Eleganketal a, a highly oxygenated dibenzospiroketal from the marine-derived fungus Spicaria elegans KLA03

Article abstract of DOI:10.1021/np500458a

Eleganketal A (1), a naturally occurring aromatic polyketide possessing a rare highly oxygenated spiro[isobenzofuran-1,3′-isochroman] ring system, was isolated from the fungus Spicaria elegans KLA03 by culturing it in a modified mannitol-based medium. The

Full text of DOI:10.1021/np500458a

Article
pubs.acs.org/jnp
Eleganketal A, a Highly Oxygenated Dibenzospiroketal from the
Marine-Derived Fungus Spicaria elegans KLA03
Yepeng Luan,† Hongjuan Wei,† Zhenpei Zhang,† Qian Che,† Yankai Liu,† Tianjiao Zhu,† Attila Mandi,‡
́
Tibor Kurtan,‡ Qianqun Gu,† and Dehai Li*,†
́
†Key Laboratory of Marine Drugs, Chinese Ministry of Education, School of Medicine and Pharmacy, Ocean University of China,
Qingdao 266003, People’s Republic of China
‡Department of Organic Chemistry, University of Debrecen, POB 20, H-4010 Debrecen, Hungary
S
* Supporting Information
ABSTRACT: Eleganketal A (1), a naturally occurring aromatic polyketide possessing a rare highly oxygenated
spiro[isobenzofuran-1,3′-isochroman] ring system, was isolated from the fungus Spicaria elegans KLA03 by culturing it in a
modified mannitol-based medium. The structure of 1 including the absolute configuration was determined by combining
spectroscopic analysis, synthesis of the racemic permethylated analogue, chiral-phase HPLC separation, and TDDFT-ECD
analysis.
produce structurally diverse cytochalasin-type polyketides,
more fermentation conditions were tested, and it was found
that the HPLC-UV profile of the EtOAc extract changed
dramatically when cultured in a modified mannitol-based
medium (NH4Cl as nitrogen source). From the broth,
eleganketal A (1), possessing a rare substituted 3H-spiro-
[isobenzofuran-1,3′-isochroman]-4′(1′H)-one skeleton, togeth-
er with two biogenetically related known compounds, 4,5,6-
trihydroxy-7-methyl-1,3-dihydroisobenzofuran (2)9 and flavi-
mycin A (3),10 was isolated. The new structure of 1 including
the absolute configuration was determined by a combination of
spectroscopic analysis, chemical synthesis, chiral-phase HPLC
analysis, and TDDFT-ECD calculations.
arine-derived fungi are a powerful resource to produce
Munique bioactive structures attracting the attention of
chemists and biologists.1 Although thousands of compounds
with various chemical structures have been disclosed, most of
the gene clusters encoding secondary metabolites are silent
under a single culture condition.2,3 To maximize the chemical
diversity of fungal metabolites, the so-called “OSMAC”
approach has proven to be a convenient and useful method
by altering culture parameters, feeding precursors, adding
enzyme inhibitors, etc.3
During our exploration for bioactive compounds from
marine-derived fungi, the cytochalasins Z9−Z15 (glucose-based
medium under static conditions) have been isolated from the
fungus Spicaria elegans KLA03, which derives from marine
sediments collected in Jiaozhou Bay, China.4 Guided by the
OSMAC approach, 14 new polyketides of this family were
further isolated including spicochalasin A and aspochalasins
M−Q (starch and soybean-based medium upon shaking for 8
days),5 7-deoxycytochalasins Z7 and Z9 (adding a cytochrome
P-450 inhibitor to glucose-based medium under static
conditions),6 aspochalasins R−T (starch and soybean-based
medium, shaking for 14 days),7 and cytochalasins Z21−Z23
(adding D- and L-tryptophan to glucose-based medium, static
conditions).8 Attracted by the amazing ability of this strain to
RESULTS AND DISCUSSION
■
The fermented whole broth (20 L) was obtained by culturing S.
elegans KLA03 with shaking at 28 °C for 8 days in the modified
mannitol medium. The EtOAc extract (20 g) was subjected to
repeated silica gel and LH-20 column chromatography to yield
the new compound 1 (8.0 mg).
Received: June 5, 2014
Published: June 26, 2014
© 2014 American Chemical Society and
American Society of Pharmacognosy
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a
Scheme 1. Synthesis of Compound ( )-1a
Eleganketal A (1), an amorphous, yellow powder, possessed
the molecular formula C18H16O9 according to the negative
HRESIMS peak at m/z 375.0710. The 1D NMR data indicated
two methyls, two methylenes, one ketal (δC 107.5, C-8), one
ketone (δC 192.2, C-8′), and 14 quaternary carbons including
12 aromatic ones with six of them oxygenated, suggesting the
presence of two hexasubstituted benzene rings.
a
Reagents and conditions: (a) Br2, AcOH, DCM (89%); (b) 1,3-
propanediol, p-TsOH, toluene (95%); (c) n-BuLi, MeI, THF; (d)
saturated citric acid (68% for c and d); (e) CF3COOAg, I2, CHCl3
(80%); (f) i. TMSA, Pd(PPh3)4, CuI, TEA. ii. K2CO3, MeOH (57%
for two steps); (g) Pd(PPh3)4, CuI, 7, TEA (82%); (h) NaBH4,
MeOH, rt. (98%); (i) PBr3, THF (99%); (j) RuCl3, KIO4, H2O, CCl4,
MeCN (55%); (k) KOH, THF/H2O, reflux (57%). TMSA:
(Trimethylsilyl)acetylene.
The planar structure was tentatively proposed by analysis of
the 2D NMR data (Figure 1). The 1,6-dioxaspiro[4.5]decane
ybenzaldehyde in the presence of a catalytic amount of acetic
acid at room temperature (rt). The resultant aldehyde was
protected with 1,3-propanediol, affording the acetal 5, which
underwent methylation followed by deprotection to give 6 in
68% yield. Iodination of 6 gave the precursor 7 of the cross-
coupling in 80% yield. The reaction of 7 with (trimethylsilyl)-
acetylene was carried out by a Pd/Cu-catalyzed Sonogashira
cross-coupling. After desilylation of the initial aryl-
(trimethylsilyl)acetylene derivative, the symmetrical diary-
lalkyne 8 was prepared in a second Sonogashira coupling
with 7. The dibromide derivative 9 could be generated in a
reduction and bromination sequence from 8. Oxidation of 9 in
the presence of RuCl3 and KIO4 furnished diketone 10 in 55%
yield. With the key precursor 10 in hand, we were able to
induce cyclization with KOH to get the desired ketal quaternary
carbon center, and ( )-1a was obtained with a total yield of
7%. The identical 1H and 13C NMR data of the natural derivate
1a and the racemic synthetic derivative ( )-1a further
confirmed the planar structure of compound 1.
The enantiomers of the synthetic racemic ( )-1a were
separated by HPLC using a chiral stationary phase (Chiralpak
ADH) (Figure 2). The enantiomer of 1a that eluted second
showed the same negative sign for the specific rotation as the
natural derivative (−)-1a. For the determination of the absolute
configuration, the solution ECD spectra of the two enantiomers
were compared with the computed TDDFT-ECD spectrum of
the randomly selected conformer (8S)-1a (Figure 3), which
showed a similar ECD curve to the first-eluting (+)-1a with
positive Cotton effects (CE) at 358 and 267 nm and negative
ones at 312, 229, and 202 nm.11 The MMFF conformational
search of the arbitrarily selected (8S)-1a followed by DFT
reoptimization at the B3LYP/6-31G(d) level afforded eight
conformers above 2% population (Figure S33, Supporting
Information). In all of the conformers, the isochroman ring
adopted conformations with an axial C-8−O (isobenzofuran)
bond and P-helicity [positive ωC‑7′,C‑8′,C‑8,O(isochroman) dihedral
Figure 1. Key HMBC correlations of 1 and 1a.
subunit was deduced by HMBC correlations from H-1 (δH
5.05, 4.93) to C-2 (δC 131.0), C-7 (δC 115.2), C-8, and C-8′,
from H-1′ (δH 5.00, 4.81) to C-2′ (δC 131.4), C-7′ (δC 106.2),
and C-8, and from OH-6′ (δH 11.84) to C-6′ (δC 150.0), C-7′,
and C-8′, together with the chemical shifts of C-1 (δC 72.6), C-
8 (δC 107.5), and C-1′ (δC 60.4). The two methyls were located
at C-3 and C-3′ according to the HMBC correlations from H3-
9 (δH 1.98) to C-2, C-3 (δC 107.6), and C-4 (δC 146.3) and
from H3-9′ (δH 1.95) to C-2′, C-3′ (δC 111.0), and C-4′ (δC
152.5). According to the molecular formula, five hydroxy
groups remained. Finally, the structure of 1 was established by
assigning the hydroxy groups to C-4, C-5 (δC 132.6), C-6 (δC
139.7), C-4′, and C-5′ (δC 130.2) according to the chemical
shifts9,10 and HMBC correlations of the methoxy groups to the
corresponding carbons in the permethylated product 1a
(Figure 1).
The appearance of several key 4J HMBC correlations (Figure
1) led to the ambiguity in the structure elucidation, and
compound 1 was quite unstable. In order to confirm the planar
structure and determine the absolute configuration of 1
unambiguously, the total synthesis of the permethylated
racemic derivate ( )-1a was carried out in 11 steps starting
from 3,4,5-trimethoxybenzaldehyde (4) (Scheme 1). The
synthesis started with the bromination of the 3,4,5-trimethox-
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2,3-dihydroquinolin-4(1H)-one chromophore in the recently
isolated brocaeloid A.16 The sign of the high-wavelength n−π*
CE of these chromophores was correlated with the helicity of
their heterocyclic ring by helicity rules, according to which P-
helicity of the heterocyclic ring adopting an envelope or half-
chair conformation is manifested in a positive n−π* CE above
300 nm.17 Because the isochroman-4-one chromophore is an
analogue of the above chromophores, a similar helicity rule is
expected between the sign of the n−π* CE and the helicity of
the heterocyclic ring of the isochroman-4-one moiety. Our
conformational analysis revealed that the isochroman-4-one
ring of (8S)-1a has P-helicity, which should result in a positive
n−π* CE in accordance with the observed positive CE for the
highest wavelength ECD band of (+)-1a. TDDFT-ECD spectra
were calculated for low-energy conformers above 2%
population with various functionals (B3LYP, BH&HLYP,
PBE0) and the TZVP basis set to confirm the above
configurational assignment by calculations. The computed
ECD spectra of all of the conformers showed a positive CE for
the lowest energy ECD transition (358 nm), which could be
assigned to the n−π* transition (see Khon−Sham orbitals in
Figure S34, Supporting Information). The 312 nm band was
assigned to the π−π* transition of the aromatic rings. In
contrast to the lowest energy ECD transition, the conformers
with half-chair and envelope conformations of the isochroman-
4-one ring had markedly different ECD curves in the higher
energy region. Except for a negative computed CE at 270 nm,
the Boltzmann-weighted ECD spectra of (8S)-1a reproduced
well the experimental ECD spectrum of (+)-1a, confirming its
absolute configuration as 8S. Thus, the absolute configuration
of the natural 1 and 1a, which had opposite configuration of
that of (+)-1a, was unambiguously determined as 8R. The
TDDFT-ECD calculations also confirmed that the same
correlation exists between the helicity of the isochroman-4-
one chromophore and the sign of the n−π* ECD transition as
those established for the chromanone and 2,3-dihydroquinolin-
4(1H)-one chromophores; a positive n−π* CE derives from P-
helicity.
Figure 2. HPLC profile for the chiral-phase separation of ( )-1a.
Figure 3. Experimental ECD spectrum (black) of (+)-1a (first-eluting
enantiomer) compared with the PBE0/TZVP-calculated ECD
spectrum of (8S)-1a (purple). Bars represent computed rotational
strengths of the lowest energy conformer.
angle]. The conformers differed in the conformation of the
fused pyran ring and orientations of the methoxy groups. The
fused pyran ring of the lowest energy conformers (conformers
A and B with 35.2% and 19.8% populations, Figure S33) had a
half-chair conformation, which is also represented by con-
formers E, F, and G with a total population of 63.0% (structure
I in Figure 4). Conformers C, D, and H with a total population
The synthesized ( )-1a and its separated enantiomers
showed no cytotoxicity against K562 and HL-60 cells (IC50
>
50 μM). The antiviral activities of synthesized (+)-1a and
(−)-1a were also evaluated against influenza A virus (H1N1) in
the cytopathic effect (CPE) inhibition assay.18 Only compound
(−)-1a exhibited activity with an IC50 = 149 μM against the
influenza A H1N1 virus (ribavirin was used as positive control,
IC50 101 μM).
Eleganketal A (1) is the first naturally occurring polyketide
having a spiro[isobenzofuran-1,3′-isochroman] ring system,
which has been reported in only a few synthetic molecules.19
Structurally, crombenine, which was isolated from the plant
Acacia crombei C. T. White, is the most similar natural product
to eleganketal A.20 Crombenine differs from compound 1 by
possessing a spiro[benzofuran-2,3′-isochroman] ring system,
and the absolute configuration was undetermined. The total
synthesis of crombenine has been attempted, but only a bicyclic
[4.3.1] derivative was formed.21 The highly substituted
aromatic rings, lack of proton signals, and low stability of 1
exerted difficulties in determining the structure by spectro-
scopic methods, especially the absolute configuration. In this
work, we determined the structure of 1 by a combination of
spectroscopic analysis, chemical synthesis of the permethylated
analogue, chiral-phase HPLC separation of a synthetic
analogue, and TDDFT ECD calculations.
Figure 4. Equilibrating conformers of (S)-1a with half-chair and
envelope conformations of the isochroman-4-one chromophore.
of 26.7% had an envelope conformation of the pyran ring
(structure II in Figure 4), in which C-8 moved into the plane of
the fused benzene ring. The isochroman-4-one chromophore of
1a belongs to the group of cyclic aryl ketones such as the
chroman-4-one chromophore in flavanones,12 2-hydroxyflava-
nones,12 2-alkylchromanones,13 and isoflavanones14,15 or the
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EXPERIMENTAL SECTION
Table 1. 1H (400 MHz) and 13C (100 MHz) NMR Data for 1
in DMSO-d6
■
General Experimental Procedures. Specific rotations were
obtained on a JASCO P-1020 digital polarimeter. UV spectra were
recorded on a Beckman DU 640 spectrophotometer. CD spectra were
measured on a JASCO J-715 spectropolarimeter. IR spectra were taken
position
δC (type)
δH mult (J in Hz)
1
72.6, CH2
131.0, C
107.6, C
146.3, C
132.6, C
139.7, C
115.2, C
107.5, C
11.9, CH3
60.4, CH2
131.4, C
111.0, C
152.5, C
130.2, C
150.0, C
106.2, C
192.2, C
9.7, CH3
5.05, d (12.0); 4.93, d (12.0)
1
2
on a Nicolet Nexus 470 spectrophotometer in KBr discs. H NMR,
13C NMR, and DEPT spectra and 2D NMR were recorded on JEOL
JNM-ECP 600, Agilent 500 MHz DD2, and Bruker AM-400
spectrometers using TMS as internal standard. ESIMS utilized a
Waters Q-TOF Ultima Global mass spectrometer and a Thermo
Scientific LTQ Orbitrap XL mass spectrometer. Semipreparative
HPLC was performed using an ODS column [HPLC (YMC-Pack
ODS-A, 10 × 250 mm, 5 μm, 4 mL/min)]. Racemic mixtures were
resolved on a Chiralpak ADH column (5 μm, 4.6 × 250 mm, hexane/
2-propanol eluent, 1 mL/min). TLC and column chromatography
(CC) were performed on plates precoated with silica gel GF254 (10−
40 μm) and over silica gel (200−300 mesh, Qingdao Marine Chemical
Factory) and Sephadex LH-20 (Amersham Biosciences), respectively.
Vacuum-liquid chromatography (VLC) was carried out over silica gel
H (Qingdao Marine Chemical Factory). All starting materials,
reagents, and solvents were purchased from commercial sources and
used as such without further purification: AcOH, MeCN, DCM,
MeOH, sodium borohydride (NaBH4), sodium bicarbonate
(Na2CO3), sodium chloride (NaCl), sodium sulfate (Na2SO4), sodium
thiosulfate (Na2S2O3), tetrachloromethane (CCl4), tetrahydrofuran
(THF), toluene (PhCH3), triethylamine (Et3N), petroleum ether
(PE). Unless otherwise stated, all reactions were carried out using
anhydrous solvents. The seawater was collected from Jiaozhou Bay,
Qingdao, China.
3
4
5
6
7
8
9
1.98, s
1′
2′
3′
4′
5′
6′
7′
8′
9′
6′-OH
5.00, d (15.6); 4.81, d (15.6)
1.95, s
11.84, s
mol, 1.1 equiv) dissolved in 50 mL of CH2Cl2, the mixture was stirred
at rt for 30 min. The reaction solution was washed with a saturated
aqueous Na2S2O3 solution. The organic layer was dried over
anhydrous Na2SO4 and purified by CC (EtOAc/PE = 1:20) to afford
2-bromo-3,4,5-trimethoxybenzaldehyde as white crystals with a yield of
Fermentation, Extraction, and Isolation of Compound 1. The
fungal strain4 S. elegans KLA03 was cultured with shaking in 500 mL
Erlenmeyer flasks each containing 150 mL of liquid medium (mannitol
2.0%, maltose 2.0%, glucose 1.0%, NH4Cl 1.0%, KH2PO4 0.05%,
MgSO4·7H2O 0.03%, yeast extract 0.3%, corn steep liquor 0.1%, and
naturally collected seawater after adjusting pH to 6.5) at 28 °C. After 8
days of cultivation, 20 L of whole broth was extracted with EtOAc (10
L × 3) and concentrated under reduced pressure to give a dark brown
gum. The EtOAC extract (20 g) was subjected to vacuum liquid
chromatography over a silica gel (200−300 mesh) column using
stepwise gradient elution with mixtures of petroleum ether/CHCl3/
MeOH to give five fractions. Fraction 3 was rechromatographed on a
silica gel column, eluting with a petroleum ether/acetone gradient, to
provide 10 subfractions (fractions 3.1−3.10). Fraction 3.10 was further
purified on Sephadex LH-20 (MeOH) to give compound 1 (8.0 mg).
Eleganketal A (1): yellow, amorphous powder; [α]20D −90 (c 0.10,
MeOH); 1H and 13C NMR data, see Table 1; HRESIMS m/z
375.0710 [M − H]− (calcd for C18H15O9, 375.0716).
1
89%: mp 65−66 °C; H NMR δH (600 MHz, CDCl3) 10.28 (1H, s),
7.29 (1H, s), 3.97 (3H, s), 3.90 (3H, s), 3.89 (3H, s); 13C NMR δC
(150 MHz, CDCl3) 191.2, 153.1, 150.9, 148.8, 128.9, 115.7, 107.5,
61.4, 61.3, 56.4; ESIMS (m/z) 275/277 [M + H]+.
Preparation of 2-(2-Bromo-3,4,5-trimethoxyphenyl)-1,3-di-
oxane (5). The 2-bromo-3,4,5-trimethoxylbenzaldehyde (5.48 g,
20.00 mmol, 1.0 equiv), 1,3-propanediol (4.57 g, 60.00 mmol, 3.0
equiv), and a catalytic amount of p-toluene sulfonic acid were
dissolved in 200 mL of toluene in a 250 mL round-bottom flask
equipped with a Dean−Stark apparatus. The solution was heating to
reflux for 24 h. After cooling to rt, the mixture was washed with
saturated Na2CO3 solution three times, and the organic layer was dried
over anhydrous Na2SO4. Toluene was removed to give compound 5 as
1
a colorless oil (6.35 g; 95% yield): H NMR NMR δH (600 MHz,
CDCl3) 7.08 (1H, s), 5.75 (1H, s), 4.24−4.27 (2H, dd), 4.00−4.04
(2H, td), 3.89 (3H, s), 3.87 (3H, s), 3.85 (3H, s), 2.23 (1H, m), 1.50
(1H, m); 13C NMR δC (150 MHz, CDCl3) 153.1, 150.7, 143.7, 133.1,
109.1, 106.6, 100.9, 67.7, 61.2, 56.2, 25.7; MSESI (m/z) 333/335 [M
+ H]+.
Preparation of the Methylated Derivative 1a from 1.
Eleganketal A (1, 4.0 mg) was stirred with iodomethane (15 μL)
and potassium carbonate (10 mg) in refluxing anhydrous acetone
overnight. The reaction mixture was filtered and washed with acetone.
The acetone solution was evaporated under vacuum and further
Preparation of 3,4,5-Trimethoxy-2-methylbenzaldehyde (6).
Compound 5 (3.33 g, 10.00 mmol, 1.0 equiv) was dissolved with 100
mL of freshly redistilled anhydrous THF in a two-necked round-
bottom flask under an atmosphere of argon. After cooling to −78 °C,
1.6 N n-butyllithium (9.38 mL, 15.00 mmol, 1.5 equiv) was added
through a syringe. After stirring for 1 h, methyl iodide (935.0 μL, 15.00
mmol, 1.5 equiv) was added dropwise. The reaction mixture was
slowly warmed to rt and stirred overnight. The reaction was quenched
with saturated aqueous citric acid solution, washed with EtOAc three
times, and dried over anhydrous Na2SO4. Compound 6 was purified
by CC (EtOAc/PE = 1:8) and was obtained as a yellow oil (1.42 g;
68% yield): 1H NMR δH (600 MHz, CDCl3) 10.24 (1H, s), 7.20 (1H,
s), 3.95 (3H, s), 3.89 (3H, s), 3.83 (3H, s), 2.51 (3H, s); 13C NMR δC
(150 MHz, CDCl3) 190.9, 152.2, 151.7, 147.9, 129.5, 128.8, 108.3,
61.0, 60.9, 56.1, 10.2; MSESI (m/z) 211 [M + H]+.
purified on HPLC (tR = 9.8 min, 75% MeOH/H2O) to afford 1a (1
mg): pale yellow oil; [α]20 −50 (c 0.10, MeOH); H NMR δH (600
1
D
MHz, CDCl3) 5.21 (1H, d, J = 15.8 Hz; H-1), 5.20 (1H, d, J = 12.4
Hz; H-1′), 5.09 (1H, d, J = 12.4 Hz; H-1′), 4.89 (1H, d, J = 15.8 Hz;
H-1), 3.95 (3H, s; 4′-OCHH3), 3.91 (6H, s; 4-OCHH3 and 6′-
OCHH3), 3.88 (3H, s; 5-OCHH3), 3.87 (3H, s; 6-OCHH3), 3.85 (3H,
s; 5′-OCHH3), 2.09 (3H, s; H-9′); 13C NMR δC (150 MHz, CDCl3)
186.0 (C-8′), 156.8 (C-4′), 154.4 (C-4′), 154.3 (C-4), 146.8 (C-6),
146.1 (C-5), 145.9 (C-6′), 136.7 (C-3′), 135.2 (C-3), 125.1 (C-7),
122.0 (C-2′), 119.7 (C-2), 118.8 (C-7′), 108.9 (C-8), 73.4 (C-1), 61.9
(6′-OCHH3), 61.7 (OCH3-4), 61.4 (6-OCHH3), 61.0 (5-OCHH3 and
4′-OCHH3), 60.9 (C-1′ and 5′-OCHH3), 12.4 (C-9′), 10.7 (C-9);
LRESIMS [M + H]+ m/z 461.
Preparation of 2-Iodo-3,4,5-trimethoxy-6-methyl-
benzaldehyde (7). Compound 6 (3.00 g, 14.28 mmol, 1.0 equiv)
was dissolved in 100 mL of CHCl3 followed by the addition of 3.63 g
of CF3COOAg (16.42 mmol, 1.15 equiv). To this suspension was
added dropwise over 30 min 3.63 g of iodine (14.28 mmol, 1.0 equiv)
Total Synthesis of ( )-1a. Preparation of 2-Bromo-3,4,5-
trimethoxybenzaldehyde. 3,4,5-Trimethoxybenzaldehyde (19.60 g,
0.10 mol, 1.0 equiv) was dissolved in 200 mL of CH2Cl2 followed by
adding 500 μL of AcOH. After the addition of bromide (5.64 mL, 0.11
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dissolved in 150 mL of CHCl3. The silver iodide formed was filtered
away, and the organic layer was washed by a saturated solution of
Na2S2O3 and brine. After drying over anhydrous Na2SO4, the crude
product was purified by CC (EtOAc/PE = 1:10), and 7 was obtained
(8:8:1, v/v, 20 mL) was added KIO4 (0.31 g, 1.35 mmol, 4.1 equiv)
followed by RuCl3 (1.5 mg, 7.25 μmol, 0.022 equiv). After vigorously
stirring for 1 h at rt, the reaction mixture was diluted with H2O and
extracted with CH2Cl2 three times. The organic layer was collected
and dried by anhydrous Na2SO4. The crude compound 10 was
purified by CC (EtOAc/PE = 1:10) and obtained as a white snowflake
1
as yellow crystals with a yield of 80% (3.84 g): H NMR δH (600
MHz, CDCl3) 10.20 (1H, s), 3.98 (3H, s), 3.87 (3H, s), 3.81 (3H, s),
1
2.42 (3H, s); MSESI (m/z) 337 [M + H]+.
solid with a yield of 55%: mp 156−157 °C; H NMR δH (600 MHz,
CDCl3) 4.94 (4H, s), 3.91 (6H, s), 3.83 (6H, s), 3.69 (6H, s), 2.32
(6H, s); 13C NMR δC (150 MHz, CDCl3) 190.9, 156.8, 154.4, 145.7,
134.6, 128.7, 121.6, 60.9, 60.6, 58.5, 28.4, 11.0; MSESI (m/z) 602/
604/606 [M + H]+.
Preparation of 2-Ethynyl-6-methyl-3,4,5-trimethoxy-
benzaldehyde. To an oven-dried 50 mL round-bottomed flask
were added compound 7 (0.60 g, 1.80 mmol) and anhydrous TEA
(18.00 mL). After 20 min of purging with nitrogen, CuI (34.9 mg, 0.18
mmol) and Pd(PPh3)4 (72.5 mg, 0.063 mmol) were added.
Trimethylsilylacetylene (0.50 mL, 3.60 mmol) was added very slowly,
and the mixture was stirred at 60 °C for 20 h. After cooling and
filtration, a saturated NaCl solution was added, and the mixture was
extracted with EtOAc three times. The organic layer was dried by
anhydrous Na2SO4, and EtOAc was removed under vacuum. The
3,4,5-trimethoxy-2-methyl-6-((trimethylsilyl)ethynyl)benzaldehyde
was dissolved in MeOH (20 mL), and oven-dried K2CO3 (0.26 g, 1.90
mmol) was added in one portion. The flask was placed under a
nitrogen atmosphere, and the mixture was stirred for 15 min. Then
H2O was added to the solution, which was extracted with EtOAc three
times. After concentration in vacuo, 2-ethynyl-6-methyl-3,4,5-
trimethoxybenzaldehyde was purified by CC (EtOAc/PE = 1:10)
and obtained as a white snowflake solid (0.24 g) with a yield of 57%:
Preparation of the Racemic Methylated Eleganketal ( )-1a.
Compound 10 (0.10 g, 0.17 mmol) was dissolved in a mixture of 4 mL
of THF and 1 mL of H2O. The mixture was stirred under reflux for 24
h after addition of KOH (93.0 mg, 1.66 mmol, 10 equiv). The solution
was diluted with 20 mL of H2O and extracted with EtOAc three times.
The organic layer was collected, washed with brine, and dried over
anhydrous Na2SO4. The crude product was purified by CC (EtOAc/
PE = 1:8) to give the pure target compound (44.5 mg) as a colorless
oil with a yield of 57%: 1H NMR δH (600 MHz, CDCl3) 5.193 (1H, d,
J = 16.0 Hz), 5.189 (1H, d, J = 12.1 Hz), 5.07 (1H, d, J = 12.1 Hz),
4.88 (1H, d, J = 15.4 Hz), 3.93 (3H, s), 3.90 (6H, s), 3.87 (3H, s), 3.86
(3H, s), 3.84 (3H, s), 2.08 (3H, s); 13C NMR δC (150 MHz, CDCl3)
186.0, 156.8, 154.4, 154.2, 146.8, 146.1, 145.9, 136.7, 135.2, 125.1,
122.0, 119.7, 118.8, 108.9, 73.4, 61.9, 61.7, 61.3, 61.0, 60.9, 12.4, 10.7;
HRESIMS (m/z) 461.1808 [M + H]+ (calcd for C24H29O9, 461.1806).
Chiral-Phase Separation of the ( )-1a. The racemic ( )-1a
(27.0 mg) was injected onto a chiral-phase (CHIRALPAK ADH)
HPLC column. Eluting with hexane/2-propanol (70/30), the two
enantiomers (+)-1a (9.1 mg) and (−)-1a (14.4 mg) were obtained,
with rotation values (c 0.10, MeOH) of +40 and −40, respectively.
ECD (2.23 × 10−4 M, MeCN) of (−)-1a: λmax (Δε) 358 (2.25), 312
(−1.75), 267 (0.88), 229 sh (−12.29), 202 (−21.62).
Computational Section. Mixed torsional/low mode conforma-
tional searches were carried out by means of the Macromodel
9.9.22322 software using the Merck molecular force field (MMFF)
with an implicit solvent model for chloroform applying a 21 kJ/mol
energy window. The resultant 56 conformers were reoptimized at the
B3LYP/6-31G(d) level in vacuo. DFT optimizations and TDDFT
calculations were performed with Gaussian 0923 using various
functionals (B3LYP, BH&HLYP, PBE0) and the TZVP basis set.
ECD spectra were generated as the sum of Gaussians with 1800 cm−1
half-height width (corresponding to ∼7 at 200 nm), using dipole-
velocity-computed rotational strengths.24 Boltzmann distributions
were estimated from the ZPVE-corrected B3LYP/6-31G(d) energies.
The MOLEKEL25 software package was used for visualization of the
results.
1
mp 64−65 °C; H NMR δH (600 MHz, CDCl3) 10.61 (1H, s), 3.98
(3H, s), 3.94 (3H, s), 3.82 (3H, s), 3.63 (1H, s), 2.46 (3H, s); 13C
NMR δC (150 MHz, CDCl3) 193.1, 154.0, 153.1, 150.7, 131.2, 131.1,
118.0, 88.3, 75.6, 61.6, 61.1, 61.0, 12.4; MSESI (m/z) 235 [M + H]+.
Preparation of 6,6′-(Ethyne-1,2-diyl)bis(3,4,5-trimethoxy-2-
methyl benzaldehyde) (8). Compound 7 (148.0 mg, 0.44 mmol)
was added to the TEA solution of Pd(PPh3)4 (25.0 mg) and CuI (5.0
mg) at rt, and the mixture was stirred for 15 min under the protection
of nitrogen. The TEA (5.0 mL) solution of 2-ethynyl-6-methyl-3,4,5-
trimethoxybenzaldehyde (93.6 mg, 0.4 mmol) was added by syringe
over 2 min. The mixture was heated to 60 °C and stirred for 10 h.
During the reaction process, a solid precipitated. After cooling to rt,
the solid was filtered and washed with PE and H2O three times,
respectively, which led to recovery of compound 7 as a white solid
1
(158.9 mg; yield 82%): mp 194−195 °C; H NMR δH (600 MHz,
CDCl3) 10.79 (2H, s), 4.01 (6H, s), 3.99 (6H, s), 3.85 (6H, s), 2.51
(6H, s); 13C NMR δC (150 MHz, CDCl3) 193.3, 153.4, 153.2, 150.8,
131.2, 130.5, 119.0, 92.3, 61.6, 61.2, 61.1, 12.4; MSESI (m/z) 443 [M
+ H]+.
Preparation of 6,6′-(Ethyne-1,2-diyl)bis(3,4,5-trimethoxy-2-
methylbenzyl alcohol). NaBH4 (68.8 mg, 1.81 mmol, 4 equiv) was
added to the solution (5.0 mL MeOH) of compound 8 (0.2 g, 0.45
mmol), and the mixture was stirred for 8 h. After removal of MeOH,
10.0 mL of H2O was added and filtration provided a white solid of
6,6′-(ethyne-1,2-diyl)bis(3,4,5-trimethoxy-2-methylbenzyl alcohol)
Anti-influenza A (H1N1) Assay. The antiviral activity against
H1N1 was evaluated by the CPE inhibition assay. Confluent MDCK
cell monolayers were incubated with influenza virus (A/Puerto Rico/
8/34 (H1N1), PR/8) at 37 °C for 1 h. After removing the virus
dilution, cells were maintained in infecting media (RPMI 1640, 4 μg/
mL of trypsin) containing different test compounds at 37 °C. After 48
h incubation at 37 °C, the cells were fixed with 100 μL of 4%
formaldehyde for 20 min at rt. After removal of the formaldehyde, the
cells were stained with 0.1% crystal violet for 30 min. The plates were
washed and dried, and the intensity of crystal violet staining for each
well was measured in a microplate reader (Bio-Rad) at 570 nm. The
IC50 was calculated as the compound concentration required to inhibit
influenza virus yield at 48 h postinfection by 50%. Ribavirin was used
as the positive control in the CPE inhibition assay.
1
(792.0 mg; yield 98%): mp 138−139 °C; H NMR δH (600 MHz,
CDCl3) 4.88 (4H, s), 4.01 (6H, s), 3.91 (6H, s), 3.85 (6H, s), 2.31
(6H, s); 13C NMR δC (150 MHz, CDCl3) 153.0, 152.9, 145.8, 136.80,
127.1, 113.8, 91.4, 61.5, 61.1, 60.9, 60.7, 11.7; MSESI (m/z) 447 [M +
H]+.
Preparation of 6,6′-(Ethyne-1,2-diyl)bis(3,4,5-trimethoxy-2-
methylbenzyl bromide) (9). The 6,6′-(ethyne-1,2-diyl)bis(3,4,5-
trimethoxy-2-methylbenzyl alcohol) (0.98 g, 0.22 mmol) was dissolved
in 10 mL of anhydrous THF under a nitrogen atmosphere and was
cooled in an ice bath. After the addition of phosphorus tribromide
(0.90 mmol, 4.0 equiv), the solution was stirred at rt for 1 h. Removing
THF under vacuum gave the compound 9 as a white solid (124.5 mg)
with a yield of 99%: mp 167−168 °C; 1H NMR δH (600 MHz,
CDCl3) 4.91 (4H, s), 4.05 (6H, s), 3.93 (6H, s), 3.87 (6H, s), 2.31
(6H, s); 13C NMR δC (150 MHz, CDCl3) 153.2, 153.0, 146.8, 133.2,
127.8, 114.6, 91.4, 61.9, 61.0, 60.9, 31.0, 11.7; MSESI (m/z) 570/572/
574 [M + H]+.
ASSOCIATED CONTENT
■
S
* Supporting Information
1D and 2D NMR and MS spectra of 1 and 1a; NMR spectra of
the key intermediates for the total synthesis of ( )-1a. These
materials are available free of charge via the Internet at http://
pubs.acs.org.
Preparation of 1,2-Bis(2-(bromomethyl)-4,5,6-trimethoxy-3-
methylphenyl)ethan-1,2-dione (10). To a solution of compound 9
(0.19 g, 0.33 mmol, 1.0 equiv) in a mixture of CCl4, MeCN, and H2O
1722
dx.doi.org/10.1021/np500458a | J. Nat. Prod. 2014, 77, 1718−1723

Journal of Natural Products
Article
(16) Zhang, P.; Meng, L.-H.; Man
Li, X.; Li, C.-S.; Wang, B.-G. Eur. J. Org. Chem. 2014, in press, DOI:
10.1002/ejoc.201400067.
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(17) Kurtan, T.; Antus, S.; Pescitelli, G. In Comprehensive Chiroptical
Spectroscopy: Applications in Stereochemical Analysis of Synthetic
Compounds, Natural Products, and Biomolecules; Berova, N.;
Polavarapu, P. L.; Nakanishi, K.; Woody, R. W., Eds.; John Wiley:
Hoboken, NJ, 2012; Vol. 2, Chapter 3, pp 73−114.
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di, A.; Kurtan
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, T.; Li, X.-M.; Liu, Y.;
AUTHOR INFORMATION
■
Corresponding Author
*Tel: 0086-532-82031619. Fax: 0086-532-82033054. E-mail:
dehaili@ouc.edu.cn.
Notes
The authors declare no competing financial interest.
(18) (a) Peng, J.; Lin, T.; Wang, W.; Xin, Z.; Zhu, T.; Gu, Q.; Li, D. J.
Nat. Prod. 2013, 76, 1133−1140. (b) Gao, H.; Guo, W.; Wang, Q.;
Zhang, L.; Zhu, M.; Zhu, T.; Gu, Q.; Wang, W.; Li, D. Bioorg. Med.
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ACKNOWLEDGMENTS
■
This work was financially supported by the National Natural
Science Fundation of China (Nos. 41176120 and 21372208),
the NSFC-Shandong Joint Fund (No. U1406402), the National
High Technology Research and Development Program of
China (No. 2013AA092901), the Program for New Century
Excellent Talents in University (No. NCET-12-0499), the
Public Projects of State Oceanic Administration (No.
2010418022-3), and the Program for Changjiang Scholars
and Innovative Research Team in University (No. IRT0944).
T.K. and A.M. thank the Hungarian National Research
Foundation (OTKA K105871) for financial support and the
National Information Infrastructure Development Institute
(NIIFI 10038) for CPU time.
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