bicyclic cyclohexenones as inhibitors of NF-κB signaling

Article abstract of DOI:10.1021/ml300034a

A series of structurally simplified cryptocaryone analogues were synthesized by a facile Pd-catalyzed acetoxylation of alkyne-tethered cyclohexadienones and evaluated as inhibitors of NF-κB signaling. Compounds 10 and 11 were found to possess low micromolar inhibitory properties toward induced NF-κB activity by blocking p50/p65 nuclear protein through a covalent inhibition mechanism. Both compounds were able to inhibit NF-κB-induced IL-8 expression and exhibited antiproliferative activity against two model cancer cell lines. These analogues constitute a promising new scaffold for the development of novel NF-κB inhibitors and anticancer agents.

Full text of DOI:10.1021/ml300034a

Letter
pubs.acs.org/acsmedchemlett
Bicyclic Cyclohexenones as Inhibitors of NF-κB Signaling
Joseph K. Hexum,† Rodolfo Tello-Aburto,‡ Nicholas B. Struntz,† Andrew M. Harned,*,‡
and Daniel A. Harki*,†
†Department of Medicinal Chemistry, University of Minnesota, 717 Delaware Street SE, Minneapolis, Minnesota 55414, United
States
‡Department of Chemistry, University of Minnesota, 207 Pleasant Street SE, Minneapolis, Minnesota 55455, United States
S
* Supporting Information
ABSTRACT: A series of structurally simplified cryptocaryone analogues were
synthesized by a facile Pd-catalyzed acetoxylation of alkyne-tethered cyclohexadienones
and evaluated as inhibitors of NF-κB signaling. Compounds 10 and 11 were found to
possess low micromolar inhibitory properties toward induced NF-κB activity by blocking
p50/p65 nuclear protein through a covalent inhibition mechanism. Both compounds were
able to inhibit NF-κB-induced IL-8 expression and exhibited antiproliferative activity
against two model cancer cell lines. These analogues constitute a promising new scaffold
for the development of novel NF-κB inhibitors and anticancer agents.
KEYWORDS: Natural product analogues, Pd-catalyzed acetoxylation, NF-κB inhibitors, anticancer agents
ransient induction of the nuclear factor-κB (NF-κB)
transcription factors is an essential step in the immune
used to modulate aberrant NF-κB signaling. Due to this
promising activity, enantioselective total syntheses of crypto-
caryone have been recently described by both Fujioka21 and
Franck.22
T
response to pathogens, resulting in the expression of genes
associated with cellular proliferation, differentiation, and
survival, as well as activating the cellular inflammatory
response.1−3 Equally important is the ability of cells to down-
regulate or terminate this activity.2 Constitutive NF-κB
activation has been observed in a spectrum of human cancers,
such as acute and chronic myeloid leukemias, prostate, breast,
lung, and brain cancers.4 Chronic inflammation resulting from
constitutive NF-κB activation has been strongly implicated in
the carcinogenesis of tissues from these organ sites.5−8
Consequently, the NF-κB signaling pathway has become an
important therapeutic target for developing the next-generation
of small molecule anticancer agents.9
There have been numerous efforts aimed at identifying
natural products with activity against the NF-κB pathway.10−13
Many of these natural products contain reactive moieties (e.g.,
enones) that can capture nucleophiles and inhibit signaling
through a covalent mechanism.14,15 Drugs that function
through a covalent mechanism are widely used in the
pharmaceutical industry, and at least 39 FDA-approved
medicines can be classified as “covalent drugs”.16 One recently
identified natural product NF-κB inhibitor with a reactive
enone moiety is cryptocaryone (1).17−19 Researchers at the
National Cancer Institute reported that 1 inhibits degradation
of the NF-κB repressor protein IκBα in B lymphocytes with
high constitutive IKK (IκB kinase complex) activity.20 Activated
IKKs phosphorylate IκB repressor proteins, targeting them for
ubiquitination and degradation by the 26S proteosome. Freed
p50/p65 (NF-κB) heterodimers can then translocate to the
nucleus and activate the expression of NF-κB target genes.1,8,9
Accordingly, inhibiting the degradation of IκB proteins by small
molecules such as cryptocaryone (1) is a strategy that can be
Our interest in cryptocaryone stems from its structural
similarity to a series of compounds we have recently prepared
via Pd-catalyzed acetoxylation of alkyne-tethered cyclohexadie-
nones (e.g., 2→3, Figure 1A).23 While 3 does not have the
cinnamoyl side chain present in 1, we hypothesized that the
structurally similar enone moiety that is present in both 1 and 3
may yield similar biological activity profiles for both
compounds. Previous studies with sesquiterpene lactones that
inhibit NF-κB signaling have revealed covalent Michael adduct
formation to exposed cysteine residues on essential NF-κB
proteins, thereby eliminating their enzymatic activity and
disrupting the signaling pathway.13 While cryptocaryone’s
exact protein target(s) and mechanism of binding (e.g.,
covalent or noncovalent) are unknown at this time, it is
possible that it similarly captures accessible cysteine sulfhydryl
groups on key protein(s) involved in NF-κB signaling and
abolishes their function. If this is the case, then structurally
analogous compounds to cryptocaryone (1), such as 3, should
also possess NF-κB inhibitory properties. The presence of the
fully substituted carbon atom at the γ-position of the enone in 3
does provide some additional steric hindrance that may deter
Michael adduct formation in comparison to 1. Undeterred, we
investigated the NF-κB inhibitory activity of several bicyclic
enones similar to 3 and we also studied their antiproliferative
activities against protypical leukemia and prostate cancer cell
lines.
Received: February 8, 2012
Accepted: May 4, 2012
Published: May 11, 2012
© 2012 American Chemical Society
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Figure 1. (A) Structure of cryptocaryone (1) and methodology for the
preparation of the bicyclic enones of interest. (B) Analogues 4−12
(racemic) screened for NF-κB inhibitory activity.
The compounds investigated during this study are shown in
Figure 1B and were prepared using methods previously
described.23 Compounds 4−12 were all prepared as racemic
samples. Initial screening of 4−11 was carried out by utilizing a
standard NF-κB luciferase reporter assay in A549 human lung
cancer cells. This cell line bears a stably transfected luciferase
reporter construct downstream of six repeats of the consensus
NF-κB binding site. Induction of NF-κB signaling is achieved
by treatment with TNF-α, and cell permeable small molecules
can inhibit this induced activity. We performed a preliminary
screen by treating induced cells with 50 μM concentrations of
4−11. This concentration was selected on the basis of
cryptocaryone’s ability to inhibit the degradation of IκBα at
16 μM.20 Results from our study found 4 to be poorly soluble
under the assay conditions, yielding unreliable values, and
compounds 5−9 failed to inhibit induced NF-κB activity
(Supporting Information Figure S1). Gratifyingly, analogues 10
and 11 both inhibited induced NF-κB activity (Figure 2A and
Table 1). At a concentration of 50 μM, both compounds
inhibited induced NF-κB activity, with 11 (36% residual NF-κB
activity) exhibiting more potent inhibition than 10 (62%
residual NF-κB activity) following treatment. The NF-κB
inhibitory activity was not attributable to nonspecific cell
death, as only 10% (for 10) and 5% (for 11) decreases in cell
viability were observed following 50 μM treatment with each
compound under identical assay conditions (Supporting
Information Figure S2). Furthermore, 11 completely inhibited
NF-κB activity to noninduced levels at a 100 μM treatment and
was more potent than 10 at all concentrations examined.
Encouraged by these results, we evaluated compounds 10
and 11 for their cytotoxicity in two standard human cancer cell
lines, DU-145 and CCRF-CEM. DU-145 is a model for
hormone-independent prostate cancer, and CCRF-CEM is a
Figure 2. (A) Cellular NF-κB luciferase reporter assay in A549 cells.
NI = noninduced control cells, I = cells induced with TNF-α.
Compound-treated cells are induced with TNF-α and treated with
analogues 10−12 at the following concentrations: 100 μM, 50 μM,
and 10 μM. (B−E) Cytotoxicity of 10−12 against (B) CCRF-CEM,
(C) DU-145, (D) Vero, and (E) RWPE-1 cells. Cells were treated
with 10 (open triangles), 11 (closed circles), and 12 (open squares) at
various concentrations, and cell viability was measured by Alamar Blue
staining.
model for childhood T-cell acute lymphoblastic leukemia. DU-
145 cells are known to possess constitutive NF-κB activity.24,25
As shown in Figure 2, 10 and 11 exhibited similar cytotoxicities
against DU-145 and CCRF-CEM. Both compounds were
slightly more potent against CCRF-CEM cells (IC50: 10, 11 =
26 μM) than DU-145 cells (IC50: 10 = 34 μM, 11 = 36 μM) in
a 48-h cytotoxicity assay (Figure 2B−C and Table 1). The
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Table 1. Results of NF-κB Reporter and Cellular Cytotoxicity Assays Performed with 10−12
a
b
% NF-κB activity
50 μM
IC50 (μM)
DU-145
c
compd
100 μM
10 μM
CCRF-CEM
Vero
RWPE-1
SI
10
11
12
33.9 10.8
17.3 5.1
76.1 10.9
61.6 10.7
36.2 8.2
94.4 13.1
90.9 13.5
93.7 15.5
109.4 12.0
26.4 7.3
26.4 3.1
>500
33.7 5.8
35.9 4.8
>500
>500
>500
>500
14.8 2.6
98.8 15.1
>500
0.44
2.75
a
Percentage (%) NF-κB activity at various doses is shown relative to the induced, DMSO treated control (arbitrarily assigned 100% activity). Mean
SD values are shown. Cellular cytotoxicity as measured by Alamar Blue staining. IC50 values shown are the mean SD. The selectivity indices
b
c
(SI) were calculated by dividing the IC50 values obtained for compound-treated RWPE-1 cells by the IC50 values obtained for compound-treated
DU-145 cells.
observed IC50 values for 10 and 11 against DU-145 are
approximately 15-fold higher than the reported IC50 value for
cryptocaryone (IC50 = 2.3 μM) in the same cell line.26
cules.33 A solution of 10 in DMSO-d6 was dissolved in aqueous
(D2O) phosphate buffered saline (PBS), and the vinyl protons
were observed by 1H NMR (Figure 3B). Addition of two molar
We next evaluated whether 10 and 11 may exhibit any
selectivity toward cancerous versus noncancerous cells.
Recently, the enone-containing natural product piperlongumine
has been found to possess remarkable selectivity for promoting
the death of cancer cells versus normal cells.27 To determine if
10 and 11 are similarly selective, we measured the cellular
cytotoxicities of both compounds against Vero and RWPE-1
cells (Figure 2D-E and Table 1). Vero is a noncancerous kidney
epithelial cell line derived from the African green monkey and is
widely utilized for screening bioactive candidate molecules for
toxicity toward normal cells.28,29 To our delight, both 10 and
11 exhibited no appreciable toxicity toward Vero cells (IC50
values > 500 μM). We then screened both molecules for
toxicity against RWPE-1, which is an immortalized human
prostate epithelial cell line derived from noncancerous prostate
tissue.30−32 Since 10 and 11 exhibited reasonable potency
against DU-145 prostate cancer cells, we deemed that
measuring their tissue-specific selectivities for targeting cancer-
ous versus noncancerous prostate cells may yield useful
information that could inform future development of this
class of molecules as prostate cancer therapeutics. Interestingly,
10 was found to be toxic toward RWPE-1 cells (IC50 = 14.8
μM) and exhibited no selectivity toward cancerous DU-145
cells versus noncancerous RWPE-1 cells (selectivity index, SI =
0.44). Conversely, 11 was significantly less toxic than 10 toward
normal RWPE-1 cells (IC50 = 98.8 μM) and had modest
selectivity toward the cancerous DU-145 cell line (SI = 2.75).
Consequently, 11 may represent a better lead molecule for
future development than constitutional isomer 10.
To determine if 10 and 11 function through a covalent
mechanism of inhibition, the enone moiety of compound 10
was reduced to produce the respective cyclohexanone 12
(Figure 1). If covalent capture of proteins is the mechanism of
biological activity for this class of molecules, then 12 should
exhibit diminished NK-κB inhibitory activity and be signifi-
cantly less cytotoxic to cancer cells than enone-containing
molecules 10 and 11. Utilizing the NK-κB luciferase reporter
assay, we found that 12 only reduced NF-κB activity by 24% at
the highest concentration tested (100 μM), which is a ∼4-fold
decrease in potency compared to the case of 11 (Figure 2A and
Table 1). Furthermore, 12 exhibited no appreciable toxicity
toward any of the cell lines tested (IC50 values could not be
accurately calculated due to lack of potency). Therefore, the
presence of the enone on compounds 10 and 11 is required for
biological potency.
Figure 3. (A) Analogue 10 forms Michael adduct 13 upon reaction
with cysteamine in aqueous buffer. The reaction shown in panel A was
1
1
monitored by H NMR spectroscopy and analytical HPLC. (B) H
NMR analysis of vinyl protons Ha and Hb from 10 before (t = 0 min)
and after addition of cysteamine. Reaction was judged to be nearly
complete after 5 min. (C) HPLC analysis of 10 before (top
chromatogram) and after addition of cysteamine (t = 10 min, bottom
chromatogram). Adduct 13 was characterized by mass spectrometry.
H/D for 13 is dictated by whether the reaction is performed in D2O
(for NMR analysis, panel B) or H2O (for HPLC analysis, panel C).
equivalents of cysteamine resulted in the disappearance of the
doublets at 6.06 ppm (Hb of 10) and 6.75 ppm (Ha of 10)
within 5 min, suggesting Michael addition of the cysteamine
thiol to the enone of 10 and formation of adduct 13 (Figure
3A). To verify the formation of 13, the cysteamine reaction was
performed again (in H2O) and the reaction products were
analyzed by HPLC (Figure 3C). Analysis of the major reaction
product by mass spectrometry confirmed the identity of newly
We further characterized the ability of compounds of this
class to capture cellular nucleophiles by performing a recently
described, NMR-based thiol reactivity assay of small mole-
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ACS Medicinal Chemistry Letters
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formed adduct 13. Together with the biological data for
cyclohexanone 12, these results show that 10 and 11 can
capture cellular nucleophiles through their electrophilic enones
and that reactivity is required for their inhibitory activities
toward NK-κB signaling and cancer cell growth.
To study the biochemical mechanism of NF-κB inhibition by
cryptocaryone analogues, we fractionated NF-κB-induced and
compound-treated DU-145 and CCRF-CEM cells and then
immunoblotted for p65 protein in the nuclear lysate (Figure 4A
degradation of IκB repressor proteins, whereas no change in
nuclear p65 levels should be observed if 10 and 11 modulate
NF-κB by directly targeting the p50 or p65 protein (e.g.,
alkylation of NF-κB proteins by the small molecule abolishes
DNA-binding capability). Both 10 and 11 completely inhibited
nuclear translocation of p65 at a 100 μM treatment in NF-κB-
induced DU-145 (Figure 4A) and CCRF-CEM (Supporting
Information Figure S3) cell lines. Partial inhibition of p65
nuclear translocation was observed at a 25 μM treatment of 10
and 11 in both cell lines. Immunoblotting of our prepared
nuclear fractions for α-tubulin, an abundant cytosolic protein,
revealed no detectable signal, which verifies that our nuclear
p65 lysate samples are not contaminated with cytosolic p65
(Supporting Information Figure S3). On the basis of these
results, we can conclude that cryptocaryone analogues such as
10 and 11 modulate NF-κB activity by inhibiting IκB
degradation and p50/p65 heterodimer translocation to the
nucleus.
To further evaluate the ability of our molecules to regulate
NF-κB activities in cells, we tested their ability to inhibit the
activation of IL-8, a well-known proinflammatory regulator. IL-
8 is a chemokine whose gene expression is activated by NF-κB
binding to the IL-8 promoter.34−36 DU-145 cells were treated
with 10−12, and NF-κB was induced by addition of TNF-α.
Secreted IL-8 levels in cell media were measured by ELISA.
Additionally, we further correlated IL-8 protein levels to IL-8
mRNA levels by semiquantitative (traditional) RT-PCR
analysis. As expected, analysis of IL-8 mRNA and protein
levels revealed significant increases in both following NF-κB
induction with TNF-α (Figure 4B−C). Compound 11 was
found to inhibit the production of IL-8 mRNA to visibly lower
levels than that of 10 at the lowest concentration tested (33
μM). The changes in mRNA levels observed with 10 and 11
were mirrored in the amount of secreted IL-8 protein
measured. Compound 10 abolished IL-8 production to
noninduced levels, while compound 11 depleted IL-8 protein
levels to nearly undetectable levels at the highest concentration
tested. Additionally, control compound 12 at 100 μM had an
insignificant effect (4,900 pg/mL) on IL-8 levels as compared
to 10 and 11 at a 33 μM dose (3,480 pg/mL and 2,970 pg/mL,
respectively). These results further support our findings that
the formation of Michael adducts is vital to the mechanism of
NF-κB pathway inhibition for bicyclic cyclohexenones 10 and
11 and establish the anti-inflammatory features of molecules of
this class.
Figure 4. (A) Western blot analysis of p65 and histone H2B proteins
from nuclear lysate fractions of compound-treated DU-145 cells. Cells
were dosed with either vehicle control (DMSO; NI and I lanes) or
analogues 10 or 11 (100 μM and 25 μM), followed by induction of the
NF-κB pathway with TNF-α. (B) RT-PCR analysis of IL-8 and
GAPDH mRNA from DU-145 cells. (C) Secreted IL-8 protein levels
as measured by ELISA. Data shown is mean SEM. (B−C) Cells
were dosed with either vehicle control (DMSO; NI and I lanes) or
analogues 10 or 11 (100 μM and 33 μM) or 12 (100 μM), followed
by induction of the NF-κB pathway with TNF-α. (A−C) NI =
noninduced cells; I = induced cells.
and Supporting Information Figure S3). Decreased p50/p65
nuclear translocation is expected if 10 and 11 inhibit the
Figure 5. Enhanced potency of enantioenriched 11*. (A) Synthesis of 11*. (B) Cellular NF-κB luciferase reporter assay in A549 cells. NI =
noninduced control wells; I = cells induced with TNF-α. Compound-treated cells are induced with TNF-α and treated with analogue 11* at the
following concentrations: 100 μM, 50 μM, and 10 μM. (C) Cytotoxicity of 11* against CCRF-CEM (open triangles) and DU-145 human prostate
cancer cells (closed circles). Cell viability was measured by Alamar Blue staining.
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With another goal of beginning to establish the stereo-
isomeric preferences of these compounds, we carried out the
enantioselective reaction shown in Figure 5. To our delight, the
use of the chiral bipyridine ligand (−)-iso-PINDY37 allowed us
to produce enone 11* in 63% ee from precursor 14, with the
major enantiomer being the one shown (Figure 5A). To
determine if enantioenriched 11 (which we have denoted as
11*) possesses enhanced biological potency compared with
racemic 11, we repeated the NF-κB reporter assay and cancer
cell cytotoxicity experiments with 11*. Interestingly, 11*
completely abolished induced NF-κB signaling to noninduced
levels (Figure 5B), resulting in 18% relative NF-κB activity at
50 μM. This constitutes a 2-fold enhancement in potency
compared with the case of racemic 11. An enhancement in
potency with 11* versus racemic 11 was observed in
cytotoxicity assays (Figure 5C) against CCRF-CEM cells
(IC50 = 19 μM), whereas 11* was slightly less active toward
DU-145 cells (IC50 = 46 μM). Unfortunately, (−)-iso-PINDY
has not proven to be generally useful for preparing other
bicyclic products with high selectivity; therefore, a more
thorough study into this effect will have to wait until a more
effective ligand is identified.
In conclusion, we have identified a new class of oxygenated
bicyclic enones with activity against the NF-κB signaling
pathway. While the cytotoxic and NF-κB inhibitory activities of
these compounds are modest, they are sufficiently strong to
view these compounds as interesting lead compounds for
further development. In addition to establishing a more
comprehensive structure−activity relationship, we are working
to further clarify the exact molecular target(s) of these
compounds. All of these results will be reported in due course.
Tian (Institute for Therapeutics Discovery and Development,
University of Minnesota) for technical assistance.
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*D.A.H.: tel, 612-625-8687; fax, 612-626-6318; e-mail,
daharki@umn.edu. A.M.H.: tel, 612-625-1036; fax, 612-626-
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