(±)-Peniorthoesters A and B, Two Pairs of Novel Spiro-Orthoester en-antiomers With an Unusual 1,4,6-Trioxaspi-ro[4.5]decane-7-One Unit From Penicillium minioluteum

(±)-Peniorthoesters A and B (±1 and ±2), two pairs of unprecedented spiro-orthoester enantiomers with a 1,4,6-trioxaspiro[4. 5]decane-7-one unit, were obtained from Penicillium minioluteum. Their structures were determined by spectroscopic methods, X-ray diffraction analyses, and ECD calculations. (±)-Peniorthoesters A and B are the first examples of spiro-orthoester enantiomers, and they represent the first spiro-orthoesters originating from fungi. All compounds showed potential inhibitory activities comparable to dexamethasone against NO production with IC50 values ranging from 14.2 to 34.5 μM.


INTRODUCTION
Orthoesters, a special functional group characterized by three alkoxy groups attached to a single carbon atom, are unusual structural subunits in natural products (Liao et al., 2009). Natural occurring orthoesters include several major types, such as daphnane diterpenoid orthoesters (He et al., 2002), limonoid orthoesters (Roy and Saraf, 2006), steroid orthoesters (Steyn and van Heerden, 1998), and coumarinoid orthoesters (Santana et al., 2004). In our previous study on the plant Wikstroemia chamaedaphne, three new daphnane type diterpenoids with orthoester group were isolated (Guo et al., 2015). A literature investigation revealed that most of these orthoesters originate from plants, and only a few originate from fungi, such as novofumigatonin, a meroterpenoid orthoester from Aspergillus novofumigatus (Rank et al., 2008). As a special class of natural products, orthoesters have attracted great attention due to their diverse structures and biological properties (Liao et al., 2009;Bourjot et al., 2014;Li et al., 2015;Liu et al., 2017).
Fungi have historically played an important role in drug discovery. The genus Penicillium has been shown to be a rich source of structurally unique and biologically active secondary metabolites (Meng et al., 2016;Sun et al., 2016;Luo et al., 2017) and many metabolites from Penicillium are clinically used drugs with penicillin as a representative compound. Previous studies on the secondary metabolites of Penicillium minioluteum have resulted in the identification of scores of bioactive metabolites, including isomeric furanones with cytotoxic activities against HeLa cell lines (Tang et al., 2015), sesquiterpene-conjugated amino acids with cytotoxic activities against HepG2 cells (Ngokpol et al., 2015), and hybrid polyketide-terpenoids (Iida et al., 2008). This fungus was also used to produce clovane derivatives, which are the raw materials for the synthesis of rumphellclovane A (Gontijo de Souza et al., 2012), and an enzyme from this fungus was used in the bioconversion (Kmiecik and Zymanczyk-Duda, 2017). During our ongoing search for structurally unique and biologically interesting constituents from fungi Chen et al., 2017;Zhou et al., 2017), P. minioluteum, obtained from China General Micro-biological Culture Collection Center (CGMCC), was phytochemically investigated, and two pairs of new orthoesters (Figure 1, compounds ±1 and ±2) were isolated along with their biosynthetic intermediate, sclerotinin A (3) (Xiao et al., 2009). The structures and absolute configurations of (±)-1 and (±)-2 were determined by a combination of spectroscopic methods, X-ray diffraction analyses, and ECD calculations. (±)-Peniorthoesters A (±1) and B (±2) are the first examples of spiro-orthoester enantiomers and represent the first spiro-orthoesters originating from fungi.

Fungal Material and Fermentation
The strain in this work was bought from China General Microbiological Culture Collection Center (CGMCC). A voucher Specimen was preserved in the herbarium of Huazhong University of Science and Technology, China. The fungal strain was cultured on potato dextrose agar (PDA) at 28 • C for 8 days to prepare the seed culture. Then the strain was inoculated into 200 Erlenmeyer flasks (1 L each) which had previously been sterilized by autoclaving. Each flask contained 250 g rice and 200 mL distilled water. The flasks were incubated at 20 • C for 26 days.

Computational Details
The crystal structure of 9R,10S,11S-1, and 9R,10R,11R-2 were optimized at the B3LYP/6-31G(d) level in acetonitrile with the IEFPCM solvation model using Gaussian 09 program. The harmonic vibrational frequencies were calculated to confirm the stability of the optimized structure. The electronic FIGURE 1 | Structures of (±)-peniorthoesters A (±1) and B (±2). circular dichroism (ECD) spectrum were calculated using the TDDFT methodology at the LC-wPBE/6-311++G(d,p) level with acetonitrile as solvent by the IEFPCM solvation model implemented in Gaussian 09 program. The ECD spectra was simulated using a Gaussian function with a bandwidth σ of 0.3 eV. The UV correction was applied to generate the final spectra (Zhu, 2015).

Determination of No Production
RAW 264.7 cells were obtained from the Boster Biological Technology Co., Ltd (Wuhan, China) and maintained in DMEM containing 10% heat-inactived fetal bovine serum (FBS) (Gibco BRL Co, Grand Island, NY, United States) at 37 • C in humidified incubator containing 5% CO 2 . All tested compounds were dissolved in DMSO (the final concentration of DMSO was  <0.25% in assay). RAW 264.7 cells were seeded into 48-well plates (1 × 10 5 cells/well) for 24 h and then pretreated with different concentrations (1-40 µM) of test compounds. After being incubated for 3 h, the cells were stimulated with 100 ng/ml LPS (final concentration) for another 24 h. Dexamethasone was used as the positive control in the experiment. NO content in the supernatant was measured using Griess reagent. The absorbance at 540 nm was measured on a microplate reader. All the experiments were performed in three independent replicates.

Cytotoxic Activity
Cell lines were cultured in RPMI-1640 or DMEM medium (HyClone, USA), supplemented with 10% fetal bovine serum (HyClone, USA) at 37 • C in a humidified atmosphere with 5% CO 2 . For cell viability assay, cells were plated into 96-well plates in 50 µl of medium and then compounds were serially diluted in medium and delivered to the cells as 2 × solutions in 50 µl of medium. After 48 h, cell viability was detected by a CCK-8 Kit (Dojindo,Japan) according to the instruction. Growth relative to untreated cells was calculated (positive control, anticancer drug VP16), and this data was used for the dose-response curve, the IC 50 (50% inhibiting concentration) of compounds to each cell lines were calculated by SPSS.

RESULTS AND DISCUSSIONS
Compound 1 was isolated as a white powder. Its UV spectrum exhibited absorption maxima at 216 and 260 nm. Its IR spectrum indicated the presence of an OH functionality (3,435 cm −1 ), a conjugated carbonyl group (1,661 cm −1 ), and an aromatic ring (1,612 and 1,456 cm −1  123.7, 123.5, and 103.5], one oxygenated quaternary carbon (δ C 124.0), six methyl groups and three methines (including two oxygenated ones). The above analyses confirmed the presence of an ester carbonyl group and a hexa-substituted benzene ring, which account for five degrees of unsaturation, indicating the presence of two additional rings. With the aid of the HSQC spectrum, all protons were unambiguously assigned to their respective carbons. The planar structure of 1 was elucidated on the basis of 1 H-1 H COSY and HMBC experiments (Figure 2). The HMBC spectrum of 1 displayed correlations from H 3 -14 to C-3, C-4, and C-5; from H 3 -13 to C-2, C-3, and C-4; from H 3 -12 to C-1, C-2, and C-3; and from H-9 to C-1, and C-6, which together with the HMBC correlations from the OH to C-4, C-5, and C-6 constructed the hexa-substituted benzene ring. In addition, two spin systems of H 3 -17/H-11/H-10/H 3 -16 and H-9/H 3 -15 were established from the 1 H-1 H COSY spectrum. Therefore, the HMBC correlations from H-9 to C-1, C-6, and C-8 and from H 3 -15 to C-1, C-8, and C-9 suggested the C-15/C-9/C-8 fragment was connected to the benzene ring via C-9. Moreover, the ester carbonyl (δ C 170.3) was connected to C-6 based on the chemical shifts of C-6 (δ C 103.5), C-1 (δ C 137.8), and C-3 (δ C 145.7). Combined with the chemical shifts of C-10 (δ C 81.7) and C-11 (δ C 79.3), the C-17/C-11/C-10/C-16 fragment was proposed to be a 2,3butanediol unit, which should be linked with C-8 and form a 4,5-dimethyl-1,3-dioxolane moiety. Finally, a lactone ring was proposed between C-7 and C-8 to satisfy the above deduced tricyclic ring system as well as the chemical shift of C-8 (δ C 124.0). This planar structure satisfied all of the correlations observed in the 2D NMR spectra and the chemical shifts in the 1D NMR spectra.
A NOESY experiment was performed on 1, but no interactions useful for determining the relative configuration were observed. Unfortunately, the relative configuration of H-10 and H-11 could also not be determined from their coupling constants because they were located on a five-membered ring. To confidently assign the configuration of 1, we tried to crystallize it so we could use X-ray single-crystal analysis. After a number of attempts, a high-quality single-crystal of 1 was finally obtained from a mixture of methyl alcohol and water. The X-ray crystallography data (CCDC 1840165) obtained with Cu Kα radiation confirmed the structure of 1 (Figure 3). However, because it has a centrosymmetric monoclinic space group of chiral P2 1 /c, indicating the crystal is racemic, the absolute configuration of 1 could not be determined. After analyses by using various types of chiral columns, the racemic nature of this solution was further confirmed by the presence of two peaks in the HPLC chromatogram acquired using a chiral Daicel IC column (Figure 4). Finally, compounds (+)-1 and (-)-1 were successfully obtained, and they showed specific rotations with opposite signs {(+)-1: [α] 20 D +37 (c 0.1, CH 2 Cl 2 ); (-)-1: [α] 20 D -36 (c 0.1, CH 2 Cl 2 )}. In addition, the ECD spectra of (+)-1 and (-)-1 displayed similar signal intensities but mirror-image Cotton effects (Figure 5).
Compound 2, obtained as a white powder, possesses the same molecular formula (C 17 H 22 O 5 ) as that of compound 1 based on its HRESIMS data with an [M + H] + ion peak at m/z 307.1534 (calcd for C 17 H 23 O 5 , 307.1545). A detailed comparison of its NMR spectroscopic data with those of 1 indicated that the main differences between 1 and 2 were tiny changes in the chemical shifts of C-10 and C-11 as well as their protons [δ C 81.1 (C-10), 79.8 (C-11); δ H 3.84 (1H, dq, J = 8.1, 6.2 Hz, H-10), 4.36 (1H, dq, J = 8.1, 6.2 Hz, H-11) in 2; δ C 81.7 (C-10), 79.3(C-11); δ H 3.91 (1H, dq, J = 8.5, 6.1 Hz, H-10), 4.18 (1H, dq, J = 8.5, 6.1 Hz, H-11) in 1]. These findings, combined with the 2D NMR data, implied that 2 has the same planar structure as 1, and it should be a stereoisomer of 1. The planar structure of 2 was further confirmed by analyses of its 1 H-1 H COSY and HMBC spectra. Unfortunately, the NOESY experiment of 2 also did not show any NOESY correlations useful in for the elucidation of the relative configuration of compound 2.
To the best of our knowledge, (±)-1 and (±)-2 are the first examples of spiro-orthoester enantiomers with an unusual 1,4,6-trioxaspiro[4.5]decane-7-one unit, and they represent the first spiro-orthoesters originating from fungi. The proposed biosynthetic pathway of 1 and 2 was outlined in Figure 6. First, the condensation of acetyl-CoA and four molecules of malonyl-CoA by a polyketide synthase formed i, which underwent cycloaddition and methylations to form precursor sclerotinin A (3). Then, sclerotinin A underwent isomerization and hydrolytic cleavage to afford iv, which further formed vi by a H + mediated double bond isomerization. After that, intermediate vii was produced by an aldol condensation, which further generated the key intermediates viii and x via an esterification reaction with 2R,3R-butanediol and 2S,3Sbutanediol (Ji et al., 2011), respectively. Finally, compounds (±)-1 and (±)-2 were formed via condensation and lactonization reactions.

CONCLUSION
In conclusion, two pairs of new spiro-orthoester enantiomers, (±)-peniorthoesters A and B (±1 and ±2), were isolated from P. minioluteum. These compounds, characterized by an unexpected 1,4,6-trioxaspi-ro[4.5]decane-7-one unit, are the first examples of spiro-orthoester enantiomers, and they represent the first spiro-orthoesters originating from fungi. All of them showed potential inhibitory activities against NO production in activated macrophages with IC 50 values ranging from 14.2 to 34.5 µM, which are comparable to the positive control, dexamethasone. Their highly functionalized structures and promising biological activities will attract considerable attention from the pharmacological, synthetic, and biosynthetic communities.

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.

AUTHOR CONTRIBUTIONS
XL and CC conducted the main experiments and wrote the manuscript. YiZ, MZ, and QZ carried out bioassays. JL did the ECD calculations. JW and ZL analyzed the spectroscopic data. HZ and YoZ initiated and oversaw all research. All authors reviewed the manuscript.

SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fchem. 2018.00605/full#supplementary-material The Supplementary Material includes Full NMR, HRESIMS, UV, and IR spectra of 1 and 2; detailed of the ECD calculations of 1 and 2; X-ray data of 1 and 2 (PDF); and crystallographic data (CIF).