2. Results and Discussion
Alternaria sp., a microorganism from A. crassispina, was cultured at 27 °C for 6 weeks in a medium (70 L) containing 1% glucose, 1% malt extract, and 0.05% peptone in artificial seawater adjusted to pH 7.5. After incubation, the EtOAc extract of the culture filtrate was purified via bioassay-directed fractionation by using a stepwise combination of silica-gel column and Sephadex LH-20 chromatography, followed by reverse-phase HPLC, affording altercrasins A (1) (10.3 mg, 0.020%), B (2) (6.5 mg, 0.012%), C (3) (4.6 mg, 0.009%), D (4) (1.3 mg, 0.002%), and E (5) (5.3 mg, 0.010%) as pale yellow oils.
As it has been observed for previously reported
1 [
6], altercrasin B (
2) also exhibited the molecular formula C
24H
33NO
5 as established by the [M + Na]
+ peak in HRFABMS. The IR spectrum exhibited absorption bands at 3478, 1720, and 1710 cm
−1 characteristic of hydroxyl and carbonyl groups. The close inspection of the
1H and
13C NMR spectra of
2 (
Table 1 and
Table S1) by using distortionless enhancement by polarization transfer (DEPT) and
1H–
13C correlation spectroscopy (HMQC) revealed the presence of two secondary methyl groups (C-17 and C-23, respectively); a tertiary methyl (C-24) group; three sp
3-hybridized methylene groups (C-1, C-2, and C-4, respectively); six sp
3-methine groups (C-3, C-5, C-8, C-10, C-13, and C-16, respectively); two quaternary sp
3-carbon groups (C-9 and C-12, respectively); four sp
2-methine groups (C-6, C-7, C-14, and C-15, respectively); and three carbonyl groups (C-11, C-18, and C-22, respectively), including an amide carbonyl (C-22). The
1H–
1H COSY analysis of
2 led to three partial structures, including a hydroxy butylene group (H-13/H-14, H-14/H-15, H-15/H-16, H-16/16-OH, and H-16/H-17) and a hydroxyethyl group (H-19/H-20, H-20/20-OH, and H-20/H-21), as indicated by the bold-faced lines shown in
Figure 2. The key HMBC correlations shown in
Figure 2 verified the connection of these three units and the remaining functional groups, indicating that the planar structure of
2 is the same as that of
1.
The stereochemistry of
2 was deduced from NOESY experiments and the comparison of NMR and ECD spectral data with
1, where the absolute configuration of
1 was determined by the application of the modified Mosher’s method after the NOESY experiment of the acetonide derivative following the stereoselective reduction at the carbonyl C-18 group in
1.
4 The observed NOESY correlations of
2 (H-1β/H-3, H-1β/H-5, H-1β/H-24, H-5/H-24, H-8/H-14, H-8/H-24, and H-10/H-13) revealed that the relative configuration of the decalin moiety is the same as that of
1; i.e.,
2 was a diastereomer of
1 at C-12, C-16, C-19, or C-20. In
1H NMR spectrum of
2 (
Table 1 and
Table S1), the general features closely resembled those of
1, except for the
1H NMR signals for H-19 (
1;
δH 3.66 and
2;
δH 3.93)). In addition, the coupling constant for the H-19 signal was different (
1;
J19,20 = 6.8 Hz and
2;
J19,20 = 4.2 Hz). These differences were supposedly related to the magnetic anisotropic effect of the carbonyl group (C-18) that reflected the change of a dihedral angle between H-19 and H-20, indicating that the absolute configuration at C-19 in
2 is opposite to that of
1. The chemical shifts for the
1H and
13C NMR signals at C-16 and C-20 in
2 resembled those of
1, indicative of identical chirality at C-16 and C-20 between
1 and
2 (
Table 1). On the other hand, the ECD Cotton effects were expected to be induced by the carbonyl groups (C-11, C-18, and C22) around C-12. Hence, the experimental observation that the ECD spectrum of
2 shows good agreement with that of
1 revealed identical chirality at C-12 between
1 and
2 (
Figure 3). The above evidence suggested that
2 is an epimer of
1 at C-19.
According to HRFABMS data, altercrasin C (
3) was assigned the molecular formula C
22H
27NO
4. The general features of its IR spectrum matched those of
1 and
2. By the accurate inspection of the NMR spectrum of
3, the signals corresponding to the hydroxyethyl moiety (C-20–C-21) and the carbonyl (C-18) group observed in
1 and
2 were replaced by amide carbonyl (C-19 (
δC 173.3)) and a quaternary sp
3 carbon groups with a hydroxyl group (C-18 (
δC 81.2)) (
Table 1 and
Table S2). In addition, the geometrical configuration of the side-chain olefin moiety (C-14 to C-15) as
cis from the
1H NMR coupling constant (
J14, 15 8.8 Hz) was revealed to be less than that of
1 (
J14, 15 15.5 Hz). The above evidences and the
1H–
1H COSY and HMBC correlation shown in
Figure 4 and
Table S3 led to the planar structure of
3, which had a cyclic imide. For the stereochemistry of
3, the NOESY correlations (H-1/H-3, H-1/H-5, H-1/H-24, H-4/H-10, H-5/H-24, H-8/H-24, and H-10/H-13) for the decalin moiety revealed that the relative configurations among C-3, C-5, C8, C-9, C-10, and C-13 are the same as
1 and
2. In addition, the NOESY correlation between H-13 and H-17 revealed that the 16-methyl group is oriented
cis to H-13 (
Figure 5). The absolute configurations in the decalin moiety were hypothesized as those of the above compounds in terms of the biosynthetic pathway. To determine the absolute configuration of the remaining chiral centers (C-12 and C-18), the ECD spectrum of
3 following a conformational consideration by the NOESY experiment and the building of an HGS molecular model was recorded. For all possible combinations,
3a (12
S, 18
R),
3b (12
S, 18
S),
3c (12
R, 18
R), and
3d (12
R, 18
S) (
Figure 6), the
trans ring junction at C-12 and C-18 such as
3a and
3d led to a large distortion of the five-membered cyclic imide; hence, the possibility of
3 exhibiting the
3a and
3d stereostructures is excluded in addition to some contradiction of NOESY correlation; i.e., the correlation between H-17 and H-13 should not be observed in
3a. In ECD spectra of
1,
2, and
3, the Cotton effect at 285 nm corresponded to the carbonyl groups (
n→π* interaction); hence, the absolute configuration at C-12 is assigned (
Figure 3). The ECD spectra of
3 showed a negative Cotton effect at 285 nm; hence, the absolute configuration at C-12 in
3 is deduced to be 12
R, i.e.,
3 exhibited the stereostructure of
3c.
Altercrasins D (
4) and E (
5) were assigned the same molecular formula C
24H
31NO
4 based on the deduction according to HRFABMS data.
1H and
13C NMR spectra of
4 and
5 revealed similar features except that the differences for the chemical shift between
4 (proton signals: H-1α (
δH 1.67), H-10 (
δH 1.70), H-13 (
δH 3.71), H-14 (
δH 5.52), and H-19 (
δH 3.49); carbon signals: C-11 (
δC 208.4), C-12 (
δC 70.5), C-13 (
δC 43.6), C-14 (
δC 124.6), C-18 (
δC 208.4), and C-22 (
δC 168.1)) and
5 (proton signals: H-1α (
δH 1.91), H-10 (
δH 1.37), H-13 (
δH 3.96), H-14 (
δH 5.70), and H-19 (
δH 3.81); carbon signals: C-11 (
δC 205.2), C-12 (
δC 72.7), C-13 (
δC 41.5), C-14 (
δC 126.9), C-18 (
δC 204.7), and C-22 (
δC 170.7)) were observed (
Table 2,
Tables S3 and S4). Analyses of HMBC correlations confirmed that
4 and
5 have the same planar structure (
Figure 7). In the NOESY experiments of
4 and
5 (
Table 2,
Tables S3 and S4), the observed correlations clearly indicated their homologous stereochemistry except for the spiro γ-lactam moiety; i.e., diastereomeric relationship at C-12, C-19, or C-20 between
4 and
5 was revealed.
Their absolute stereostructure was considered to be based on the relationship between the ECD Cotton effects and the absolute configuration at C-12 as described above. The Cotton effects at 285 nm in the CD spectra of
4 and
5 clearly revealed the absolute configuration at C-12, i.e.,
4 possessed 12
S, and
5 possessed 12
R (
Figure 8). For the
1H and
13C NMR signals at C-20 in
4 and
5, the chemical shifts were in good agreement with those of
1 and
2. Based on this evidence and the fact that these were isolated from the same fungal metabolites, we guessed that
4 and
5 exhibit a 20
R configuration as
1 and
2. On the other hand, the
1H NMR signals for H-19 in
4 and
5 demonstrated the same difference in the chemical shifts and spin–spin coupling constants (
4:
δH 3.49 (7.2 Hz) and
5:
δH 3.81 (4.8 Hz)) as those of
1 and
2 (
Table 1 and
Table 2); hence, the dihedral angle between H-19 and H-20 in
4 and
5 is thought to be the same as those of
1 and
2, respectively, i.e.,
4 and
5 exhibited the 19
S and 19
R configurations, respectively. Thus, the absolute configuration of
4 (12
S,19
S,20
R) and
5 (12
R,19
R,20
R) is proposed. These stereostructural hypotheses will be positively supported by synthetic research or X-ray crystal structure analysis in the future.
In this study, the cancer cell growth inhibitory property of the metabolites was examined using murine P388 leukemia, human HL-60 leukemia, and murine L1210 leukemia cell lines.
Table 3 summarizes the results.
4 and
5 bearing a diene moiety (C-6 to C-8) exhibited significant cytotoxic activity against these cancer cells. In particular, the activity of
4 was equal to that of 5-fluorouracil. Contrary to our expectations, the difference in the stereochemistry was not related to the activity. Currently, investigation of related compounds isolated from this fungal metabolite is underway, as well as their structure-activity relationships.
3. Materials and Methods
3.1. General Experimental Procedures
NMR spectra were recorded on an Agilent-NMR-vnmrs (Agilent Technologies, Santa Clara, CA, USA) 600 with tetramethylsilane (TMS) as an internal reference. FABMS was recorded using a JEOL JMS-7000 mass spectrometer (JEOL, Tokyo, Japan). IR spectra was recorded on a JASCOFT/IR-680 Plus (Tokyo, Japan). Optical rotations were measured using a JASCO DIP-1000 digital polarimeter (Tokyo, Japan). Silica gel 60 (230–400 mesh, Nacalai Tesque, Inc., Kyoto, Japan) was used for column chromatography with medium pressure. ODS HPLC was run on a JASCO PU-1586 (Tokyo, Japan) equipped with a differential refractometer RI-1531 (Tokyo, Japan) and Cosmosil Packed Column 5C18-MSII (25 cm × 20 mm i.d., Nacalai Tesque, Inc., Kyoto, Japan). Analytical TLC was performed on precoated Merck aluminum sheets (DC-Alufolien Kieselgel 60 F254, 0.2 mm, Merck, Darmstadt, Germany) with the solvent system CH2Cl2-MeOH (19:1) (Nacalai Tesque, Inc., Kyoto, Japan), and compounds were viewed under a UV lamp (AS ONE Co., Ltd., Osaka, Japan) and sprayed with 10% H2SO4 (Nacalai Tesque, Inc., Kyoto, Japan) followed by heating.
3.2. Fungal Material
A strain of Alternaria sp. OUPS-117D-1 was initially isolated from the sea urchin Anthocidaris crassispana, collected in Osaka bay, Japan in May 2010. The fungal strain was identified by Techno Suruga Laboratory Co., Ltd., Shizuoka, Japan. The sea urchin, which wiped its surface with EtOH, was cracked by a scalpel and its inside was applied to the surface of nutrient agar layered in a Petri dish. Serial transfers of one of the resulting colonies provided a pure strain of Alternaria sp.
3.3. Culturing and Isolation of Metabolites
The fungal strain was cultured at 27 °C for six weeks in a liquid medium (70 L) containing 1% glucose, 1% malt extract and 0.05% pepton in artificial seawater adjusted to pH 7.5. The fungal strain filtrated culture broth was extracted thrice with MeOH (Nacalai Tesque, Inc., Kyoto, Japan). The combined extracts were evaporated in vacuo to afford a mixture of crude metabolites (52.8 g). The MeOH extract was chromatographed on a silica gel (Nacalai Tesque, Inc., Kyoto, Japan) column with a CH2Cl2-MeOH (Nacalai Tesque, Inc., Kyoto, Japan) gradient as the eluent. The MeOH-CH2Cl2 (5:95) eluate (22.6 g) was chromatographed again on a silica gel column with a hexane-EtOAc-MeOH (Nacalai Tesque, Inc., Kyoto, Japan) gradient as the eluent. The MeOH–EtOAc (2:98 and 5:95) eluate (F1 (1.9 g), F2 (1.3 g), respectively) were chromatographed on LH-20 (GE Healthcare Japan, Tokyo, Japan) using MeOH-CH2Cl2 (1:1) as the eluent. The fraction exhibiting cytotoxicity from F1 (105.1 mg) was purified by HPLC using MeOH-H2O (80:20) as the eluent to afford 3 (4.6 mg), 4 (1.3 mg), and 5 (5.3 mg). The fraction exhibiting cytotoxicity from F2 (794.5 mg) was purified by HPLC using MeOH-H2O (80:20) as the eluent to afford 1 (10.3 mg) and 2 (6.5 mg).
Altercrasin A (
1). colorless oil; IR (film)
νmax 3345, 1730 1671 cm
−1; NMR data, see
Table 1 and
Table S0, and the previous report
4; HRFABMS [M + H]
+ m/z 416.2436 (calcd for C
24H
34NO
5: 416.2437).
Altercrasin B (
2). colorless oil; IR (film)
νmax 3368, 1729 1701 cm
−1; NMR data, see
Table 1 and
Table S1; HRFABMS [M + H]
+ m/z 416.2430 (calcd for C
24H
34NO
5: 416.2437).
Altercrasin C (
3). colorless oil; IR (film)
νmax 3478, 1720 1710 cm
−1; NMR data, see
Table 1 and
Table S2; HRFABMS [M + Na]
+ m/z 392.1839 (calcd for C
22H
27NO
4Na: 392.1837).
Altercrasin D (
4). colorless oil; IR (film)
νmax 3318, 2917, 1693 cm
−1; NMR data, see
Table 2 and
Table S3; HRFABMS [M + H]
+ m/z 398.2332 (calcd for C
24H
32NO
4: 398.2332).
Altercrasin E (
5). colorless oil; IR (film)
νmax 3318, 2918 1701 cm
−1; NMR data, see
Table 2 and
Table S4; HRFABMS [M + H]
+ m/z 398.2332 (calcd for C
24H
32NO
4: 398.2332).
3.4. Assay for Cytotoxicity
Cytotoxic activities of 1–5 and 5-fluorouracil were examined with the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) method. P388, HL-60, and L1210 cells were cultured in RPMI 1640 Medium (10% fetal calf serum) at 37 °C in 5% CO2. The test materials were dissolved in dimethyl sulfoxide (DMSO) to give a concentration of 10 mM, and the solution was diluted with the Essential Medium to yield concentrations of 200, 20, and 2 μM, respectively. Each solution was combined with each cell suspension (1 × 10−5 cells/mL) in the medium, respectively. After incubating at 37 °C for 72 h in 5% CO2, grown cells were labeled with 5 mg/mL MTT in phosphate-buffered saline (PBS), and the absorbance of formazan dissolved in 20% sodium dodecyl sulfate (SDS) in 0.1 N HCl was measured at 540 nm with a microplate reader (MTP-310, CORONA electric). Each absorbance values were expressed as percentage relative to that of the control cell suspension that was prepared without the test substance using the same procedure as that described above. All assays were performed three times, semilogarithmic plots were constructed from the averaged data, and the effective dose of the substance required to inhibit cell growth by 50% (IC50) was determined.
