Calamene-Type Sesqui-, Mero-, and Bis-sesquiterpenoids from Cultures of Heimiomyces sp., a Basidiomycete Collected in Africa

New meroterpenoids bis-heimiomycins A–D (1–4) and heimiomycins D and E (5 and 6) were isolated from solid rice cultures of Heimiomyces sp., while new calamene-type sesquiterpenoids heimiocalamene A (7) and B (8) were isolated from shake cultures, respectively. Structures of the metabolites were elucidated by 1D and 2D NMR in addition to HRESIMS data. While relative configurations were assigned by ROESY data, absolute configurations were derived from the structurally related, previously described calamenes, which we herein name heimiocalamenes C–E (9–11). A plausible biosynthetic pathway was proposed for 1–6, with a radical reaction connecting their central para-benzoquinone building block to calamene-sesquiterpenoids. Based on the assumption of a common biosynthesis, we reviewed the structure of the known nitrogen-containing derivative 11, calling the validity of the originally proposed structure into question. Subsequently, the structure of 11 was revised by analysis of HMBC and ROESY NMR data. Only heimiomycin D (5) displayed cytotoxic effects against cell line KB3.1.

B asidiomycota represent the second largest phylum in the kingdom of fungi and are well known to show both a high biological diversity by comprising more than 35,000 species 1 and a high chemical diversity of their secondary metabolites, leading to the identification of many different classes of natural products and especially important bioactive molecules like strobilurins and pleuromutilins. 2 In particular, these species can be found in partly untapped ecosystems that have not been exhaustively explored yet. For this reason, Heimiomyces sp. (MUCL 56078, collected from Mount Elgon National Reserve, Kenya) was evaluated for its secondary metabolite profile, and previous studies led to the identification of several new secondary metabolites (9−14). 3 In the course of this work, the presence of a vast amount of secondary metabolites was observed within the extracts, which led to further studies on this strain. Herein, we present the isolation, structure elucidation, and biological evaluation of new meroterpenoids bis-heimiomycins A−D and heimiomycins D and E (1−6) from solid rice cultures, as well as the new calamene-type sesquiterpenoids heimiocalamenes A (7) and B (8) from shaking cultures of Heimiomyces sp. Furthermore, known compounds heimiomycin B (13), hispidin (15), and hypholomin B (16) (Figures 2 and S4) were isolated from both liquid and solid cultures. Heimiomycin B (13) was recently published together with heimiomycins A and C, as well as three new calamene derivatives (Figure 2), which we propose to name heimiocalamenes C−E (9−11), after isolation from liquid cultures of Heimiomyces sp., 3 emphasizing that this species shows a very diverse secondary metabolite profile.
Bis-heimiomycin B (2), C (3), and D (4) were isolated as closely related congeners of 1. With the molecular formula C 36 H 40 O 9 , bis-heimiomycin B (2) implies the loss of an oxygen in comparison to 1. Proton NMR (Table 1) and HSQC data of 2 indicated the presence of an additional oxymethylene (H 2 -15″). HMBC correlations from H 2 -15″ to C-5′/C-6″/C-7″/C-8″/C-9″/C-10″ revealed a ring closure at C-15″ after elimination of an oxygen. In the 1D and 2D NMR spectra of bis-heimiomyin C (3) the presence of another oxymethylene group (H 2 -15) and the absence of the aldehyde H-15 occurred as a key difference in 2. Interactions from H 2 -15 to C-7/C-8/ C-9 obtained from HMBC data were consistent with the reduction of 3 at C-15″. Finally, bis-heimiomycin D (4) with the molecular formula C 36 H 40 O 8 implied the loss of another oxygen in comparison to 2, affording a second ring closure at C-15, leading to a symmetric molecule as observed for 1. This was confirmed by the replacement of the aldehyde H-15/H-15″ by an oxymethylene H 2 -15/H 2 -15″. Relative and absolute configurations of 2−4 were deduced from 1 due to comparison of ECD spectra ( Figure S1), 13 C NMR, and ROESY data to those of 1 and other calamene-type compounds described before, 3 as well as the common biological source of these compounds.
Heimiomycin D (5) was isolated as a red oil from extracts of solid rice cultures. It was shown to possess the molecular formula C 22 H 24 O 7 by HRESIMS data according to the molecular ion cluster at m/z 401.1593 [M + H] + requiring 11 degrees of unsaturation. The proton ( Table 2) and HSQC NMR spectra of 5 were highly similar to those of heimiomycin C (14). 3 The key difference is an additional hydroxy group at position C-5′. The relative and absolute configurations were deduced as 2S,5R from 14 by comparison of ECD spectra and 13 C and ROESY data ( Figure S2) to those of 14 3 and due the common source of both compounds.  For heimiomycin E (6) a molecular formula of C 21 H 22 O 7 was identified by HRESIMS data, indicating the formal loss of a CH 2 fragment in comparison to 5. Highly similar NMR spectra showed the absence of methyl group C-7′, leaving an sp 2 -hybridized methine (H-6′) as the difference between both. In contrast to 5, the signals for C-1′, C-2′, C-4′, and C-5′ were not visible, most likely due to tautomerism.
New calamene-type sesquiterpenoids heimiocalamenes A (7) and B (8) were isolated from the mycelial extracts of liquid cultures. The molecular formula of 7 was assigned as C 15 H 22 O 2 according to the molecular ion cluster at m/z 235.1689 [M + H] + in the HRESIMS spectrum. The NMR spectra of 7 were highly similar to those of 9. Key differences are the loss of the hydroxy group at C-4, resulting in a methylene (H 2 -4) and the replacement of the oxygenated sp 2 -hybridized carbons at C-9 and C-10 by two methylenes (H 2 -9 and H 2 -10). The relative and absolute configurations were deduced as 2S,5R by analogy to assignments for 9, 3 as these compounds were isolated from the same biological source (Heimiomyces sp. MUCL 56078) and showed similar ROESY correlations. Heimiocalamene B (8) was identified as the 3-hydroxy derivative of 10 due to close similarities of their NMR data. 13 C ( Table 2) and HMBC data revealed the presence of the oxygenated sp 2 -hybridized carbon at position C-3. We propose to name compounds 9−11 as heimiocalamenes C−E, because they have been isolated from the same biological source and show structural similarities to 7 and 8.
Minor isomers were observed in the HPLC-MS and NMR data for compounds 1 and 2. After purification via preparative HPLC, results from the analytical HPLC of compound 1 showed the presence of two peaks with the same molecular mass ( Figure S5) in a ratio of 9:1. Since this ratio adjusted spontaneously, it is most likely caused by interconversion of 1 between two different forms of the compound. The presence of two peaks with the same molecular mass was also observed in the HPLC-MS data of compound 2 ( Figure S6). This is also reflected in the 1 H and 13 C spectra of both 1 and 2, where additional weak signals of the minor isomers can be observed (Tables S4 and S6). However, two possible explanations to cause the presence of these minor isomers can be taken into account. On the one hand, the quinone substructure of 1−6 could possess a para-or ortho-orientation, while both would show similar features, and on the other hand, there is the possibility of atropisomerism within the molecules that would lead to different stereoisomers.
Compounds 1−6 were described to possess a para-quinone substructure due to comparison of their UV spectra ( Figure  S7) and  to the ones of structurally related para-and ortho-quinones previously described in the literature. 4 Especially, absorption maxima at lower wavelengths (maxima with strongest intensity at λ max = 240−300 nm and with medium intensity at λ max = 285−440 nm), characteristic for para-quinones, were observed, while the absorption maximum at 500−580 nm, characteristic for ortho-quinones, was not observed. However, UV spectra of 1b and 4 slightly differed from the ones of the other compounds. Therefore, IR spectra of compounds 1b and 4 ( Figures S8 and S9) were measured and compared to the ones of similar quinones described in the literature to support the structural assignment of the para-quinone, since for ortho-quinones a characteristic Journal of Natural Products pubs.acs.org/jnp Article and well-separated carbonyl band around 1680−1700 cm −1 was not observed. 4 For compounds 1−6 there is the possibility of hindered rotation around the C-3′/C-7 and C-6′/C-7″ bonds. In the case of bis-heimiomycin A (1) and heimiomycins D and E (5 and 6) a tautomerism within the p-benzoquinone substructure is preventing atropisomerism, while compounds 2−4 did not show any effects in their ECD data that indicate the presence of atropisomers. The rapid conversion of possible atropisomers can be rationalized by low rotational barriers of the C-7/C-3′ and C-6′/C-7″ bonds. An effective radius of only 1.53 pm and rotational barrier of 27.1 kJ/mol had been determined by Bott et al. for the hydroxy group. 5 Thus, we assume the keto and hydroxy substituents to be small enough for allowing rotation of the C-7/C-3′ and C-6′/C-7″ bonds.
Additionally, the previously described compounds heimiomycin B (13), 3 hispidin (15), 6 and hypholomin B 7 (16) were observed in both liquid and solid cultures (Figures 2 and S4). Hispidin and its derivatives, including hypholomin B, are reported to show antioxidant effects. 8−10 The variety of secondary metabolites produced by Heimiomyces sp. MUCL 56078 can be explained by the combination of calamene-type sesquiterpenoid precursors with various oxidized building blocks. In the case of 1−4, the resulting intermediate undergoes another linkage reaction to a second calamene-type sesquiterpenoid precursor. These reactions might follow a radical mechanism, similar to the biosynthesis of the bibenzoquinone oosporein, 11 or an electrophilic aromatic substitution mechanism ( Figure S5).
Both proposed mechanisms are expected to leave the configurations of carbon centers C-2 and C-5 unaffected. Nevertheless, two sesquiterpenoids being linked via a pbenzoquinone is an uncommon feature for natural products. 12 So far only a few similar compounds, like popolohuanones F− H 13,14 and nakijiquinone E, 15 have been described in the literature.
However, the structure of the nitrogen-containing derivative 11, isolated in the preceding study, did not match this logic. In particular, the biogenetic origin of the Schiff base carbon C-15 could not be mechanistically derived from the parental calamene scaffold. Therefore, we carefully reviewed the NMR data of 11 (Table S13) and observed HMBC correlations from H-15 to C-7, C-8, and C-9. By contrast, a correlation from H-15 to C-6 was not observed, which would have been expected for the structure proposed in our earlier study. In addition, ROESY correlations were observed between H-15/9-OMe and H 3 -13/H-5′, respectively, indicating that the linkages of C-15 and C-8 to the calamene moiety have to be exchanged ( Figure 2). This assignment does explain the addition of an amino-p-benzoquinone to a calamene precursor as a possible biosynthetic pathway to 11.
All isolated compounds were evaluated for their antimicrobial activities in a serial dilution assay against several Gram positive and Gram negative bacteria as well as fungal strains, but were mostly inactive (Table S1). Furthermore, they were tested for cytotoxicity against the human cervical cancer cell line KB3.1 and the murine fibroblast cell line L929, 16 where only heimiomycin D (5) showed cytotoxic effects against KB3.1 with an IC 50 of 6.3 μM (Table S2) and therefore was tested against further cell lines (Table S3), resulting in effects on breast cancer cell line MCF-7 (IC 50 of 2.5 μM), ovarian cancer cell line SKOV-3 (IC 50 of 3 μM), and skin cancer cell line A431 (IC 50 of 4.25 μM). Quinone derivatives have been reported to be an important class of molecules, showing a number of various biological activities, presumably due to their ability to undergo nucleophilic attacks and electron reductions. 17,18 For this reason, compounds 1−6 should be considered as candidates for various other targets, such as antiviral or antioxidant assays.
In summary, cultivation of a Heimiomyces sp. led to the isolation and identification of new meroterpenoids (1−6) and two new calamene-type sesquiterpenoids (7 and 8), as well as the previously described hispidin (15), hypholomin B (16), and heimiomycin B (13), from shaking and solid rice cultures. Together with the compounds isolated in the preceding study (9−14), 3 this Heimiomyces sp. showed a vast chemical diversity of its secondary metabolite profile, which emphasizes that Basidiomycota, especially unexplored species from the tropics, should be explored as potentially rich sources of novel natural products in the ongoing search for new drug leads. d 6 ( 1 H: 2.05 ppm, 13 C: 29.32 ppm) and MeOH-d 4 ( 1 H: 3.31 ppm, 13 C: 49.15 ppm), respectively. HRESIMS mass spectra were measured using the Agilent 1200 series HPLC-UV system in combination with an ESI-TOF-MS (Maxis, Bruker). Measurements were performed with a 2.1 × 50 mm, 1.7 μm, C18 Acquity UPLC BEH (Waters) column, using Milli-Q H 2 O + 0.1% formic acid as solvent A and MeCN + 0.1% formic acid as solvent B (gradient: 5% B for 0.5 min increasing to 100% B in 19.5 min and maintaining 100% B for 5 min, flow rate: 0.6 mL/min, UV detection: 200−600 nm).
Fungal Material. Heimiomyces sp. (MUCL 56078) was collected from Mount Elgon National Reserve (1°7′6″ N, 34°31′30″ E) in Kenya by C. Decock and J. C. Matasyoh. Identification of the genus and deposition of a dried specimen were carried out as described by Cheng et al. 3 Fermentation and Extraction. Cultures of Heimiomyces sp. were maintained on YM6.3 agar plates.
For the seed cultures, three 50 mm 2 sized pieces of well-grown mycelium from YM6.3 agar plates were transferred into a 500 mL Erlenmeyer shape culture flask containing 200 mL of YM6.3 medium (10 g/L malt extract, 4 g/L D-glucose, 4 g/L yeast extract, pH 6.3). The incubation was performed at 23°C and 140 min −1 on a rotary shaker. After 23 days of cultivation the culture broth was homogenized with an Ultra-Turrax (T25 easy clean digital, IKA), equipped with a S 25 N − 25 F dispersing tool, at 8000 rpm for 10− 20 s.
Solid Rice Cultures. The inoculum (8 mL per flask) was transferred into four 500 mL Erlenmeyer shape culture flasks containing BRFT medium (1 g/L yeast extract, 0.5 g/L sodium tartrate, 0.5 g/L K 2 HPO 4 , 100 mL of the solution added to 28 g of brown rice). Afterward, the medium was loosened with a spatula to make it accessible for oxygen and homogeneously distribute the inoculum. The incubation was performed at 23°C in an incubator. After 72 days the fermentation process was stopped. At first, the medium and the mycelium were covered with acetone. Following this, the medium was loosened with a spatula and mixed with the acetone. Extraction was carried out by using ultrasonication for 30 min. Liquid and solid phase were separated by filtration. This procedure was repeated, followed by evaporation (40°C) of the organic solvent with a rotary evaporator. The remaining aqueous phase was extracted with EtOAc (1:1) in a separatory funnel, twice. The organic phase was evaporated to dryness (40°C). Furthermore, the extract was dissolved in 5 mL of MeOH. Afterward, 50 mL of a 1:1 mixture of heptane and MeOH/H 2 O (1:1) was added. Extraction was carried out in a separatory funnel twice, and the heptane and aqueous phases were collected separately. Finally, both were evaporated to dryness (40°C). This led to the isolation of 476 mg of aqueous extract and 206 mg of heptane extract. A second fermentation of six solid rice cultures was performed using the same conditions, leading to the isolation of 1071 mg of aqueous extract (no heptane extraction).
Liquid Cultures. The inoculum (3 mL per flask) was transferred into 21 500 mL Erlenmeyer shape culture flasks containing 200 mL of YM6.3 medium and five 500 mL Erlenmeyer shape culture flasks containing 200 mL of MOF medium (75 g/L mannitol, 16.2 g/L MES, 15 g/L oat flour, 5 g/L yeast extract, 4 g/L L-glutamic acid, pH 6.0). The incubation was performed at 23°C and 140 min −1 on a rotary shaker. Glucose consumption was monitored using test strips (Medi-Test Glucose, Macherey-Nagel). The fermentation process was stopped 2 days after the culture broth tested negative for glucose (33 days in total for YM6.3 cultures and 27 days in total for MOF cultures). Mycelium and supernatant were separated by centrifugation at 5100 min −1 for 15 min (lab centrifuge 4-16KS, Sigma Laborzentrifugen GmbH). The mycelium was overlaid with acetone and afterward extracted in an ultrasonic bath for 30 min, twice. Solid and liquid phases were separated by filtration, followed by evaporation (40°C) of the organic solvent with a rotary evaporator. The remaining aqueous phase was diluted with H 2 O and extracted against EtOAc. Following this, the organic phase was evaporated to dryness (40°C). The supernatant was extracted with EtOAc (1:1) twice in a separatory funnel. The organic phase was kept and evaporated to dryness (40°C). This led to the isolation of 567 mg of extract from the mycelium and 651 mg of extract from the supernatant of YM6.3 cultures, as well as 211 mg of extract from the mycelium and 339 mg of crude extract from the supernatant of MOF cultures. Filtration of the extracts was performed by using an SPME Strata-X 33 μm Polymeric RP cartridge (Phenomenex, Inc.).
Analytical HPLC. The obtained extracts were dissolved in acetone to yield a concentration of 10 mg/mL. Solvation was aided by ultrasonication at 40°C for 10 min. Samples were analyzed by an analytical HPLC device (Dionex UltiMate 3000 series) coupled to an ion trap mass spectrometer (amazon speed by Bruker) to conduct the measurements. HPLC grade H 2 O and HPLC grade MeCN supplemented by 0.1% formic acid were used as mobile phase. With a flow rate of 600 μL/min, 2 μL of the injected samples was separated over an ACQUITY-UPLC BEH C18 column (50 × 2.1 mm; particle size: 1.7 μm) by Waters. Starting with 5% of MeCN, the amount was increased to 100% in 20 min and retained for 5 min at 100%. The obtained chromatograms were evaluated with the appropriate Bruker analysis software (Data Analysis 4.4).
Isolation of Compounds 1−8. After evaluation of the analytical data, the extracts were separated via RP HPLC using a Gilson PLC 2250 purification system.
Solid Rice Cultures (BRFT). The extract obtained from the aqueous phase of the solid rice cultures of the first fermentation was purified using a Gemini LC column 250 × 50 mm, 110 Å, 10 μm (Phenomenex); solvent A: Milli-Q H 2 O + 0.1% formic acid, solvent B: MeCN + 0.1% formic acid, flow rate: 60 mL/min, gradient: 5 min B at 25%, increasing to 80% B in 55 min, increasing to 100% B in 10 min, maintaining 100% B for 10 min. The fraction at 60.5−61.5 min led to 2.76 mg of 3, the fraction at 63.5−64.5 min led to 6.8 mg of compound 4, and the fraction at 65.5−66.5 min led to 2.8 mg of compound 2. The extract obtained from the solid rice cultures of the second fermentation was purified using a Synergi Polar RP 250 × 50 mm, 80 Å, 10 μm (Phenomenex) column; solvent A: Milli-Q H 2 O + 0.1% formic acid, solvent B: MeCN + 0.1% formic acid, flow rate: 60 mL/min, gradient: 5 min B at 20%, increasing to 40% B in 5 min, increasing to 85% B in 50 min, increasing to 100% B in 5 min, maintaining 100% B for 10 min. The fraction at 29.5−30.5 min led to 7.4 mg of 6, the fraction at 37.0−37.75 min led to 4.5 mg of compound 5, and the fraction at 48.0−48.75 min led to 43.2 mg of compound 1.
Acetylation of 1. Acetylation was performed as previously described by Duncan et al.; 19 16 mg of 1 was dissolved in 4.8 mL of pyridine, and afterward 2.4 mL of acetic anhydride (resulting in a 2:1 mixture) was added. The solution was left at room temperature for 3−4 h. Following this, the reagents were removed by evaporation (40°C) with a rotary evaporator. The product was dissolved in acetone and analyzed by HPLC/MS. Due to side product formation, a purification was performed via RP HPLC using a Gilson PLC 2050 purification system. The sample was purified using an XBridge Prep C18 column, 19 × 250 mm, 5 μm (Waters); solvent A: Milli-Q H 2 O + 0.1% formic acid, solvent B: MeCN + 0.1% formic acid, flow rate: 20 mL/min, gradient: 5 min B at 70%, increasing to 90% B in 35 min, increasing to 100% B in 5 min, maintaining 100% B for 5 min. The fraction at 20.5−21.5 min led to 3.3 mg of compound 1b.