Colorectal cancer has emerged as a major cause of death in Western countries. Down-regulation of β-catenin expression has been considered a promising approach for cytotoxic drug formulation. Eight 4,9-friedodrimane-type sesquiterpenoids (1-8) were acquired using the oxidative potential of Verongula rigida on bioactive metabolites from two Smenospongia sponges. Compounds 3 and 4 contain a 2,2-dimethylbenzo[d]oxazol-6(2H)-one moiety as their substituted heterocyclic residues, which is unprecedented in such types of meroterpenoids. Gauge-invariant atomic orbital NMR chemical shift calculations were employed to investigate stereochemical details with support of the application of advanced statistics such as CP3 and DP4. Compounds 2 and 8 and the mixture of 3 and 4 suppressed β-catenin response transcription (CRT) via degrading β-catenin and exhibited cytotoxic activity on colon cancer cells, implying that their anti-CRT potential is, at least in part, one of their underlying antineoplastic mechanisms.
Colorectal cancer has emerged as a major cause of death in Western countries. Down-regulation of β-catenin expression has been considered a promising approach for cytotoxic drug formulation. Eight 4,9-friedodrimane-type sesquiterpenoids (1-8) were acquired using the oxidative potential of Verongula rigida on bioactive metabolites from two Smenospongia sponges. Compounds 3 and 4 contain a 2,2-dimethylbenzo[d]oxazol-6(2H)-one moiety as their substituted heterocyclic residues, which is unprecedented in such types of meroterpenoids. Gauge-invariant atomic orbital NMR chemical shift calculations were employed to investigate stereochemical details with support of the application of advanced statistics such as CP3 and DP4. Compounds 2 and 8 and the mixture of 3 and 4 suppressed β-catenin response transcription (CRT) via degrading β-catenin and exhibited cytotoxic activity on colon cancer cells, implying that their anti-CRT potential is, at least in part, one of their underlying antineoplastic mechanisms.
Approximately 140 000
people are diagnosed annually with colorectal cancer, and among them
50 000 patients have died of this malignant disease in the
U.S. alone during the past decade.[1] Molecular
lesions in the adenomatous polyposis coli (APC) gene, the largest
structural core protein of the β-catenin destruction complex,
are observed frequently in colorectal cancer.[2,3] This
alteration results in stabilization of cytosolic β-catenin,
a multifunctional protein capable of binding to the T-cell factor/lymphocyte
enhancer factor (Tcf/LEF) family of transcription factors to induce
gene expression.[3] The gene targets of this
expression, activated by β-catenin response regulated transcription
(CRT), include c-Myc and cyclin D1, which play pivotal roles in colorectal tumorigenesis and malignancy.[3] Since APC is a negative regulator directly upstream
of β-catenin,[3] only a few signaling
components of the Wnt/β-catenin pathway can be targeted for
development of clinical intervention for such oncogenicity. Among
these candidates, an accelerated turnover of β-catenin has been
recognized as a pertinent protocol for cytotoxic agent development.Diverse pharmacophores have been derived from natural products,
providing evidence for structural prerequisites for the disruption
of the oncogenic β-catenin function. For instance, we demonstrated
that galangin, a flavanone abundantly present in propolis, suppresses
CRT via expediting degradation of cytosolic β-catenin, verifying
the disruptive action on Tcf/β-catenin complexes as at least
one of its cytotoxic mechanisms on certain humancarcinomas.[4] Moreover, ellagitannins (ETs) were shown to retard
Tcf/LEF-dependent transcription activated by a mutant β-catenin
construction, corroborating that the consumption of an ET-enriched
diet results in a decline of colon cancer.[5] Unlike terrestrial organisms,[4,5] marine organisms producing
effective cytotoxic secondary metabolites[6] have been underutilized for exploration of potential modulators
of the oncogenic Wnt/β-catenin pathway. For instance, sponge-derived
4,9-friedodrimanesesquiterpenoids exhibited potent antiproliferative
activities via activation of DNA damage-inducible gene 153 and hypoxia-inducible
factor-1.[7,8] Yet, the inhibitory potential on β-catenin-associated
cellular pathways as the underlying cytotoxic mechanisms of the meroterpenoids
has rarely been investigated.[9]Biotransformation
is a rational approach to provide a diverse and
new series of derivatives that could be employed for the generation
of chemical libraries to implement preliminary lead optimization and
SAR studies.[10] Particularly, sponges in
the order Verongida, featuring unusual biochemical profiles exemplified
by the absence of terpenoids and the production of sterols and brominated
compounds, exhibited strong oxidative potential. Such characteristics
render those sponges as optimal sources to derivatize bioactive molecules
for drug screening.In ongoing efforts targeting the search
for sponge-derived potent
anticancer prototypes,[9,11] the isolation and structural
characterization of eight 4,9-friedodrimane-type sesquiterpenoids
generated by biotransformation utilizing the oxidative potential of
a Verongida sponge (Verongula rigida) on 4,9-friedodrimanes,
typically isolated from Smenospongia sponges, are
discussed herein. The potential of those meroterpenoids to be developed
into colon cancer inhibitory agents capable of suppressing CRT via
promoting the degradation of β-catenin is also addressed.
Results
and Discussion
It was reported recently that ilimaquinone,
a 4,9-friedodrimane-type
sesquiterpenoid, and its analogues exhibited antiproliferative activity
via suppressing β-catenin expression.[9] These results initiated an exploration of new 4,9-friedodrimane
derivatives capable of down-regulating expression of the oncogenic
protein from marine resources. In an effort to generate new 4,9-friedodrimanes,
the aggressive oxidative properties of a Verongida sponge, which are
well established to undergo rapid oxidation and rearrangements when
exposed to the atmosphere, were employed.[12]Smenospongia sponges producing 4,9-friedodrimanes
(Smenospongia aurea and S. cerebriformis) and V. rigida were homogenized and incubated together
at room temperature to facilitate the formation of new analogues from
this series.[13] This approach was successful
in generating a number of new 4,9-friedodrimane derivatives for bioassay.
The LC-MS analysis of the incubated mixtures of these three sponges
exhibited unreported quasimolecular ions (m/z 384.2 and 398.2; Figure S1, Supporting
Information) from identified friedodrimanes, which prompted
further purification of the extract for identification of possible
new inhibitors of β-catenin expression. Fractionation and purification
of the extract afforded eight new 4,9-friedodrimane-type sesquiterpenoids
(1–8) (Figure 1). This report reveals the unique utility that the Verongida sponges
may have on metabolism, lead optimization, and SAR studies.
Figure 1
Structures
of compounds 1–8. The
ethoxy groups in 5–8 could be derived
from ethanol used for extraction.
Structures
of compounds 1–8. The
ethoxy groups in 5–8 could be derived
from ethanol used for extraction.The HRFABMS of compound 1, which exhibited a
quasimolecular
ion at m/z 384.2540 (calcd [M +
H]+, m/z 384.2539), and
its 13C NMR data led to its molecular formula being established
as C24H33NO3. The 1H and 13C NMR data (Table 1) exhibited resonances
for a pentasubstituted aromatic moiety (δH 6.98;
δC 98.8, 109.2, 132.3, 143.7, 144.6, 146.6, 162.0),
an exomethylene (δH 4.36, 4.32; δC 102.7, 160.5), a methoxy (δH 3.90; δC 56.6), and four methyl groups (δH 0.91,
1.04, 1.04, 2.54; δC 14.6, 18.5, 17.7, 20.7). Inspection
of the 2D NMR spectra indicated the presence of a 4,9-friedodrim-4(11)-ene-type
sesquiterpenoid framework[13] and a benzoxazole
residue. The heterocyclic residue was identical to that of 5-epi-nakijinol C, a recently isolated sesquiterpenoid from
the Indonesian sponge Dactylospongia metachromia,[14] based on the deshielded 13C NMR chemical
shift values of C-20 (δC 132.3), C-21 (δC 146.6), and C-22 (δC 162.0) and the HMBC
correlation from H3-23 (δH 2.54) to C-22
(δC 162.0).[14] HMBC correlations
between H2-15 (δH 2.80, 2.89) and C-16,
C-17, and C-21 (δC 109.2, 143.7, 146.6) indicated
that the benzoxazole moiety is connected to C-15. The relative configuration
of the core framework was established on the basis of analysis of
the NOESY spectrum. Key NOESY correlations were detected from H-1ax (δH 1.51) to H3-12 (δH 1.04) and H3-14 (δH 0.91), from
H-2ax (δH 1.22) to H-10 (δH 0.92), and from H3-12 (δH 1.04) and
H3-13 (δH 1.04) to H3-14 (δH 0.91), demonstrating that compound 1 is related
structurally to the trans-4,9-friedodrimanes (Figure 2).[13] This NOESY-based
configurational analysis was corroborated by the shielded 13C NMR chemical shift value of C-12 (δC 20.7), in
view of previous studies verifying that 13C NMR chemical
shift values of C-12 in trans-decalins are more shielded
than those in cis-decalins (Δ ca. 10 ppm).[14−16] The absolute configuration of 1 was deduced from the
consistent absolute configuration of trans-4,9-friedodrimanemeroterpenoids obtained from natural sources and confirmed based on
the similar specific rotation value of 1 with those of
reported meroterpenoids (Figure 3).[13] The trivial name (−)-nakijinol E was
assigned to 1.
Table 1
1H (600 MHz) and 13C NMR (150
MHz) Data for Compounds 1–8 in CDCl3
1
2
3
4
5
6
7
8
position
δH, mult. (J in Hz)
δC
δH, mult. (J in Hz)
δC
δH, mult. (J in Hz)
δC
δH, mult. (J in Hz)
δC
δH, mult. (J in Hz)
δC
δH, mult. (J in Hz)
δC
δH, mult. (J in Hz)
δC
δH, mult. (J in Hz)b
δC
1
ax
1.51, m
23.4
1.94, m
23.0
1.42, m
23.0
1.85, m
22.7
1.40, m
22.1
1.81, m
21.6
1.91, m
21.5
1.84, m
22.7
eq
2.27, m
2.30, m
2.13, m
2.19, m
2.09, m
1.54, m
2.14, m
2.10, tda
2
ax
1.22, m
28.9
1.82, m
25.3
1.20, m
28.8
1.77,
m
25.2
1.29, m
28.5
1.72, m
24.7
1.74, m
24.8
1.65, m
25.1
eq
1.87, m
1.68, m
1.84,
m
1.65, m
1.85, m
1.61,
m
1.66, m
1.76, m
3
ax
2.33, t (7.5)
33.2
2.44, dd (14, 5.4)
32.2
2.29, dt (14, 5.2)
33.2
2.42, dt (14, 6.6)
32.2
2.27, m
33.2
2.44, m
32.1
2.45, m
32.0
2.10,
tda
32.1
eq
2.02, m
2.11, dt
(14, 6.6)
2.04, m
2.10, dd (14, 4.8)
2.06, m
2.09, m
2.11, m
2.41, td (13.2, 6.0)
4
160.5
153.9
161.0
153.7
160.7
153.5
153.9
153.7
5
40.6
39.6
40.5
39.6
40.3
39.4
39.5
39.7
6
ax
1.19, m
36.5
0.93,
dt (14, 3.3)a
37.8
1.30, m
36.9
1.01,
dt (14, 3.0)a
38.2
1.46, m
37.0
1.19,
m
38.3
1.19, m
38.2
1.07, td (14.1, 3.0)a
38.1
eq
1.43, m
1.92, m
1.47,
m
1.97, br d (14)
1.55, ma
2.06, m
2.06, m
1.99, br d (13.8)
7
ax
1.39, m
28.2
1.50, m
28.0
1.36, m
28.1
1.46, m
28.0
1.40, m
27.6
1.52, m
27.7
1.52,
m
27.6
1.19, m
28.0
eq
1.17,
m
1.15, m
1.45, m
1.24,
m
1.24, m
1.48, m
8
1.40, m
37.4
1.33, m
38.7
1.22, m
38.1
1.11, m
39.1
1.40,
m
37.0
1.29, m
38.6
1.39, m
38.2
1.19, m
39.7
9
43.1
44.5
42.9
44.2
39.7
41.1
41.2
45.0
10
0.92, m
49.4
1.40, d (6.6)
47.6
0.80, m
50.0
1.17, d (6.0)a
47.8
0.87, br d (10.2)
49.0
1.43, d (6.0)
47.0
1.25, d (6.0)a
47.0
1.19, m
48.7
11
a
4.36, br s
102.7
4.69, br s
105.8
4.40, br s
102.4
4.68, br s
105.8
4.45 br s
102.6
4.70, br s
106.1
4.70, br s
105.9
4.68, br s; 4.65, br s
105.9
b
4.32, br s
4.66, br s
4.38, br s
4.65, br s
4.68, br
s
4.68, br s
12
1.04, s
20.7
0.98, s
33.2
1.01, s
20.7
1.01, s
33.3
1.03, s
20.6
1.12, s
33.0
1.09, s
33.2
1.04, s
33.4
13
1.04, da
18.5
1.01, d (6.6)
18.8
0.97, d (6.6)
18.0
0.95, d (6.6)
18.4
0.72, da
16.5
0.90, d (6.6)
16.6
0.71, d (6.6)
16.7
0.92, d (6.0)
18.5
14
0.91, s
17.7
0.95, s
19.0
0.81, s
17.5
0.85, s
19.0
0.72, s
17.8
0.80, s
19.2
0.79, s
19.3
0.86, s
18.8
15
2.80, d (14)
34.6
2.83, d (14)
34.7
2.37, d (14)
32.7
2.47, d (14)
32.5
1.00,
dd (15.6, 4.2)
33.9
1.25,
dd (16, 4.8)a
33.9
1.05, dd (16, 3.6)
33.9
2.47, d (13.2)
33.0
2.89, d (14)
2.96,
d (14)
2.53, d (14)
2.51, d (14)
2.04, dd (15.0, 3.6)a
1.99, dd (16, 3.6)
2.07, dd (16, 4.8)a
2.57, d (13.2)
16
109.2
109.4
110.3
110.1
2.61, t (4.2)
54.9
2.68, t (4.8)a
54.8
2.67, t (4.2)
54.7
117.7
17
143.7
143.6
181.6
181.57
80.6
80.6
80.5
153.3
18
144.6
144.5
158.9
158.9
5.92, s
121.9
5.93, s
122.0
5.93, s
121.9
182.7
19
6.98, s
98.8
6.98, s
98.8
6.14, s
96.9
6.16, s
96.89
158.9
159.0
159
5.82, s
102.4
20
132.3
132.5
155.6
155.6
198.5
198.4
198.7
161.2
21
146.6
146.7
161.4
161.5
174.4
174.4
174.4
182.3
22
162.0
162.0
116.2
116.19
4.15,
m
63.4
4.17, m
63.4
4.17, m
63.4
4.04, q (7.2)
66.1
4.26, m
4.26, m
4.26, m
23
2.54, s
14.6
2.54, s
14.6
1.58,
s
25.9
1.61, s
25.8
1.21, t (7.2)
14.3
1.21, ta
14.3
1.21, ta
14.3
1.49, t (7.2)
14.0
24
1.60, s
26.0
1.61, s
25.8
OH
5.87, s
5.85, s
3.93, s
3.95, br s
3.95, br s
7.42, s
OCH3
3.90, s
56.6
3.91, s
56.7
3.83, s
56.4
3.84, s
56.5
3.72, s
57.4
3.73,
s
57.4
3.73, s
57.4
Partially overlapped.
Diastereotopic protons may
be interchangeable.
Figure 2
NOESY analysis to establish trans- and cis-4,9-friedodrim-4(11)-ene moieties found
in this study.
Minimization was implemented at the MMFF level employing MacroModel
(Schrodinger LLC), and yellow dotted lines indicate NOE correlations.
Figure 3
Establishment of absolute configuration of optically
pure compounds
in this study (i.e., compounds 1, 2, 5, and 8). The trans-friedodrimanes
(−)-nakijinol E (1) and (−)-dactylospongenone
E (5) displayed negative specific rotations in accord
with those of (−)-ilimaquinone[8,25] and (−)-dactylospongenone
A.[17] Inversion of the C-5 absolute configuration
with formation of the cis-friedodrimane core structure
led to a significant conformational change that is exemplified by
a consistent change in the sign of the optical rotation. Thus, the
specific rotations of (+)-5-epi-nakijinol E (2) and (+)-5-epi-ethylsmenoquinone (8) are consistent with those of the analogous (+)-5-epi-ilimaquinone.[15,26]
NOESY analysis to establish trans- and cis-4,9-friedodrim-4(11)-ene moieties found
in this study.
Minimization was implemented at the MMFF level employing MacroModel
(Schrodinger LLC), and yellow dotted lines indicate NOE correlations.Establishment of absolute configuration of optically
pure compounds
in this study (i.e., compounds 1, 2, 5, and 8). The trans-friedodrimanes
(−)-nakijinol E (1) and (−)-dactylospongenone
E (5) displayed negative specific rotations in accord
with those of (−)-ilimaquinone[8,25] and (−)-dactylospongenone
A.[17] Inversion of the C-5 absolute configuration
with formation of the cis-friedodrimane core structure
led to a significant conformational change that is exemplified by
a consistent change in the sign of the optical rotation. Thus, the
specific rotations of (+)-5-epi-nakijinol E (2) and (+)-5-epi-ethylsmenoquinone (8) are consistent with those of the analogous (+)-5-epi-ilimaquinone.[15,26]Partially overlapped.Diastereotopic protons may
be interchangeable.The
molecular formula of compound 2 was established
as C24H33NO3 based on 13C NMR and HRFABMS data (obsd [M + H]+, m/z 384.2542; calcd [M + H]+, m/z 384.2539), implying that compound 2 is an isomer of 1. The 1D NMR spectra were
similar to those of 1 except for the more deshielded
C-12 resonance (δC 33.2) compared to 1 (δC 20.7). Such deshielding of the methyl carbon
in 4,9-friedodrim-4(11)-ene sesquiterpenoids is indicative of inversion
of configuration of the C-5 stereogenic center, validating that compound 2 is the C-5 epimer of 1. Key NOESY correlations
were detected from H3-14 (δH 0.95) to
H-2ax (δH 1.82) and H3-13 (δH 1.01), from H-7ax (δH 1.50) to
H-11b (δH 4.66), and from H3-12 (δH 0.98) to H-10 (δH 1.40),
confirming the presence of a cis-4,9-friedodrimane
architecture in 2 (Figure 2).[13] The absolute configuration of compound 2 was established as for 1 (Figure 3), and the trivial name (+)-5-epi-nakijinol
E was assigned to 2.Compounds 3 and 4 were obtained as a
1:2 mixture, with the ratio being established via the integration
of corresponding 1H NMR resonances for each compound. The
HRFABMS data of this mixture displayed a quasimolecular ion at m/z 398.2696, which, in conjunction with
the 13C NMR data, led to assignment of their same molecular
formula as C25H35NO3 (calcd [M +
H]+, m/z 398.2695). The 1H and 13C NMR data of compounds 3 and 4 exhibited several common features to those of 1 and 2 aside from the presence of a resonance for an
additional methyl group (δH 1.58, 1.61; δC 25.8, 25.9) in 3 and 4. This suggested
that compounds 3 and 4 possess 4,9-friedodrim-4(11)-ene
scaffolds with different C-15 substituents. The HMBC correlations
from H2-15 to C-16, C-17, and C-21 and from H3-23 and H3-24 to C-22 (Figure 4) revealed that the C-23 methyl groups of the benzoxazole moieties
in 1 and 2 are replaced with gem-dimethyl moieties in 3 and 4. The 13C NMR chemical shift values of C-12 were observed as δC 20.7 and 33.3, establishing trans- and cis-junctions of the decalin rings in 3 and 4, respectively. This was validated by the NOESY analysis
shown in Figure 2. When considered in conjunction
with the consistent biosynthetic pathways toward the 4,9-friedodrim-4(11)-ene-type
meroterpenoids derived from natural sources,[13] the absolute configurations of compounds 3 and 4 are presumably identical to those of the trans- and cis-4,9-friedodrim-4(11)-enes, respectively.
Thus, these substances, based on 4,9-friedodrimanes with a 2,2-dimethylbenzo[d]oxazol-6(2H)-one moiety, possess unique
scaffolds, and the trivial names nakijinone A and 5-epi-nakijinone A were assigned to compounds 3 and 4, respectively.
Figure 4
Selected HMBC correlations for 3 and 4.
Selected HMBC correlations for 3 and 4.The HRFABMS data of compound 5 displayed an
[M + H]+ ion at m/z 405.2638
(calcd
[M + H]+, m/z 405.2641),
which, in conjunction with the 13C NMR data, led to assignment
of the molecular formula, C24H36O5. The 1D NMR data were similar to those of compounds 1–4, substantiating the presence of a 4,9-friedodrimane
framework. The substituted moieties in 5 showed close
similarities with the cyclopentenone moieties in dactylospongenones,[15,17] similar merosesquiterpenoids from the Palau and Fijian sponges Dactylospongia spp., based on the observed resonances for
an isolated olefinic methine group (δH 5.92, δC 121.9), keto and ester carbonyl groups (δC 198.5, 174.4), a methoxy group (δH 3.72, δC 57.4), and an oxygenated tertiary carbon (δC 80.6). Compound 5 exhibited resonances corresponding
to an ethoxy group (δH 1.21, 4.15, 4.26; δC 14.3, 63.4) instead of the methoxy groups in dactylospongenones,
implying that the substituted heterocyclic moiety in 5 carries an ethyl ester group. This was verified by HMBC correlations
from H2-22 (δH 4.15, 4.26) to C-21 (δC 174.4) and C-23 (δC 14.3) and from H-18
(δH 5.92) and H-16 (δH 2.61) to
C-21 (δC 174.4). The HMBC and COSY spectra indicated
that the planar structure of 5 is identical with those
of dactylospongenones A–D,[15,17] except for
the ethyl ester moiety. The shielded C-12 resonances (δC 20.6) indicated that the new meroterpenoid has a trans-decalin moiety, and this was corroborated further
by the NOESY correlations shown in Figure 2. The similar specific rotation and NMR data of 5 to
those of dactylospongenone A (Figure 3), except
for resonances derived from the ethoxy moiety, imply that 5 is the ethyl ester equivalent of the methyl ester, dactylospongenone
A. Thus, compound 5 was assigned the trivial name (−)-dactylospongenone
E.Compounds 6 and 7 were obtained
as a
2:3 mixture based on the integration of associated 1H NMR
resonances for each compound. The HRFABMS data exhibited a single
quasimolecular ion at m/z 405.2637
(calcd [M + H]+, m/z 405.2641).
In conjunction with the 13C NMR data, this indicated that
these components are isomers with a C25H36O5 molecular formula. The 1D NMR spectra showed close similarities
with those of 5 except for the deshielded 13C NMR chemical shift values of the C-12 resonances in compounds 6 and 7 (δC 33.0, 33.2), indicating
that they are structurally based on cis-friedodrim-4(11)-ene
with C-15 cyclopentenone moieties.[15,17]The
stereochemical assignment of the C-15 cyclopentenone moieties
was attempted by utilizing gauge-invariant atomic orbital (GIAO) NMR
chemical shift calculations coupled with advanced statistics CP3 and
DP4 analyses,[18,19] given characteristically variable
chemical shift values of diastereomers associated with a cyclopentenone
moiety. Prior to application of the computational approach, the method
was verified for the investigation of stereochemical details of avarane-type
sesquiterpenoids. Each pair of epimers addressed in this study (i.e., 1 vs 2, 3 vs 4) was
used for the validation process employing GIAO NMR chemical shift
calculations coupled with CP3. Major conformers were generated by
conformational searches using Macromodel (Schrodinger LLC) and subjected
to GIAO shielding constant calculations at the B3LYP/6-31G(d,p) level
employing the Gaussian 09 package (Gaussian Inc.) (computational details
and Table S1, Supporting Information).
These calculated chemical shift values were Boltzmann-averaged according
to their relative MMFF94 potential energy, and the averaged values
were provided for the calculation of CP3 analysis (computational details,
Tables S1–S3, Supporting Information). CP3 supported the NMR-based assignment made for each pair of epimers
with 100% probability (Figures S2 and S3, Supporting
Information), validating that the application of such advanced
statistics is a relevant tool in establishing stereochemical details
of sesquiterpenoids addressed in this study. With the in silico method
corroborated, the four cis-4,9-friedodrimane-based
diastereomers associated with the C-15 cyclopentenone moieties of 6 and 7 (I: 16R, 17S; II: 16S, 17R; III: 16R, 17R; IV: 16S, 17S, Figure 5) were considered and subjected to chemical shift
calculation coupled with DP4 analysis because experimental data were
available for only two diastereomers. DP4 application with calculated
chemical shift values of diastereomers I–IV (Table S4, Supporting Information) supported the structural equivalence of diastereomer I with 7 with 100% probability (Figure S4, Supporting Information), and the consecutive
application with the remaining candidates (II–IV) suggested with 100% certainty (Figure S6, Supporting Information) that the absolute configuration
of 6 was identical to diastereomer II. These
new merosesquiterpenoids were assigned the trivial names 5-epi-dactylospongenones E (6) and F (7).
Figure 5
Plausible diastereomers of four cis-4,9-friedodrimane-based
diastereomers associated with the C-15 cyclopentenone moieties of 6 and 7.
Plausible diastereomers of four cis-4,9-friedodrimane-based
diastereomers associated with the C-15 cyclopentenone moieties of 6 and 7.The molecular formula of compound 8 was deduced
as
C23H32O4 based on 13C
NMR data and HRFABMS analysis (obsd [M + H]+, m/z 373.2381; calcd [M + H]+, m/z 373.2379). The NMR data displayed the
presence of a 5α-methyl-4,9-friedodrimane moiety (δC 33.4) carrying a C-15 dioxygenated-1,4-benzoquinone moiety
(δH 5.82; δC 102.4, 117.7, 153.3,
161.2, 182.3, 182.7) and, hence, showing structural resemblance with
5-epi-ilimaquinone.[15] The
NMR data of these two compounds differ in resonances of an ethoxy
group (δH 1.49, 4.04; δC 14.0, 66.1)
in 8 instead of a methoxy moiety in 5-epi-ilimaquinone. This suggests that compound 8 is an ethyl
ether homologue of 5-epi-ilimaquinone, which is confirmed
by the HMBC correlations from H2-22 (δH 4.04) to C-20 (δC 161.2). The specific rotation
of compound 8 is similar to that of 5-epi-ilimaquinone. This permitted the definition of an absolute configuration
identical to that of 5-epi-ilimaquinone (Figure 3). The trivial name (+)-5-epi-20-O-ethylsmenoquinone was assigned to 8.Over the past two decades, many natural products with hydroquinone/benzoquinone-type
moieties appended to a sesquiterpene or diterpene skeleton have been
reported from marine sponges.[13] The isolation
of 4,9-friedodrimane-type sesquiterpenoids 5, and 6 and 7 indicate that a competitive intramolecular
Michael addition might be associated in the formation of these secondary
metabolites. As illustrated in Figure 6, the
intramolecular addition of enolate B onto the C-20 carbonyl
would result in the generation of 5 and 7 or 6, depending on re- or si-face addition (path A in Figure 6). Alternatively, the addition of the enolate onto the C-21 carbonyl
group and tautomerization followed by transetherification would lead
to the formation of 8 (path B in Figure 6). In addition to these oxidative metabolites, natural products
such as nakijiquinones, smenospongine, and smenospongidine, featuring
hydroquinones attached to a sesquiterpene or diterpene scaffold, are
also known to be generated from marine organisms.[13] In this regard, reductive amination of D followed
by Schiff’s base formation with acetone or acetaldehyde and
consecutive isomerization would generate F and G, respectively. Finally, the oxidation of these two intermediates
would lead to the more stable benzoxazole moieties in 1–4, which might be facilitated by the oxidative
properties of V. rigida utilized in the current biotransformation
study.
Figure 6
Plausible biosynthetic pathways involved in the generation of 1–8.
Plausible biosynthetic pathways involved in the generation of 1–8.Aberrant activation of the Wnt/β-catenin pathway accompanied
by accumulation of intracellular β-catenin and subsequent activation
of CRT is important in the development and progression of colorectal
cancer.[2−4] Owing to limited amounts of isolated compounds, only
compounds 2, 3, 4, and 8 were evaluated for their potential to serve as prototypes
of new anticolorectal cancer agents capable of tuning the Wnt/β-catenin
pathway. To evaluate their inhibitory activities on the target pathway,
HEK293-firefly luciferase (FL) reporter cells stably transfected were
constructed with TOPflash, a synthetic β-catenin/Tcf-dependent
FL reporter, and humanFrizzled-1 (hFz-1) expression plasmids.[20] Incubation of HEK293-FL reporter cells with
different concentrations of compounds 2 and 8 and the mixture of 3 and 4 decreased the
CRT levels that had been activated upon Wnt3a-conditioned medium (Wnt3a-CM)
treatment (Figure 7A).
Figure 7
(A) HEK293-FL reporter
cells were incubated with the active molecules
in the presence of Wnt3a-CM for 15 h, and luciferase activity was
determined. (B) Cytosolic proteins were prepared from HEK293-FL reporter
cells treated with the vehicle or the active meroterpenoids in the
presence of Wnt3a-CM for 15 h and subjected to Western blotting. (C)
Two CRT-positive colon cancer cell lines were incubated with different
concentrations of the active meroterpenoids, and cell viability was
determined by Cell-Titer-Glo assay. *p < 0.05
and **p < 0.01, compared with the vehicle control
group.
(A) HEK293-FL reporter
cells were incubated with the active molecules
in the presence of Wnt3a-CM for 15 h, and luciferase activity was
determined. (B) Cytosolic proteins were prepared from HEK293-FL reporter
cells treated with the vehicle or the active meroterpenoids in the
presence of Wnt3a-CM for 15 h and subjected to Western blotting. (C)
Two CRT-positive colon cancer cell lines were incubated with different
concentrations of the active meroterpenoids, and cell viability was
determined by Cell-Titer-Glo assay. *p < 0.05
and **p < 0.01, compared with the vehicle control
group.Based on previous studies corroborating
that CRT is predominantly
dependent on expression levels of intracellular β-catenin,[4] Western blot analysis with anti-β-catenin
antibody in HEK293 reporter cells treated with the active compounds 2, 3, 4, and 8 was
conducted to examine whether these secondary metabolites can reduce
intracellular β-catenin levels. Consistent with previous results,[4] β-catenin expression was increased upon
treatment with Wnt3a-CM (Figure 7B). Incubation
of HEK293 cells with those active molecules mitigated up-regulated
cytosolic β-catenin expression (Figure 7B). Interestingly, the inhibitory potency of the compounds on β-catenin
expression was in accord with their suppressing effects on Wnt3a-CM-activated
CRT, suggesting that the active compounds may inhibit the Wnt/β-catenin
pathway via promoting cytosolic β-catenin degradation.The specific reduction of β-catenin has been shown to suppress
the proliferation of CRT-positive colon cancer cells.[4] To validate if the active sesquiterpenoids retard proliferation
of colon cancer cells via the observed inhibition on the Wnt/β-catenin
pathway, two CRT-positive colon cancer cell lines, SW480 and HCT116,
were treated with various concentrations of the compounds. Compound 2 and the mixture of compounds 3 and 4 started to exert antiproliferative activity at concentrations more
than 20 μM, while compound 8 began to inhibit tumor
growth at 0.75 and 1.5 μM against SW480 (IC50 = 3.24
μM) and HCT116 cells (IC50 = 2.95 μM), respectively
(Figure 7C), demonstrating that 8 displayed more potent cytotoxic activity on the CRP-positive cancer
cells than the other compounds. It is thus plausible that the underlying
mechanism of the antineoplastic properties of the tested meroterpenoids
is closely involved in their mitigating activities on the Wnt/β-catenin
pathway considering that the observed cytotoxic potency on the colon
cancer cells is consistent with their down-regulating potential against
β-catenin expression elevated by Wnt3a-CM.In conclusion,
marine invertebrates could be a promising resource
in providing drug frameworks modulating the oncogenic Wnt/β-catenin
pathway, which may warrant further exploration of such marine organisms
for the discovery of colon cancer inhibitory leads. The current study
demonstrates the potential of chemical oxidation of the cell contents
of a Verongida sponge to expand the diversity of 4,9-friedodrimane
metabolites capable of suppressing the Wnt/β-catenin signaling
pathway. Such an approach may also be applicable in similar studies
with other marine organisms.The current findings show that
the 4,9-friedodrimane chemotype
could serve as a drug template capable of exerting antineoplastic
properties via inhibition of β-catenin expression. Further structural
refinements in selective anticancer drug design can build on this
study, presumably in conjunction with insights acquired from well-characterized
biochemical mechanisms of colorectal tumorigenesis.
Experimental Section
General Experimental Procedures
Optical rotations were
measured on a JASCO DIP-1000 automatic digital polarimeter (Tokyo,
Japan). UV–vis spectra were measured on an Agilent 1100 series
diode array and multiple-wavelength detectors. The NMR spectra were
recorded on a Varian 600 MHz (VNS 600) spectrometer equipped with
a 5 mm direct detection pulsed field gradient probe. All NMR experiments
were conducted at 294 K using CDCl3 as a solvent and referenced
by residual solvent signals for CHCl3 (δH = 7.24, δC = 77.16). HRFABMS data were obtained
from the Korea Basic Science Institute (Daegu) on a JEOL JMS 700 high-resolution
mass spectrometer, and LC-MS profiling was performed utilizing an
Agilent 1100 HPLC system with a Phenomenex Luna 5 μm C18 column (4.6 × 250 mm) eluted with an MeOH–H2O gradient solvent system and a Bruker Daltonics microTOF mass spectrometer.
Vacuum-liquid chromatography (VLC) was performed on Merck silica gel
(70–230 mesh). Medium-pressure liquid chromatography (MPLC)
was carried out using a Biotage Isolera equipped with a reversed-phase
C18 SNAP cartridge KP-C18-HS (120 g, Biotage, Charlotte,
NC, USA). High-performance liquid chromatography (HPLC) was implemented
on a Gilson system using Luna C18 (250 × 21.20 mm,
10 μm), Luna C18 (250 × 4.60 mm, 5 μm),
YMC C18 (250 × 4.60 mm, 5 μm), and Mightysil
C18 columns (150 × 4.60 mm, 5 μm). Thin-layer
chromatography (TLC) was executed on glass plates precoated with silica
gel F254 (20 × 20 cm, 200 μm, 60 Å, Merck).
Animal
Material
Smenospongia aurea (08FL-20-B), Smenospongia cerebriformis (08FL-20),
and Verongula rigida (08FL-20-A) were collected from
Key Largo, FL, in October 2008. The sponges were collected from a
shallow coral reef habitat between 3 and 24 m depth[11] and identified by Dr. Michelle Kelly (National Institute
of Water and Atmospheric Research, Auckland, New Zealand). Voucher
specimens under the aforementioned codes have been deposited in the
University of Mississippi.
Extraction and Isolation
Smenospongia aurea and S. cerebriformis were homogenized and incubated
with V. rigida in ethanol for a week. The dried ethanol
extract (3.6 kg) of the mixture of three sponges was subjected to
silica gel VLC (36 kg, 14 (H) × 17.5 (D) cm) and eluted with
a stepwise gradient of hexanes (100%), hexanes–acetone (80:20,
60:40, 50:50, 40:60, 20:80), acetone (100%), acetone–MeOH (80:20,
60:40, 50:50), MeOH (100%), MeOH–H2O (50:50), and
H2O (100%) to give 13 fractions (Fr. 1–13). Fraction
10 (39.3 g) was further divided into nine fractions (Fr. 10-1–10-9)
using silica gel VLC (12 (H) × 17.5 (D) cm) with a mixture of
hexanes–acetone (95:5, 90:10, 85:15, 80:20), MeOH (100%), and
MeOH–H2O (50:50). Fraction 10-7 (3.7 g) was applied
to C18 MPLC (15.5 × 4 cm) with an isocratic condition
of MeOH–H2O (85:15) to yield six subfractions (Fr.
10-7-1–10-7-6). Fraction 10-7-3 (115.8 mg) was chromatographed
over C18 HPLC (250 × 21.20 mm, 10 μm) with MeOH–H2O (83:17) to produce three fractions (Fr. 10-7-3-1–10-7-3-3).
Fraction 10-7-3-2 (12.4 mg) was purified using C18 HPLC
(250 × 4.60 mm and 150 × 4.60 mm, 5 μm, connected
in line) with MeOH–H2O (75:25) to obtain 2 (2.2 mg, tR = 223 min) and a mixture
of 3 and 4 (4.6 mg, tR = 208 min). Fraction 10-7-3-3 (9.7 mg) was subjected to C18 HPLC (250 × 4.60 mm and 150 × 4.60 mm, 5 μm,
connected in line) with MeOH–H2O (78:22) followed
by C18 HPLC (250 × 4.60 mm, 5 μm) with gradient
conditions of MeOH–H2O (80:20 → 100:0) for
120 min to afford 1 (1.8 mg, tR = 151 min). Fraction 10-7-1 was chromatographed over C18 HPLC (250 × 21.20 mm, 10 μm) with MeOH–H2O (78:22) to afford a mixture of 6 and 7 (1.6 mg, tR = 92 min) as well as 5 (1.5 mg, tR = 100 min). Fraction
10-5 was combined with Fr. 10-6 based on their similar TLC patterns
and fractionated to generate seven subfractions (Fr. 10-6-1–10-6-7)
by C18 MPLC (15.5 × 4 cm) with an isocratic condition
of MeOH–H2O (85:15). Fraction 10-6-2 was sequentially
purified using C18 HPLC (250 × 21.20 mm, 10 μm)
with MeOH–H2O (78:22), C18 HPLC (250
× 4.60 mm, 5 μm) with MeOH–H2O (75:25),
and C18 HPLC (250 × 4.60 mm, 5 μm) with MeOH–H2O (73:27) to give 8 (1.1 mg).
pale yellow, amorphous
solid; [α]D25 +22 (c 0.1, MeOH); UV (MeOH) λmax 294 nm; 1H and 13C NMR, see Table 1; HRFABMS m/z 384.2542
[M + H]+ (calcd for C24H34NO3, 384.2539).
Mixture of nakijinone A and 5-epi-nakijinone
A: 18-methoxy-22,22-dimethyl-16-[{(5R,8S,9R,10S)- and (5S,8S,9R,10S)-5,8,9-trimethyl-4-methylenedecahydronaphthalen-9-yl}methyl]benzo[d]oxazol-17(2H)-one (3 and 4):
yellow, amorphous solid; [α]D25 +21 (c 0.1, MeOH); UV (MeOH) λmax 297 nm; 1H and 13C NMR, see Table 1; HRFABMS m/z 398.2696 [M + H]+ (calcd for C25H36NO3, 398.2695),
420.2509 [M + Na]+ (calcd for C25H35NO3Na, 420.2515).
yellow, amorphous solid; [α]D25 −91 (c 0.1, MeOH); UV (MeOH) λmax 246, 283 nm; 1H and 13C NMR, see Table 1; HRFABMS m/z 405.2638 [M + H]+ (calcd for C24H37O5, 405.2641),
427.2464 [M + Na]+ (calcd for C24H36O5Na, 427.2460).
Mixture of 5-epi-dactylospongenones E and F:
ethyl (16R,17S)- and (16S,17R)-17-hydroxy-19-methoxy-20-oxo-16-[{(5R,8S,9R,10S)-5,8,9-trimethyl-4-methylenedecahydronaphthalen-9-yl}methyl]cyclopent-18-ene-17-carboxylate
(6 and 7):
yellow, amorphous solid;
[α]D25 +5 (c 0.1, MeOH); UV (MeOH) λmax 247, 287 nm; 1H and 13C NMR, see Table 1; HRFABMS m/z 405.2637
[M + H]+ (calcd for C24H37O5, 405.2641), 427.2463 [M + Na]+ (calcd for C24H36O5Na, 427.2460).
dark yellow, amorphous solid; [α]D25 +39 (c 0.1, MeOH); UV (MeOH) λmax 288 nm; 1H and 13C NMR, see Table 1; HRFABMS m/z 373.2381 [M + H]+ (calcd for C23H33O4, 373.2379),
395.2203 [M + Na]+ (calcd for C23H32O4Na, 395.2198).
Computational Details[18,19,21]
All conformational searches
were implemented using the
Macromodel (version 9.9, Schrodinger LLC) program with “mixed
torsional/low mode sampling” in the MMFF force field. The searches
were conducted in the gas phase with a 50 kJ/mol energy window limit
and 10 000 maximum number of steps to thoroughly examine all
low-energy conformers. The Polak–Ribiere conjugate gradient
method was utilized for minimization processes with 10 000
maximum iterations and a 0.001 kJ (mol Å)−1 convergence threshold on the rms gradient. Conformers within 10
kJ/mol of each global minimum of compounds (i.e., compounds 1–4 in Figure 1 and diastereomers I–IV in Figure 5) were used for GIAO shielding constant calculations
without geometry optimization employing the Gaussian 09 package (Gaussian
Inc.) at the B3LYP/6-31G(d,p) level in the gas phase. Calculated chemical
shift values were acquired based on the following equation: δcalcd = (σ0 – σ)/(1 – σ0/106), in which
δcalcd is the calculated chemical shift value for nucleus x (e.g., 1H or 13C) and σ and σ0 are the calculated
isotropic constants for nucleus x and tetramethylsilane
(TMS), respectively. In particular, the geometry of TMS was optimized
using the B3LYP/6-31G(d,p) level in the gas phase using the Gaussian
09 package (Gaussian Inc.) for unbiased and consistent comparison
of calculated chemical shift values with those of other molecules
in this study according to the original authors’ recommendation.[19] These calculated chemical shift values of compounds 1–4 and diastereomers I–IV were averaged based on their Boltzmann populations (Tables
S1–S4, Supporting Information) and
used for calculations of CP3 and DP4 analysis employing applets available
at http://www-jmg.ch.cam.ac.uk/tools/nmr/ and http://www-jmg.ch.cam.ac.uk/tools/nmr/DP4/, respectively. In application of the statistical analyses, calculated
and experimental chemical shift values of diastereotopic protons were
compared with their closest matches based on the original author’s
recommendation.[19] The 3D images in Figure 2 were illustrated utilizing Pymol 1.6.x (Schrödinger
LLC.).
Cell Culture, Reporter Assays, and Chemicals
HEK293
(humanembryonic kidney cell), HCT116 (humancolon carcinoma), and
SW480 (humancolon adenocarcinoma) cells were acquired from the American
Type Culture Collection (Manassas, VA, USA) and maintained in Dulbecco’s
modified Eagle’s medium supplemented with 10% fetal bovine
serum, 120 μg/mL penicillin, and 200 μg/mL streptomycin.
HEK293-FL reporters (TOPFlash) and controls (FOPFlash) were established
as described previously.[22,23] The Wnt3a-CM was prepared
as described previously.[22] The luciferase
assay was implemented using the Dual Luciferase Assay kit (Promega,
Fitchburg, WI, USA).
Plasmid Constructs, siRNA, and Transfection
The hFz-1
cDNA was constructed with reference to a previous protocol.[20] The pTOPFlash and pFOPFlash reporter plasmids
were purchased from Millipore Corporation (Billerica, MA, USA).
Western Blots
The cytosolic fraction was generated
as described previously.[24] Proteins were
separated utilizing SDS-polyacrylamide gel electrophoresis in a 4%
to 12% gradient gel (Invitrogen, Grand Island, NY, USA) and transferred
to nitrocellulose membranes (Bio-Rad Laboratories, Hercules, CA, USA).
The membranes were blocked with 5% nonfat milk and probed with anti-β-catenin
(BD Transduction Laboratories, Lexington, KY, USA) and anti-actin
antibodies (Cell Signaling Technology, Danvers, MA, USA). The membranes
were incubated with horseradish-peroxidase-conjugated anti-mouse IgG
or anti-rabbit IgG and visualized employing the enhanced chemiluminescence
(ECL) system (Santa Cruz Biotechnology, Santa Cruz, CA, USA).
Cell Viability
Assay
The HCT116 and SW480 cells were
inoculated into 96-well plates and treated with compounds 1–8 for 48 h. The cell viability from each treated
sample was assessed in triplicate using a CellTiter-Glo assay kit
(Promega) according to the manufacturer’s instructions.
Authors: S Satoh; Y Daigo; Y Furukawa; T Kato; N Miwa; T Nishiwaki; T Kawasoe; H Ishiguro; M Fujita; T Tokino; Y Sasaki; S Imaoka; M Murata; T Shimano; Y Yamaoka; Y Nakamura Journal: Nat Genet Date: 2000-03 Impact factor: 38.330
Authors: Maina Lepourcelet; Ying-Nan P Chen; Dennis S France; Huisheng Wang; Phillip Crews; Frank Petersen; Charles Bruseo; Alexander W Wood; Ramesh A Shivdasani Journal: Cancer Cell Date: 2004-01 Impact factor: 31.743
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7