Inhibition of IκB kinase-β and anticancer activities of novel chalcone adamantyl arotinoids

Article abstract of DOI:10.1021/jm800285f

On the basis of the observations that chalcone 7 (MX781) and some related adamantyl arotinoids (AdArs) inhibit IκBα kinase β (IKKβ) activity, inhibit cell growth, and induce apoptosis in cancer cells, a new series of AdArs structurally related to 7 have b

Full text of DOI:10.1021/jm800285f

J. Med. Chem. 2008, 51, 5431–5440
5431
Inhibition of IKB Kinase-ꢀ and Anticancer Activities of Novel Chalcone Adamantyl Arotinoids
,†
Paula Lorenzo,† Rosana Alvarez,† Maria A. Ortiz,‡ Susana Alvarez,† F. Javier Piedrafita,*,‡ and Angel R. de Lera*
´
Departamento de Qu´ımica Orga´nica, UniVersidade de Vigo, Lagoas-Marcosende, 36310 Vigo, Spain, and Sidney Kimmel Cancer Center,
10905 Road to the Cure, San Diego, California 92121
ReceiVed March 14, 2008
On the basis of the observations that chalcone 7 (MX781) and some related adamantyl arotinoids (AdArs)
inhibit IκBR kinase ꢀ (IKKꢀ) activity, inhibit cell growth, and induce apoptosis in cancer cells, a new
series of AdArs structurally related to 7 have been designed and synthesized. Modifications were intended
to reduce or eliminate RAR activity, and we evaluated the effect of the novel analogues of 7 on IKKꢀ
activity and proliferation of a variety of cancer cell lines (leukemia, Jurkat; prostate, PC-3; breast carcinomas,
T47D, MDA-MB-468). Consistent with the design principles, the biological activities of these AdArs do
not appear to be RAR-mediated, since most analogues are unable to activate RAR-mediated transactivation
and exhibit significantly diminished antagonist activity. All compounds are capable of inducing apoptosis
in Jurkat cells, as demonstrated by elevated DEVDase activity and externalization of phosphatidylserine.
Several of the analogues elicit stronger growth inhibitory activity against prostate (PC-3) and breast (MDA-
MB-468) carcinoma cells, which contain elevated basal IKK activity; this antiproliferative activity correlates
with increased inhibition of recombinant IKKꢀ in vitro, suggesting that the anticancer activities of these
AdArs might be related to the inhibition of IKK/NFκB signaling.
Introduction
(6-[(3-adamant-1-yl)-4-hydroxy-phenyl]-2-naphthoic acid, CD437,
also called AHPN) are classical examples of RRMs (see Figure
1) that induce growth arrest and apoptosis in a variety of cancer
cell lines. Within this class, the adamantyl arotinoids (AdArs),
of which 3 is the prototype lead compound, have been the most
thoroughly studied. Novel derivatives of 3 (including 5-Cl-
AHPN 4 and 3-Cl-AHPC 6; see Figure 1) have been obtained
that preserve apoptotic activity but lack RAR transactivation
activity.12-14
The retinoids, natural and synthetic analogues of retinol
(vitamin A), are key regulators of many biological events
including cell growth and differentiation, development, homeo-
stasis, and carcinogenesis.1-3 Most of these biological functions
are mediated by the complexes obtained after binding of ligands
to the retinoic acid receptors (RARs,a subtypes R, ꢀ, and γ)
and retinoid X receptors (RXRs, subtypes R, ꢀ, and γ), which
are members of the nuclear receptor superfamily.4-6 Structural
information is available on both unbound apo- and holoretinoid
receptors bound to a variety of ligands,7 which has facilitated
the design of more potent modulators of retinoid receptor action
such as antagonists, inverse agonists, and agonists selective for
a particular RAR subtype,8 as well as RXR-selective retinoids
or rexinoids. Some of these selective retinoids or rexinoids show
enhanced antiproliferative activity against cancer cells, which
correlates with their ability to induce cell differentiation, growth
arrest, and apoptosis.9 The highly promising potential of vitamin
A and its derivatives as cancer preventive and chemotherapeutic
agents has, however, not yet been fully realized because of the
toxic side effects observed, which somehow limit their thera-
peutic use.10
The mechanism of RRM-mediated cell death, in particular
the induced apoptosis by 3, has been investigated by numerous
laboratories and seems to be largely dependent on cell type.
RRMs have been shown to inhibit cell growth by caspase-
15-17
dependent and independent mechanisms
and induce
apoptosis via the intrinsic pathway,18,19 although activation of
the death receptor-mediated extrinsic pathway has also been
demonstrated in some cases.20 Even though the majority of the
RRMs bind and activate the nuclear RARs, they seem to induce
apoptosis independently of RARs,11 most likely by binding to
and modulating the activity of other cellular receptors yet to be
identified. Compound 3 and other RRM analogues induced a
strong and sustained activation of cJun N-terminal kinase (JNK)
and p38 stress kinases in several cancer cell lines independently
of caspase activity21 (and our unpublished observations). In
Jurkat T cells, pharmacological inhibition of both JNK and p38
MAPK prevented the release of cytochrome c and subsequent
induction of apoptosis.21 Other groups have reported contradic-
tory results with different MAPK inhibitors and various cancer
cell lines. Thus, inhibition of p38 reduced the apoptosis induced
by 3 in ovarian carcinoma cells,22 whereas inhibition of JNK
partially prevented the induction of cell death in AML cells by
6.23 In contrast, several MAPK inhibitors had no effect on RRM
activity in various leukemia cells.13,24,25 In addition to inducing
MAPK activity, RAR antagonist 7 ((E)-4-{3-[(3-adamant-1-yl)-
4-(2-methoxyethoxymethoxy)-phenyl]-3-oxoprop-1-en-1-
yl}benzoic acid, MX781) and RAR agonist 8 (CD2325)
substantially inhibited the activity of IKK isolated from TNFR-
stimulated HeLa cells, although only the antagonist displayed
A subgroup of retinoids, termed collectively atypical retinoids
or retinoid related molecules (RRMs),10,11 does not fit into the
conventional concept of retinoid action. Although inspired on
the structure of classical retinoids, either they do not bind to
the retinoid receptors or their strong growth inhibitory or
apoptogenic activities do not appear to be retinoid receptor-
mediated. Anhydroretinol 1, 4-hydrophenylretinamide 2, and 3
* To whom correspondence should be addressed. For F.J.P.: phone, +1-
858-410-4188; fax, +1-858-450-3251; e-mail, jpiedrafita@skcc.org. For
´
A.R.d.L.: phone, +34-986-812316; fax, +34-986-811940; e-mail, qolera@
uvigo.es.
†
Universidade de Vigo.
Sidney Kimmel Cancer Center.
‡
a Abbreviations: AdArs, adamantyl arotinoids; ATRA: all-trans-retinoic
acid; IKK, IκB kinase; JNK, cJun N-terminal kinase; PI, propidium iodine;
RAR, retinoic acid receptor; RRMs, retinoid-related molecules; RXR,
retinoid X receptor.
10.1021/jm800285f CCC: $40.75
2008 American Chemical Society
Published on Web 08/14/2008

5432 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17
Lorenzo et al.
Figure 1. Structure of selected RRMs including those with chalcone and adamantyl groups. A chalcone retinoid (10) in its RARγ-bound s-trans
conformation is also shown.
effective and consistent inhibition of IKK/NFκB signals in a
number of cancer cell lines.26 Targeting IKK/NFκB pathways
by means of kinase dead IKK mutants or a nonphosphorylable
form of IκBR was sufficient to induce cell death, further
supporting an important role for IKK and NFκB activity in cell
survival by inhibiting apoptosis.27-29 Our observations contrast
with recent reports showing that 3 and 6 induced cell death via
activation of NFκB in prostate and breast carcinoma cells.30,31
The scarcity of SAR studies on AdArs,32 focusing primarily
on binding to and transactivation through the RAR subtypes,
precludes obtaining insights concerning the structural determi-
nants that could mediate their anticancer activity. It is quite
puzzling that 7 is the only AdAr known to significantly inhibit
TNFR-mediated activation of IKK in cell-based assays.26 The
presence of both the chalcone and the adamantyl groups might
be responsible for this profile, although each of these substruc-
tures is also present in other RRMs that show different biological
profiling (Figure 1). For example, 9 (AGN193198) is a novel
RAR antagonist that induces apoptosis with IC50 at nanomolar
concentrations,33 although its effect on IKK activity has not been
reported. Compound 9 exhibits structural similarities with 7,
with a central chalcone and a long alkyl chain (the N-heptyl
substituent) that resembles the acetal OMEM side chain of 7.
However, 9 lacks the adamantyl group, which could be
considered partly responsible for the binding of 7 to IKK.
Nevertheless, it is clear that the presence of this group is not
sufficient given the fact that several related AdArs do not
the flexibility of the chalcone function in both 7 and 9 might
counteract the effect to a certain degree. In fact, retinoid agonist
10 (BMS181156, Figure 1), which shares the chalcone function,
adopts an s-trans conformation of the chalcone C(O)-CdC
bond when bound to the RARγ receptor, as shown in the crystal
structure of the complex.36 We therefore planned to further
distort the planarity around the chalcone unit of 7, reduce its
conformational flexibility, and disfavor the s-trans conformation
by incorporating an additional substituent ortho to the carbonyl
group (i.e., OR in 11, Scheme 1), an approach complementary
to the one described for 4 and 6 relative to the parent system.12,14
We report herein the synthesis of novel analogues of chalcone
7 (11, 24) along with their biological characterization. Some of
these AdArs surpass the lead compound in the inhibition of cell
growth of several cancer cell lines, including leukemia, prostate,
and estrogen-dependent and independent breast cancer cell lines.
Because RAR transactivation was not detected, the antiprolif-
erative activity of the novel AdArs must be independent of the
activation of the retinoid receptors. Since the inhibition of
recombinant IKKꢀ by several analogues is higher than that
exhibited by 7 and correlates with increased growth inhibitory
and apoptotic activities, it is possible that this antikinase activity
of the analogues might be partially involved in their anticancer
effect on cell lines that express high levels of basal IKK.
Synthesis
26
efficiently inhibit IKK in vitro or in intact cells.
Preserving the location of the adamantyl and OMEM sub-
stituents of the parent structure, derivatives of 7 having alkyl
chains of increasing length attached to the phenol vicinal to
the chalcone unit were designed and synthesized as shown in
Scheme 1. Additionally ethers incorporating unsaturated (allyl,
propargyl) or aryl substituents were included and prepared using
the same strategy. The Claisen-Schmidt condensation of methyl
4-formylbenzoate 23 and the ortho-substituted acetophenone
derivative 22 was selected as the key step for the convergent
construction of the chalcone functionality of the analogues
substituted with hydroxy and alkoxy groups at the C2′ position
(structures 24 and 11, Scheme 1).
Given that IKK has been validated as a cancer therapeutic
target by means of pharmacological and genetic inhibitors34 and
apoptotic AdArs can inhibit IKK in vitro and in cell assays,26
we set out to synthesize a series of derivatives of 7 and examine
their activities as IKK inhibitors to gain information on the
structural determinants of this novel antikinase activity of
AdArs. As a general objective, the designed AdArs should have
reduced binding to the RARs and/or exhibit RAR antagonist
profile. Comparative structural analysis suggests that the OMEM
group of 7 or the N-heptyl substituent of 9 might be responsible
for the RAR-antagonistic effects. The large substituent would
displace the helix H12 from its agonist conformation and
preclude binding of coactivators, in keeping with the classical
mechanism of nuclear receptor antagonistic action.35 However,
The previously described32 Friedel-Crafts-type alkylation of
2,4-dihydroxybenzaldehyde 14 and adamantan-1-ol 15 using
sulfuric acid as catalyst was quite capricious in our hands, and

Inhibition of IKKꢀ by MX781 Analogues
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17 5433
Scheme 1a
a Reagents and reaction conditions: (a) adamant-1-ol 15, H2SO4, CH2Cl2, ultrasounds, 50 °C, 16 h (80%); (b) NaH, MEMCl, DMF, 25 °C, 19 h (98%);
(c) (i) MeLi, Et2O, 0 °C, 1.5 h; (ii) CrO3/Py, CH2Cl2, 25 °C, 3 h (74%); (d) BCl3, CH2Cl2, -78 °C, 18 h (74%); (e) HNa, DMF, 0 °C; then R-X 20 and
21, 25 °C (59-95%); (f) NaOH, MeOH, 70 °C (22-58%).
the yields were highly variable (but not greater than 24%).
Fortunately, the ultrasound irradiation of the heterogeneous
mixture allowed the reaction to reach completion and improve
performance (80% yield of 16). This was followed by exhaustive
phenol protection by treatment of 16 with NaH in DMF at 0
°C followed by trapping with MEMCl to afford 17 in 98% yield.
The corresponding acetophenone 18 was acquired by addition
of MeLi at 0 °C to 17 and oxidation of the secondary benzyl
alcohol with CrO3/pyridine in CH2Cl2 (74% combined yield,
Scheme 1).37
contrast, when cells were stimulated with 1 µM all-trans-retinoic
acid (ATRA), strong induction of luciferase activity was seen,
in particular with the RARR fusion protein. We then examined
the antagonistic activity of the chalcone analogues. CV-1 cells
transfected with Gal4-RARR, RARꢀ, or RARγ were stimulated
with 0.2 µM ATRA alone or together with 2 µM AdArs, a 10-
fold molar excess of the candidate antagonists. For comparison,
the antagonist activity of 7 and pan-inhibitor UVI2024
(BMS204493)39 was also tested. Figure 2B illustrates that
UVI2024 was a robust inhibitor of all three RARs whereas 7
strongly inhibited RARR. The inhibition of RARꢀ and RARγ
mediated by 7 was substantially lower but still significant
(30-40%). Among the analogues, 24 was a slightly better
inhibitor of RARR than 7, while it inhibited RARꢀ and RARγ
with similar potency as 7. The 2-ethoxy substitution in 11a
decreased RAR antagonist activity, and increasing the length
of the alkyl chain substituent gradually eliminated the RAR
antagonistic activity to almost completion in 11e. Other allyl,
propargyl, and aryl substitutions in position C2′ similarly
hindered RAR antagonistic activity (Figure 2B). Of note, 11h
inhibited RARR but not RARγ, whereas 11g slightly inhibited
RARγ and RARR but not RARꢀ. In general, a stronger
inhibition of RARR was seen in the presence of the AdArs and
only modest inhibition of RARꢀ was detected, which could be
explained by the higher affinity of RARꢀ and RARγ for their
natural ligand ATRA. None of the synthesized analogues
activated RXRR-driven luciferase activity or inhibited 9-cis-
retinoic acid or LGD1069-mediated activation of RXR (data
not shown).
Selective deprotection32 of the C2 phenol of 18 was achieved
in 74% yield using 1 equiv of BCl3 in CH2Cl2 at -78 °C. This
outcome is presumably due to the coordinating ability of the
vicinal ketone and the more hindered nature of the OMEM group
at the alternative C4 position. The same method described above
for phenol protection was selected for the alkylation of 19 with
alkyl (20a-f), allyl (21g,j), propargyl (21h), or benzyl halides
(21i, 21k), reactions that took place in moderate to good yields
(59-95%).
For the preparation of chalcones 11 and 24, ketones 22 and
19, respectively, were treated with methyl 4-formylbenzoate 23
using the classical Claisen-Schmidt condensation conditions
described for the parent system (NaOH in MeOH at 70 °C).38
The basic reaction conditions induced the saponification of the
esters, and therefore, the desired carboxylic acids were obtained
as final products. The yield decreased steadily upon increasing
the length of the alkyl chain, and 20f was unreactive. Likewise,
halogenated benzylethers exhibited low reactivity, and 11k
(3.3% yield) was not used in the biological assays.
Anticancer Activity of the AdArs. To evaluate the antipro-
liferative activity of the novel analogues, we first tested their
effect on the RRM-sensitive Jurkat T leukemia cell line using
a MTT colorimetric assay. With the exception of phenol 24,
all other compounds elicited stronger antiproliferative activity
compared to parent 7. Compound 7 inhibited growth of Jurkat
cells with an IC50 of 1.20 µM, whereas 11c and 11i were almost
4 times more active with IC50 values of 0.38 and 0.35 µM,
Biology
RAR Transactivation Profile. We first evaluated the RAR
transactivation activity of the chalcones using CV-1 cells
expressing Gal4-RAR fusion proteins following transient trans-
fection of the corresponding expression vectors. Figure 2A
illustrates that none of the compounds were capable of enhanc-
ing RAR-driven luciferase activity when tested alone. In

5434 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17
Lorenzo et al.
Figure 2. RAR transactivation profile of the novel AdArs. CV-1 cells were cotransfected with 100 ng of UAS-luciferase reporter, 25 ng of
ꢀ-galactosidase, and 10 ng of Gal4-RAR/RXR expression vectors. Sixteen hours after transfection, cells were treated with 1 µM ATRA (atRA) or
2 µM of the indicated compounds (A). To evaluate the antagonistic activity of the analogues, cells were stimulated with 0.2 µM ATRA alone
(hatched column) or together with 2 µM of the indicated AdArs (in gray scale) (B). Luciferase and ꢀ-galactosidase activities were measured after
6 (RARR,ꢀ) or 20 h (RARγ) of AdAr stimulation following standard procedures. Normalized luciferase activity was calculated and referenced to
percentage of maximum activity obtained in the presence of ATRA. The average ( SD of at least two experiments performed in triplicate is shown.
Table 1. Inhibition of Cancer Cell Growth by AdArsa
Jurkat
PC-3
T-47D
MB-468
7
24
1.20 ( 0.12
1.89 ( 0.14
1.14 ( 0.14
0.80 ( 0.11
0.38 ( 0.08
0.50 ( 0.08
0.74 ( 0.10
0.79 ( 0.12
0.58 ( 0.09
0.35 ( 0.08
1.01 ( 0.10
1.48 ( 0.40
2.51 ( 0.17
1.86 ( 0.44
1.32 ( 0.45
0.74 ( 0.10
1.31 ( 0.27
1.35 ( 0.11
1.16 ( 0.30
0.76 ( 0.04
1.27 ( 0.42
2.36 ( 1.21
3.27 ( 0.17
8.00 ( 0.40
5.01 ( 0.09
5.09 ( 0.24
1.41 ( 0.15
2.19 ( 0.08
3.16 ( 0.09
1.98 ( 0.08
1.97 ( 0.15
1.27 ( 0.06
1.40 ( 0.12
2.11 ( 0.21
6.08 ( 0.42
1.79 ( 0.29
1.36 ( 0.11
0.93 ( 0.23
1.08 ( 0.20
1.86 ( 0.21
1.28 ( 0.11
1.03 ( 0.11
0.93 ( 0.01
1.76 ( 0.51
11a
11b
11c
11d
11e
11g
11h
11i
11j
a IC50 values (in µM) were calculated after 24 h (Jurkat) or 48 h (PC-3,
T-47D, MB-468) of treatment with increasing concentrations (0.125-8 µM)
of the indicated analogues. The average ( SD of two experiments performed
in triplicate is shown. Lowest values for each cell line are in boldface.
Figure 3. Time course of action of the AdArs. MDA-MB-468 cells
were incubated with 2 µM of the indicated AdArs for 24, 48, or 72 h,
when cell proliferation was measured by MTT. The experiment was
repeated twice with triplicate time points, and the results of one
representative experiment are shown.
respectively. Other analogues, 11b, 11d, 11e, 11g, and 11h, also
inhibited Jurkat cell proliferation with IC50 values below 1 µM
(Table 1). We next evaluated the growth inhibitory activity of
AdArs against cell lines derived from solid tumors. We chose
one prostate carcinoma cell line (PC-3) that has elevated basal
IKK/NFκB activity26 and two breast cancer cell lines, one
estrogen-dependent (T-47D) and one estrogen-independent
(MDA-MB-468). High IKK/NFκB activity has been reported
to increase with tumor progression in breast carcinomas.40-42
As shown in Table 1, 7 exhibited enhanced growth inhibitory
activity against PC-3 and MDA-MB-468 cells compared to
T-47D. As we observed in Jurkat T cells, most of the analogues
with the exception of 24 were more effective than the parental
compound 7 and they all showed IC50 values within the same
range. Derivatives 11c and 11h were most active against PC-3
cells, whereas compounds 11c, 11d, 11h, and 11i were highly
active against MDA-MB-468 cells, with IC50 values about 2-fold
lower than that obtained with 7.
We then evaluated the time course of AdAr-mediated growth
inhibitory activity, and Figure 3 illustrates the results obtained
in MDA-MB-468 cells. As expected, the antiproliferative
activity of the AdArs significantly increased with the time of
incubation and highly active derivatives (11c, 11h) reached
maximum activity after only 48 h of treatment. Less effective
compounds exhibited increased growth inhibition with prolonged
periods of exposure (72 h). Similar results were obtained with

Inhibition of IKKꢀ by MX781 Analogues
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17 5435
Figure 4. AdArs induce apoptosis in Jurkat T leukemia cells. (A) Induction of DEVDase activity by the MX781 analogues. Jurkat cells were
incubated with 2 (light gray) or 4 (dark gray) µM of the indicated AdArs for 4 h, when DEVDase activity (arbitrary units) was evaluated as
indicated in the Experimental Section. The experiment was performed twice with triplicates and the average ( standard deviation is shown. (B)
Externalization of phosphatidylserine. Jurkat cells were incubated with 2 µM of the indicated compounds for 3 h, when cells were harvested and
stained with annexin V-FITC and PI, followed by flow cytometry analysis. The experiment was performed three times with almost identical outcome,
and the percentage of annexin V-positive/PI-negative cells of one representative experiment performed in duplicate is shown.
the rest of analogues, including 24, which inhibited cell
proliferation by ∼40% after 72 h of treatment with 4 µM (not
shown).
We previously reported that 7 and related RRMs rapidly
induced caspase-dependent apoptosis in Jurkat T cells.19,21 To
evaluate the proapoptotic activity of the derivatives of 7, we
therefore analyzed the activation of caspases 3 and 7 by
measuring the amount of DEVDase activity in cell extracts
obtained from AdAr-stimulated Jurkat cells. All AdAr com-
pounds induced significant levels of DEVDase activity in Jurkat
T cells (Figure 4A) after 4 h of treatment. When tested at lower
concentrations (1 µM), only the most active analogues (11c,
11d, 11h, and 11i) induced substantial DEVDase activity (not
shown). The enhanced caspase activation achieved by 11c and
11i paralleled the higher antiproliferative activity observed in
Jurkat as well as in prostate and breast cancer cell lines (see
Figure 5. Effect of the MX781 analogues on recombinant IKKꢀ.
Table 1). We then quantitated the induction of apoptosis at the
cellular level by analyzing externalization of phosphatidylserine
following staining with annexin V. Figure 4B illustrates that
all analogues of 7 were at least as effective as the parent
compound in increasing the percentage of annexin V-positive/
PI-negative cells, which relates to early stages of apoptosis. As
observed with DEVDase activity, compounds 11c and 11h
induced the highest apoptotic rate when used at 2 µM.
Purified His-HA-IKKꢀ was incubated in the absence (none, black
column) or in the presence of 20 µM of the indicated AdArs (gray
columns). The white column (no IκBR) indicates basal IKK activity
obtained in the absence of substrate. For comparison, the kinase was
incubated with 10 µM BMS345541 (BMS345) or 20 µM SC-514.
Kinase activity was determined after incubation at 30 °C for 30 min as
specified in Experimental Section, and the ratio relative to control
sample (none) was estimated. Shown here are the average ( SD of
two to four experiments performed in triplicate.
Inhibition of IKKꢀ by the Novel AdArs. We have shown
that 7 is a strong inhibitor of IKK activity in vitro as well as in
cell based assays.26 A preliminary kinase profiler using recom-
binant kinases performed by Upstate demonstrated that 7
selectively inhibited IKKꢀ with an IC50 of 7 µM, with no effect
on IKKR (data not shown). We therefore evaluated the inhibition
of purified recombinant IKKꢀ expressed in baculovirus-infected
Sf9 cells by the synthesized AdAr analogues. Figure 5 illustrates
that 20 µM 7 inhibited IKKꢀ by ∼65% in the presence of an
ATP concentration corresponding to its Km value. This inhibition
is slightly lower but comparable to that originally observed in
the Upstate’s profile analysis, which might be likely due to the
use of different peptide substrates, ATP concentrations, and/or
enzyme preparations. With the exception of compounds 24 and
11j, which showed partial inhibition of IKKꢀ, all other
analogues 11a-i elicited enhanced inhibition of the kinase
compared to parent compound 7 and similar to that observed
with two well characterized inhibitors of IKK (BMS345541 and
SC-514) (Figure 5). In general, this increased inhibition of IKKꢀ
parallels the growth inhibitory profile of the AdArs found in
IKK-overexpressing cells (Jurkat, PC-3, and MDA-MB-468).
Furthermore, the highest inhibition of IKKꢀ by 11c and 11h
correlates well with the strongest induction of apoptosis observed
in Jurkat cells (see Figure 4). More interestingly, compounds
11e and 11i exhibit improved anti-IKKꢀ and growth inhibitory
activities and have lost their RAR-dependent transactivation
function. These results clearly substantiate our assumption that
modifications can be designed on the scaffold of 7 to increase
antikinase activity while reducing or eliminating RAR-trans-
activation activity.
Discussion
Overwhelming evidence supports a critical role for IKK/NFκB
activity in cancer progression, cell survival, and inhibition of
TNFR-induced apoptosis.27-29,40 Numerous inhibitors of IKK/
NFκB activity have been described that act at different levels,
including inhibitors that function upstream of the IKK complex,
inhibitors of IKK activity, or inhibitors of NFκB nuclear
functions.43 Because IKKꢀ is the catalytic subunit essential for
the activation of the classical NFκB pathway and is required
for the reduced apoptosis rates observed in cancer cells,44,45

5436 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17
Lorenzo et al.
the development of IKKꢀ inhibitors is a promising strategy for
novel cancer therapies, and several IKK inhibitors have indeed
been described and are under clinical evaluation.46 Some
compounds inhibit IKKꢀ with an IC50 of 4-70 nM, but many
of them are active at micromolar concentrations,46 which is
within the same range as 7 and analogues reported here. Using
pharmacological and genetic inhibitors, we have established a
correlation between inhibition of IKK/NFκB activity and
bound to mediate RRM-induced apoptosis, like SHP and protein
phosphatases, the identification of these targets and their
validation in cancer cells and in animal models will be absolutely
critical for the optimization of RRMs/AdArs as effective
anticancer drugs to evaluate in a clinical setting.
Here, we report on the biological activity of a series of
analogues of 7 and their effect on recombinant IKKꢀ and cancer
cell proliferation. Analogues were designed to have reduced
RAR transactivation activity. The activities of the number of
AdArs evaluated indicate that a hydroxy or an alkoxy group
on the ring adjacent to the chalcone unit (24, 11a-j) retain the
activity of the parent system and are unable to activate RAR-
mediated transcription. All compounds evaluated with the
exception of 24 show reduced RAR binding, as evidenced by
their significantly reduced antagonist activity. From the pre-
liminary studies, the alkoxy group should be longer than a three-
carbon atom, since the ethoxy derivative (11a) shows undesired
RAR binding, although lower than the parent compound
(MX781) 7. Most analogues, including 11c and the propargyl
ether 11h and 11c afforded the strongest inhibition of IKKꢀ,
concurring with the strongest induction of apoptosis in Jurkat
cells and growth inhibitory activity against PC-3 and MDA-
MB-468 cells. In general, there was a good correlation between
the inhibition of recombinant IKKꢀ and the inhibition of cell
growth in leukemia (Jurkat), prostate (PC-3), and breast (MDA-
MB-468) cancer cells, all of which contain elevated basal IKK
activity. These data suggest that IKKꢀ inhibition could mediate
AdAr anticancer activity, although we do not rule out that other
alternative mechanisms (targeting of SHP, protein phosphatases,
other kinases) might be as critical for the anticancer activity of
AdArs as blocking IKK/NFκB activity. Most importantly, the
anticancer activity of these AdArs is completely independent
of RAR transactivation activity (neither activation nor antago-
nism), thus confirming some of the design principles. The data
reported here clearly demonstrate that we can design novel AdAr
analogues lacking RAR activity that show improved anti-IKKꢀ
and anticancer activities. Further verifying the inhibition of IKK
signaling in cell-based assays and identifying novel targets of
AdAr action will certainly facilitate clinical advancement of
AdAr like molecules as alternative therapies for the chemopre-
vention and treatment of cancers.
26
induction of apoptosis in lung and prostate carcinoma cells.
Furthermore, RAR antagonist 7 and the RARγ/ꢀ agonist 8
inhibit in vitro the activity of IKK immunopurified from TNFR-
stimulated HeLa cells,26 and using recombinant IKKR and IKKꢀ
proteins, we have now demonstrated that 7 is a selective inhibitor
of IKKꢀ. Together, these data suggest that IKKꢀ is most likely
involved in the anticancer activity mediated by 7 and validate
IKKꢀ as an AdAr target. Although 7 inhibits recombinant IKKꢀ
with an IC50 of 7 µM, it is very effective in cell-based assays,
in which a 6 µM dose completely prevented activation of IKK
by TNFR.26 This contrasts with other typical kinase inhibitors
such as AS602868, which normally require higher concentra-
tions in cells to induce apoptosis than those required in vitro to
inhibit purified kinase.47 The reason for this discrepancy is
unknown, but numerous factors such as cellular accumulation
of the compound, its stability in culture medium, and most likely
the targeting of alternative cell signaling molecules (see below)
may certainly affect the anticancer activity of one compound
in cell-based assays.
In contrast to the inhibition of IKK/NFκB signaling by 7, it
has recently been shown that 3 and its analogue 6 induce NFκB
activity in prostate carcinoma cells, and this NFκB activation
is necessary for apoptosis induction.30,31 Compound 6 seems
to activate IKKR in prostate carcinoma cells,30 although the
exact mechanism of IKK/NFκB activation by these AdArs
remains unsolved. These observations were certainly unex-
pected; one could anticipate that retinoid agonists like 3 would
inhibit NFκB transcriptional activity in a RAR-dependent
manner. This effect has been observed with all-trans-retinoic
acid (ATRA)26 and could be explained by a ligand-dependent
interaction of RARs with NFκB subunits, which would inhibit
NFκB transcriptional activity in the absence of a direct effect
on IKK activity. Besides IKK, a different target has been
reported for 6 and related compounds, which bind the nuclear
hormone receptor small heterodimer partner (SHP) and modulate
SHP interaction with the Sin3A repressor.48 Binding of 6 to
SHP seems to mediate apoptosis, since SHP-deficient mouse
embryo fibroblasts and breast carcinoma cells expressing
reduced levels of SHP exhibit significantly lower degrees of
apoptosis in response to this AdAr. In addition to IKK and SHP,
some AdArs can also target certain protein phosphatases in vitro,
including MKP-1 and the protein tyrosine phosphatases CD45
and SHP-2.49,50 Inhibition of protein phosphatases has not yet
been demonstrated in cell-based assays, and their relevance to
AdAr anticancer activity needs to be addressed.
Experimental Section
(E)-4-[3-(5-(Adamant-1-yl)-2-hydroxy-4-(2-methoxyethoxy-
methoxy)-phenyl)-3-oxoprop-1-en-1-yl]benzoic Acid 24. General
Procedure for the Claisen-Schmidt Condensation. To a solution
of 1-[5-(adamant-1-yl)-2-ethoxy-4-(2-methoxyethoxymethoxy)phe-
nyl]ethan-2-one 19 (0.34 g, 0.91 mmol) and methyl 4-formylben-
zoate 23 (0.18 g, 1.097 mmol) in MeOH (8.1 mL) was added a 1
M aqueous NaOH solution (5.4 mL), and the reaction mixture was
heated to 70 °C for 18 h. After cooling to ambient temperature,
the mixture was treated with a 10% aqueous HCl solution until
acidic pH was attained and the mixture was extracted with AcOEt
(3×). The combined organic layers were dried (Na2SO4), and the
solvent was removed. The residue was purified by chromatography
(silica gel, 98:2 CH2Cl2/MeOH) to afford 0.286 g (60%) of a yellow
solid identified as (E)-4-[3-(5-adamant-1-yl)-2-hydroxy-4-(2-meth-
oxyethoxymethoxy)phenyl)-3-oxoprop-1-en-1-yl]benzoic acid 24.
Mp 200 °C. Anal. (C30H34O7): C, 71.13; H, 6.76. Found: C, 71.06;
For the optimal development of novel RRMs with kinase
inhibitory profiles with therapeutic potential, their RAR-
dependent transcriptional activity should be reduced to a
minimum because of the associated side effects of classical
retinoids. In this regard, accumulating structural data on retinoid
receptors have provided very efficient modulators (agonists,
partial agonists, inverse agonists, and antagonists) of retinoid
action and the current guidelines for optimizing their potency
can be used in the reverse sense to minimize RAR binding and
transactivation.7,36,51,52 Whether protein kinases such as IKKꢀ
are the main targets of RRM action or other receptors are also
1
H, 7.16. H NMR (400.13 MHz, CDCl3): δ 8.15 (d, J ) 8.2 Hz,
2H), 7.87 (d, J ) 15.4 Hz, 1H), 7.73 (d, J ) 8.3 Hz, 2H), 7.67 (s,
1H), 7.64 (d, J ) 15.4 Hz, 1H), 6.69 (s, 1H), 5.36 (s, 2H), 3.9-3.8
(m, 2H), 3.6-3.5 (m, 2H), 3.39 (s, 3H), 2.1-2.0 (m, 9H), 1.8-1.7
(m, 6H) ppm. 13C NMR (100.62 MHz, CDCl3): δ 191.7 (s), 169.1
(s), 164.6 (s), 163.3 (s), 142.4 (s), 139.9 (d), 130.8 (d, 2×), 130.6
(s), 128.4 (d, 2×), 128.2 (d), 123.5 (s), 114.3 (s), 103.1 (d), 93.2

Inhibition of IKKꢀ by MX781 Analogues
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17 5437
(t), 71.5 (t), 68.6 (t), 59.1 (q), 40.9 (t, 3×), 37.0 (t, 3×), 36.7 (s),
29.0 (d, 3×) ppm. MS (FAB+): m/z (%) 507 (78), 506 (30), 392
(28), 391 (99), 307 (21), 167 (26), 155 (31), 154 (100). HRMS
(FAB+): calcd for C30H35O7, 507.2383; found, 507.2384. IR (NaCl):
ν 2910-2840 (s), 1690 (s), 1642 (s) cm-1. UV (MeOH): λmax 321
nm.
3.6-3.5 (m, 2H), 3.40 (s, 3H), 2.1-2.0 (s, 9H), 1.8-1.7 (m, 8H),
1.45 (q, J ) 7.5 Hz, 2H), 0.87 (t, J ) 7.4 Hz, 3H) ppm. 13C NMR
(100.62 MHz, CDCl3): δ 190.3 (s), 171.1 (s), 161.3 (s), 158.4 (s),
140.9 (s), 139.2 (d), 131.4 (s), 130.6 (d, 2×), 130.3 (d), 130.0 (s),
129.9 (d), 128.0 (d, 2×), 121.3 (s), 99.5 (d), 93.2 (t), 71.5 (t), 68.5
(t), 67.9 (t), 59.0 (q), 40.8 (t, 3×), 37.0 (t, 3×), 36.6 (s), 31.41 (t),
29.0 (d, 3×), 19.5 (t), 13.4 (q) ppm. MS (FAB+): m/z (%) 563
(10), 562 (28), 486 (15), 473 (21), 175 (14), 89 (100). HRMS
(FAB+): calcd for C34H42O7, 562.2931; found, 562.2935. IR (NaCl):
ν 2910-2840 (s), 1718 (m), 1691 (m), 1607 (s) cm-1. UV (MeOH):
λmax 309 nm.
(E)-4-[3-(5-(Adamant-1-yl)-4-(2-methoxyethoxymethoxy)-2-(pen-
tyl-1-oxy)phenyl)-3-oxoprop-1-en-1-yl]benzoic Acid 11d. Following
the general procedure for the Claisen-Schmidt condensation the
reaction of 1-[5-(adamant-1-yl)-4-(2-methoxyethoxymethoxy)-2-
(pentyl-1-oxy)phenyl]ethan-2-one 22d (0.13 g, 0.29 mmol), methyl
4-formylbenzoate 23 (0.057 g, 0.35 mmol), and a 1 M aqueous
NaOH solution (1.7 mL) in MeOH (2.6 mL) afforded, after
purification by chromatography (silica gel, 97:3 CH2Cl2/MeOH),
0.058 g (35%) of a yellow solid identified as (E)-4-[3-(5-(adamant-
1-yl)-4-(2-methoxyethoxymethoxy)-2-(pentyl-1-oxy)phenyl]-3-oxo-
prop-1-en-1-yl]benzoic acid 11d. Mp: 149 °C. Anal. (C35H44O7),
calcd: C, 72.89; H, 7.69. Found: C, 72.87; H, 7.73. 1H NMR (400.13
MHz, CDCl3): δ 8.11 (d, J ) 8.0 Hz, 2H), 7.75 (d, J ) 15.8 Hz,
1H), 7.73 (s, 1H), 7.68 (d, J ) 15.8 Hz, 1H), 7.65 (d, J ) 8.0 Hz,
2H), 6.80 (s, 1H), 5.38 (s, 2H), 4.04 (t, J ) 6.1 Hz, 2H), 3.9-3.8
(m, 2H), 3.6-3.5 (m, 2H), 3.40 (s, 3H), 2.1-2.0 (s, 9H), 1.9-1.8
(m, 2H), 1.8-1.7 (s, 6H), 1.5-1.4 (m, 2H), 1.3-1.2 (m, 2H), 0.79
(t, J ) 7.2 Hz, 3H) ppm. 13C NMR (100.62 MHz, CDCl3): δ 190.3
(s), 171.1 (s), 161.3 (s), 158.4 (s), 140.9 (s), 139.2 (d), 131.4 (s),
130.6 (d), 130.3 (d), 130.1 (s), 130.0 (d, 2×), 128.0 (d, 2×), 121.3
(s), 99.5 (d), 93.2 (t), 71.5 (t), 68.9 (t), 67.9 (t), 59.0 (q), 40.9 (t,
3×), 37.0 (t, 3×), 36.6 (s), 29.2 (t), 29.0 (d, 3×), 28.4 (t), 22.4 (t),
13.8 (q) ppm. MS (FAB+): m/z (%) 577 (100), 576 (20), 393 (12),
307 (24), 289 (14), 263 (15), 165 (12), 155 (32), 154 (95). HRMS
(FAB+): calcd for C35H45O7, 577.3165; found, 577.3180. IR (NaCl):
ν 2910-2840 (s), 1719 (m), 1692 (m), 1607 (s) cm-1. UV (MeOH):
λmax 311 nm.
(E)-4-[3-(5-(Adamant-1-yl)-2-(hexyl-1-oxy)-4-(2-methoxy-
ethoxymethoxy)phenyl)-3-oxoprop-1-en-1-yl]benzoic Acid 11e. Fol-
lowing the general procedure for the Claisen-Schmidt condensation
the reaction of 1-[5-(adamant-1-yl)-2-(hexyl-1-oxy)-4-(2-methoxy-
ethoxymethoxy)phenyl]ethan-2-one 22e (0.11 g, 0.24 mmol), methyl
4-formylbenzoate 23 (0.048 g, 0.29 mmol), and a 1 M aqueous
NaOH solution (1.4 mL) in MeOH (2.6 mL) afforded, after
purification by chromatography (silica gel, 97:3 CH2Cl2/MeOH),
0.031 g (22%) of a yellow solid identified as (E)-4-[3-(5-(adamant-
1-yl)-2-(hexyl-1-oxy)-4-(2-methoxyethoxymethoxy)phenyl)-3-oxo-
prop-1-en-1-yl]benzoic acid 11e. Mp: 131 °C. Anal. (C36H46O7),
calcd: C, 73.19; H, 7.85. Found: C, 72.88; H, 7.75. 1H NMR (400.13
MHz, CDCl3): δ 8.11 (d, J ) 8.8 Hz, 2H), 7.75 (d, J ) 16.0 Hz,
1H), 7.73 (s, 1H), 7.69 (d, J ) 16.0 Hz, 1H), 7.66 (d, J ) 8.0 Hz,
2H), 6.80 (s, 1H), 5.38 (s, 2H), 4.04 (t, J ) 6.6 Hz, 2H), 3.9-3.8
(m, 2H), 3.6-3.5 (m, 2H), 3.40 (s, 3H), 2.1-2.0 (s, 9H), 1.9-1.8
(m, 4H), 1.8-1.7 (s, 6H), 1.5-1.4 (m, 2H), 1.2-1.1 (m, 2H), 0.79
(t, J ) 7.4 Hz, 3H) ppm. 13C NMR (100.62 MHz, CDCl3): δ 190.3
(s), 170.8 (s), 161.3 (s), 158.4 (s), 140.9 (s), 139.1 (d), 131.4 (s),
130.6 (d, 2×), 130.3 (d), 130.0 (d), 129.9 (s), 128.0 (d, 2×), 121.3
(s), 99.5 (d), 93.2 (t), 71.5 (t), 68.9 (t), 67.9 (t), 59.0 (q), 40.8 (t,
3×), 37.0 (t, 3×), 36.6 (s), 31.5 (t), 29.4 (t), 29.0 (d, 3×), 26.0 (t),
22.5 (t), 13.9 (q) ppm. MS (FAB+): m/z (%) 591 (100), 590 (23),
307 (32), 289 (16), 155 (29), 154 (95). HRMS (FAB+): calcd for
C36H47O7, 591.3322; found, 591.3306. IR (NaCl): ν 2910-2840
(s), 1716 (m), 1692 (m), 1650 (m), 1607 (s) cm-1. UV (MeOH):
λmax 288 nm.
(E)-4-[3-(5-(Adamant-1-yl)-2-ethoxy-4-(2-methoxyethoxymethox-
y)phenyl)-3-oxoprop-1-en-1-yl]benzoic Acid 11a. Following the
general procedure for the Claisen-Schmidt condensation the
reaction of 1-[5-(adamant-1-yl)-2-ethoxy-4-(2-methoxyethoxymethox-
y)phenyl]ethan-2-one 22a (0.14 g, 0.33 mmol), methyl 4-formyl-
benzoate 23 (0.066 g, 0.4 mmol), and a 1 M aqueous NaOH solution
(2 mL) in MeOH (2 mL) afforded, after purification by chroma-
tography (silica gel, 97:3 CH2Cl2/MeOH), 0.1 g (56%) of a yellow
solid identified as (E)-4-[3-(5-(adamant-1-yl)-2-ethoxy-4-(2-meth-
oxyethoxymethoxy)phenyl)-3-oxoprop-1-en-1-yl]benzoic acid 11a.
1
Mp: 182 °C. H NMR (400.13 MHz, CDCl3): δ 8.10 (d, J ) 8.0
Hz, 2H), 7.77 (d, J ) 15.5 Hz, 1H), 7.73 (s, 1H), 7.66 (d, J ) 15.5
Hz, 1H), 7.65 (d, J ) 8.5 Hz, 2H), 6.81 (s, 1H), 5.37 (s, 2H), 4.12
(t, J ) 6.9 Hz, 2H), 3.9-3.8 (m, 2H), 3.6-3.5 (m, 2H), 3.40 (s,
3H), 2.1-2.0 (m, 9H), 1.8-1.7 (m, 6H), 1.44 (t, J ) 7.0 Hz, 3H)
ppm. 13C NMR (100.62 MHz, CDCl3): δ 190.2 (s), 168.0 (s), 161.3
(s), 158.3 (s), 140.9 (s), 139.1 (d), 131.5 (s), 130.7 (d, 2×), 130.2
(d), 130.0 (d), 129.8 (s), 128.0 (d, 2×), 121.4 (s), 99.8 (d), 93.2
(t), 71.5 (t), 67.9 (t), 64.6 (t), 59.0 (q), 40.9 (t, 3×), 37.1 (t, 3×),
36.7 (s), 29.0 (d, 3×), 14.9 (q) ppm. MS (FAB+): m/z (%) 535
(89), 534 (18), 391 (12), 307 (26), 289 (15), 155 (32), 154 (100).
HRMS (FAB+): calcd for C32H39O7, 535.2696; found, 535.2694.
IR (NaCl): ν 2910-2840 (s), 1692 (s), 1608 (s) cm-1. UV (MeOH):
λmax 313 nm.
(E)-4-[3-(5-Adamant-1-yl-4-(2-methoxyethoxymethoxy)-2-(prop-
1-oxy)-phenyl)-3-oxoprop-1-en-1-yl]benzoic Acid 11b. Following
the general procedure for the Claisen-Schmidt condensation the
reaction of 1-[5-(adamant-1-yl)-4-(2-methoxyethoxymethoxy)-2-
(prop-1-oxy)phenyl]ethan-2-one 22b (0.077 g, 0.19 mmol), methyl
4-formylbenzoate 23 (0.038 g, 0.23 mmol), and a 1 M aqueous
NaOH solution (1.1 mL) in MeOH (1.7 mL) afforded, after
purification by chromatography (silica gel, 97:3 CH2Cl2/MeOH),
0.057 g (56%) of a yellow solid identified as (E)-4-[3-(5-(adamant-
1-yl)-4-(2-methoxyethoxymethoxy)-2-(prop-1-oxy)phenyl)-3-oxo-
prop-1-en-1-yl]benzoic acid 11b. Mp: 189 °C. Anal. (C33H40O7),
calcd: C, 72.24; H, 7.35. Found: C, 71.55; H, 7.31. 1H NMR (400.13
MHz, CDCl3): δ 8.11 (d, J ) 8.3 Hz, 2H), 7.76 (d, J ) 15.8 Hz,
1H), 7.73 (s, 1H), 7.68 (d, J ) 15.8 Hz, 1H), 7.66 (d, J ) 8.3 Hz,
2H), 6.81 (s, 1H), 5.38 (s, 2H), 4.01 (t, J ) 6.4 Hz, 2H), 3.9-3.8
(m, 2H), 3.6-3.5 (m, 2H), 3.40 (s, 3H), 2.1-2.0 (s, 9H), 1.9-1.8
(m, 2H), 1.6-1.7 (s, 6H), 1.01 (t, J ) 7.4 Hz, 3H) ppm. 13C NMR
(100.62 MHz, CDCl3): δ 190.3 (s), 170.8 (s), 161.3 (s), 158.4 (s),
140.9 (s), 139.2 (d), 131.4 (s), 130.6 (d, 2×), 130.3 (d), 130.0 (d),
129.9 (s), 128.0 (d, 2×), 121.4 (s), 99.6 (d), 93.2 (t), 71.5 (t), 70.4
(t), 67.9 (t), 59.0 (q), 40.9 (t, 3×), 37.0 (t, 3×), 36.6 (s), 29.0 (d,
3×), 22.7 (t), 10.7 (q) ppm. MS (FAB+): m/z (%) 549 (100), 548
(20), 391 (34), 307 (18), 289 (13), 167 (15), 165 (12), 155 (30),
154 (89), 151 (12). HRMS (FAB+): calcd for C33H41O7, 549.2852;
found, 549.2851. IR (NaCl): ν 2910-2840 (s), 1691 (m), 1607 (m)
cm-1. UV (MeOH): λmax 312 nm.
(E)-4-[3-(5-(Adamant-1-yl)-2-(but-1-oxy)-4-(2-methoxy-
ethoxymethoxy)-phenyl)-3-oxoprop-1-en-1-yl]benzoic Acid 11c. Fol-
lowing the general procedure for the Claisen-Schmidt condensation
the reaction of 1-[5-(adamant-1-yl)-2-(but-1-oxy)-4-(2-methoxy-
ethoxymethoxy)phenyl]ethan-2-one 22c (0.11 g, 0.30 mmol), methyl
4-formylbenzoate 23 (0.06 g, 0.36 mmol), and a 1 M aqueous NaOH
solution (1.8 mL) in MeOH (2.6 mL) afforded, after purification
by chromatography (silica gel, 95:5 CH2Cl2/MeOH), 0.091 g (58%)
of a yellow solid identified as (E)-4-[3-(5-(adamant-1-yl)-2-(but-
1-oxy)-4-(2-methoxyethoxymethoxy)phenyl)-3-oxoprop-1-en-1-yl-
]benzoic acid 11c. Mp: 156 °C. Anal. (C34H42O7), calcd: C, 72.57;
H, 7.52. Found: C, 72.09; H, 7.48. 1H NMR (400.13 MHz, CDCl3):
δ 8.11 (d, J ) 8.0 Hz, 2H), 7.76 (d, J ) 16.0 Hz, 1H), 7.69 (s,
1H), 7.67 (d, J ) 16.0 Hz, 1H), 7.65 (d, J ) 8.0 Hz, 2H), 6.81 (s,
1H), 5.38 (s, 2H), 4.05 (t, J ) 6.1 Hz, 2H), 3.9-3.8 (m, 2H),
(E)-4-[3-(5-(Adamant-1-yl)-4-(2-methoxyethoxymethoxy)-2-(prop-
2-en-1-oxy)phenyl)-3-oxoprop-1-en-1-yl]benzoic Acid 11g. Follow-
ing the general procedure for the Claisen-Schmidt condensation
the reaction of 1-[5-(adamant-1-yl)-4-(2-methoxyethoxymethoxy)-
2-(prop-2-en-1-oxy)phenyl]ethan-2-one 22g (0.086 g, 0.21 mmol),
methyl 4-formylbenzoate 23 (0.041 g, 0.25 mmol), and a 1 M

5438 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17
Lorenzo et al.
aqueous NaOH solution (1.2 mL) in MeOH (1.8 mL) afforded, after
purification by chromatography (silica gel, 97:3 CH2Cl2/MeOH),
0.064 g (57%) of a yellow solid identified as (E)-4-[3-(5-(adamant-
1-yl)-4-(2-methoxyethoxymethoxy)-2-(prop-2-en-1-oxy)phenyl)-3-
oxoprop-1-en-1-yl]benzoic acid 11g. Mp: 182 °C. Anal. (C33H38O7),
calcd: C, 72.51; H, 7.01. Found: C, 72.40; H, 7.02. 1H NMR (400.13
MHz, CDCl3): δ 8.11 (d, J ) 8.2 Hz, 2H), 7.75 (d, J ) 15.8 Hz,
1H), 7.72 (s, 1H), 7.69 (d, J ) 15.8 Hz, 1H), 7.66 (d, J ) 8.2 Hz,
2H), 6.84 (s, 1H), 6.05 (tdd, J ) 15.8, 10.3, 5.3 Hz, 1H), 5.44 (dd,
J ) 15.8, 1.4 Hz, 1H), 5.38 (s, 2H), 5.27 (dd, J ) 10.3, 1.4 Hz,
1H), 4.6-4.5 (m, 2H), 3.9-3.8 (m, 2H), 3.6-3.5 (m, 2H), 3.40 (s,
3H), 2.1-2.0 (m, 9H), 1.8-1.7 (m, 6H) ppm. 13C NMR (100.62
MHz, CDCl3): δ 190.3 (s), 171.1 (s), 161.2 (s), 157.7 (s), 140.8
(s), 139.4 (d), 132.5 (d, 2×), 131.8 (s), 130.6 (d), 130.0 (d), 129.9
(s), 129.9 (d), 128.1 (d, 2×), 121.6 (s), 118.2 (t), 100.0 (d), 93.2
(t), 71.5 (t), 69.6 (t), 67.8 (t), 59.0 (q), 40.8 (t, 3×), 37.0 (t, 3×),
36.6 (s), 29.0 (d, 3×) ppm. MS (FAB+): m/z (%) 547 (98), 546
(19), 307 (27), 289 (15), 175 (15), 155 (31), 154 (100), 151 (12).
HRMS (FAB+): calcd for C33H39O7, 547.2696; found, 547.2691.
IR (NaCl): ν 2910-2840 (m), 1715 (m), 1691 (m), 1606 (s) cm-1
UV (MeOH): λmax 315 nm.
.
(E)-4-[3-(5-(Adamant-1-yl)-4-(2-methoxyethoxymethoxy)-2-(E)-
3-phenylprop-2-en-1-oxy)phenyl)-3-oxoprop-1-en-1-yl]benzoic Acid
11j. Following the general procedure for the Claisen-Schmidt
condensation the reaction of 1-[5-(adamant-1-yl)-4-(2-methoxy-
ethoxymethoxy)-2-(E)-3-phenylprop-2-en-1-oxy)phenyl]ethan-2-
one 22j (0.13 g, 0.27 mmol), methyl 4-formylbenzoate 23 (0.053
g, 0.32 mmol), and a 1 M aqueous NaOH solution (1.6 mL) in
MeOH (2.4 mL) afforded, after purification by chromatography
(silica gel, 98:2 CH2Cl2/MeOH), 0.038 g (23%) of a yellow solid
identified as (E)-4-[3-(5-(adamant-1-yl)-4-(2-methoxyethoxymethoxy)-
2-(E)-3-phenylprop-2-en-1-oxy)phenyl)-3-oxoprop-1-en-1-yl]ben-
zoic acid 11j. 1H NMR (400.13 MHz, CDCl3): δ 8.10 (d, J ) 8.1
Hz, 2H), 7.87 (d, J ) 8.1 Hz, 2H), 7.82 (s, 1H), 7.78 (d, J ) 8.1
Hz, 2H), 7.67 (d, J ) 15.8 Hz, 1H), 7.59 (d, J ) 8.2 Hz, 2H), 7.57
(d, J ) 15.8 Hz, 1H), 6.89 (s, 1H), 6.75 (d, J ) 16.0 Hz, 1H), 6.41
(td, J ) 15.9, 5.7 Hz, 1H), 5.40 (s, 2H), 4.77 (d, J ) 5.5 Hz, 2H),
3.9-3.8 (m, 2H), 3.6-3.5 (m, 2H), 3.38 (s, 3H), 2.1-2.0 (m, 9H),
1.8-1.7 (m, 6H) ppm. MS (FAB+): m/z (%) 623 (14), 549 (11),
393 (17), 392 (26), 391 (87), 369 (17), 330 (14), 322 (13), 307
(13), 279 (18), 221 (14), 207 (13), 203 (20), 191 (10). HRMS
(FAB+): calcd for C39H43O7, 623.3009; found, 623.2996. IR (NaCl):
ν 2910-2840 (s), 1718 (s), 1605 (s) cm-1. UV (MeOH): λmax 311
nm.
Cell Proliferation Assays. All cancer cell lines were obtained
from ATCC and grown according to ATCC’s suggestions. AdArs
were prepared in DMSO as 4 mM stock solutions and diluted in
basic DMEM as 20× dilutions. Cell proliferation assays were
performed in 96-well plates. PC-3 cells, T-47D, and MDA-MB-
468 (5000 cells per well) were seeded in complete growing medium
the day before treatment and allowed to attach. Medium was
changed to 0.5% FBS containing medium just before AdAr
exposure. Jurkat T cells (10 000 cells per well) were resuspended
(105 cells/mL) in RPMI supplemented with 0.5% FBS and seeded
in 96-well plates immediately before AdAr treatment. Cells were
treated with increasing concentrations of the AdArs, and when
indicated, an amount of 10 µL of a 5 mg/mL 3-[4,5-dimethylthiazol-
2-yl]-2,5-diphenyltetrazolium bromide (MTT) solution was added.
After 4 h of incubation at 37 °C, an amount of 100 µL of 10%
SDS in 10 mM HCl was added to solubilize the formazan crystals.
The absorbance at 595 nm was measured in a Molecular Devices
SpectraMax reader, and the background from wells with no cells
was subtracted. The percentage of cell viability was calculated with
respect to controls (100%), which were incubated with an equivalent
amount of solvent DMSO (e0.1% v/v). All experiments were
performed at least twice with triplicate points. IC50 values were
calculated using GraphPad Prism software.
IR (NaCl): ν 2910-2850 (s), 1717 (m), 1691 (s), 1607 (s) cm-1
UV (MeOH): λmax 314 nm.
.
(E)-4-[3-(5-(Adamant-1-yl)-4-(2-methoxyethoxymethoxy)-2-(prop-
2-yn-1-oxy)phenyl)-3-oxoprop-1-en-1-yl]benzoic Acid 11h. Follow-
ing the general procedure for the Claisen-Schmidt condensation
the reaction of 1-[5-(adamant-1-yl)-4-(2-methoxyethoxymethoxy)-
2-(prop-2-yn-1-oxy)phenyl]ethan-2-one 22h (0.068 g, 0.17 mmol),
methyl 4-formylbenzoate 23 (0.032 g, 0.20 mmol), and a 1 M
aqueous NaOH solution (1.0 mL) in MeOH (1.5 mL) afforded, after
purification by chromatography (silica gel, 97:3 CH2Cl2/MeOH),
0.045 g (50%) of a yellow solid identified as (E)-4-[3-(5-(adamant-
1-yl)-4-(2-methoxyethoxymethoxy)-2-(prop-2-yn-1-oxy)phenyl)-3-
oxoprop-1-en-1-yl]benzoic acid 11h. 1H NMR (400.13 MHz,
CDCl3): δ 8.12 (d, J ) 8.0 Hz, 2H), 7.75 (d, J ) 15.6 Hz, 1H),
7.74 (s, 1H), 7.71 (d, J ) 7.8 Hz, 2H), 7.69 (d, J ) 15.6 Hz, 1H),
6.91 (s, 1H), 5.39 (s, 2H), 4.80 (s, 2H), 3.9-3.8 (m, 2H), 3.6-3.5
(m, 2H), 3.41 (s, 3H), 2.57 (s, 1H), 2.1-2.0 (m, 9H), 1.8-1.7 (m,
6H) ppm. 13C NMR (100.62 MHz, CDCl3): δ 190.0 (s), 170.8 (s),
161.0 (s), 156.5 (s), 140.8 (s), 139.9 (d), 132.7 (s), 130.6 (d, 2×),
130.0 (d), 129.9 (s), 128.2 (d, 2×), 122.0 (s), 100.6 (d), 93.4 (t),
78.0 (d), 76.1 (d), 71.5 (t), 67.9 (t), 59.0 (q), 56.8 (t), 40.8 (t, 3×),
37.0 (t, 3×), 36.7 (s), 29.0 (d, 3×) ppm. MS (FAB+): m/z (%) 545
(63), 544 (10), 307 (33), 289 (17), 155 (31), 154 (100), 151 (10).
HRMS (FAB+): calcd for C33H37O7, 545.2539; found, 545.2547.
IR (NaCl): ν 3246 (w), 2910-2850 (s), 1692 (s), 1606 (s) cm-1
UV (MeOH): λmax 313 nm.
.
(E)-4-[3-(5-(Adamant-1-yl)-4-(2-methoxyethoxymethoxy)-2-phe-
nylmethoxy-phenyl)-3-oxoprop-1-en-1-yl]benzoic Acid 11i. Fol-
lowing the general procedure for the Claisen-Schmidt condensation
the reaction of 1-[5-(adamant-1-yl)-4-(2-methoxyethoxymethoxy)-
2-phenylmethoxyphenyl]ethan-2-one 22i (0.13 g, 0.28 mmol),
methyl 4-formylbenzoate 23 (0.094 g, 0.56 mmol), and a 1 M
aqueous NaOH solution (1.6 mL) in MeOH (2.5 mL) afforded, after
purification by chromatography (silica gel, 95:5 CH2Cl2/MeOH),
0.078 g (47%) of a yellow solid identified as (E)-4-[3-(5-(adamant-
1-yl)-4-(2-methoxyethoxymethoxy)-2-phenylmethoxy-phenyl)-3-
oxoprop-1-en-1-yl]benzoic acid 11i. Mp: 167 °C. Anal. (C37H40O7),
calcd: C, 74.37; H, 6.76. Found: C, 73.89; H, 6.81. 1H NMR (400.13
MHz, CDCl3): δ 7.97 (d, J ) 8.1 Hz, 2H), 7.83 (s, 1H), 7.74 (d,
J ) 15.7 Hz, 1H), 7.64 (d, J ) 15.7 Hz, 1H), 7.45 (d, J ) 7.0 Hz,
2H), 7.38 (d, J ) 6.8 Hz, 2H), 7.36 (d, J ) 6.8 Hz, 2H), 7.33 (t,
J ) 7.6 Hz, 1H), 6.96 (s, 1H), 5.40 (s, 2H), 5.13 (s, 2H), 3.9-3.8
(m, 2H), 3.6-3.5 (m, 2H), 3.41 (s, 3H), 2.1-2.0 (m, 9H), 1.8-1.7
(m, 6H) ppm. 13C NMR (100.62 MHz, CDCl3): δ 189.6 (s), 171.0
(s), 161.4 (s), 158.1 (s), 140.8 (s), 139.5 (d), 136.0 (s), 131.9 (s),
130.5 (d, 2×), 130.3 (d), 130.2 (d), 129.7 (s), 128.8 (d, 2×), 128.4
(d), 128.1 (d, 2×), 128.0 (d, 2×), 121.2 (s), 99.9 (d), 93.3 (t), 71.5
(t), 71.1 (t), 67.9 (t), 59.1 (q), 40.8 (t, 3×), 37.0 (t, 3×), 36.7 (s),
31.5 (t), 29.0 (d, 3×) ppm. MS (FAB+): m/z (%) 597 (32), 596
(9), 308 (11), 307 (41), 289 (18), 155 (31), 154 (100), 151 (9).
HRMS (FAB+): calcd for C37H41O7, 597.2852; found, 597.2862.
Measurement of DEVDase Activity. To measure caspase activity,
we adapted a fluorescence enzymatic assay17 to 96-well plates. Jurkat
T cells (40 000 cells per well, 106 cells/mL) in RPMI containing 0.5%
FBS were seeded and immediately treated with AdArs. Alter 4 h of
treatment, cells were lysed by adding 5× CE buffer (25 mM PIPES,
pH 7, 25 mM KCl, 5 mM EGTA, 1 mM DTT, 10 µM cytochalasin
B, 0.5% NP-40, and a mixture of protease inhibitors consisting of 1
mM PMSF, 1 µg/mL leupeptine, and 1 µg/mL aprotinin) followed by
30 min of incubation at 4 °C. Cell extracts were then diluted with 2×
caspase buffer (50 mM HEPES, pH 7.4, 100 mM NaCl, 1 mM EDTA,
5 mM DTT, 0.1% CHAPS, and 10% sucrose) containing 100 µM
Ac-DEVD-AFC (Enzyme System Products). Mixtures were incubated
at 37 °C for 30 min, and the release of free AFC was measured every
2 min at 510 nm emission upon excitation at 390 nm, using a Victor2
microplate reader (Perkin-Elmer).
Staining with Annexin-FITC and PI. Jurkat cells (106 cells) were
resuspended at a density of 500 000 cells/mL in medium containing
0.5% FBS and stimulated with 2 µM AdArs for 3 h. DMSO (0.1%
v/v) was added to control cells. Following treatment, cells were
washed with PBS and stained with annexin-V-FITC (BD Bio-
sciences) and propidium iodine (PI) in binding buffer (10 mM
Hepes, pH 7.4, 140 mM NaCl, 2.5 mM CaCl2) during 15 min at
room temperature and protected from light. Cells were subsequently

Inhibition of IKKꢀ by MX781 Analogues
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17 5439
Academic Press: San Diego, CA, 2002.
analyzed by flow cytometry (FACS Calibur) for apoptosis (FITC)
and viability (PI).
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Leid, M.; de Lera, A. R.; Lotan, R.; Mangelsdorf, D. J.; Gronemeyer,
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RAR/RXR Transactivation Profiling. Gal4-LBD fusion proteins
contain residues 1-147 encoding the DNA binding domain of Gal4
fused to the LBDs of the retinoid receptors: hRARR (residues
156-462), hRARꢀ (residues 149-448), hRARγ (residues 158-454),
and mRXRR (residues 218-467). CV-1 cells (20 000 cells per well)
were transfected in 96-well plates with 10 ng of each of the Gal4-
LBD expression vectors together with 100 ng of UAS-luciferase
reporter and 25 ng of ꢀ-galactosidase expression vector (pCH110)
using Superfect transfection reagent. Sixteen hours after transfection,
the medium was removed and fresh medium containing 5%
charcoal-treated FBS was added, followed by AdAr stimulation for
6 h (20 h with RARγ). Cells were washed once with PBS, lysed,
and assayed for luciferase and ꢀ-galactosidase activities using a
Dual-Light chemiluminescent assay (Tropix).
IKKꢀ Expression, Purification, and Kinase Assays. (His)6-HA-
IKKꢀ was cloned into the pBAC-2 cP baculovirus expression vector
(Novagen) and transfected into Sf9 cells using a BacVector-3000
DNA kit according to the manufacturer’s instructions. Recombinant
viruses were identified by PCR and subsequently purified and
amplified to evaluate protein expression. Large scale protein
expression and purification were carried out by infecting 1 L of
Sf9 culture (2 × 106 cells/mL) with 25 mL of a high titer viral
preparation. Sixty hours after infection, cells were harvested, washed
with ice-cold PBS, and lysed in 10 mM Tris-HCl, pH 7.5, 150
mM NaCl, 1% NP-40 containing a mixture of protease and
phosphatase inhibitors. (His)6-HA-IKKꢀ was purified with 5 mL
of ProBond resin (Invitrogen) under native conditions following
the supplier’s recommendations and dyalized against 50 mM Tris-
HCl, pH 7.6, 270 mM sucrose, 150 mM NaCl, 1 mM benzamidine,
0.1 mM EGTA, 0.1% ꢀ-mercaptoethanol, 0.03% Brij-35, 0.2 mM
PMSF.
To measure kinase activity, an amount of 100 ng of (His)6-HA-
IKKꢀ was diluted in a final volume of 25 µL with kinase buffer (8
mM MOPS, pH 7.0, 0.25 mM EDTA) containing 5 µg of GST-
IκBR, 10 mM MgCl2, 6.25 µM ATP, 2 µCi 32P-γ-ATP, and 100
ng BSA. Reactions were incubated for 30 min at 30 °C, and an
amount of 25 µL of 1% phosphoric acid was added to stop the
reaction. Samples were transferred to a MultiScreen-PH (Millipore)
filter plate, washed five times with 200 µL of 0.5% phosphoric
acid, and air-dried. A total of 50 µL of OptiPhase Supermix
scintillation liquid was added, and radioactivity was counted in a
MicroBeta Trilux scintillation counter. Samples containing no IKKꢀ
enzyme were used to subtract the background. When including
inhibitors, these were added to an enzyme/substrate containing
buffer mixture prior to the addition of an ATP/MgCl2 cocktail. IKK
inhibitors (BMS345541 and SC-514) were purchased from EMD
Biosciences.
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Acknowledgment. This work was supported by funds from
´
the Spanish MEC (Grant SAF04-07131, FEDER to A.R.d.L.),
´
Xunta de Galicia (Grant PGIDIT05PXIC31403PN to A.R.d.L.),
and NIH (Grant CA55681 to F.J.P.). We thank Ming Zhao for
technical assistance and Sandy Marsh for secretarial support.
Supporting Information Available: Additional details on
experimental procedures and analytical and spectral characterization
data for compounds 14-22. This material is available free of charge
via the Internet at http://pubs.acs.org.
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