Two New Terpenes Isolated from Dictyostelium Cellular Slime Molds

We report a protoilludane-type sesquiterpene, mucoroidiol, and a geranylated bicyclogermacranol, firmibasiol, isolated from Dictyostelium cellular slime molds. The methanol extracts of the fruiting bodies of cellular slime molds were separated by chromatographic methods to give these compounds. Their structures have been established by several spectral means. Mucoroidiol and firmibasiol are the first examples of more modified and oxidized terpenoids isolated from cellular slime molds. Mucoroidiol showed moderate osteoclast-differentiation inhibitory activity despite demonstrating very weak cell-proliferation inhibitory activity. Therefore, cellular slime molds produce considerably diverse secondary metabolites, and they are promising sources of new natural product chemistry.


Introduction
Natural products, particularly those derived from microorganisms such as fungi and bacteria, have long played an essential role in the development of novel drugs [1] However, pharmaceutical research into natural products has recently declined because of factors such as increased difficulty in identifying new compounds with skeletally novel structures [2,3]. Thus, novel natural resources for natural products are required.
Cellular slime molds are a group of soil microorganisms that belong to the eukaryotic kingdom Amoebozoa, which is taxonomically distinct from fungi [4,5] The cellular slime mold Dictyostelium discoideum has been used as a model organism for studying eukaryotic cell functions because of its simple developmental pattern and ease of handling [6][7][8][9] Vegetative cells of D. discoideum grow as single ameba by eating bacteria. When these cells are starved, they initiate a developmental program of morphogenesis, forming a slug-shaped multicellular aggregate. This aggregate differentiates into two cell types, prespore and prestalk cells, which are precursors to spores and stalk cells, respectively. At the end of its development, the aggregate forms a fruiting body consisting of spores and a multicellular stalk [10].
We have focused on the utility of cellular slime molds as a source of natural compound [11] and have isolated α-pyronoids [12][13][14] amino sugar derivatives [15], and aromatics [16][17][18][19] with unique structures and various biological activities. For example, brefelamide [16] and its derivatives exhibit inhibitory effects on osteopontin expression [20,21] and immune checkpoint PD-L1 expression [22]. The above results indicate that cellular slime molds are an important source of lead compounds for drug discovery.

Isolation and Structural Elucidation of Mucoroidiol
Multicellular fruiting bodies (80 g dry weight) of D. mucoroides Dm7 were cultured on agar plates in the presence of 0.5 mM ZnCl 2 [23]. They were extracted three times with methanol at room temperature to yield an extract (10 g), which was then partitioned between ethyl acetate and water. The fraction soluble in ethyl acetate (2.8 g) was separated by silica-gel column chromatography and gel permeation column chromatography to afford mucoroidiol (1) (1.3 mg).
HRFAB-MS (m/z 239.2011 [M+H] + ) indicated the molecular formula of 1 as C 15 H 26 O 2 . The NMR spectra of 1 are shown in Supplementary Materials (Pages S2~S4). The 13 C NMR spectrum of 1 showed the presence of two quaternary, four methine, seven methylene, and two methyl carbons ( Table 1). The 1 H-1 H COSY correlations revealed the connectivity of C-1-C-2-C-9(-C-10)-C-8-C-7(-C-14)-C-6-C-5-C-4. The HMBC correlations of H 3 -15 to C-2, C-3, C-4 and C-6; H 2 -12 to C-1, C-10 and C-11; and H 3 -13 to C-1, C-10 and C-11 confirmed the protoilludane skeleton of 1 (Figure 2A). The cross-peaks of H-2 to H-4α, H-9, and H 3 -13, as well as those of H-9 to H-4α and H-7, revealed that these protons faced the α-plane, and that the relative configurations at C-2, C-7, C-9 and C-11 are determined as S*, R*, R* and R*, respectively. Conversely, correlations from H 3 -15 to H-1β and H-5β in the NOESY spectrum revealed that these protons faced the β-plane, and that the relative configurations at C-3 and C-6 are determined as R* and S*, respectively ( Figure 2B). The yield of 1 was so small that its absolute configuration could not be determined by chemical conversion. On the other hand, it was reported that the incubation of DdTPS6 (a terpene cyclase of D. discoideum) with farnesyl diphosphate afforded (2S,3R,6S,7R,9S)-protoillud-7-ene (3) via carbocation intermediate A (Scheme 1) [24]. Some terpene cyclases of D. mucoroides would be evolutionary common with those of D. discoideum, indicating that the absolute configurations of 1 should be common with 3. Thus, the absolute configurations of 1 are assumed to be 2S, 3R, 6S, 7R, 9R, and 11R.

Isolation and Structural Elucidation of Firmibasiol
Multicellular fruiting bodies (48 g dry weight) of the cellular slime mold (D. firmibasis 91HO-33) were cultured on plates and extracted three times with methanol at room temperature to yield an extract (11 g), which was partitioned between ethyl acetate and water. The fraction soluble in ethyl acetate (2.3 g) was separated by repeated column chromatography over silica gel and octadecyl silica gel to yield firmibasiol (2) (1.8 mg).
HREI-MS (m/z 358.3258 [M] + ) indicated the molecular formula of 2 as C 25 H 42 O. The NMR spectra of 2 are shown in Supplementary Materials (Pages S5~S7). The 13 C NMR spectrum of 2 showed the presence of six olefinic, two quaternary, three methine, seven methylene, and seven methyl carbons ( Table 2). The HMBC correlations of H 3 -15 to C-2, C-3 and C-4; H 3 -14 to C-6, C-7 and C-8; H 3 -12 to C-1, C-10 and C-11; and H 3 -13 to C-1, C-10 and C-11 connect the partial structures confirmed by the 1 H-1 H COSY spectrum established the bicyclogermacrane moiety of 2 ( Figure 3A). In addition, the HMBC correlations of H 3 -10 to C-2 , C-3 and C-4 ; and H 3 -9 to C-6 , C-7 and C-8 revealed the 1-geranylated bicyclogermacrane structure of 2. The cross-peaks of H 3 -12-H-10, H 3 -12-H-1, H-1-H 3 -15, and H 3 -15-H-5β in the NOESY spectrum revealed that these protons faced the β-plane ( Figure 3B). Since H-1, H-10, and H 3 -12 direct to the same side, the relative configurations at C-1 and C-10 are determined as R* and S*, respectively. Conversely, the cross-peaks of H 3 -13-H-2, H-2-H-6, and H-2-H-9α indicated that these protons faced the α-plane, determining that the olefin between C-2 and C-3 has an E-configuration, and the relative configuration at C-6 is S*. In addition, the cross-peaks of H-9β-H 3 -14 and H 3 -14-H 2 -1 indicated that these protons faced the same direction ( Figure 3C), indicating that the relative configuration at C-7 is S*. In addition, the NOESY cross-peak between H-1 and H 3 -10 revealed that the olefin between C-2 and C-3 also has an E-configuration. The yield of 2 was very small such that its absolute configuration could not be determined by chemical conversion. On the other hand, it was reported that the incubation of DdTPS8 (a terpene cyclase of D. discoideum) with farnesyl diphosphate afforded (S)-hedycaryol (4) via carbocation intermediate B (Scheme 2) [25]. Firmibasiol (2) is presumed to be biosynthesized from intermediate B, which would be converted into (1R,10S)-bicyclogermacrene (5). Subsequently, the C-6-C-7 olefin attacks geranyl diphosphate, and water addition to the carbocation intermediate C at C-7 produces 2. Through this plausible biosynthetic route, the absolute configurations of C-10 in 2 should be retained from carbocation intermediate B. Therefore, the absolute configurations of 2 are assumed to be 1R, 6S, 7S, and 10S. Although firmibasiol (2) contains five isoprene units (C 25 ), it is not a sesterterpene, but rather a geranylated sesquiterpene. Prenylated terpenoids such as 2 are very rare types of natural compounds; only one compound has been reported so far [26].

Biological Activity of Compounds 1 and 2
Mucoroidiol (1) and firmibasiol (2) were screened to investigate several types of biological activities.
Osteoclasts are multinucleated cells that resorb bone tissue. They are formed by the fusion of mononuclear monocyte/macrophage lineage precursor cells. Excessive bone resorption often results in osteoporosis and rheumatoid arthritis [27]. Firmibasiol (2) showed moderate receptor activator of NF-κB ligand (RANKL)-induced osteoclast-differentiation inhibitory activity (IC 50 28 µM) by measurement of activity of tartrate-resistant acid phosphatase (TRAP) [28] (Figure 4), while mucoroidiol (1) did not show remarkable inhibitory activity. On the other hand, mucoroidiol (1) and firmibasiol (2) exhibited weak anti-proliferative activity against HeLa cells (IC 50 > 40 µM), but did not show apparent anti-bacterial activities both in Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli) bacteria (Table 3). Molecules 2020, 25, x FOR PEER REVIEW 4 of 10 at C-1 and C-10 are determined as R* and S*, respectively. Conversely, the cross-peaks of H3-13-H-2, 98 H-2-H-6, and H-2-H-9α indicated that these protons faced the α-plane, determining that the olefin 99 between C-2 and C-3 has an E-configuration, and the relative configuration at C-6 is S*. In addition, 100 the cross-peaks of H-9β-H3-14 and H3-14-H2-1′ indicated that these protons faced the same direction 101 ( Figure 3C), indicating that the relative configuration at C-7 is S*. In addition, the NOESY cross-peak 102 between H-1′ and H3-10′ revealed that the olefin between C-2′ and C-3′ also has an E-configuration. 103 The yield of 2 was very small such that its absolute configuration could not be determined by

136
The data are expressed as percentages in relation to the mean value of the control cells. The bars 137 indicate the standard deviation of the three wells. The statistical significance of the differences was 138 determined by Welch's t-test. *p < 0.05 vs. control. 139 Table 3. Antitumor and anti-bacterial activities of 1 and 2 a .

Discussion 144
Recently, a phylogenetic analysis revealed that terpene cyclase genes exist in several species of 145 cellular slime molds [29]. D. discoideum emits volatiles containing several types of terpenoid 146 hydrocarbons. These hydrocarbons were produced by the incubation of the recombinant terpene 147 a Half maximal (50%) inhibitory concentration (IC 50 ) (versus HeLa cells) and minimum inhibitory concentration (MIC) (versus S. aureus and E. coli) of 1 and 2 were assessed as described in the Section 4.
The data are expressed as percentages in relation to the mean value of the control cells. The bars indicate the standard deviation of the three wells. The statistical significance of the differences was determined by Welch's t-test. * p < 0.05 vs. control.

Discussion
Recently, a phylogenetic analysis revealed that terpene cyclase genes exist in several species of cellular slime molds [29]. D. discoideum emits volatiles containing several types of terpenoid hydrocarbons. These hydrocarbons were produced by the incubation of the recombinant terpene cyclase with farnesyl or geranylgeranyl diphosphate [24,25,30]. However, unlike when only terpene cyclase acted, the multicellular fruiting bodies of a cellular slime mold should synthesize several types of modified terpenes converted by various biosynthetic enzymes in vivo. Mucoroidiol (1) is the first example of a terpene diol obtained from cellular slime molds. Firmibasiol (2) is a type of prenylated terpenoid, which is a very rare type of natural compound. Because the isolated amounts of compounds 1 and 2 were very small, their absolute configuration could not be determined. Instead, they are assumed by biosynthetic similarity with terpenes obtained by terpene cyclases of D. discoideum. The determination of their absolute configuration should be made by de novo synthetic studies in the future. On the other hand, firmibasiol (2) showed moderate osteoclast-differentiation inhibitory activity, and can be used as a seed compound for anti-osteoporosis drugs. Therefore, these cellular slime molds are promising sources of new natural product chemistry.

Isolation of Mucoroidiol (1)
The fruiting bodies (dry weight 80 g) of D. mucoroides Dm7 were collected after cultured in A-medium with 0.5 mM Zinc (II) chloride. They were extracted three times with methanol at room temperature to give an extract (10 g), which was then partitioned between ethyl acetate and water to yield ethyl acetate solubles (2.8 g). The ethyl acetate solubles were chromatographed over silica gel, and the column was eluted with hexane-ethyl acetate mixtures with increasing polarity to afford hexane-ethyl acetate (1:3) eluent (fraction A, 67 mg). Fraction A was further separated by octadecyl silica gel column using water-acetonitrile solvent system to give water-acetonitrile (1:1) elutant (fraction B, 28 mg). Fraction B was subjected to recycle preparative HPLC (column, GPC-T-2000 (φ 20 mm × 600 mm, YMC Co., Ltd.); solvent, ethyl acetate) to give mucoroidiol (1)

Isolation of Firmibasiol (2)
The fruiting bodies (dry weight 48 g) of D. firmibasis 91HO-33 were collected after cultured in A-medium. They were extracted three times with methanol at room temperature to give an extract (11 g), which was then partitioned with ethyl acetate and water to yield ethyl acetate solubles (2.3 g). The ethyl acetate solubles were chromatographed over silica gel and the column was eluted with hexane-ethyl acetate mixtures of increasing polarity to afford hexane-EtOAc (17:3) eluent (fraction C, 356 mg). Fraction C was separated by ODS column using water-acetonitrile solvent system to give water-acetonitrile (1:4) elutant (fraction D, 20 mg). Fraction D was further separated by silica gel column using chloroform to give firmibasiol (2)

Measurement of Minimum Inhibitory Concentration (MIC)
The Gram-positive bacteria methicillin-susceptible Staphylococcus aureus (MSSA; strain ATCC29213), methicillin-resistant S. aureus (MRSA; ATCC43300) and the Gram-negative bacterium Escherichia coli (ATCC25922) were used in this study. The bacteria suspended in Mueller-Hinton broth (5 × 10 5 CFU/mL; 0.1 mL/well) were incubated for 24 h at 37 • C in 96-well plates (Corning, NY, USA) in the presence of vehicle, various concentrations of serially diluted test compounds, or known antibiotics; MIC was defined as the lowest concentration of the additives that inhibited visible bacterial growth.

Osteoclast Differentiation Experiments
RAW264 cells were grown in α-MEM containing 10% fetal bovine serum and passaged every 3 days. To differentiate into osteoclast, RAW264 cells were seeded at 4 × 10 3 cells/well in 96-well plates and cultured in the presence of RANKL (50 ng/mL) and each compound for 4 days. The cells were sequentially fixed with 10% formalin for 10 min and ethanol for 1 min, and then dried. To measure the activity of tartrate-resistant acid phosphatase (TRAP), which is a marker enzyme of osteoclastogenesis, fixed cells were incubated in 100 µL of citrate buffer (50 mM, pH 4.6) containing 10 mM tartrate and 5 mM p-nitrophenylphosphate for 30-60 min and the reaction mixtures were transferred into another well containing 100 µL of 0.1 M NaOH solution. The absorbances at 405 nm were measured as TRAP activity.

Conflicts of Interest:
The authors declare no conflict of interest.