Phomanolides A and B from the Fungus Phoma sp.: Meroterpenoids

Article pubs.acs.org/jnp

Phomanolides A and B from the Fungus Phoma sp.: Meroterpenoids Derived from a Putative Tropolonic Sesquiterpene via Hetero-Diels− Alder Reactions Jinyu Zhang,†,§,# Ling Liu,†,# Bo Wang,‡ Yang Zhang,‡ Lili Wang,‡ Xingzhong Liu,† and Yongsheng Che*,‡ †

State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, People’s Republic of China ‡ State Key Laboratory of Toxicology & Medical Countermeasures, Beijing Institute of Pharmacology & Toxicology, Beijing 100850, People’s Republic of China § University of Chinese Academy of Sciences, Beijing 100049, People’s Republic of China S Supporting Information *

ABSTRACT: Phomanolides A (1) and B (2), unique meroterpenoids with new pentacyclic and tetracyclic skeletons, respectively, and phomanoxide (3), the double-epoxidation product of a putative biosynthetic precursor of 1 and 2, were isolated from the solid substrate fermentation cultures of the fungus Phoma sp., along with the known compound eupenifeldin (4). The structures of 1−3 were elucidated based on NMR spectroscopic data and electronic circular dichroism calculations and further secured by X-ray crystallography. Biogenetically, compounds 1 and 2 could be derived from a hypothetical monotropolonic sesquiterpene intermediate via hetero-Diels−Alder reactions. Compound 4 showed potent antiproliferative effects against three human glioma cell lines, with IC50 values of 0.08−0.13 μM.

T

specifically inhibit the proliferation of the glioma cells is urgently needed. In an ongoing search for natural products effective against the human glioma and tumor cells from the rarely studied fungi of unique niches,14 a strain of Phoma sp. isolated from a soil sample collected from the Qinghai-Tibetan plateau, Tibet, P. R. China, was investigated. An EtOAc extract prepared from the solid substrate fermentation cultures showed potent inhibitory effects against the proliferation of SH-SY5Y (neuroblastoma), U251 (glioma stem-like), and H4 (brain glioma) glioma cells. Bioassayguided separation of the crude extract afforded the active principle, eupenifeldin (4).1−4 Further chemical investigations directed by both bioactivity and chemistry led to the isolation of phomanolides A (1) and B (2), two meroterpenoids possessing new pentacyclic and tetracyclic skeletons, respectively, and a new sesquiterpenoid, phomanoxide (3), the double-epoxidation product of a putative biosynthetic precursor of 1 and 2. Details of the structure elucidation, biological evaluation, and plausible biogenesis of compounds 1−3 are reported herein.

ropolonic sesquiterpenes are a unique class of naturally occurring meroterpenoids with profound biological effects1−7 and therefore have attracted attention of synthetic chemists.8 Generally, monotropolone6,9 and bistropolone sequiterpenoids1−5 are the two frequently encountered subclasses, with epolone B6 and eupenifeldin (4)1−4 as the respective representatives. The bistropolone meroterpenoid eupenifeldin (4) was first discovered from the fungus Eupenicillium brefeldianum and showed potent in vitro cytotoxic effects against the human tumor cell lines HCT-116 and HCT-VM461 and in vivo antitumor activity in a P388 leukemia mouse model.1,2 The compound was also isolated from other fungi as anthelmintic, antimalarial, antifungal, and cytotoxic agents.3,4 Gliomas are the most common and aggressive human brain tumors in both adult and pediatric patients.10 Despite progress in finding lead compounds, the agents entered into clinical trials have failed to prove their efficacies.11 To date, the widely accepted therapy for gliomas is surgery, followed by adjunctive radiotherapy (RT) and chemotherapy using the drug temozolomide (TMZ).12 However, the prognosis of patients remains poor, and the median survival rate is low after the treatment.13 Therefore, the discovery of small-molecule natural products that © XXXX American Chemical Society and American Society of Pharmacognosy

Received: October 28, 2015

A

DOI: 10.1021/acs.jnatprod.5b00969 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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Article

RESULTS AND DISCUSSION Phomanolide A (1) was assigned the molecular formula C32H40O8 (13 degrees of unsaturation) by HRESIMS (m/z 553.2797 [M + H]+). Analysis of its NMR data (Table 1) revealed the presence of two exchangeable protons (δH 9.99 and 4.96, respectively), six methyl groups, five methylenes, three methines including one oxymethine, 12 olefinic or aromatic carbons with six protonated, four sp3 quaternary carbons with three oxygenated, and two carboxylic carbons (δC 165.1 and 170.5, respectively). These data accounted for all the NMR resonances of 1 except for one unobserved exchangeable proton and required 1 to be a pentacyclic compound. Analysis of the 1 H−1H COSY NMR data of 1 defined three isolated spinsystems of C-2−C-4, C-6−C-25 (via C-7), and C-9−C-12. Interpretation of the NMR data of 1 revealed a tetrasubstituted aryl ring (E) as confirmed by the HMBC correlations from H-28 Table 1. NMR Data for 1 and 2 (CDCl3) 1

a

no.

δC,a mult.

1 2a 2b 3 4 5 6a 6b 7 8 9 10a 10b 11 12a 12b 13 14 15 16 17 18a 18b 19 20 21 22 23 24 25a 25b 26 27 28 29 30 31 32 OH-9 OH-29

88.8, qC 46.7, CH2 123.8, CH 146.2, CH 35.2, qC 46.5, CH2 32.4, CH 80.2, qC 70.8, CH 31.6, CH2 40.3, CH 37.0, CH2 86.7, qC 160.5, qC 105.1, CH 165.1, qC 170.5, qC 119.9, CH 166.8, qC 21.0, CH3 29.7, CH3 27.2, CH3 15.5, CH3 14.3, CH3 29.7, CH2 111.0, qC 153.6, qC 101.5, CH 154.8, qC 110.2, CH 139.7, qC 19.5, CH3

δHb (J in Hz)

2 δC,a mult.

HMBC

2.76, dd (13.0, 11.0) 2.66, dd (13.0, 4.4) 5.61, ddd (15.5, 11.0, 4.4) 5.87, d (15.5)

1, 3, 4, 11, 20 1, 3, 4, 11, 20 1, 2, 5 2, 5, 6, 21, 22

1.74, d (14.8) 0.82, dd (14.8, 4.2) 1.71, dd (4.2, 4.9)

4, 5, 7, 8, 21, 22 4, 5, 7, 8, 21, 22 5, 6, 8, 25, 26

4.07, d (11.6) 2.35, dd (14.8, 5.6) 1.31, ddd (14.8, 11.6, 2.0) 2.86,dddd (13.9, 5.6, 2.3,2.0) 2.98, dd (15.0, 13.9) 1.57, dd (15.0, 2.3)

8, 10, 11, 23 8, 11, 12 1, 8, 9, 11, 12 1, 9, 10, 20 1, 11, 13, 17 1, 10, 11, 13, 14

5.10, s

13, 14, 16

5.98, d (1.3)

13, 17, 19, 24

1.61, s 1.04, s 1.11, s 1.01, s 2.19, d (1.3) 2.68, dd (16.2, 4.9) 2.28, d (16.2)

1, 2, 11 4, 5, 6, 22 4, 5, 6, 21 7, 8, 9 13, 17, 18 6, 7, 8, 26, 27, 31 6, 7, 8, 26, 27, 31

6.13, d (2.3)

26, 27, 30

6.31, d (2.3)

26, 28, 29, 32

2.15, s 4.96, br s 9.99, br s

26, 30, 31

86.1, qC 46.4, CH2 119.8, CH 146.1, CH 37.5, qC 41.8, CH2 127.1, CH 136.8, qC 67.9, CH 37.0, CH2 31.3, CH 34.8, CH2 78.7, qC 162.0, qC 98.5, CH 163.1, qC 171.5, qC 41.3, CH2 85.6, qC 22.0, CH3 29.3, CH3 24.8, CH3 18.9, CH3 19.9, CH3

δHb (J in Hz)

HMBC

2.55, dd (13.6, 6.8) 2.36, dd (13.6, 7.4) 5.18, ddd (15.8, 7.4, 6.8) 5.40, d (15.8)

1, 3, 4, 11, 20 1, 3, 4, 11, 20, 14 2, 5 2, 5, 6, 21, 22

2.11, dd (12.7, 11.9) 1.83, dd (12.7, 5.9) 5.50, dd (11.9, 5.9)

4, 5, 7, 8, 21, 22 4, 5, 7, 8, 21, 22 6, 9, 23

4.67, dd (8.2, 5.4) 1.98, m 1.38, ddd (14.6, 8.2, 4.1) 2.32, m 1.97, dd (8.6, 5.8) 1.90, d (8.6)

7, 8, 10, 11, 23 1, 8, 9, 11, 12 1, 8, 9, 11, 12 1, 2, 9, 10, 12, 13, 20 1, 11 1, 10, 11, 13, 14

5.51, s

13, 14

2.97, d (17.6) 2.76, d (17.6)

13, 17, 19 17, 24, 16

1.34, s 1.09, s 1.05, s 1.66, s 1.58, s

1, 2, 11 4, 5, 6, 22 4, 5, 6, 21 7, 8, 9 13, 18, 19

Recorded at 150 MHz. bRecorded at 600 MHz. B



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to C-26, C-27, and C-30, from H-30 to C-26, C-28, C-29, and C32, and from H3-32 to C-26, C-30, and C-31. HMBC correlations from H3-20 to C-1, C-2, and C-11, H3-21 and H3-22 to C-4, C-5, and C-6, H3-23 to C-7, C-8, and C-9, and H2-6 and H2-25 to C-8 established the humulene unit (C). Correlations from H2-25 to C-26, C-27, and C-31, plus the chemical shift values for C-8 (δC 80.2) and C-27 (δC 153.6), led to the postulation that C-8 and C27 were both attached to the same oxygen atom to form a dihydro-2H-pyran ring (D). In turn, correlations from H2-12 to C-13 and C-14 and from H-15 to C-13, C-14, and C-16, plus the chemical shift values for C-1 (δC 88.8) and C-14 (δC 160.5), implied that C-13 was located between C-12 and C-14, and C-1 and C-14 were both connected to the same oxygen atom to form the second dihydropyran ring (B), with the C-16 carboxylic carbon attached to the C-14/C-15 exocyclic olefin at C-15. The cross-peaks from H-18 to C-13, C-17, C-19, and C-24 and from H3-24 to C-13, C-18, and C-19 permitted completion of the C17−C-19 subunit, with C-13 and C-24 connected to the C-18/C19 olefin at C-19. Considering the chemical shift values for C-13 (δC 86.7) and C-17 (δC 170.5) and the unsaturation requirement for 1, the two carbons were attached to the same oxygen to form the furan-2(5H)-one ring (A) by default, although no additional evidence for this linkage was provided by the HMBC data. Fusion of the 1,7-dioxaspiro[4.5]dec-3-en-2-one moiety (A and B) with the humulene unit (C) at C-1/C-11 formed a spiro[cycloundeca[b]pyran-3,2′-furan]-5′-one skeleton (A−C), which fused to the benzopyran unit (rings D and E) at C-7/C-8 to complete the core structure of 1. The three exchangeable protons were located at C-9, C-16, and C-29, respectively, by default, which were partially supported by the chemical shift values for C-9 (δC 70.8), C-16 (δC 165.1), and C-29 (δC 154.8). Collectively, the planar structure of 1 was tentatively assigned as shown. The relative configuration of 1 was deduced by analysis of the 1 H−1H coupling constants and NOESY data (Figure 1). The C-

Figure 2. Experimental CD spectrum of 1 in MeOH and the calculated ECD spectra of 1a and 1b.

(1S,7R,8S,9S,11R,13S)-1 (1a) and (1R,7S,8R,9R,11S,13R)-1 (1b) enantiomers. The MMFF94 conformational search followed by B3LYP/6-31G(d) TDDFT reoptimization afforded five lowest energy conformers for 1a (Figure S19). The overall calculated ECD spectra of 1a and 1b were then generated by Boltzmann weighting of the conformers (Figure 2). The experimental CD curve of 1 was nearly identical to the calculated ECD spectrum of 1a, suggesting that compound 1 has the absolute configuration of 1S,7R,8S,9S,11R,13S. Finally, the proposed structure for 1 was confirmed by singlecrystal X-ray diffraction analysis using Cu Kα radiation (Figure 3). In addition, the presence of a relatively high percentage of

Figure 1. Key NOESY correlations for 1 and 2.

3/C-4 olefin was assigned the E-geometry based on the large J value of 15.5 Hz observed for H-3/H-4. The large trans-diaxialtype J values of 11.6 and 13.9 Hz for H-9/H-10b and H-11/H12a, respectively, revealed their axial orientations. NOESY correlations of H3-20 with H-9 and H-12a and of H-12a with H3-24 indicated that these protons are on the same face of the ring system, whereas those of H3-23 with H-7 and H-10b placed the protons on the opposite face of the molecule. The C-14/C-15 and C-17/C-18 olefins were both assigned the Z-geometry based on NOESY correlations of H3-24 with H-15 and H-18. The absolute configuration for 1 was initially deduced by comparison of the experimental and simulated electronic circular dichroism (ECD) spectra (Figure 2) generated by timedependent density functional theory (TDDFT)15 for the

Figure 3. Thermal ellipsoid representation of 1.

oxygen in 1 and the value of the Flack parameter, 0.09(13),16 also permitted assignment of the absolute configuration of 1, which was consistent with that deduced from the ECD data. Phomanolide B (2) was determined to have a molecular formula of C24H32O6 (nine degrees of unsaturation) based on HRESIMS (m/z 417.2274 [M + H]+). Analysis of its NMR data (Table 1) revealed the presence of the same humulene and dihydropyran (B and C) as found in 1, except that the benzopyran moiety in 1 (D and E) was replaced by the C-7/ C



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C-8 olefin, which was confirmed by relevant HMBC correlations. In addition, signals for the C-18/C-19 olefin of the furan-2(5H)one in 1 (A) were replaced by those for an oxygenated sp3 quaternary carbon (δC 85.6) and a methylene unit (δH/C 2.97; 2.76/41.3) in the spectra of 2, which were supported by the HMBC correlations from H2-18 to C-13, C-17, C-19, and C-24 and from H3-24 to C-13, C-18, and C-19. A weak but distinctive four-bond correlation17 from H-18b to C-16 suggested that the C-16 carboxylic carbon acylated the C-19 oxygen to form an ester linkage, completing the cycloundeca[5,6]pyrano[3,2-c]furo[3,2b]pyran-2,5-dione core in 2. The relative configuration of 2 was deduced on the basis of NOESY data (Figure 1) and was confirmed by X-ray crystallography using Cu Kα radiation (Figure 4), from which the absolute configuration was deduced to be 1S,9S,11R,13S,19R based on the value of the Flack parameter, 0.08(16).16



Phomanoxide (3) was assigned a molecular formula of C16H22O3 by HRESIMS (m/z 253.1798 [M + H]+). Its NMR data were nearly identical to those for syn-(1R,2R,4S,5S,10R)1,2;4,5-diepoxy-10-hydroxyhumulene, a biotransformation product of humulene by fungi.18 Detailed NMR data comparison revealed that they were C-10 epimers, as supported by the 2D NMR data of 3 (Table S1) and confirmed by X-ray crystallography using Cu Kα radiation (Figure 5). The 1R,2R,4S,5S,10S absolute configuration was also assigned based on the value of the Flack parameter, −0.16(17).16 Compound 4 was identified as eupenifeldin by comparison of its optical rotation value, MS, and NMR data (Table S2) with those reported.1,3,4 Although its relative configuration was first assigned by X-ray crystallography1 and it was later reported from other fungi,3,4 the absolute configuration remained unsolved. Thus, it is necessary to assign the absolute configuration of 4 to correlate biogenesis of the co-isolated metabolites. The absolute configuration was deduced by comparison of the experimental and calculated ECD spectra for the enantiomers (1S,7R,8S,9S,11S)-4 (4a) and (1R,7S,8R,9R,11R)-4 (4b) (Figure 6). The MMFF94 conformational search followed by TDDFT reoptimization at the B3LYP/6-31G(d) basis set level afforded the two lowest energy conformers for 4a (Figure S21). The experimental CD spectrum of 4 was nearly identical to the calculated ECD curve of 4a, suggesting the 1S,7R,8S,9S,11S absolute configuration. Compounds 1−4 were first tested for antiproliferative effects against the human glioma cell lines SH-SY5Y, U251, and H4 (Table 2). Compound 4 potently inhibited proliferation of the

Figure 6. Experimental CD spectrum of 4 in MeOH and the calculated ECD spectra of 4a and 4b.

three cell lines, with IC50 values of 0.08−0.13 μM (the positive control TMZ was estimated to have IC50 values greater than 100 μM). Compound 1 also showed an inhibitory effect against the SH-SY5Y cells, with an IC50 value of 81.1 μM, which is more active than the control drug TMZ. In addition, compounds 1−4 were also evaluated against four human tumor cell lines, HeLa (cervical carcinoma), A549 (lung adenocarcinoma), T24 (bladder carcinoma), and HCT116 (colorectal carcinoma). As expected, compound 4 showed potent inhibitory effects against the tumor cells, with IC50 values of 8.2−31.2 nM, while the positive control cisplatin showed IC50 values of 8.3−22.3 μM (Table 2). The new meroterpenoid phomanolide A (1) showed an antiproliferative effect only to HeLa cells, with an IC50 value of 14.3 μM, which is comparable to that of cisplatin. Compounds 2 and 3 did not show detectable effects against the tested cell lines at 20 μg/mL. Phomanolides A (1) and B (2) are new meroterpenes with unique structural features. Although several natural products with partial structural similarity to 1 and 2 have been reported integrating either a decahydro-11H-cycloundeca[1,2-b:5,6-b′]dichromene4,19 or an octahydro-3H-cyclohepta[b]cycloundeca[e]pyran-3-one skeleton,6 compounds 1 and 2 possess a D


(12.5 ± 1.3) × 10−3 10.2 ± 0.6

(31.2 ± 2.15) × 10−3 20.37 ± 1.13

previously undescribed pentacycle and tetracycle, respectively. Structurally, compound 1 incorporated a cycloundeca[b]pyran core, which not only cis-fused to a benzopyran moiety (rings D and E) at C-7/C-8 but also formed a spiro-junction with a furan2(5H)-one ring (ring A) at C-13, completing the unique spiro[cycloundeca[b]pyran-3,2′-furan]-5′-one skeleton. Compound 2 also incorporated a cycloundeca[b]pyran core, which fused to the furo[3,2-b]pyran-2,5-dione unit to complete a unique skeleton of cycloundeca[5,6]pyrano[3,2-c]furo[3,2-b]pyran-2,5-dione. Compound 3 is the C-10 epimer of a reported biotransformation product,18 and we assigned its absolute configuration using X-ray crystallography. In addition, the absolute configuration of 4 was also deduced for the first time by ECD calculations. Phomanolides A (1) and B (2) are likely derived from tropolonic sesquiterpenoids. Natural products biosynthesized via hetero-Diels−Alder additions of tropolone/benzene derivatives and α-humulenes were classified into five types: bistropolone− (eupenifeldin1,2 and pycnidione5,6), monotropolone− (epolone B6 and the congeners9), bisbenzopyranyl− (ramiferin4 and lucidene19), and monobenzopyranyl−humulene (pughiinin A20), as well as monobenzopyranyl−humulene−monotropolone (noreupenifeldin3 and epolone A6). In a kinetic study for the production of epolone B and pycnidione during a fermentation process, the ratio of pycnidione to epolone B changed from 1:4 (day 7) to 3:1 (day 11),6a suggesting that epolone B could be the biosynthetic precursor of pycnidione.6a Synthesis of lucidene, analogues of epolone B, and pycnidione via hetero-Diels−Alder additions of orthobenzoquinone methides/tropolone orthoquinone and α-humulene provided further support for their biogeneses via hetero-Diels−Alder reactions.8,21 Diels−Alder reactions are not only powerful in forming carbon−carbon bonds in organic syntheses but are also considered to be involved in biosynthesis of various cyclohexene-containing natural products.22 However, only a few enzymes were previously shown to catalyze the reactions in nature,23 which typically showed multiple functions, but lacked catalytic efficiency and/or participated in reactions that proceed spontaneously.24−27 In a study of the biosythesis for spinosyn A, SpnF, a cyclase, has been characterized and its structure has been resolved,28 which represents the first example characterized in vitro of a standalone enzyme solely committed to the catalysis of a Diels−Alder cyclization reaction. Recently, dialkyldecalin and spiro-conjugated synthases, two new types of monofunctional cyclases that catalyze pure enzymatic cyclizations, have been characterized in a study of biosynthesis for pyrroindomycins.29 Biogenetically, eupenifeldin (4), a diastereomer of pycnidione, could be derived via hetero-Diels−Alder additions of the hypothetical precursor humulenol (3′) and tropolone orthoquinone methide (b′) (Scheme 1). Tropolone stipitaldehyde (b) could be generated from a methylbenzaldehyde precursor (a) via a series of reactions30−32 and then converted to the more reactive b′ (Scheme 1). Hetero-Diels−Alder addition of 3′ and b′ could then generate a hypothetical intermediate (c), a monotropolonic sesquiterpene with the same planar structure as epolone B,6 as suggested by the HRESIMS and 1H NMR data (Figures S22 and S23). However, the exact structure of c could not be determined due to sample limitations. Starting from intermediate c, compounds 1, 2, and 4 could be generated via different reaction routes including hetero-Diels−Alder additions, while compound 3 could be derived from double-epoxidation of the hypothetical precursor humulenol (3′; Scheme 1). In addition, a Diels−Alder reaction between 3′ and b′ favored the exo-transition state in the

(30.6 ± 2.6) × 10−3 22.3 ± 0.4 >100 >100 >100

No activity was detected for compounds 2 and 3 at 20 μg/mL. a

(100 ± 5.3) × 10−3 81.1 ± 3.3 (80 ± 2.8) × 10−3

1 4 cisplatin temozolomide

(130 ± 3.3) × 10−3

T24 tumor cell lines A549 U251

glioma cell lines

SH-SY5Y compound

H4

HeLa

IC50 (μM)

Table 2. Antiproliferative Effects for Compounds 1 and 4a

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14.3 ± 1.4 (8.2 ± 1.2) × 10−3 8.3 ± 1.4

HCT116


E



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Scheme 1. Hypothetical Biosynthetic Pathways for 1−4

morphology and sequence (GenBank Accession No. KP896486) analysis of the ITS region of the rDNA. The strain was cultured on slants of potato dextrose agar at 25 °C for 10 days. Agar plugs were cut into small pieces (about 0.5 × 0.5 × 0.5 cm3) under aseptic conditions, 15 pieces were used to inoculatein three Erlenmeyer flasks (250 mL), each containing 50 mL of media (0.4% glucose, 1% malt extract, and 0.4% yeast extract), and the final pH of the media was adjusted to 6.5. After sterilization, three flasks of the inoculated media were incubated at 25 °C on a rotary shaker at 170 rpm for 5 days to prepare the seed culture. Spore inoculum was prepared by suspending the seed culture in sterile, distilled H2O to give a final spore/cell suspension of 1 × 106/mL. Fermentation was carried out in 60 Fernbach flasks (500 mL), each containing 80 g of rice. Distilled H2O (120 mL) was added to each flask, and the contents were soaked overnight before autoclaving at 15 psi for 30 min. After cooling to room temperature, each flask was inoculated with 5.0 mL of the spore inoculum and incubated at 25 °C for 40 days. Extraction and Isolation. The fermented material was extracted repeatedly with EtOAc (4 × 4.0 L), and the organic solvent was evaporated to dryness under vacuum to afford the crude extract (62.5 g), which was fractionated by silica gel VLC using petroleum ether− CH2Cl2−MeOH gradient elution. The fraction (1.1 g) eluted with 400:11 CH2Cl2−MeOH was separated by silica gel column chromatography (CC) using petroleum ether−acetone gradient elution, and the resulting subfraction (76.0 mg) eluted with 18:7 petroleum ether− acetone was further purified by semipreparative RP HPLC (Agilent Zorbax SB-C18 column; 5 μm; 9.4 × 250 mm; 65% MeOH in H2O for 15 min, followed by 65−75% over 25 min; 2 mL/min) to afford 1 (19.4 mg, tR 38.32 min) and 2 (1.6 mg, tR 31.82 min). The fraction (3.9 g) eluted with 100:2 CH2Cl2−MeOH was separated by silica gel CC using CH2Cl2−MeOH gradient elution, and the resulting subfractions were purified by RP HPLC to afford 3 (27.2 mg, tR 12.30 min; 75% MeOH in H2O for 20 min; 2 mL/min), 4 (1.9 g, tR 13.25 min; the same condition as 3), and c (0.7 mg, tR 12.50 min; 78% CH3CN in H2O for 15 min; 2 mL/min). Phomanolide A (1): colorless needles (MeOH−H2O); mp 256 °C (dec); white powder; [α]25D +112 (c 0.05, MeOH); UV (MeOH) λmax (log ε) 212 (4.47), 277 (3.54) nm; CD (c 1.8 × 10−4 M, MeOH) λmax (Δε) 209 (+52.7), 242 (−13.1) nm; IR (neat) νmax 3339 (br), 2955, 2868, 2360, 1741, 1620, 1463, 1390, 1142, 1087 cm−1; 1H and 13C NMR and HMBC data see Table 1; NOESY correlations (CDCl3, 600 MHz) H-3 ↔ H3-21; H-4 ↔ H3-22; H-6a ↔ H-9; H-6b ↔ H3-22; H-7 ↔ H322, H3-23; H-9 ↔ H-6a, H3-20; H-10b ↔ H3-23; H-12a ↔ H3-20, H324; H-12b ↔ H3-24; H-15 ↔ H-18, H3-24; H-18 ↔ H-15, H3-24; H3-20 ↔ H-9, H-12a; H3-21 ↔ H-3; H3-22 ↔ H-4, H-7; H3-23 ↔ H-7, H-10b; H3-24 ↔ H-12a, H-12b, H-15, H-18; HRESIMS m/z 553.2797 (calcd for C32H41O8, 553.2796). X-ray Crystallographic Analysis of 1.33 Upon crystallization from MeOH−H2O (10:1) using the vapor diffusion method, colorless

proposed biosynthetic pathways, suggesting that the reaction could be catalyzed by a Diels−Alderase. Phomanolides A (1) and B (2) are new meroterpenoids showing interesting and unique structural features, which could be derived from a putative tropolonic sesquiterpene via heteroDiels−Alder reactions. Compound 1 not only possesses a unique spiro-lactone skeleton but also displays an inhibitory effect on SH-SY5Y glioma cells, while compound 4 showed potent antiproliferative effects against human glioma cells, implying that the compound could be further evaluated as a potential lead for the treatment of gliomas. In the current work, the absolute configuration of 4 was also assigned for the first time by ECD calculations. The isolation of compound 3 and the putative biosynthetic intermediate c, together with the results from previous synthesis of the tropolonic sesquiterpenes,8,21 provided support for the proposed biosynthetic pathways for compounds 1, 2, and 4 via hetero-Diels−Alder reactions. This study not only laid the foundation to further investigate compounds 1 and 4 for their potential as antiglioma leads but also provided hints for their biogenesis.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured on a Rudolph Research Analytical automatic polarimeter, and UV data were obtained on a Shimadzu Biospec-1601 spectrophotometer. CD spectra were recorded on a JASCO J-815 spectropolarimeter. IR data were recorded using a Nicolet Magna-IR 750 spectrophotometer. 1H and 13C NMR data were acquired with Varian Mercury-500 and NMR system-600 spectrometers using solvent signals (CDCl3: δH 7.28/δC 77.0) as references. The HMQC and HMBC experiments were optimized for 145.0 and 8.0 Hz, respectively. ESIMS and HRESIMS data were obtained using an Agilent Accurate-Mass-QTOF LC/MS 6520 instrument equipped with an electrospray ionization (ESI) source. The fragmentor and capillary voltages were kept at 125 and 3500 V, respectively. Nitrogen was supplied as the nebulizing and drying gas. The temperature of the drying gas was set at 300 °C. The flow rate of the drying gas and the pressure of the nebulizer were 10 L/ min and 10 psi, respectively. All MS experiments were performed in positive ion mode. Full-scan spectra were acquired over a scan range of m/z 100−1000 at 1.03 spectra/s. HPLC separations were performed on an Agilent 1260 instrument (Agilent, USA) equipped with a variablewavelength UV detector. Fungal Material. The culture of Phoma sp. was isolated from a soil sample collected from the Qinghai-Tibetan plateau, Tibet, P. R. China. The isolate was identified by one of the authors (X.L.) based on F



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crystals were obtained for 1. A crystal (0.50 × 0.50 × 0.45 mm) was separated from the sample and mounted on a glass fiber, and data were collected using the Agilent CrysAlisPro software (version 1.171.35.11) with graphite-monochromated Cu Kα radiation, λ = 1.541 84 Ǻ at 100(1) K. Crystal data: C32H41.4O8.7, M = 565.35,34 space group orthorhombic, P2(1)2(1)2(1); unit cell dimensions a = 10.0518(2) Ǻ , b = 13.1222(2) Ǻ , c = 21.7957(5) Ǻ , V = 2874.89(11) Ǻ 3, Z = 4, Dcalcd = 1.306 mg/m3, μ = 0.772 mm−1, F(000) = 1212. The structure was solved by direct methods using SHELXL-9735 and refined by using full-matrix least-squares difference Fourier techniques. All non-hydrogen atoms were refined with anisotropic displacement parameters, and all hydrogen atoms were placed in idealized positions and refined as riding atoms with the relative isotropic parameters. Absorption corrections were performed using spherical harmonics, implemented in the SCALE3 ABSPACK scaling algorithm.36 The 10 327 measurements yielded 5409 independent reflections after equivalent data were averaged and Lorentz and polarization corrections were applied. The final refinement gave R1 = 0.0365 and wR2 = 0.0939 [I > 2σ(I)]. Phomanolide B (2): colorless needles (MeOH−H2O); mp 220−222 °C (dec); [α]25D +26.0 (c 0.05, MeOH); UV (MeOH) λmax (log ε) 203 (3.77), 247 (3.68) nm; CD (c 2.4 × 10−4 M, MeOH) λmax (Δε) 205 (+17.2), 254 (−8.0) nm; IR (neat) νmax 3527, 2948, 2927, 2863, 1783, 1718, 1641, 1381, 1190, 1026 cm−1; 1H and 13C NMR and HMBC data see Table 1; NOESY correlations (CDCl3, 600 MHz) H-3 ↔ H3-21; H4 ↔ H3-22; H-6a ↔ H-9; H-7 ↔ H3-22, H3-23; H-9 ↔ H-6a, H3-20; H12a ↔ H3-20; H-12b ↔ H3-24; H-18a ↔ H3-24; H-18b ↔ H-12b, H324; H3-20 ↔ H-9, H-12a; H3-21 ↔ H-3; H3-22 ↔ H-4, H-7; H3-23 ↔ H-7; H3-24 ↔ H-12b, H2-18; HRESIMS m/z 417.2274 (calcd for C24H33O6, 417.2272). X-ray Crystallographic Analysis of 2.37 Upon crystallization from MeOH−H2O (10:1) using the vapor diffusion method, colorless crystals were obtained for 2. A crystal (0.35 × 0.35 × 0.06 mm) was separated from the sample and mounted on a glass fiber, and data were collected using the Agilent CrysAlisPro software (version 1.171.35.11) with graphite-monochromated Cu Kα radiation, λ = 1.541 84 Ǻ at 99(8) K. Crystal data: C24H32O6, M = 416.50, space group orthorhombic, P2(1)2(1)2(1); unit cell dimensions a = 6.30022(10) Ǻ , b = 15.0054(2) Ǻ , c = 23.3482(12) Ǻ , V = 2207.27(9) Ǻ 3, Z = 4, Dcalcd = 1.253 mg/m3, μ = 0.725 mm−1, F(000) = 896. The structure was solved by direct methods using SHELXL-9735 and refined by using full-matrix leastsquares difference Fourier techniques. All non-hydrogen atoms were refined with anisotropic displacement parameters, and all hydrogen atoms were placed in idealized positions and refined as riding atoms with the relative isotropic parameters. Absorption corrections were performed using spherical harmonics, implemented in the SCALE3 ABSPACK scaling algorithm.36 The 7594 measurements yielded 4186 independent reflections after equivalent data were averaged and Lorentz and polarization corrections were applied. The final refinement gave R1 = 0.0391 and wR2 = 0.1001 [I > 2σ(I)]. Phomanoxide (3): colorless needles (MeOH−H2O); mp 139−141 °C; [α]25D +12.0 (c 0.05, MeOH); UV (MeOH) λmax (log ε) 202 (3.42) nm; IR (neat) νmax 3432 (br), 2968, 2928, 1480, 1462, 1386, 1255, 1093, 1028 cm−1; 1H and 13C NMR, HMBC, and NOESY data see Table S1; HRESIMS m/z 253.1798 (calcd for C15H24O3, 253.1800). X-ray Crystallographic Analysis of 3.38 Upon crystallization from MeOH−H2O (9:2) using the vapor diffusion method, colorless crystals were obtained for 3. A crystal (0.40 × 0.35 × 0.25 mm) was separated from the sample and mounted on a glass fiber, and data were collected using the Agilent CrysAlisPro software (version 1.171.35.11) with graphite-monochromated Cu Kα radiation, λ = 1.541 84 Ǻ at 98(8) K. Crystal data: C15H26O4, M = 270.36,39 space group orthorhombic, P2(1)2(1)2(1); unit cell dimensions a = 8.28899(19) Ǻ , b = 11.8587(2) Ǻ , c = 15.3047(3) Ǻ , V = 1504.40(6) Ǻ 3, Z = 4, Dcalcd = 1.194 mg/m3, μ = 0.686 mm−1, F(000) = 592. The structure was solved by direct methods using SHELXL-9735 and refined by using full-matrix least-squares difference Fourier techniques. All non-hydrogen atoms were refined with anisotropic displacement parameters, and all hydrogen atoms were placed in idealized positions and refined as riding atoms with the relative isotropic parameters. Absorption corrections were performed using spherical harmonics, implemented in the SCALE3 ABSPACK scaling

algorithm.36 The 4998 measurements yielded 2829 independent reflections after equivalent data were averaged and Lorentz and polarization corrections were applied. The final refinement gave R1 = 0.0350 and wR2 = 0.0921 [I > 2σ(I)]. Eupenifeldin (4): [α]25D +160.0 (c 0.1, CDCl3); CD (c 1.8 × 10−3 M, CDCl3) λmax (Δε) 206 (+1.54), 239 (−0.36), 259 (+2.84), 314 (+0.19) nm; 1H and 13C NMR, HMBC, and NOESY data see Table S2; NMR and MS data were consistent with literature values.1 Computational Details. Systematic conformational analyses for 1 and 4 were performed via the Molecular Operating Environment (MOE) ver. 2009.10. (Chemical Computing Group, Canada) software package using the MMFF94 molecular mechanics force field calculation. The MMFF94 conformational analyses were further optimized using TDDFT at the B3LYP/6-31G(d) basis set level. The stationary points have been checked as the true minima of the potential energy surface by verifying they do not exhibit vibrational imaginary frequencies. The 30 lowest electronic transitions were calculated, and the rotational strengths of each electronic excitation were given using both dipole length and dipole velocity representations. ECD spectra were stimulated using a Gaussian function with a half-bandwidth of 0.45 and 0.3 eV for compound 1 and 4, respectively. Equilibrium populations of conformers at 298.15 K were calculated from their relative free energies (ΔG) using Boltzmann statistics. The overall ECD spectra were then generated according to Boltzmann weighting of each conformer. The systematic errors in the prediction of the wavelength and excited-state energies are compensated for by employing UV correlation. All quantum computations were performed using the Gaussian0340 package. Cell Cultures. Three human glioma cell lines, SH-SY5Y (neuroblastoma), U251 (glioma stem-like), and H4 (brain glioma), and four human tumor cell lines, HeLa (cervical carcinoma), A549 (lung adenocarcinoma), T24 (bladder carcinoma), and HCT116 (colorectal carcinoma), were obtained from American Type Culture Collection (Rockville, MD, USA). The A549, T24, and HCT116 cells were maintained in McCoy’s 5A medium, the HeLa cells were grown in RPMI 1640 medium, the H4 and U251 cells were maintained in MEM-EBSS (MEM Eagle’s with Earle’s balanced salts) medium, and the SH-SY5Y cells were grown in Dulbecco’s modified Eagle’s medium (DMEM). All cells were supplemented with 10% fetal bovine serum and incubated at 37 °C with 5% CO2. MTT Assay. In 96-well plates, each well was plated with 104 cells. After cell attachment overnight, the medium was removed, and each well was treated with 50 μL of medium containing 0.2% DMSO or appropriate concentrations of test compounds (10 mg/mL as stock solution of a compound in DMSO and serial dilutions). Cells were treated at 37 °C for 4 h in a humidified incubator at 5% CO2 first, and then were allowed to grow for another 48 h after the medium was changed to fresh DMEM. MTT (Sigma) was dissolved in serum-free medium or phosphate-buffered saline at 0.5 mg/mL and sonicated briefly. In the dark, 50 μL of MTT/medium was added into each well after the medium was removed from wells and incubated at 37 °C for 3 h. Upon removal of MTT/medium, 100 μL of DMSO was added to each well and agitated at 60 rpm for 5 min to dissolve the precipitate. The assay plate was read at 540 nm using a microplate reader.

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ASSOCIATED CONTENT

S Supporting Information *

X-ray data of 1−3 (CIF files). The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.5b00969. NMR spectra of 1−3, NMR data of 3 and 4, CD calculations for 1 and 4, and CD spectrum of 2 (PDF) X-ray data of 1 (CIF) X-ray data of 2 (CIF) X-ray data of 3 (CIF) G



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AUTHOR INFORMATION

Corresponding Author

*Tel/Fax: +86 10 66932679. E-mail: [email protected]. Author Contributions #

J. Zhang and L. Liu Contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We gratefully acknowledge financial support from the National Natural Science Foundation of China (81273395 and Z121102002512046) and the National Program of Drug Research and Development (2012ZX09301-003), the Program of the Excellent Youth Scientists of CAS, and the Youth Innovation Promotion Association of CAS (2011083).

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(34) Due to the presence of an extra 0.7 unit of water in the X-ray crystallographic structure of 1, the actual formula of 1 was C32H41.4O8.7, with a formula weight of 552. (35) Sheldrick, G. M. SHELXL-97, Program for X-ray Crystal Structure Solution and Refinement; University of Göttingen: Göttingen: Germany, 1997. (36) CrysAlisPro Version 1.171.35.11; Oxford Diffraction Ltd.: Oxfordshire, U.K., 2011. (37) Crystallographic data for 2 have been deposited with the Cambridge Crystallographic Data Centre (deposition number CCDC 1055005). Copies of the data can be obtained, free of charge, on application to the director, CCDC, 12 Union Road, Cambridge CB2 1EZ, UK (fax: +44 1223 336033 or e-mail: [email protected]). (38) Crystallographic data for 3 have been deposited with the Cambridge Crystallographic Data Centre (deposition number CCDC 1054990). Copies of the data can be obtained, free of charge, on application to the director, CCDC, 12 Union Road, Cambridge CB2 1EZ, UK (fax: +44 1223 336033 or e-mail: [email protected]). (39) Due to the presence of an extra unit of water in the X-ray crystallographic structure of 3, the actual formula of 3 was C15H26O4, with a formula weight of 252. (40) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Montgomery, J. A., Jr.; Vreven, T.; Kudin, K. N.; Burant, J. C.; Millam, J. M.; Iyengar, S. S.; Tomasi, J.; Barone, V.; Mennucci, B.; Cossi, M.; Scalmani, G.; Rega, N.; Petersson, G. A.; Nakatsuji, H.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Klene, M.; Li, X.; Knox, J. E.; Hratchian, H. P.; Cross, J. B.; Bakken, V.; Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Ayala, P. Y.; Morokuma, K.; Voth, G. A.; Salvador, P.; Dannenberg, J. J.; Zakrzewski, V. G.; Dapprich, S.; Daniels, A. D.; Strain, M. C.; Farkas, O.; Malick, D. K.; Rabuck, A. D.; Raghavachari, K.; Foresman, J. B.; Ortiz, J. V.; Cui, Q.; Baboul, A. G.; Clifford, S.; Cioslowski, J.; Stefanov, B. B.; Liu, G.; Liashenko, A.; Piskorz, P.; Komaromi, I.; Martin, R. L.; Fox, D. J.; Keith, T.; Al-Laham, M. A.; Peng, C. Y.; Nanayakkara, A.; Challacombe, M.; Gill, P. M. W.; Johnson, B.; Chen, W.; Wong, M. W.; Gonzalez, C.; Pople, J. A. Gaussian 03, Revision E. 01; Gaussian, Inc.: Wallingford, CT, 2004.

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Phomanolides A and B from the Fungus Phoma sp.: Meroterpenoids

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