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Spirograterpene A, a Tetracyclic Spiro-Diterpene with a Fused 5/5/5/ 5 Ring System from the Deep-Sea-Derived Fungus Penicillium granulatum MCCC 3A00475 Siwen Niu,† Zuo-Wang Fan,† Chun-Lan Xie,† Qingmei Liu,‡ Zhu-Hua Luo,† Guangming Liu,‡ and Xian-Wen Yang*,† †

State Key Laboratory Breeding Base of Marine Genetic Resources, Key Laboratory of Marine Genetic Resources, Fujian Key Laboratory of Marine Genetic Resources, South China Sea Bio-Resource Exploitation and Utilization Collaborative Innovation Center, Third Institute of Oceanography, State Oceanic Administration, 184 Daxue Road, Xiamen 361005, People’s Republic of China ‡ College of Food and Biological Engineering, Jimei University, 43 Yindou Road, Xiamen 361021, People’s Republic of China S Supporting Information *

ABSTRACT: A novel spiro-tetracyclic diterpene, spirograterpene A (1), was isolated from the deep-sea-derived fungus Penicillium granulatum MCCC 3A00475, together with two biosynthetically related cyclopianes, conidiogenone I (2) and conidiogenone C (3). The relative configuration of 1 was elucidated by extensive spectroscopic analyses, and the absolute structure was established by the modified Mosher’s method. Compound 1 is the second example of a diterpene featuring a 5/5/5/5 spirocyclic carbon skeleton. It showed modest antiallergic activity.

C

yclopianes are tetracyclic diterpenes featuring a highly fused and strained 6/5/5/5 ring system. They were first reported from the fungus Penicillium cyclopium in 2002.1 Heretofore, a total of 10 cyclopianes have been obtained in nature, solely from the Penicillium species.1−3 Although very few have been found, they have shown significant biological activities. For example, under the concentration of 20 ng/mL, conidiogenone and conidiogenol exhibited potent conidiationinducing activity.1 Conidiogenone C exhibited potent selective cytotoxicity against human leukemia (HL-60) cells, with an IC50 value of 38 nM.2 Conidiogenone B showed significant antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA) with an MIC value of 8 μg/mL.3 Provoked by the novelty of their structures and the potent bioactivities, conidiogenone, conidiogenol, and conidiogenone B were synthesized.4 In our continuing endeavor to discover biologically important and structurally unique secondary metabolites from deep-sea-derived microorganisms,5−7 the extract of Penicillium granulatum MCCC 3A00475 showed promising antiallergic activity. Subsequently, this strain was subjected to a large-scale fermentation, followed by a bioassay-guided isolation and purification. As a result, a rare spirocyclic diterpene, spirograterpene A (1), was obtained along with two known related compounds, conidiogenone I (2) and conidiogenone C (3).2,3 Herein, we report the isolation, structure elucidation, and antiallergic activities of these three compounds. © 2017 American Chemical Society and American Society of Pharmacognosy

Spirograterpene A (1) was obtained as a colorless oil. Its molecular formula was established as C20H30O3 on the basis of the protonated molecule peak at m/z 319.2242 [M + H]+ in its HRESIMS spectrum, requiring six degrees of unsaturation. The 1 H NMR spectrum exhibited two methyl doublets (δH 0.87, d, J = 6.9 Hz, Me-17; 1.00, d, J = 6.9 Hz, Me-16), two methyl singlets (δH 1.17, s, Me-19; 1.22, s, Me-18), and one oxygenated methine (δH 3.80, dt, J = 8.1, 7.0 Hz, H-3). The 13 C NMR spectrum in association with the HSQC spectrum indicated 20 carbon signals ascribed to four methyls (δC 13.5, 14.9, 21.7, and 30.4, for Me-16, 17, 19, and 18, respectively), six sp3 methylenes, four sp3 methines (one oxygenated at δC 78.5, C-3), and six nonprotonated carbons, including one carbonyl (δC 182.3, C-20), two olefinic (δC 147.3, C-6; 149.1, C-7), and three aliphatic (δC 54.2, C-14; 57.7, C-11; 61.0, C-5) carbons Received: June 3, 2017 Published: July 10, 2017 2174

DOI: 10.1021/acs.jnatprod.7b00475 J. Nat. Prod. 2017, 80, 2174−2177

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In the NOESY spectrum, correlations from Me-18 to H-15 and the absence of a correlation between Me-18 and Me-19 revealed the relative configurations of C-11, C-14, and C-15 were identical to conidiogenone I (2) and conidiogenone C (3). Meanwhile, NOESY correlations from Me-18 to Me-17/H10b (δH 2.00), from Me-19 to Me-16/H-8a (δH 2.28), from H3 to H-10a (δH 2.12), and from H-1 to H-4 deduced Me-18, Me-17, M-16, and H-3 were on the same plane, which was opposite Me-19 (Figure 1). Therefore, the relative configuration of 1 was defined as 1R*, 3R*, 4R*, 5R*, 11R*, 14S*, and 15R*, respectively. In order to determine the absolute configuration of 1, the modified Mosher’s method was performed, which established the 3R configuration (Figure 2).8,9 Accordingly, the remaining stereogenic centers were

(Table 1). Because one carbonyl group and two olefinic carbons accounted for two degrees of unsaturation, a four-ring Table 1. 1H (600 MHz) and 13C (150 MHz) NMR Spectroscopic Data of 1 in CD3OD position 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

δC, type 44.0, 42.6, 78.5, 54.8, 61.0, 147.3, 149.1, 30.6, 39.4, 48.6, 57.7, 40.6, 39.4, 54.2, 63.0, 13.5, 14.9, 30.4, 21.7, 182.3,

CH CH2 CH CH C C C CH2 CH2 CH2 C CH2 CH2 C CH CH3 CH3 CH3 CH3 C

δH, mult (J in Hz) 2.06, m 1.74−1.76, m 3.80, dt (8.1, 7.0) 1.68, m

2.28, m; 2.16, m 2.14−2.19, m 2.12, m; 2.00, dt (16.1, 2.5) 1.65, m; 1.57, m 2.17, m; 1.57, m 2.81, 1.00, 0.87, 1.22, 1.17,

d (1.8) d (6.9) d (6.9) s s

skeleton was required for the structure of 1. In the COSY spectrum, three isolated spin systems were observed as Me-17 (δH 0.87)/H-1 (δH 2.06)/H2-2 (δH 1.74−1.76)/H-3 (δH 3.80)/ H-4 (δH 1.68)/Me-16 (δH 1.00), H2-8 (δH 2.28, 2.16)/H2-9 (δH 2.17), and H2-12 (δH 1.65, 1.57)/H2-13 (δH 2.14−2.19, 1.57). Therefore, three structural fragments were deduced as C-17/C1/C-2/C-3/C-4/C-16, C-8/C-9, and C-12/C-13 (Figure 1). In the HMBC spectrum, the key correlations from both Me-16 and Me-17 to C-5 (δC 61.0, s) established the presence of ring A. The HMBC cross-peaks from H2-8 to C-5/C-6 (δC 147.3)/ C-7 (δC 149.1) and from H2-9 to C-6/C-7 constructed ring B. In addition, HMBC correlations from H2-10 (δH 2.12, 2.00) and H-15 (δH 2.81) to C-6 and C-7 revealed the presence of ring C. Finally, ring D was deduced by the HMBC correlations from Me-18 to C-10 (δC 48.6)/C-11 (δC 57.7)/C-12 (δC 40.6)/C-15 (δC 63.0) and from Me-19 to C-13 (δC 39.4)/C14 (δC 54.2)/C-15/C-20 (δC 182.3). Therefore, the planar structure of 1 was established as a rare spirocyclic diterpene with a 5/5/5/5 ring system.

Figure 2. ΔδR−S data of the MPA esters for 1 in CDCl3.

assigned as 1R, 4R, 5R, 11R, 14S, and 15R. Interestingly, a compound bearing the same carbon skeleton, spiroviolene, was reported very recently.10 The only difference lies in the configuration at C-4 bearing the 16-methyl group. In 1 the methyl groups on ring A are cis, whereas in spiroviolene they are oriented trans to each other. This is very interesting, as spiroviolene was obtained from a bacterial terpene synthase, whereas spirograterpene A was isolated from a fungal culture. A plausible biosynthesis pathway for the formation of 1 and 2 was proposed in Scheme 1. They might be synthesized from the same intermediate 4 resulting from cyclization of GGPP.10 Spirograterpene A (1) could be constructed by deprotonation of 4, followed by hydroxylation and oxidation. Ring expansion of 4, a subsequent methyl shift, trapping of the carbocation with H2O, and further oxidation finally could produce conidiogenone I (2).

Figure 1. Key COSY, HMBC, and NOESY correlations of 1. 2175

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MeOH in H2O and then extracted with petroleum ether (PE) four times. The MeOH layer was concentrated to provide a defatted extract (4.2 g). The extract was chromatographed over ODS using gradient elution of H2O−MeOH (5% → 100%) to get four fractions (Fr.1− Fr.4). Fr.2 (206 mg) was subjected to CC on Sephadex LH-20 (CHCl3−MeOH, 1:1), followed by repeated CC over silica gel using a gradient of CHCl3−MeOH (20:0 → 5:1) and CHCl3−acetone (15:1 → 1:1) to get subfraction Fr.2-1. Final purification by preparative TLC (PTLC, CHCl3−acetone, 5:1) yielded 2 (2.5 mg). Fr.3 (636 mg) was separated subsequently by CC over Sephadex LH-20 (CHCl3− MeOH, 1:1) and silica gel (CHCl3−MeOH, 20:0 → 5:1) and finally purified by CC over silica gel with the mobile phase of CHCl3− acetone (25:1 → 5:1) to obtain 1 (10.5 mg). Fr.4 (346 mg) was first separated by CC over Sephadex LH-20 (CHCl3−MeOH, 1:1), then CC eluting with PE−EtOAc (20:1 → 1:1) followed by CHCl3− acetone (50:1 → 10:1), and finally purified using PTLC (PE−EtOAc, 5:1) to get 3 (3.1 mg). Spirograterpene A (1): colorless oil; [α]25D −22.2 (c 0.35, MeOH); IR (CHCl3) νmax 3684, 3622, 3288, 3011, 2976, 2400, 2259, 1632, 1324, 1233, 1088, 782, 641 cm−1; 1H and 13C NMR data, Table 1; HRESIMS m/z 319.2242 [M + H]+ (calcd for C20H31O3, 319.2273). Conidiogenone I (2): colorless oil; [α]25D −19.2 (c 0.13, MeOH), lit. [α]20D −19.6 (c 0.33, MeOH);3 HRESIMS m/z 343.2236 [M + Na]+ (calcd for C20H32O3Na, 343.2249). Conidiogenone C (3): colorless oil; [α]25D −11.1 (c 0.10, MeOH), lit. [α]20D −11.9 (c 0.04, MeOH);2 HRESIMS m/z 325.2141 [M + Na]+ (calcd for C20H32O2Na, 325.2143). Preparation of (R)- and (S)-MPA Ester Derivatives of 1. Spirograterpene A (1) (1.0 mg) was dissolved in 600 μL of anhydrous CHCl3. Then N,N′-dicyclohexylcarbodiimide (DCC, 2.0 mg), 4dimethylaminopyridine (DMAP, 2.2 mg), and (R)-MPA (1.5 mg) were added. The reaction mixture was stirred at room temperature for 16 h. The reaction products were separated by CC over silica gel (PE− acetone, 4:1) to get the (R)-MPA ester 1a (0.8 mg). Similarly, the (S)MPA ester (1b, 1.0 mg) was prepared using (S)-MPA (1.5 mg). (R)-MPA ester of 1 (1a): 1H NMR (CDCl3, 400 MHz) δH 7.33− 7.47 (5H, m, phenyl protons), 4.76 (1H, s, CH of MPA), 4.76 (1H, dt, J = 8.9, 3.5 Hz, H-3), 3.43 (3H, s, OMe of MPA), 2.78 (1H, d, J = 1.9 Hz, H-15), 2.17 (1H, m, H-8a), 2.16 (3H, m, H2-9, H-13a), 2.12 (1H, m, H-8b), 2.07 (1H, m, H-10a), 2.01 (1H, m, H-1), 1.95 (1H, m, H10b), 1.86 (1H, m, H-2a), 1.85 (1H, m, H-4), 1.75 (1H, m, H-2b), 1.59 (1H, m, H-12a), 1.56 (2H, m, H-12b, H-13b), 1.19 (3H, s, Me18), 1.12 (3H, s, Me-19), 0.82 (3H, d, J = 6.8 Hz. Me-17), 0.72 (3H, d, J = 6.8 Hz. Me-16); HRESIMS m/z 467.2783 [M + H]+ (calcd for C29H39O5, 467.2797). (S)-MPA ester of 1 (1b): 1H NMR (CDCl3, 400 MHz) δH 7.34− 7.46 (5H, m, phenyl protons), 4.82 (1H, dt, J = 9.2, 3.5 Hz, H-3), 4.76 (1H, s, CH of MPA), 3.42 (3H, s, OMe of MPA), 2.79 (1H, brs, H15), 2.19 (1H, m, H-8a), 2.17 (2H, m, H2-9, H-13a), 2.13 (1H, m, H8b), 2.10 (1H, m, H-10a), 1.95 (2H, m, H-1, H-10b), 1.93 (1H, m, H4), 1.77 (1H, m, H-2a), 1.61 (1H, m, H-12a), 1.57 (2H, m, H-12b, H13b), 1.51 (1H, m, H-2b), 1.19 (3H, s, Me-18), 1.15 (3H, s, Me-19), 0.90 (3H, d, J = 6.9 Hz. Me-16), 0.78 (3H, d, J = 6.9 Hz. Me-17); HRESIMS m/z 489.2612 [M + Na]+ (calcd for C29H38O5Na, 489.2617). Antiallergy Assay. The antiallergic activity was measured for the efficiency of the RBL-2H3 cell degranulation inhibition rate using an IgE-mediated mast cell allergic reaction. Briefly, RBL-2H3 cells were seeded into 96-well cell culture plates to incubate with anti-DNP-IgE. IgE-sensitized RBL-2H3 cells were pretreated with tested compounds (20 μg/mL) for 1 h and stimulated with DNP-BSA. The βhexosaminidase activity was quantified by measuring the fluorescence intensity of the hydrolyzed substrate in a fluorometer.

Scheme 1. Postulated Biogenetic Pathway for 1

Compound 1 showed an antiallergic effect on immunoglobulin E (IgE)-mediated rat mast RBL-2H3 cells with 18% inhibition compared to 35% inhibition for the positive control, loratadine, at the same concentration of 20 μg/mL. Compounds 2 and 3 exhibited weak effects with inhibition of 4% and 10%, respectively, at 20 μg/mL. In summary, from the deep-sea-derived fungus Penicillium granulatum MCCC 3A00475, one new (1) and two known (2 and 3) cyclopiane diterpenes were obtained. Spirograterpene A (1) is the second example of a diterpene bearing a 5/5/5/5 spiro-tetracyclic skeleton, which provides additional evidence demonstrating the deep-sea-derived fungi as unique sources of structurally novel compounds.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were recorded on a Rudolph IV Autopol automatic polarimeter at 25 °C. The IR spectrum was obtained with a Bruker Tensor 27 FTIR spectrophotometer in CHCl3. NMR spectra including 1H, 13C, DEPT, HSQC, COSY, HMBC, and NOESY were measured on a Bruker Avance 600 MHz spectrometer. Chemical shifts were expressed in δ referenced to the solvent peaks of CD3OD at δH 3.31 for 1H and at δC 49.0 for 13C, while CDCl3 at δH 7.27 for 1H and at δC 77 for 13C. HRESIMS data were measured on a Xevo G2 Q-TOF mass spectrometer (Waters). Column chromatography (CC) was performed on ODS (50 μm, Daiso), silica gel (Qingdao Marine Chemistry Co. Ltd.), and Sephadex LH-20 (Amersham Pharmacia Biotech AB). Solvents for isolation were analytical grade. Fungal Material. The strain 3A00475 was isolated from a sediment sample (E 68.00°, S 66.83°) at a depth of 2284 m from the Prydz Bay of Antarctica. Genomic DNA of the strain 3A00475 was isolated using standard methods and was amplified using PCR with the ITS primers ITS1 and ITS3. The ITS sequence of 541 bp was compared to sequences in the GenBank database. The strain 3A00475 showed the highest sequence similarity of 100% to Penicillium granulatum isolate 732 DQ681334. The ITS sequence of 3A00475 was deposited in GenBank (accession no. KM460923). The voucher strain of this fungus (MCCC 3A00475) is preserved at the Marine Culture Collection of China. Fermentation, Extraction, and Isolation. The strain P. granulatum was cultured on a PDA plate at 25 °C for 3 days. The fresh mycelia and spores were inoculated to 250 mL Erlenmeyer flasks (×10) containing 30 mL of PDB medium, which were incubated in a 180 rpm rotary shaker at 28 °C for 5 days. Then the spore cultures were used to inoculate 25 × 1 L Erlenmeyer flasks containing rice medium (80 g of rice and 120 mL of distilled H2O for each flask) to perform the large-scale fermentation. After 25 days, the fermented broth was extracted with EtOAc three times. The organic solvent was evaporated under reduced pressure to afford an organic extract (14 g). This extract was partitioned by 90%

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.7b00475. 2176

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1D and 2D NMR spectra for 1 and its MPA esters (PDF)

AUTHOR INFORMATION

Corresponding Author

*Tel/fax: +86-592-2195319. E-mail: [email protected] (X. W. Yang). ORCID

Guangming Liu: 0000-0002-8689-0504 Xian-Wen Yang: 0000-0002-4967-0844 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was supported by grants from the Science & Technology Research Program of Fujian Province, China (2017Y0060), and the National Natural Science Foundation of China (41606185, 41676130, and 21372233).

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REFERENCES

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