Introduction

The isolation of clonostachydiol, an anthelmintic 14-membered bislactone, from the fungus Clonostachys cylindrospora (strain FH-A 6607) was reported in 1993 by Zeeck and co-workers [1, 2]. The planar structure was presented in the original isolation paper. Its stereochemistry was determined in 1995 by asymmetric synthesis as to be 1a (Fig. 1) by Rama Rao and co-workers [3, 4]. Many years later, the second and third asymmetric synthesis of clonostachydiol had been reported by independent research groups [5, 6]. A confusing issue was that the 1H NMR spectroscopic data in DMSO-d6 are apparently different between those in original isolation paper [1]/patent [2] and those in synthesis papers [3, 6]. Clonostachydiol was also isolated from Gliocladium sp [7]. and Xylaria obovata ADA-228 [8]. In both reports, NMR data are not presented as it was handled as a known compound. We also isolated this compound from Xylaria sp. BCC 4297 [9]. The 1H NMR spectrum in DMSO-d6 matched with the synthetic compound [3], and the optical rotation data of our sample was consistent with both reported data for the natural product [1, 2, 7] ([α]20D + 103, c 1.0, MeOH; original isolation paper) and synthetic samples [3, 5, 6]. From Gliocladium sp., the C-4 ketone derivative, named 4-keto-clonostachydiol, was also isolated along with clonostachydiol by Munro and co-workers [7]. They performed chemical correlation: NaBH4/CeCl3 reduction of the ketone to give clonostachydiol and its C-4 epimer. On the basis of the optical rotation data of the reduction product, the authors assigned the stereochemistry of the natural products from the fungus as 1a and 2a. Recently, She and co-workers reported asymmetric synthesis of 4-keto-clonostachydiol, both the reported structure 2a and its enantiomer 2b [10]. On the basis of the optical rotation data, they concluded that the absolute configuration of natural 4-keto-clonostachydiol (from Gliocladium sp.) needs to be revised to be 2b. She’s group did not perform chemical correlation to clonostachydiol. However, taking together with the works by Munro’s group [7], it leads to the conclusion that natural clonostachydiol should be 1b. This is inconsistent with the reports of asymmetric synthesis by three independent Indian research groups (1a), therefore, either one should be wrong.

Fig. 1
figure 1

Structures of macrobislactones

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In our continuing search for novel bioactive compounds from invertebrate pathogenic fungi, a stereoisomer of clonostachydiol, cordybislactone (3), and its hydrolyzed derivative (4), have been isolated from cultures of the hopper pathogen, Cordyceps sp. BCC 49294. In this paper we describe the isolation and structure elucidation of these new compounds. In addition, for solving above mentioned queries on the stereochemistry of clonostachydiol, we have conducted re-isolation of the “presumed” clonostachydiol from Xylaria sp. BCC 4297, and determined its absolute configuration.

Results and Discussion

Cordybislactone (3) was isolated as a colorless amorphous solid, and the molecular formula was determined as C14H20O6 by HRESIMS. The 1H and 13C NMR, DEPT135, and HSQC spectroscopic data indicated the presence of 14 carbons categorized as two ester carbonyl carbons, four sp2 methines composing two trans olefins, four oxygenated methines, two methylenes, and two methyl groups (Table 1). In addition, the 1H NMR spectrum exhibited resonances of two hydroxyl groups. The planar structure, same as clonostachydiol, was elucidated on the basis of COSY and HMBC correlations. Thus, two ester linkages constructing a bislactone was indicated by HMBC correlations from H-5, H-8, and H-9 to C-7 (δC 165.0), and from H-2, H-3, and H-13 to C-1 (δC 165.6).

Table 1 NMR spectroscopic data for cordybislactone (3, isolated from Cordyceps sp. BCC 49294) and clonostachydiol (1b, isolated from Xylaria sp. BCC 4297)
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The molecular formula of 4 was determined by HRESIMS as C14H22O7, a (partial) hydrolysate of 3. The significant difference of the NMR spectra was the upfield shift of H-5 (δH 3.75, m; in acetone-d6) when compared with 3 (H-5, δH 5.03, m; in acetone-d6). The ester linkage involving C-1 (δC 166.4) was indicated by HMBC correlations from H-2, H-3, and H-13 to the carbonyl carbon. Consequently, compound 4 was identified as the C-7 ester hydrolyzed derivative. It was further confirmed by conversion of 3 into this compound. Treatment of 3 with 0.4 M aqueous K2CO3/dioxane at room temperature gave 4 as the major product.

Clonostachydiol was re-isolated [9] from Xylaria sp. BCC 4297. Its specific rotation, [α]23D + 102 (c 1.05, MeOH), was consistent with all reported data, both isolation and synthesis. NMR spectra were taken in four different solvents, DMSO-d6, CDCl3, acetone-d6, and CD3OD. Confusingly, the 1H NMR data reported in the original isolation paper (from Clonostachys cylindrospora strain FH-A 6607, in DMSO-d6) were consistent with those of the compound from BCC 4297 taken in CDCl3, while the 13C NMR data of the original paper (in DMSO-d6) matched well with the data of our isolated compound taken in DMSO-d6. On the other hand, 1H NMR data of the synthetic compound (first and third total synthesis) [3, 6] in DMSO-d6 were consistent with those of our isolated compound in DMSO-d6. On the basis of these results, it was concluded that wrong 1H NMR solvent was recorded in the original isolation paper [1] and corresponding patent [2]. It should also be noted that the 1H NMR spectrum of the second total synthesis compound (Yadav et al.) was taken in CDCl3 [5], and the recorded data were different from any of our 1H NMR spectra of clonostachydiol (four solvents). It suggested that the synthetic compound may not be clonostachydiol. Consequently, the remained important question was whether natural clonostachydiol is 1a or 1b.

With enough quantities of samples for derivatizations in hand, absolute configurations of cordybislactone (3, from BCC 49294) and clonostachydiol (from BCC 4297) were determined. The absolute configurations of the secondary alcohol carbons, C-4 and C-10, were determined by application of the modified Mosher’s method [11]. Thus, cordybislactone (3) was acylated with (R)- and (S)-MTPA-Cl to obtain bis-(S)- and bis-(R)-MTPA ester derivatives 5a and 5b, respectively. The Δδ-values indicated the 4 R,10 S configuration of 3 (Fig. 2). However, there was only one small inconsistency with the rule of the sign of Δδ, + 0.06 for Ha-11. For application of the modified Mosher’s method of a linear mono secondary alcohol (typical case), it is generally requested to perfectly match with the Δδ-value rule. As for 3, the small mismatch is not so surprising, since 5a/5b are bis-MTPA esters of a macrocyclic diol. Similarly, clonostachydiol was also converted into bis-MTPA esters 6a and 6b, whose 1H NMR spectroscopic data unambiguously revealed the 4 R,10 S configuration.

Fig. 2
figure 2

Δδ-Values (δS–δR) of the bis-MTPA esters 5a/5b and 6a/6b (Color figure online)

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Cleavage of the two ester bonds of 3 was achieved by treatment with NaOMe in dry MeOH to furnish methyl ester fragments 7 and 8 (Fig. 3). Similarly, methanolysis of clonostachydiol gave the fragments 7 and 9. The shorter diol (7) was identical to that from 3 (1H NMR). Preparation of the acetonide derivative of 7 met with failure. However, selective methanolysis of 3 (K2CO3, MeOH) and subsequent treatment of the mono-methanolysis product with p-TsOH·H2O in 2,2-dimethoxypropane gave an acetonide derivative 10 (72%, over 2 steps). The NOESY correlations of 10 (Fig. 4) indicated that it is a cis acetonide. Taking together with the 4 R configuration as described above, both 3 and clonostachydiol were shown to possess 4 R,5 S configuration.

Fig. 3
figure 3

Structures of the methanolysis products

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Fig. 4
figure 4

Key NOESY correlations of the acetonide 10 (Color figure online)

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The only remained question was the absolute configuration of C-13 for 3 and clonostachydiol, which was also deduced by application of the modified Mosher’s method for the diol fragments 8 and 9 (Fig. 5). Since their 4 S configuration (corresponding to C-10 of 3 and clonostachydiol) has already been established, one of these diols should have 4 S,7 R configuration, while the other should be the 4 S,7 S isomer. Diol 8, the longer fragment of 3, was derivatized with Mosher reagents to afford bis-(S)- and bis-(R)-MTPA esters 11a and 11b, respectively. The Δδ-values were consistent with the 7 R isomer (corresponding to 13 R configuration of 3), showing positive sign Δδ for H3-8, and negative sign large Δδ-values for the four methylene protons H2-5 and H2-6. This result, in turn, demonstrated the 4 S,7 S configuration of the other diol 9. To further support this conclusion, bis-(S)- and bis-(R)-MTPA esters 12a and 12b were synthesized from 9. The Δδ-values, in particular, negative sign large Δδ-value for H3-8 (−0.18) and positive sign Δδ-value for H-4 ( + 0.10) were consistent with the 7 S configuration (corresponding to 13 S configuration of clonostachydiol). Because the 4-O-MTPA group should contribute to the negative sign Δδ for H2-5 and H2-6, the large chemical shift effects resulting positive sign Δδ-values for H2-5 should be the major contribution of the 7-O-MTPA group and the data suggested 7 S configuration. There are often cases in the Mosher method analysis that chemical shift effects (shielding by the phenyl group of MTPA) for β-position protons of a secondary alcohol (Mosher ester) are larger than those of α-position protons, especially when a linear carbon chain preferably adopt zigzag conformations. Therefore, the Δδ-values for the four methylene protons H2-5 and H2-6 are also in good agreement with the 4 S,7 S configuration. It should also be noted that the reversed configurational assignment between 8 and 9 (4 S,7 S for 8; 4 S,7 R for 9) is totally inconsistent with the Mosher method rule. Taking all results together, the absolute configuration of cordybislactone (3) was determined to be 4 R,5 S,10 S,13 R. Clonostachydiol was identified to be the 4 R,5 S,10 S,13 S isomer 1b, which is the enantiomer of the previously reported structure (1a).

Fig. 5
figure 5

Δδ-Values (δS–δR) of the bis-MTPA esters 11a/11b and 12a/12b (Color figure online)

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Our conclusion is consistent with the report of She’s group [10], and we propose here the revision of the previously reported absolute configuration of clonostachydiol. Natural clonostachydiol, isolated from Clonostachys cylindrospora (strain FH-A 6607), Gliocladium sp., and Xylaria sp. BCC 4297, all showing positive sign optical rotation, should be 1b.

Clonostachydiol is reported to exhibit no significant activity in antibacterial, antifungal, antiviral, and antiprotozoal assays, while it was shown to possess weak cytostatic activity and anthelmintic activity [1]. Compounds 3 and 1b were subjected to several biological assays: cytotoxicity to tumor cell-lines (KB, NCI-H187, and MCF-7), antimycobacterial activity (Mycobacterium tuberculosis H37Ra), and antibacterial activity (Bacillus cereus, Enterococcus faecium, Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae, and Acinetobacter baumannii). Cordybislactone (3) showed weak cytotoxicity: KB, IC50 43 μg/ml; NCI-H187, IC50 14 μg/ml; MCF-7, IC50 > 50 μg/ml. These results were similar to those of clonostachydiol (1b): KB, IC50 39 μg/ml; NCI-H187, IC50 17 μg/ml; MCF-7, IC50 > 50 μg/ml. Both compounds showed week antimycobacterial activity with the same MIC of 50 μg/ml, while they were inactive in all antibacterial assays.

Methods

General experimental procedures

Optical rotations were measured with a JASCO P-1030 digital polarimeter. UV spectra were recorded on an Analytik-jena SPEKOL 1200 spectrophotometer. FTIR spectra were taken on a Bruker ALPHA spectrometer. NMR spectra were recorded on Bruker DRX400 and AV500D spectrometers. ESITOF mass spectra were measured with a Bruker micrOTOF mass spectrometer.

Fungal material

Cordyceps sp. (Cordycipitaceae) was isolated from an unidentified hopper (Hemiptera) collected in Chiang Dao Wildlife Sanctuary, Chiang Mai Province, Thailand, on August 17, 2011, and it was deposited in the BIOTEC Culture Collection as BCC 49294. Identification of this fungus is based on the morphology and ITS rDNA sequence data (GenBank accession number: KT919971). Xylaria sp. (Xylariaceae) was isolated from an unidentified dead wood in Hala Bala Wildlife Sanctuary, Narathiwat Province, Thailand, and it was deposited in the BIOTEC Culture Collection as BCC 4297 on February 24, 2000. Original identification based on the morphology was later confirmed by the ITS5-4 rDNA sequence data (GenBank accession number: MF784453).

Fermentation, extraction, and isolation: Cordyceps sp. BCC 49294

The fungus BCC 49294 was fermented in 60 × 1000 ml Erlenmeyer flasks containing 250 ml of M102 medium (sucrose 30 gl−1, malt extract 20 gl−1, Bacto-peptone 2.0 gl−1, yeast extract 1.0 gl−1, KCl 0.5 gl−1, MgSO4·7H2O 0.5 g, KH2PO4 0.5 gl−1) at 25 °C for 26 days under static conditions. The cultures were filtered, and the filtrate (broth) was extracted with EtOAc (15 l × 3). The organic layers were concentrated under reduced pressure to obtain a brown gum (10.16 g). This broth extract was passed through a Sephadex LH-20 column chromatography (CC) (4.0 × 40 cm) eluted with MeOH to obtain five pooled fractions (Fr-1 – Fr-5), where the major fraction, Fr-3 (8.93 g) contained polyketide metabolites. Fr-3 was subjected to silica gel CC (6.5 × 16 cm, MeOH/CH2Cl2, step gradient elution from 0:100 to 14:86) and the fractions were further purified by silica gel CC (MeOH/CH2Cl2) to furnish 3 (4.87 g) and 4 (15.6 mg).

Cordybislactone (3): colorless gum; [α]24D + 153 (c 0.15, MeOH); UV (MeOH) λmax (log ε) 217 (3.57) nm; IR (ATR) νmax 3422, 1704, 1655, 1275 cm−1; 1H NMR (500 MHz) and 13C NMR (125 MHz) spectroscopic data in DMSO-d6, see Table 1; 1H NMR (400 MHz) and 13C NMR (100 MHz) spectroscopic data in acetone-d6, see Supplementary Information (Supplementary Table S1); HRMS (ESI-TOF) m/z 307.1157 [M + Na]+ (calculated for C14H20O6Na, 307.1152).

Compound 4: colorless gum; [α]26D + 4 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 217 (3.68) nm; IR (ATR) νmax 2923, 2852, 1704, 1278, 1179 cm−1; 1H NMR (500 MHz, acetone-d6) δ 7.05 (1 H, dd, J = 15.7, 4.6 Hz, H-3), 6.92 (1 H, dd, J = 15.6, 4.6 Hz, H-9), 6.06 (1 H, dd, J = 15.7, 1.7 Hz, H-2), 6.01 (1 H, dd, J = 15.6, 1.5 Hz, H-8), 4.97 (1 H, m, H-13), 4.33 (1 H, m, H-10), 4.14 (1 H, m, H-4), 3.75 (1 H, m, H-5), 1.76 (1 H, m, Ha-11), 1.66 (1 H, m, Hb-11), 1.64-1.57 (2 H, m, H-12), 1.23 (3 H, d, J = 6.3 Hz, H-14), 1.14 (3 H, d, J = 6.3 Hz, H-6); 13C NMR (125 MHz, acetone-d6) δ 167.7 (C, C-7), 166.4 (C, C-1), 152.3 (CH. C-9), 149.1 (CH, C-3), 122.0 (CH, C-2), 120.5 (CH, C-8), 75.6 (CH, C-4), 71.0 (CH, C-14), 70.7 (CH, C-5), 70.4 (CH, C-10), 33.1 (CH2, C-12), 32.4 (CH2, C-11), 20.3 (CH3, C-14), 19.0 (CH3, C-6); HRMS (ESI-TOF) m/z 325.1261 [M + Na]+ (calculated for C14H22O7Na, 325.1258).

Fermentation, extraction, and isolation: Xylaria sp. BCC 4297

The fermentation of the fungus BCC 4297 was conducted using a similar procedure as previously reported [9]. Final fermentation was carried out in 60 × 1000 ml Erlenmeyer flasks containing 250 ml of malt extract broth (MEB; malt extract 6.0 gl−1, yeast extract 1.2 gl−1, maltose 1.8 gl−1, dextrose 6.0 gl−1) at 25 °C for 54 days under static conditions. The cultures were filtered, and the filtrate (broth) was extracted with EtOAc (13 l × 3). The organic layers were concentrated under reduced pressure to obtain a brown gum (4.47 g). This broth extract was passed through a Sephadex LH-20 column chromatography (CC) (4.0 × 50 cm) eluted with MeOH to obtain two pooled fractions (Fr-1 and Fr-2). Fr-2 (4.01 g) was subjected to silica gel CC (4.0 × 15 cm, MeOH/ CH2Cl2, step gradient elution from 2:98 to 10:90) to obtain eight fractions, Fr-2-1 – Fr-2-8. Fr-2-6 (2.78 g) was further purified by silica gel CC (EtOAc/hexane, step gradient elution from 50:50 to 100:0) to obtain three fractions, Fr-2-6-1 – Fr-2-6-3. Fr-2-6-2 (2.24 g) was further purified by silica gel CC (EtOAc/CH2Cl2, step gradient elution from 50:50 to 100:0) to furnish 1b (190 mg).

Clonostachydiol (1b): colorless solid; [α]23D + 102 (c 1.05, MeOH); UV (MeOH) λmax (log ε) 220 (3.58) nm; IR (ATR) νmax 3418, 1703, 1656, 1251, 1178, 1047, 982 cm−1; for 1H NMR (500 MHz) and 13C NMR (125 MHz) spectroscopic data in DMSO-d6 and CDCl3, see Table 1; for 1H NMR (500 MHz) and 13C NMR (125 MHz) spectroscopic data in acetone-d6 and 1H NMR (500 MHz) spectroscopic data in CD3OD, see Supplementary Information (Supplementary Table S1); HRMS (ESI-TOF) m/z 307.1153 [M + Na]+ (calculated for C14H20O6Na, 307.1152).

Regioselective hydrolysis of 3

Cordybislactone (3, 7.1 mg; isolated from Cordyceps sp. BCC 49294) was treated with 0.4 M aqueous K2CO3 (0.1 ml) in 1,4-dioxane (0.1 ml) at room temperature for 48 h. The mixture was quenched with 1 M aqueous HCl, and the resulting mixture was concentrated under reduced pressure. The residue was extracted with CH2Cl2 and filtered. The filtrate was concentrated in vacuo to obtain a colorless gum (8.2 mg). 1H NMR spectrum of the crude product indicated that its major component was 4.

Preparation of the bis-MTPA esters of 3

Cordybislactone (3, 2.2 mg; isolated from Cordyceps sp. BCC 49294) was treated with (−)-(R)-MTPA-Cl (14 μl) and 4-dimethylaminopyridine (DMAP, 10 mg) in CH2Cl2 (0.2 ml) at room temperature for 4 h. The mixture was diluted with EtOAc and washed with H2O and 1 M NaHCO3, and the organic layer was concentrated in vacuo. The residue was purified by silica gel CC (EtOAc/hexane, 20:80) to afford a bis-(S)-MTPA ester 5a (4.4 mg). Similarly, bis-(R)-MTPA ester 5b (3.3 mg) was prepared from 3 (2.8 mg) and ( + )-(S)-MTPA-Cl.

Bis-(S)-MTPA ester 5a: white foam; 1H NMR (400 MHz, acetone-d6) δ 7.60-7.42 (10 H, m, phenyl of MTPA × 2), 6.88 (1 H, dd, J = 15.8, 3.6 Hz, H-9), 6.73 (1 H, dd, J = 15.7, 7.2 Hz, H-3), 6.15 (1 H, d, J = 15.7 Hz, H-2), 5.87 (1 H, dd, J = 15.8, 1.7 Hz, H-8), 5.74 (1 H, m, H-10), 5.64 (1 H, dd, J = 7.2, 6.6 Hz, H-4), 5.23 (1 H, dq, J = 6.6, 6.6 Hz, H-5), 5.02 (1 H, m, H-13), 3.57 (3 H, s, OCH3 of MTPA), 3.52 (3 H, s, OCH3 of MTPA), 2.12 (1 H, m, Ha-11), 1.69 (1 H, m, Hb-11), 1.62 (1 H, m, Ha-12), 1.58 (1 H, m, Hb-12), 1.39 (3 H, d, J = 6.6 Hz, H-6), 1.18 (3 H, d, J = 6.4 Hz, H-14); HRMS (ESI-TOF) m/z 739.1949 [M + Na]+ (calculated for C34H34O10F6Na, 739.1948).

Bis-(R)-MTPA ester 5b: white foam; 1H NMR (400 MHz, acetone-d6) δ 7.60-7.40 (10 H, m, phenyl of MTPA × 2), 6.79 (1 H, dd, J = 15.8, 3.3 Hz, H-9), 6.71 (1 H, dd, J = 16.0, 6.1 Hz, H-3), 5.84 (1 H, d, J = 16.0 Hz, H-2), 5.68 (2 H, m, H-4 and H-9), 5.56 (1 H, dd, J = 15.8, 1.8 Hz, H-8), 5.34 (1 H, m, H-5), 5.08 (1 H, m, H-13), 3.61 (3 H, s, OCH3 of MTPA), 3.60 (3 H, s, OCH3 of MTPA), 2.06 (1 H, m, Ha-11), 1.88 (1 H, m, Hb-11), 1.77 (1 H, m, Ha-12), 1.70 (1 H, m, Hb-12), 1.48 (3 H, d, J = 6.7 Hz, H-6), 1.20 (3 H, d, J = 6.4 Hz, H-14); HRMS (ESI-TOF) m/z 739.1945 [M + Na]+ (calculated for C34H34O10F6Na, 739.1948).

Preparation of the bis-MTPA esters of 1b

Using the same procedures described above, bis-(S)-MTPA ester 6a (5.8 mg) was prepared from 1b (2.7 mg; isolated from BCC 4297) and (−)-(R)-MTPA-Cl, and bis-(R)-MTPA ester 6b (4.3 mg) was prepared from 1b (2.0 mg) and ( + )-(S)-MTPA-Cl.

Bis-(S)-MTPA ester 6a: white foam; 1H NMR (400 MHz, acetone-d6) δ 7.60-7.42 (10 H, m, phenyl of MTPA × 2), 6.86 (1 H, dd, J = 16.0, 4.7 Hz, H-9), 6.59 (1 H, dd, J = 15.8, 8.9 Hz, H-3), 6.26 (1 H, d, J = 15.8 Hz, H-2), 5.99 (1 H, dd, J = 16.0, 1.4 Hz, H-8), 5.93 (1 H, m, H-10), 5.41 (1 H, t, J = 8.9 Hz, H-4), 5.11 (1 H, m, H-5), 5.08 (1 H, m, H-13), 3.59 (3 H, s, OCH3 of MTPA), 3.56 (3 H, s, OCH3 of MTPA), 2.22 (1 H, m, Ha-11), 1.91 (1 H, m, Hb-11), 1.59 (1 H, m, Ha-12), 1.38 (1 H, m, Hb-12), 1.26 (3 H, d, J = 6.3 Hz, H-6), 1.19 (3 H, d, J = 6.6 Hz, H-14); HRMS (ESI-TOF) m/z 739.1942 [M + Na]+ (calculated for C34H34O10F6Na, 739.1948).

Bis-(R)-MTPA ester 6b: white foam; 1H NMR (400 MHz, acetone-d6) δ 7.60-7.43 (10 H, m, phenyl of MTPA × 2), 6.81 (1 H, dd, J = 16.0, 4.6 Hz, H-9), 6.47 (1 H, dd, J = 15.8, 8.4 Hz, H-3), 6.15 (1 H, d, J = 15.8 Hz, H-2), 5.91 (1 H, m, H-10), 5.77 (1 H, dd, J = 16.0, 1.5 Hz, H-8), 5.45 (1 H, t, J = 8.4 Hz, H-4), 5.15 (1 H, m, H-5), 5.12 (1 H, m, H-13), 3.59 (3 H, s, OCH3 of MTPA), 3.57 (3 H, s, OCH3 of MTPA), 2.23 (1 H, m, Ha-11), 2.01 (1 H, m, Hb-11), 1.69 (1 H, m, Ha-12), 1.65 (1 H, m, Hb-12), 1.47 (3 H, d, J = 6.3 Hz, H-6), 1.23 (3 H, d, J = 6.6 Hz, H-14); HRMS (ESI-TOF) m/z 739.1941 [M + Na]+ (calculated for C34H34O10F6Na, 739.1948).

Methanolysis of 3

To a stirred solution of cordybislactone (3, 4.0 mg, 14.1 µmol) in MeOH (0.2 ml) was added NaOMe (15 mg, 0.28 mmol) at room temperature. After stirring for 1 h, the reaction mixture was quenched with 5 M aqueous HCl and extracted with EtOAc four times. The combined organic extracts were dried over anhydrous MgSO4, and concentrated under reduced pressure. The residue was purified by silica gel CC (EtOAc/CH2Cl2, 50:50) to afford 7 (1.2 mg, 7.5 µmol, 53%) and 8 (1.3 mg, 6.9 µmol, 49%). Repetition of this reaction afforded additional samples of 7 (3.6 mg) and 8 (2.2 mg) for further derivatizations.

Methyl (E,4 R,5 S)-4,5-dihydroxy-2-hexenoate (7): colorless oil; [α]24D + 20 (c 0.18, CHCl3); IR (ATR) νmax 3416, 2921, 2852, 1709, 1658, 1440, 1280, 1176, 1079, 986 cm−1; 1H NMR (400 MHz, acetone-d6) δ 7.07 (1 H, dd, J = 15.7, 4.5 Hz, H-3), 6.08 (1 H, dd, J = 15.7, 1.6 Hz, H-2), 4.24 (1 H, d, J = 5.0 Hz, 4-OH), 4.14 (1 H, m, H-4), 3.84 (1 H, d, J = 5.0 Hz, 5-OH), 3.74 (1 H, m, H-5), 3.68 (3 H, s, CO2CH3), 1.14 (3 H, d, J = 6.3 Hz, H-6); 13C NMR (125 MHz, acetone-d6) δ 167.1, 149.6, 121.1, 75.7, 70.7, 51.5, 19.1; HRMS (ESI-TOF) m/z 183.0623 [M + Na]+ (calculated for C7H12O4Na, 183.0628).

Methyl (E,4 S,7 R)-4,7-dihydroxy-2-octenoate (8): colorless oil; [α]24D −3 (c 0.11, CHCl3); IR (ATR) νmax 2921, 2852, 1709, 1278, 1171 cm−1; 1H NMR (500 MHz, acetone-d6) δ 6.95 (1 H, dd, J = 15.6, 4.5 Hz, H-3), 6.03 (1 H, dd, J = 15.6, 1.2 Hz, H-2), 4.42 (1 H, d, J = 4.6 Hz, 4-OH), 4.31 (1 H, m, H-4), 3.75 (1 H, m, H-7), 3.68 (3 H, s, CO2CH3), 1.70-1.60 (2 H, m, H-5), 1.55-1.50 (2 H, m, H-6), 1.13 (3 H, d, J = 6.1 Hz, H-8); 13C NMR (125 MHz, acetone-d6) δ 167.3, 152.7, 119.7, 70.9, 67.5, 51.5, 35.8, 34.0, 24.1; HRMS (ESI-TOF) m/z 211.0939 [M + Na]+ (calculated for C9H16O4Na, 211.0941).

Methanolysis of 1b

To a stirred solution of clonostachydiol (1b, 11.5 mg, 40.4 µmol) in MeOH (0.3 ml) was added NaOMe (44 mg, 0.81 mmol) at room temperature. After stirring for 45 min, the reaction mixture was quenched with 5 M aqueous HCl and extracted with EtOAc four times. The combined organic extracts were dried over anhydrous MgSO4, and concentrated under reduced pressure. The residue was purified by silica gel CC (MeOH/CH2Cl2, 5:95) and then silica gel CC (EtOAc/CH2Cl2, 50:50) to afford 7 (2.5 mg, 15.6 µmol, 39%) and 9 (2,5 mg, 13.3 µmol, 33%).

Methyl (E,4 S,7 S)-4,7-dihydroxy-2-octenoate (9): colorless oil; [α]23D + 13 (c 0.17, CHCl3); IR (ATR) νmax 2923, 2853, 1708, 1462, 1278 cm−1; 1H NMR (500 MHz, acetone-d6) δ 6.95 (1 H, dd, J = 15.6, 4.5 Hz, H-3), 6.03 (1 H, dd, J = 15.6, 1.0 Hz, H-2), 4.35 (1 H, d, J = 4.6 Hz, 4-OH), 4.31 (1 H, m, H-4), 3.74 (1 H, m, H-7), 3.68 (3 H, s, CO2CH3), 3.64 (1 H, d, J = 4.3 Hz, 7-OH), 1.75 (1 H, m, Ha-5), 1.59-1.53 (2 H, m, Hb-5 and Ha-6), 1.48 (1 H, m, Hb-6), 1.12 (3 H, d, J = 6.2 Hz, H3-8); 13C NMR (125 MHz, acetone-d6) δ 167.2, 152.7, 119.7, 71.0, 67.6, 51.5, 35.7, 33.9, 24.0; HRMS (ESI-TOF) m/z 211.0948 [M + Na]+ (calculated for C9H16O4Na, 211.0941).

Synthesis of the acetonide derivative 10

To a stirred solution of cordybislactone (3, 16.5 mg, 58.0 µmol) in MeOH (0.3 ml) was added K2CO3 (8.0 mg, 58.0 µmol) at room temperature. After stirring for 10 min, the reaction mixture was quenched with 1 M aqueous HCl and extracted with EtOAc four times. The combined organic extracts were dried over anhydrous MgSO4, and concentrated under reduced pressure to afford a crude triol. To a stirred solution of the crude triol in 2,2-dimethoxypropane (0.5 ml) was added p-TsOH·H2O (ca. 5 mg, 26 µmol) at room temperature. After stirring for 5 h, the reaction mixture was quenched with 1 M aqueous NaHCO3 and extracted with EtOAc three times. The combined organic extracts were dried over anhydrous MgSO4, and concentrated under reduced pressure. The residue was purified by silica gel CC (EtOAc/CH2Cl2, 20:80) to afford the acetonide derivative 10 (14.8 mg, 41.5 µmol, 72% over 2 steps).

Acetonide derivative 10: colorless gum; IR (ATR) νmax 2925, 1716, 1659, 1258, 1170, 987 cm−1; 1H NMR (400 MHz, acetone-d6) δ 6.93 (1 H, dd, J = 15.6, 4.4 Hz, H-9), 6.83 (1 H, dd, J = 15.6, 5.8 Hz, H-3), 6.05 (1 H, d, J = 15.6 Hz, H-2), 6.03 (1 H, d, J = 15.6 Hz, H-8), 4.99 (1 H, m, H-13), 4.74 (1 H, t, J = 6.2 Hz, H-4), 4.46 (1 H, dq, J = 6.2, 6.2 Hz, H-5), 4.34 (1 H, m, H-10), 4.24 (1 H, d, J = 4.0 Hz, 10-OH), 3.68 (3 H, s, CO2CH3), 1.80-1.56 (4 H, m, H-11 and H-12), 1.44 (3 H, s, H-3ʹ), 1.32 (3 H, s, H-1ʹ), 1.24 (3 H, d, J = 6.2 Hz, H-14), 1.09 (3 H, d, J = 6.3 Hz, H-6); 13C NMR (100 MHz, acetone-d6) δ 167.3, 166.0, 152.1, 144.9, 124.0, 120.5, 109.3, 78.5, 75.0, 71.5, 70.8, 51.5, 33.4, 32.6, 28.5, 25.8, 20.3, 16.7; HRMS (ESI-TOF) m/z 379.1715 [M + Na]+ (calculated for C18H28O7Na, 379.1727).

Preparation of bis-MTPA esters of 8 and 9

Using the similar procedures as described above, bis-(S)-MTPA ester 11a was synthesized from diol 8 and (−)-(R)-MTPA-Cl, and bis-(R)-MTPA ester 11b was synthesized from 8 and ( + )-(S)-MTPA-Cl. Similarly, bis-(S)-MTPA ester 12a was synthesized from 9 and (−)-(R)-MTPA-Cl, and bis-(R)-MTPA ester 12b was synthesized from 9 and ( + )-(S)-MTPA-Cl. NMR analyses were performed using the crude reaction products. Assignments of protons of these bis-MTPA ester derivatives were established on the basis of COSY data.

Bis-(S)-MTPA ester 11a: 1H NMR (400 MHz, acetone-d6) δ 7.58-7.41 (10 H, m, phenyl of MTPA × 2), 6.80 (1 H, dd, J = 15.8, 6.8 Hz, H-3), 6.02 (1 H, d, J = 15.8 Hz, H-2), 5.66 (1 H, m, H-4), 5.11 (1 H, m, H-7), 3.73 (3 H, s, CO2CH3), 3.60 (3 H, s, OCH3 of MTPA), 3.54 (3 H, s, OCH3 of MTPA), 1.67 (1 H, m, Ha-5), 1.63 (1 H, m, Hb-5), 1.53 (1 H, m, Ha-6), 1.51 (1 H, m, Hb-6), 1.29 (3 H, m, H3-8).

Bis-(R)-MTPA ester 11b: 1H NMR (400 MHz, acetone-d6) δ 7.58-7.41 (10 H, m, phenyl of MTPA × 2), 6.82 (1 H, dd, J = 15.8, 5.6 Hz, H-3), 5.87 (1 H, d, J = 15.8 Hz, H-2), 5.75 (1 H, m, H-4), 5.20 (1 H, m, H-7), 3.69 (3 H, s, CO2CH3), 3.57 (3 H, s, OCH3 of MTPA), 3.53 (3 H, s, OCH3 of MTPA), 1.96 (1 H, m, Ha-5), 1.95 (1 H, m, Hb-5), 1.82 (1 H, m, Ha-6), 1.78 (1 H, m, Hb-6), 1.28 (3 H, d, J = 6.4 Hz, H-8).

Bis-(S)-MTPA ester 12a: 1H NMR (400 MHz, acetone-d6) δ 7.55-7.43 (10 H, m, phenyl of MTPA × 2), 6.92 (1 H, dd, J = 15.8, 5.7 Hz, H-3), 6.08 (1 H, dd, J = 15.8, 1.4 Hz, H-2), 5.76 (1 H, m, H-4), 5.12 (1 H, m, H-7), 3.72 (3 H, s, CO2CH3), 3.57 (3 H, br s, OCH3 of MTPA), 3.53 (3 H, br s, OCH3 of MTPA), 1.96 (1 H, m, Ha-5), 1.87 (1 H, m, Hb-5), 1.58 (1 H, m, Ha-6), 1.57 (1 H, m, Hb-6), 1.18 (3 H, d, J = 6.2 Hz, H-8).

Bis-(R)-MTPA ester 12b: 1H NMR (400 MHz, acetone-d6) δ 7.56-7.41 (10 H, m, phenyl of MTPA × 2), 6.71 (1 H, dd, J = 15.8, 5.5 Hz, H-3), 5.78 (1 H, dd, J = 15.8, 1.4 Hz, H-2), 5.66 (1 H, m, H-4), 5.19 (1 H, m, H-7), 3.70 (3 H, s, CO2CH3), 3.57 (3 H, br s, OCH3 of MTPA), 3.54 (3 H, br s, OCH3 of MTPA), 1.72 (2 H, m, H-5), 1.70 (1 H, m, Ha-6), 1.68 (1 H, m, Hb-6), 1.37 (3 H, d, J = 6.2 Hz, H-8).

Biological assays

Cytotoxic activities against the tumor cell-lines, NCI-H187 (human small-cell lung cancer), MCF-7 (human breast cancer), and KB (oral human epidermoid carcinoma), were evaluated using the resazurin microplate assay [12]. Antimycobacterial activity against Mycobacterium tuberculosis H37Ra was evaluated using the green fluorescent protein (GFP)-based microplate assay [13]. The MIC values of the standard antituberculosis drugs (positive control), isoniazid and rifampicin, were 0.0469 and 0.0125 μg/ml, respectively.