The methanol extract of the dried mycelium was subjected to a combination of column chromatography on silica gel, Sephadex LH-20 and C18 reversed phase silica gel.
was isolated as a red amorphous powder. HR-EIMS at m/z
= 586.1471 [M]+
indicated the molecular formula C32
(calcd. for C32
, 586.1470). Compound 1
exhibited strong optical rotation
= 1.0, MeOH) which indicated the possibility of an asymmetric centre and/or axial chirality (). The IR spectrum (KBr) exhibited a weak shoulder at 1652 cm−1
and an intense band at 1638 cm−1
for carbonyl groups. The UV spectrum displayed bands at 224, 280 and 437 nm, suggesting a quinonoid chromophore. The 1
H NMR spectrum () showed a pair of chelated hydroxyl resonances (δH
= 13.61 and 13.15 ppm), four aromatic protons (δH
= 7.67, 7.55, 6.92 and 6.88 ppm), two methoxyl protons (δH
= 3.68 and 3.66 ppm), two singlet methyls (δH
= 2.18 and 1.07 ppm), two methylene protons (δH
= 2.53 and 2.72 ppm, δH
= 2.20 and 2.34 ppm), and oxygenated methine (δH
= 3.51 ppm). The 13
C NMR spectrum displayed four carbonyl signals (δC
= 183.6, 187.8, 181.1 and 186.7 ppm), twenty signs of aromatic carbons, one quaternary carbon (δC
= 69.0 ppm), one methine (δC
= 70.1 ppm) and two methylenes (δC
= 29.1 and 36.1 ppm). These data implied that compound 1
possessed a bianthranquinone scaffold, including an anthraquinone unit and a tetrahydroanthraquinone unit () [5
]. The unsubstituted carbons for two aromatic rings of the anthraquinone unit were located at C-8′ (δC
= 130.3 ppm; δH
= 7.67 ppm, d, J
= 0.8 Hz), C-5′ (δC
= 110.5 ppm; δH
= 7.55 ppm, d, J
= 0.8 Hz) and C-3′ (δC
= 103.8 ppm; δH
= 6.921 ppm, s) by the HMBC correlations (). In the tetrahydroanthraquinone unit, one aromatic proton at H-3 (δH
= 6.88 ppm, s) and the protons in the alicyclic ring, including one oxygenated methine H-5 (δH
= 3.51 ppm, ddd, J
= 5.4, 5.5, 12.5 Hz) and two methylene protons H-6 (δH
= 2.53 and 2.72 ppm) and H-7 (δH
= 2.20 and 2.34 ppm) were observed.
CD Spectra of 1. Recorded in MeOH at amibient temperature.
NMR spectroscopic data (DMSO-d6) of 1a,b.
Key HMBC, NOE and 1H-1H COSY correlations of 1–3.
A contiguous sequence of coupled signals from H-5 to H-7 in the 1H-1H COSY spectrum combined with the HMBC correlations from H-5 to C-6, C-7 and C-8a, from H-6 to C-5, C-8, and C-10a, and from H-7 to C-5, C-8, C-8a, C-9 and C-11, established the substructure of the cyclohexene ring ().
HMBC correlations from each of 4-OH (δH = 13.15 ppm) and 4′-OH (δH = 13.63 ppm) to C-4, C-3, C-4a and to C-4′, C-3′, C-4′a, respectively, indicated their location at C-4 and C-4′ of the bianthraquinone scaffold.
The presence of a bond connecting C-2 and C-2′ was suggested by the absence of two sets of ortho-coupled doublets and the presence of two singlets H-3 (δH = 6.88 ppm, s) and H-3′ (δH = 6.92 ppm, s). Moreover, HMBC correlations of H-3 with C-1, C-2, C-2′, C-4, C-4a and C-10, and of H-3′ with C-1′, C-2, C-2′, C-4′, C-4′a and C-10′, respectively () provided evidence for C-2–C-2′ linkage of 1. The HMBC spectrum showed correlations of the methoxyl proton (δH = 3.66 ppm, s, 3H, H-12) with C-1 and C-3. Likewise, correlations were observed from methoxyl proton (δH = 3.69 ppm, s, 3H, H-12′) to C-1′. These allowed us to assign two methoxyl groups to C-1 and C-1′.
The relative configuration of the chiral centers of C-5 and C-8 were deduced by 2D 1H-1H NOESY experiments () and the analysis of 1H-1H coupling constants (). A NOESY correlation between CH3-11 (δH = 1.07 ppm, s) and H-5 suggested that they were on the same side of the cyclohexene ring. The axial position of H-5 was confirmed by coupling constant JH-5,H-6a = 12.5 Hz, indicating CH3-11 to also have an axial orientation. Therefore, compound 1 was determined as (5S*,8R*)-4,4′,5,7′,8-pentahydroxy-1,1′-dimethoxy-6′,8-dimethyl-5,6,7,8-tetrahydro-[2,2′-bianthracene]-9,9′,10,10′-tetraone. We propose the trivial name alterporriol K.
were obtained as a mixture after separating with Sephadex LH-20 chromatography. Upon HPLC analysis, the mixture exhibited two partially overlapped peaks (area ratio ca.
4:1). Then compounds 2
were isolated with re-separation by preparative HPLC, respectively. Compound 2
was a red amorphous powder,
= 1.0, MeOH). The HR-ESI-TOF-MS exhibited a peak at m/z
= 601.1340 [M – H]−
indicating a molecular formula of C32
(calcd. for C32
, 601.1346). Comparison of the 1
H and 13
C NMR spectral data of 2
() with that of 1
showed a close structural relationship between both compounds, except for the presence of an additional oxymethine group proton (δH
= 4.03, d, J
= 6.7 Hz, H-8) and an additional hydroxyl group signal (δH
= 2.17 ppm, s, 6-OH) and the absence of the methylene signals corresponding to H-7 in the 1
H NMR spectrum of 2
. The substructure of the cyclohexene ring was established by HMBC correlations from H-8 to C-6, C-7, C-8a, C-9, C-10a and C-11 and 1
H COSY correlations between H-5 and H-6 (). NOE difference and analysis of 1
H coupling constants () enabled the relative of configuration of 2
to be deduced. In the NOE experiment, when the methyl signal CH3
= 1.16, s) was irradiated, no enhancement of signals H-6 and H-8 was observed. Meanwhile, irradiation of H-8 caused an enhancement of H-6. These data suggested that CH3
-11 with H-6 and H-8 was trans
-configuration in the cyclohexene ring (). A large coupling constant for H-5a/H-6 (JH-5a,H-6
= 10.0 Hz) indicated an axial location for H-6, likewise suggesting an axial location for H-8. Thus, the relative configuration at C-6, C-7, and C-8 was 6S
* and 8R
*. Compound 2
was finally defined as (6S
*)-4,4′,6,7,7′,8-hexahydroxy-1,1′-dimethoxy-6′,7-dimethyl-5,6,7,8-tetrahydro-[2,2′-bianthracene]-9,9′,10,10′-tetraone, named as alterporriol L.
NMR spectroscopic data (DMSO-d6) of 2 and 3a,b.
, obtained as a red amorphous power,
= 1.0, MeOH), had the molecular formula C32
, as determined by HR-ESI-TOF-MS at m/z
= 601.1340 [M – H]−
(calcd. for C32
, 601.1346). The MS analysis revealed 3
to be an isomer of 2
. The UV and IR absorptions, 1
H and 13
C NMR data, HMBC and 1
H COSY correlations ( and ) of 3
were almost identical with those of 2
, suggesting the two compounds to have identical carbon skeletons. However, in contrast with 2 in the NOE experiment of 3, irradiation of the methyl signal CH3
= 1.16 ppm, s) resulted in obvious enhancements of the signals for both H-6 and H-8. The coupling constant for H-5a/H-6 was measured to be JH-5a,H-6
= 9.9 Hz suggesting axial locations for H-6 and, by extension, H-8. These data suggested that 2 and 3 are epimers at C-7. Therefore compound 3 was elucidated as (6S
*)-4,4′,6,7,7′,8-hexahydroxy-1,1′-dimethoxy-6′,7-dimethyl-5,6,7,8-tetrahydro-[2,2′-bianthracene]-9,9′,10,10′-tetraone, named alterporriol M.
An interesting feature of the isolated bianthquinones is their optical activity. Ab initio
calculations of CD spectra for several phenylanthraquinones with chiral axes have recently been reported [17
]. Specific optical rotation of compounds 2
and −30, respectively. CD spectra of 2
recorded in methanol showed a near quasi-mirror image pattern (). In the case of 2
, the CD spectrum consists of a small positive band at 480 nm, followed by two moderate negative bands in the 300–400 nm regions, and a stronger negative band centered at 220 nm, then a stronger positive band at 264 nm. For 3,
the sequence of bands is similar but their signs are inverted. The CD spectra of 2
reflect contributions from a chiral axis and three chiral centers. The chiral axis occurring within the chromophore is expected to dominate the observed CD spectrum [18
] ().The chiral center C-7 is expected to have a much smaller contribution to the observed CD spectrum. With these results, compounds 2
were deduced as two diastereomers and atropisomers [20
CD Spectra of 2 and 3. Recorded in MeOH at amibient temperature.
The known compounds were identified as physcion (4
), marcrospin (5
), dactylariol (6
), tetrahydroaltersolanol B (7
), alternariol (AOH) (8
) and alternariol methyl ether (AME) (9
) by spectral analyses and comparison with reported literature data, respectively [21
]. The structure of 7
was confirmed by X-ray diffraction analysis () and its crystallographic data was reported for the first time.
Compounds 1 and 2 were evaluated for their cytotoxicity against human breast cancer cell lines MDA-MB-435 and MCF-7 by MTT assay. Compound 1 had an IC50 value of 26.97 μM against MDA-MB-435 and 29.11 μM against MCF-7 cells, respectively. Compound 2 showed activities against MDA-MB-435 (IC50 = 13.11 μM) and MCF-7 (IC50 = 20.04 μM). No biological studies were performed for compound 3 due to the limited yield.