Mycalol: A natural lipid with promising cytotoxic properties against human anaplastic thyroid carcinoma cells

Article abstract of DOI:10.1002/anie.201303039

From Antarctic waters: Human anaplastic thyroid carcinoma (ATC) cells which stably express antisense construct against nuclear high-mobility groupA (HMGA) proteins were used in a novel phenotypic assay for screening marine natural extracts. The rational search led to the isolation and identification of a novel polyoxygenated linear lipid (1) that showed important and selective cytotoxicity against ATC-derived tumor cell lines. Copyright

Full text of DOI:10.1002/anie.201303039

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DOI: 10.1002/anie.201303039
Anticancer Lead Discovery
Mycalol: A Natural Lipid with Promising Cytotoxic Properties against
Human Anaplastic Thyroid Carcinoma Cells**
Adele Cutignano, Genoveffa Nuzzo, Daniela D’Angelo, Eleonora Borbone, Alfredo Fusco,* and
Angelo Fontana*
The high-mobility group A (HMGA, types 1 and 2) proteins
are low-molecular-weight nuclear factors that orchestrate the
assembly of nucleoprotein complexes involved in gene tran-
scription, replication, and chromatin structure. HMGAs
possess oncogenic activity[1,2] and proteins of type 1
(HMGA1) have been correlated to cellular invasiveness and
drug-resistance in human malignancies.[3] In particular, block-
age of expression of these proteins significantly enhances the
responsiveness of tumor cell lines that are otherwise resistant
to cytotoxic agents. Thus, phenotypic assays based on cells
with reduced levels of HMGA are a possible tool for
a rational search of novel compounds against tumors whose
aggressiveness and resistance reduce the success of normal
screening methods.
the Austral summer of 2005. The sponge, frozen soon after
collection, was extracted with MeOH and fractionated
according to a modified Kupchan method.[6] The chloroform
extract showed no activity against FRO cells up to 50 mgmLꢀ1,
but gave
a
good response (IC50 = 7.5 mgmLꢀ1) against
HMGA1-silenced FRO cells (FRO-asHMGA), thus support-
ing the potential of a novel screening method based on
HMGA-interference. Sequential steps of silica gel radial
chromatography and reverse-phase HPLC (see the Support-
ing information) gave alkyl glyceryl ether 1 together with
a number of minor compounds that are still under study.
Herein, we report the elucidation of the structure of
mycalol (1), a novel polyoxygenated ether lipid that showed
a promising in vitro specific activity against different cell lines
derived from human anaplastic thyroid carcinoma (ATC), the
most aggressive human thyroid gland malignancy.[4] Mycalol
was identified by a novel screening method based on the
parallel use of FRO cells, which are human ATC-derived cells
with high constitutive levels of HMAG1, but not HMGA2,
and FRO-asHMGA1 cells, a genetically modified population
of FRO cells that stably express an anti-HMGA1 antisense
construct that blocks HMGA1 synthesis.[5] The effects of
extracts and fractions were measured on the paired cell lines
by MTS proliferation assay.
The HR-ESI+ MS spectrum of 1 showed a MꢀNa+ ion at
m/z 573.3959, thus accounting for the molecular formula
C29H58O9 (calcd m/z 573.3979 for C29H58O9Na) and requiring
1
only one formal unsaturation. Accordingly, H and 13C 2D
NMR data indicated a C27 linear structure with nine oxy-
genated carbons and an acetyl group (for full assignment, see
the Supporting Information, Table S1). COSY spectra iden-
tified seven of these carbons as part of a glycerol moiety (H1’
d 3.85 and 3.90; H2’ d 4.35; H3’ d 4.08) and two gem-diol spin
systems. On the other hand, the acetyl residue at C20 was
inferred on the basis of a long-range TOCSY correlation of
the down-shifted oxymethine proton at d 5.07 with the
terminal methyl at d 0.82 through three methylene groups at d
1.20 (H223), d 1.33 (H222) and d 1.56 (H221). A diagnostic
HMBC correlation of C1’ of glycerol (73.8 ppm) with the
methylene signals at d 3.97 allowed us to join the glyceryl
residue with the linear chain through an ether linkage and to
assign the last oxygenated function to C1.
Mycalol was isolated from a chloroform extract of the
sponge Mycale (Oxymycale) acerata Kirkpatrick 1907 col-
lected along the coasts of Terra Nova Bay (Antarctica) during
[*] Dr. A. Cutignano, Dr. G. Nuzzo, Dr. A. Fontana
Istituto di Chimica Biomolecolare, Consiglio Nazionale delle
Ricerche, Via Campi Flegrei 34, 80078, Pozzuoli, Napoli (Italy)
E-mail: afontana@icb.cnr.it
Homepage: http://www.icb.cnr.it
Dr. D. D’Angelo, Dr. E. Borbone, Dr. A. Fusco
This methylene group showed scalar couplings with the
diastereomeric protons at d 2.40 and d 2.16 (H22), both also
coupled to one of the oxymethine protons (d 4.18, H3) of the
two gem-diol systems. Characterization and location of these
groups were unambiguously accomplished by 2D NMR
analysis of the triacetonide derivatives 2 and 3 obtained by
treatment of 1 with deuterated acetone and dimethoxypro-
pane, respectively (Figure 1a). In particular, the deuterated
derivative 2 allowed an easier inspection of the up-field
region in 2D NMR experiments (see the Supporting Infor-
mation), which revealed two methylene groups at d 1.57 (H25)
and 1.74 (H26) between the two diol moieties. The relative
Istituto di Endocrinologia ed Oncologia Sperimentale, Consiglio
Nazionale delle Ricerche, Via Pansini 5, 80131, Napoli (Italy)
[**] We are deeply grateful to Ernesto Mollo for taxonomic identification
of the sponge. Carmine Iodice, Dominique Melck, and Maurizio
Zampa are also acknowledged for recording CD, NMR, and HR-MS
spectra, respectively. This work was supported by Italian PNRA,
Project SMART funded by Regione Campania (2009.0932025) and
Associazione Italiana per la Ricerca sul Cancro (AIRC) and the
Ministero dell’Universitꢀ e della Ricerca Scientifica e Tecnologica
MIUR (PRIN 2008). D.D’A. is recipient of a fellowship from
Fondazione Italiana per la Ricerca sul Cancro (FIRC).
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201303039.
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Figure 1. A) Synthesis of acetonide and MTPA derivatives of mycalol (1). B) Dd (dSꢀdR) values (ppm) for selected protons of (R)-MTPA ester 5
and (S)-MTPA ester 6. Reagents and conditions: a) (CD3)2CO, I2, RT, 24 h; b) (CH3)2C(OCH3)2, PPTS, 758C, 6 h; c) Na2CO3, MeOH, 558C, 28 h;
d) S-(ꢀ)- or R-(ꢀ)-MTPA chloride, DMAP, dry CH2Cl2, RT, overnight. Ac=acetyl, DMAP=4-dimethylaminopyridine, MTPA=Mosher’s acid; a-
methoxy-a-(trifluoromethyl)phenylacetic acid, PPTS=pyridinium p-toluenesulfonate.
stereochemistry of these latter functions was established by
the acetonide method on derivative 3,[7] whose NMR
spectrum in CDCl3 showed well-separated methyl acetals
(27.1 and 25.9 ppm; 26.8 and 25.4 ppm), which is in agreement
with the erythro configuration for both vic diols of 1 (see the
Supporting Information). The depicted erythro configuration
was also supported by the small coupling constants that could
be estimated between the vicinal protons in the dioxolane
rings.
stereochemistry of the isolated hydroxy group at C20 by
a modification of Mosherꢀs method.[8] Despite the near
symmetry around this stereocenter, 2D NMR analysis of
MTPA derivatives 5 and 6 clearly indicated Dd values in
agreement with an R configuration for this carbon center (see
the Supporting Information). On the other hand, the absolute
stereochemistry of the central carbon of the substituted
glycerol was deduced by the dibenzoate chirality method[9] on
derivative 9, which was obtained from the perpivaloyl
derivative 7 by mild basic hydrolysis (Scheme 1). After
purification by reverse-phase HPLC, compound 9 showed
After methanolysis to generate alcohol 4 (Figure 1),
acetonide 3 was also used to determine the absolute
Scheme 1. Selective derivatization of mycalol (1). a) PvCl, dry pyridine, RT; b) Na2CO3, MeOH RT, overnight; c) BzCl (4 equiv), dry pyridine, RT
overnight; d) (CH3)2C(OCH3)2, PPTS, 758C, 6 h; e) Na2CO3, MeOH RT, 72 h, chromatographic resolution; f) excess BzCl, dry pyridine, 508C, 24 h.
Bz=benzoyl, Pv=pivaloyl.
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Figure 2. Circular dichroism curve of 1’,2’-dibenzoyl-3,4,7,8-tetrapiva-
loyl mycalol (9).
a CD spectrum with a negative first Cotton effect (CE;
236 nm) and a positive second CE (216 nm; Figure 2). Sign
and wavelength of the Davydov splitting of 9 were identical to
those reported for mono-1-O-alkyl sn-glycerol,[10] thus defin-
ing as S the absolute configuration at C2’ of natural product 1.
Determination of the absolute configuration of the two
erythro diol systems was also addressed by the chiroptical
approach. In consideration of the difficulties in assigning the
stereochemistry of erythro diols in flexible compounds,[7c] we
first used an approach based on auxiliary chomophore
dimolybdenum tetraacetate ([Mo2(AcO)4]), in accordance
with the revised method developed by Snatzke and Frelek,[11a]
which was further confirmed by the dibenzoate technique of
Harada and Nakanishi.[9] The application of both methods
required selective protection of the two vic-diol moieties and
restriction of the conformational changes of the flexible chain
of mycalol. After several attempts with different orthogonal
protecting groups, both tasks were achieved by pivaloylation
of the hydroxy groups followed by partial hydrolysis and
chromatographic resolution of the two isomeric triols 11 and
12 (Scheme 1). The steric bulk of the pivaloyl groups
significantly increased the rotational energy barriers of the
linear chain in both compounds (see the Supporting Infor-
mation). Therefore, in situ complexation with [Mo2(AcO)4]
induced a conformational preference that, in analogy with
erythro and threo diol models reported in the literature,[11]
produced CD spectra with four major bands in the range of
500–250 nm (Figure 3). The signs of the CEs centered around
300 nm (band IV) and 400 nm (band II), as well as a minimum
or a maximum between these bands in 11 and 12, respectively,
were in full agreement with the assignment of 3R,4S and
7R,8S stereochemistry for the two diol moieties of 1 when the
molecules adopt conformations with the part containing the
pivaloyl groups antiperiplanar to the vicinal hydroxy group
(Figure 3). This interpretation of the induced CD spectra of
11 and 12 is mostly dependent on the orientation of the
Figure 3. Circular dichroism curves of chiral MO2–dimer complexes of
the isolated diol derivatives 11 (A) and 12 (B). Roman numbers
indicate the diagnostic CE bands following Snatzke and Frelek’s
nomenclature.
pivaloate-bearing substituents (see the Supporting Informa-
tion). Whereas it is intuitive that the C5–C24 chain is larger
than the dioxolane part for triol 11, the differences between
the two alkyl substituents around the C7/C8 diol in compound
12 is less obvious. To confirm that the conformational
preferences are governed by the pivaloyl residues, and to
support the configurational assignment of the erythro-vic
diols in mycalol, compounds 11 and 12 were converted into
benzoate derivatives 13 and 14 (Scheme 1). The resulting CD
spectra (Figure 4) showed Cotton effects whose Davydov
splitting, signs, and frequencies fully confirmed the 3R,4S and
7R,8S stereochemistry of the two diol systems.[9] Notably, the
CD curve of compound 14 only agrees with the helicity rule
described by Harada and Nakanishi for dibenzoate in the
conformer where the substituent at C3 is anti to the benzoate
group at C4 (see the Supporting Information), thus support-
ing the prediction about the conformational model proposed
for complexes of 11 and 12 with [Mo2(AcO)4].
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interference of 1 with the mechanisms leading to proliferation
of ATC cells. Alkylglycerols are a known class of bioactive
natural products with several biomedical and biochemical
properties.[12] Nevertheless, to the best of our knowledge, 1 is
the first member of the family to show such a wide extent of
oxidation.[13]
In conclusion, we have identified a new polyoxygenated
monoalkyl glyceryl ether, mycalol (1), with promising activity
against ATC-derived cell lines. Full determination of the
structure was deduced by a combination of chemical and
chiroptical methods applied at the micromolar scale. Partic-
ularly relevant is the characterization of the two erythro-vic
diol moieties by Snatzke and Frelekꢀs method on conforma-
tionally constrained pivaloyl derivatives of 1, which could
open the possible application of this approach to other natural
products with flexible structure. According to recent evidence
on the cytotoxic effect of ecteinascidin-743 through impair-
ment of the HMGA functions,[14] the assay based on
HMGA1-interefered cells may result in an important
method for screening of novel chemotherapeutic agents.
Figure 4. Circular dichroism curves of dibenzoyl derivatives 13 (A) and
14 (B). The inserts show the suggested chiralities of the two
dibenzoates in agreement with Harada and Nakanishi model.[9]
A,B) R1 =glycerol-containing part. A) R2 =C5–C24 alkyl chain.
B) R2 =C9–C24 alkyl chain.
Anaplastic thyroid carcinoma (ATC) causes up to 40% of
all deaths from thyroid cancer.[4] The present chemothera-
peutic treatments are mostly palliative, and currently there is
no clinical candidate for the treatment of this disease. Mycalol
exhibited considerable cytotoxicity on three ATC cell lines, Experimental Section
General procedures: Optical rotations were measured on a Jasco
with the highest effect being on the 8505c and ACT1 cells
DIP-370 digital polarimeter. CD spectra were acquired on a Jasco J-
815 polarimeter. NMR spectra were recorded on a Bruker Avance
DRX 600 equipped with a cryoprobe operating at 600 MHz for
proton or a Bruker DRX 400 operating at 400 MHz for proton.
Chemical shifts values are reported in ppm (d) and referenced to
internal signals of residual protons [C5D5N 1H d 7.19, 13C 123.5 ppm;
CDCl3 1H d7.26, 13C 77.0 ppm; C6D6 1H d7.15, 13C 128.0 ppm). Mass
spectra were acquired on a Q-Tof-micro mass spectrometer (Waters)
equipped with an ESI source and a Lock-Spray apparatus for accurate
mass measurements. Solvents were distilled prior to use.
(IC50 3.8 mm and 4.5 mm, respectively; Table 1). Notably, no
Table 1: Cytotoxic activity of mycalol (1).
Cell line
Source
IC50 [mm]
Human ATC-derived cell lines
1
2
3
4
FRO
human ATC-derived cells
15.7
7.3
4.5
3.8
FRO-HMGA1as FRO cells silenced for HMGA1
ACT1
8505c
human ATC-derived cells
human ATC-derived cells
Cell viability assay: Cells were exposed to extracts, fractions, or
pure mycalol dissolved in DMSO. After 72 h, CellTiter 96 Aqueous
One Solution (20 mL; Promega Italia, Milano, Italy) was dispensed,
and the absorbance at 490 nm measured. The results were normalized
for absorbance in the absence of compounds. Stable clones of
HMGA1-Interfered FRO cells were prepared according to Ref. [5] by
co-transfection of the plasmid together with pBabe-Puro vector
containing puromycin resistance gene.
Other solid tumor cell lines
5
6
7
8
9
GEO
human colon cancer cells
n.a.[c]
n.a.[d]
10.9
GEO+HMGA1 GEO cells over-expressing HMGA1
HCT116
OVCAR8
MCF7
human colon carcinoma cells
human ovarian cancer cells
human breast adenocarcinoma cells
n.a.[e]
n.a.[f]
Mycalol (1): Colorless oil. HR-ESIMS+ m/z 573.3959 [M+Na]+
20
(calcd for C29H58O9Na, 573.3979). ½aꢁD ¼ + 3.45 (c = 0.1, MeOH); for
Functional thyroid cells
10 FRTL5
1H and 13C NMR data, see Table S1. Further experimental details can
be found in the Supporting Information.
rat thyroid cells
>36
[a] 95% cell vitality at 36 mm; [b] 85% cell vitality at 36 mm; [c] 81% cell
vitality at 36 mm; [d] 76% cell vitality at 36 mm; [e] 92% cell vitality at
11 mm; [f] 72% cell vitality at 11 mm. n.a.=not active.
Received: April 11, 2013
Published online: July 16, 2013
Keywords: configuration determination · cytotoxicity · lipids ·
natural products · NMR spectroscopy
significant cytotoxicity was reported against differentiated rat
thyrocytes (FRTL5 cells). Furthermore, except for a weak
effect on colon carcinoma (IC50 10.9 mm), 1 did not affect the
vitality of cell lines derived from other solid tumors.
.
Overall, these results indicate that the response orches-
trated by HMGA1 proteins provides a simple and versatile
in vitro tool for searching for novel agents for the treatment of
this thyroid neoplasm. In this line of reasoning, it is note-
worthy that the absence of an effect on thyrocytes transfected
with activated ras oncogene (TL5-ky and TL5-ki; data not
shown) or HMGA1-overexpressing colon carcinoma cells
(GEO+HMGA1; Table 1) may be the result of the specific
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