Nocapyrones: α- and γ-Pyrones from a Marine-Derived Nocardiopsis sp.

One new α-pyrone (nocapyrone R (1)), and three known γ-pyrones (nocapyrones B, H and L (2–4)) were isolated from the culture extract of a Nocardiopsis strain collected from marine sediment. Structures of these compounds were determined on the basis of spectroscopic data including NMR and MS. γ-Pyrones 2–4 were found to induce adiponectin production in murine ST-13 preadipocyte cells but the α-pyrone 1 had no activity. The absolute configuration of the anteiso-methyl branching in 4 was determined by HPLC comparison of a degraded product of 4 with standard samples as a 2:3 enantiomeric mixture of (R)- and (S)-isomers.


Introduction
Natural products have been playing an important role for the development of novel therapeutics owing to their enormous and unpredictable structural diversity [1]. Specifically, actinomycetes are continuously providing structurally diverse secondary metabolites possessing pharmaceutically useful bioactivities. More than 70% of microbial antibiotics have been discovered from actinomycetes [2]. In recent years, in addition to the terrestrial species, actinomycetes from marine environments are attracting a substantial attention as a new resource of novel drug candidates [3]. The differences between the marine and terrestrial environments are likely reflected in the genetic divergences. Marine-derived actinomycetes are recognized to actually produce a range of chemically distinctive secondary metabolites that terrestrial actinomycetes are not able to produce [4]. Consequently, the number of novel metabolites from marine actinomycetes has been increasing in recent years.
The increasing incidents of type 2 insulin-resistant diabetes, as a result of growing obesity rates, are causing a serious social and economic problem [5]. Adipose tissue secretes various types of biologically active adipokines, such as free fatty acids, tumor necrosis factor-α, adiposin, resistin, and leptin, to regulate energy homeostasis [6]. Adiponectin is one of such adipokines secreted exclusively by mature adipocytes [7] and this proteineous substance functions to regulate the glucose and lipid metabolism [8]. Recent research revealed that the mRNA expression of adiponectin is reduced in obese diabetic murine model [9] and type 2 diabetic patients [10][11][12], and also plasma level of adiponcetin is significantly lower in obese diabetic mice and humans [13][14][15]. Therefore, the replenishment of adiponectin by transcriptional induction in adipocytes is believed to provide a new effective therapeutic approach to insulin resistance, type 2 diabetes, and related diseases. Thiazolidinediones represented by rosiglitazone (Avandia) and pioglitazone (Actos) are widely-used orally available drugs for type 2 diabetes. These synthetic compounds activate transcription by PPARγ (peroxisome proliferator activated receptor γ) primarily in adipose tissues and induce the elevation of adiponectin plasma level. Meanwhile, several research groups have demonstrated that serious side effects are caused by rosiglitazone; the use of this drug is associated with a 43% increase in myocardial infraction and 64% increase in the risk of death from cardiovascular causes [16]. Thus, the development of side effect-free new agents for type 2 diabetes is desperately desired. In our screening program for discovering new lead scaffolds for adiponectin inducers from natural products [17][18][19], a marine-derived Nocardiopsis strain was found to produce a new α-pyrone (1) along with three known γ-pyrones, nocapyrones B, H, and L (2-4, Figure 1). Interestingly, nocapyrone L (4) was isolated as an enantiomeric mixture, which is noticeable in marine natural products. Herein, we describe isolation, structure determination, and biological activities of these compounds.

Isolation
The producing strain TP-A0876 was isolated from a sediment sample collected at −775 m in Ishikari gulf, Hokkaido, Japan. On the basis of the result of 16S rRNA gene sequence similarity, the isolate was identified as Nocardiopsis sp. The strain TP-A0876 was cultured in A-3M medium at 30 °C for 6 days and the whole culture broth was extracted with 1-butanol. The crude extract was subjected to silica gel and ODS column chromatographies, followed by HPLC purification, to yield nocapyrone R (1) along with nocapyrones B, H, and L (2-4), three known compounds previously isolated from marine Nocardiopsis strains by other groups [20,21] S2 and S4). The NMR spectra of nocapyrone R (1) showed the resonances for a pyrone unit as well as two methyl (C-13 and C-15) and one methoxy (C-14) substituents (Table 1, Supplementary Figures S1 and S2): however, the UV spectrum showing absorption maximum at λ max 290 nm was different from that for γ-pyrone (2-4: UV λ max 252 nm). Furthermore, the ester carbonyl absorption of IR spectrum and the ester carbonyl signal of 13 C NMR suggested the presence of an ester/lactone functionality. Protons of two olefinic methyl groups (H 3 -13 and H 3 -15) had HMBC correlations with C-3, to which was correlated the methoxy protons (H 3 -14), establishing the α-pyrone substructure with a methoxy group at C-3 flanked by two olefinic methyl groups (Supplementary Figure S5). The 1 H-1 H COSY spectrum provided two fragments, H 2 -6/H 2 -7/H 2 -8/H 2 -9 and H 3 -11/ H-10/H 3 -12. These two fragments were assembled into an alkyl chain bearing an isopropyl terminus by HMBC correlations from H 3 -12 to C-9, C-10, and C-11. This was consistent with the presence of two doublet methyl proton signals for H 3 -11 and H 3 -12 ( Supplementary Figures S3 and S5). The alkyl side chain was attached to C-5 by HMBC correlations from H 2 -6 to C-4 and C-5, completing the structure of 1 (Figure 2).

Absolute Configuration of Nocapyrone L (4)
Nocapyrone L (4) has been reported as a secondary metabolite of Nocadiopsis sp. isolated from a mollusk as a symbiotic bacterium [21]. Due to the remoteness of the branched methyl chiral center (C-10) from the modifiable functional groups, the absolute configuration of 4 had not been assigned. From the structural analogy of 4 to germicidin A, we hypothesized that 4 could be biosynthesized through condensation of an amino acid-derived starter with malonate extenders [22]. Consequently, the anteiso-subunit known to be originated from L-isoleucine is assumed to have S configuration. Interestingly, the specific rotation of 4 we isolated showed

Biological Activities
Activity of compounds 1-4 to promote adiponectin-production in adipocyte cells was assessed by measuring the mRNA expression level of adiponectin-coding gene [25]. After the treatment of murine ST-13 preadipocyte cells with the compounds for 11 days, the total mRNA was subjected to quantitative real-time PCR analysis. All the γ-pyrones (2-4) enhanced the expression of adiponectin mRNA in a concentration-dependent manner while the α-pyrone (1) showed no such effect ( Figure 5). Especially, 2 and 4 more strongly induced adiponectin production at 8 μM and at the same concentration the accumulation of lipid droplets were observed ( Figure 6, oil red O staining) that indicates the differentiation to mature adipocyte cells. Compound 2 was further examined for the transcriptional activation of PPARγ that is the main target of the antidiabetic drug, thiazolidinediones. In the luciferase reporter assay, troglitazone activated the transcription through PPARγ but 2 did not (Figure 7), confirming that 2 is not a ligand for PPARγ and its mode of action is different from thiazolidinediones. These effects on adipocytes or adiponectin-inducing activity have not been described for nocapyrones and other related pyrone compounds.

Microorganism
Strain TP-A0876 was isolated from a sediment sample collected at a depth of 775 m off the island of Hokkaido (N 42°56′80″, E 140°06′32″), Japan in 2006 (August), using a piston corer. The strain was identified as a member of the genus Nocardiopsis on the basis of 100% 16S rRNA gene sequence (1455 nucleotides; GenBank accession number AB488799) similarity to Nocardiopsis sp. 123 (accession number AY036002).

Determination of the Absolute Configuration of the Anteiso-Methyl Group in 4 by Ohrui-Akasaka Method
Oxidative Degradation of 4 to Yield 5: A solution of nocapyrone L (4, 4 mg, 15 μmol) in a mixture of MeCN (320 μL) and H 2 O (240 μL) was stirred with NaIO 4 (19.2 mg, 89 μmol) until the salt was dissolved. To this solution were added CCl 4 (320 μL) and 1 mg/mL solution of RuCl 3 hydrate in 0.1 M sodium phosphate buffer (240 μL, pH 7.6) and the biphasic mixture was vigorously stirred at room temperature for 17 h. The reaction mixture was passed through Celite and the filter cake was washed with MeCN. After evaporation of the organic solvent from the filtrate, the aqueous solution was acidified with 2 M HCl and extracted with EtOAc. The EtOAc layer was concentrated and the remaining material was dissolved in MeOH (1 mL) and THF (1 mL). To this solution was added 1 M NaOH (1 mL) and the mixture was stirred at room temperature for 12 h. The reaction mixture was then acidified with 2 M HCl and extracted with EtOAc. The EtOAc layer was washed with water and brine, dried over anhydrous Na 2 SO 4 , and concentrated in vacuo to give 6-methyloctanoic acid (5, 1.2 mg) which was used for the next reaction without further purification: 1

Biological Activity Study
Adipocyte differentiation assay was carried out according to the procedure previously described [25].

Oil Red O Staining
ST-13 cells treated with nocapyrones B and L for 11 days were washed three times with phosphate-buffered saline (PBS), fixed with 10% formalin neutral buffer solution (Wako Pure Chemical, Osaka, Japan) at room temperature for 10 min, and then washed with distilled water to remove formalin solution. Furthermore, the cells were rinsed with 60% 2-propanol for 5 min, stained with 0.24% oil red O at room temperature for 20 min, and then were photographed under a phase contrast microscope (×100 magnification) equipped with a CCD camera (Leica Microsystems Japan, Tokyo, Japan).

Luciferase Reporter Assay
An expression plasmid containing the ligand binding domain of human PPARγ fused to the GAL4 DNA binding domain (pPPARγ-GAL4), and the luciferase reporter plasmid, 17m2G TATA Luc (p17m2G), were kindly donated by Dr. S. Kato (University of Tokyo, Tokyo, Japan). We transiently transfected COS-1 cells (6 × 10 5 cells) with pPPARγ-GAL4 (0.25 μg) and p17m2G (1 μg) using Effectene Transfection Reagent (QIAGEN, Tokyo, Japan). Transfections were performed in triplicate in 24-well plates according to the manufacture's instructions. After 16 h, the transfected cells received the indicated concentrations of nocapyrones B (or troglitazone), and were cultured for additional 24 h at 37 °C in a 5% CO 2 incubator. Then cells were harvested and luciferase activities were measured by a Steady-Glo ® Luciferase Assay System (Promega, Madison, WI, USA) according to manufacturer's instructions.

Conclusions
In this study, a marine-derived actinomycete Nocardiopsis sp. TP-A0876 was isolated from a sediment sample and its secondary metabolites were investigated. Purification from the 1-butanol extract of the culture broth resulted in isolation of one α-pyrone and three γ-pyrones. Based on the spectroscopic analysis, the α-pyrone was confirmed as a new compound, whereas other three γ-pyrones were identified as nocapyrones B (2), H (3), and L (4). Biological testing in a set of assays proved that γ-pyrone compounds (2-4) have ability to induce preadipocyte differentiation and adiponectin production in murine ST13 preadipocyte cells. Since the α-pyrone compound 1 showed no activity, the γ-pyrone structure is proposed to play a key role in this activity.
A large number of pyrone-containing marine natural products are known [26][27][28][29][30][31][32]. Specifically, a series of nocapyrones (A-Q) have been continuously reported from marine-derived Nocardiopsis strains with a wide range of bioactivity (Figure 8). Nocapyrones B (2) and H (3) modulate nerve cell depolarization, activate or inhibit Ca 2+ flux into a panel of human cells overexpressing various transient receptor potential (TRP) channels depending upon the agent and channel subtype, together with cytotoxicity against human breast adenocarcinoma, but have no antibacterial activity [21]. Nocapyrones E-G are described to have modest antibacterial activity against Bacillus subtilis [30]. Nocapyrone H* inhibits NO production in LPS-stimulated BV-2 microglial cells and neuro-protective effect [31], and nocapyrones H*, I*, and M* were shown to suppress gene expression in quorum sensing [32]. However, there is no report on the activity of these pyrones for preadipocyte differentiation or adiponectin production. Although the effectiveness is lower than thiazolidinediones, different mode of action of the γ-pyrones may provide an opportunity of drug development from this new therapeutic scaffold for insulin resistance type 2 diabetes. The concise synthetic strategies [33][34][35][36] and interesting bioactivities of structurally related compounds have been reported [37,38]. On the base of these studies, further synthetic and biological studies of nocapyrone families could be inspiring.
The absolute configuration of anteiso-methyl group is known and believed to be S in most cases because it is derived from L-isoleucine. However, the anteiso-methyl branching in 4 was confirmed as a mixture of (R)-and (S)-configurations in a ratio of 2:3. Along with the advances in synthetic and analytical techniques, several enantiomeric mixtures have been discovered in natural products ( Figure 9) [39][40][41]. Many of them are marine-derived: trichodenone A, an antitumor metabolite of Trichoderma harzianum isolated from marine sponge (Halichondria okadai), is an (R)-and (S)-enantiomeric mixture [39]; pericosines B and C, metabolites of Periconia byssoide isolated from sea hare Aplysia kurodai, bearing four chiral centers are also enantiomeric mixtures [40]. Recently, the enantiomeric mixture regarding to the anteiso-methyl asymmetry was found in the insect pheromone.  [41]. To the best of our knowledge, nocapyrone L (4) is the first microbial metabolite proved to comprise an enantiomeric pair of Rand S-configurations at the anteiso-methyl substituent. The sec-methyl group at C-10 of nocapyrones C [20] and M [21] are reported to be an R/S-mixture but these compounds have a hydroxyl group on the neighboring carbon [21]. Biosynthetic origin of their anteiso-methyl portion should be clarified. Further investigation, focused on the biosynthetic precursors and gene analysis with our nocapyrone H (4), are under way to identify the enzymatic reactions responsible for the formation of enantiomeric mixture [42].