Phomanolides C–F from a Phoma sp.: Meroterpenoids Generated via

May 23, 2019 - Phomanolides C–F (1–4), four new meroterpenoids, were isolated from a Phoma sp., together with the known phomanolides A (5) and B (...
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Cite This: J. Nat. Prod. 2019, 82, 1678−1685

Phomanolides C−F from a Phoma sp.: Meroterpenoids Generated via Hetero-Diels−Alder Reactions Jinyu Zhang,† Yumei Li,†,§ Fengxia Ren,† Yang Zhang,† Xingzhong Liu,⊥ Ling Liu,*,⊥ and Yongsheng Che*,†,‡

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†

State Key Laboratory of Toxicology & Medical Countermeasures, Beijing Institute of Pharmacology & Toxicology, Beijing 100850, People’s Republic of China ‡ Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, People’s Republic of China § Nanjing University of Chinese Medicine, Nanjing 210023, People’s Republic of China ⊥ State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, People’s Republic of China S Supporting Information *

ABSTRACT: Phomanolides C−F (1−4), four new meroterpenoids, were isolated from a Phoma sp., together with the known phomanolides A (5) and B (6); their structures were elucidated primarily by NMR experiments. The absolute configurations of 1−3 were assigned by electronic circular dichroism calculations, and that of 4 was established by X-ray diffraction analysis using Cu Kα radiation. Compounds 1−3 incorporate an unprecedented trioxa[4.4.3]propellane subunit in their skeletons. Compounds 2 and 4 were weakly cytotoxic.

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intermediate in the biosynthesis of 5 and 6, but its configuration could not be established due to sample limitations. To identify other minor Diels−Alder adducts and to obtain enough hypothetical intermediate for configuration assignment, the fungus was refermented on a larger scale. Fractionation of the EtOAc extract prepared from the cultures afforded four new meroterpenoids, phomanolides C−F (1−4), and the previously identified hypothetical sesquiterpene intermediate (c), together with 5 and 6. Details of the isolation, structure elucidation, cytotoxicity evaluation, and putative biogenesis of these compounds are reported herein.

eroterpenoids are natural products derived from mixed terpenoid−non-terpenoid biosynthetic pathways.1,2 As a unique class, tropolonic sesquiterpenoids have been reported mainly from fungi and can be grouped into five major subclasses: bistropolone−humulene,3−7 monotropolone−humulene,6−8 bisbenzopyranyl−humulene,6,9,10 monobenzopyranyl−humulene,11 and monobenzopyranyl−humulene−monotropolone.6,7,12 Tropolonic sesquiterpenoids show a broad spectrum of biological activities including cytotoxic,3,5−9,13 antifungal,7 antimalarial,9 and anthelmintic effects.12 Their remarkable structural diversity and profound biological activities have attracted much attention from synthetic chemists and biochemists.14,15 In an ongoing search for new cytotoxic metabolites from the rarely studied fungi inhabiting unique environments,16 a strain of Phoma sp. isolated from a soil sample collected from the Qinghai-Tibetan plateau, Tibet, People’s Republic of China, was chemically investigated, leading to the isolation of phomanolides A (5) and B (6), two meroterpenoids that could be generated via hetero-Diels−Alder reactions, and the double-epoxidation product of their putative biosynthetic precursor.13 In addition, a monotropolonic sesquiterpenoid was isolated as a minor component and proposed as the key © 2019 American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION Phomanolide C (1) was obtained as white powder and was assigned the molecular formula C32H40O8 (13 degrees of unsaturation) on the basis of its HRESIMS and NMR data (Table 1). Analysis of its NMR data revealed the presence of two exchangeable protons (δH 7.90 and 3.46, respectively), six methyl groups, six methylene units, three methines including Received: March 27, 2019 Published: May 23, 2019 1678

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one oxymethine, four oxygenated tertiary carbons with one doubly oxygenated, one sp3 quaternary carbon, 10 olefinic/ aromatic carbons with five protonated carbons, and two carboxylic carbons (δC 170.6 and 160.4, respectively). These data accounted for all of the NMR resonances and required 1 to be a hexacyclic compound. The 1H and 13C NMR spectroscopic data of 1 revealed structural features similar to those presented in phomanolide A (5), which were a tetrahydrohumulene moiety, a dihydro-2H-pyran ring, and a tetrasubstituted aryl ring, corresponding to fragments D−F, respectively. However, resonances for the remaining portion of 1 differed significantly from those of 5, indicating that 1 is related to 5 but with different partial structure. Detailed analysis of the 2D NMR data was performed to establish the left moiety of the structure. Interpretation of the 1H−1H Table 1. NMR Data for 1 and 2 1 δC, mult. 1 2a 2b 3 4 5 6a 6b 7 8 9 10a 10b 11 12a 12b 13 14 15a 15b 16 17 18 19 20 21 22 23 24 25a 25b 26 27 28 29 30 31 32 33 OH-9 OH-29

81.9, C 47.5, CH2 125.9, CH 145.6, CH 35.6, C 47.3, CH2 33.0, 81.0, 71.5, 32.5,

CH C CH CH2

40.0, CH 35.0, CH2 81.1, C 104.0, C 44.1, CH2 170.6, C 160.4, C 117.3, CH 157.1, C 21.0, CH3 29.9, CH3 27.3, CH3 15.7, CH3 16.5, CH3 30.2, CH2 110.7, C 155.0, C 102.3, CH 157.0, C 110.4, CH 139.6, C 19.4, CH3

2

δH (J in Hz)

a

b

2.47, 2.47, 5.75, 5.94,

m m ddd (15.8, 9.7, 5.8) d (15.8)

HMBC 1, 1, 2, 2,

3, 4, 11, 20 3, 4, 11, 20 5 5, 21, 22

1.78, d (14.4) 0.77, dd (14.4, 4.6) 1.87, m

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

4.10, 2.48, 1.29, 2.29, 2.63, 2.16,

8, 10, 11, 23 12 1, 9, 11, 12 1, 9, 10, 12, 20 1, 11, 13, 16, 17 1, 10, 11, 13, 14, 17

d (11.8) m ddd (14.6, 11.8, 1.9) m dd (15.0, 14.2) dd (15.0, 2.5)

3.25, d (17.1) 2.72, d (17.1)

81.0, C 46.6, CH2 125.7, CH 144.3, CH 35.1, C 46.8, CH2 32.0, 81.8, 70.9, 31.5,

CH C CH CH2

38.9, CH 33.9, CH2 80.6, C 103.2, C 43.9, CH2

1, 14, 16 13, 14, 16

5.94, q (1.4)

13, 17, 24

1.57, 1.02, 1.12, 1.04, 2.08, 2.73, 2.31,

1, 2, 11 4, 5, 6, 22 4, 5, 6, 21 7, 8, 9 13, 18, 19 6, 7, 26, 27, 31 6, 7, 8, 26, 27, 31

s s s s d (1.4) dd (16.0, 5.6) d (16.0)

δC, mult.

6.11, d (2.3)

26, 27, 29, 30

6.27, d (2.3)

26, 28, 29, 32

2.11, s

26, 30, 31

3.46, t (2.0) 7.90, br s

9, 10 28, 29, 30

δH (J in Hz)

c

170.2, C 160.2, C 116.8, CH 156.4, C 20.6, CH3 29.6, CH3 27.0, CH3 16.0, CH3 16.9, CH3 34.4, CH2 118.5, C 159.4, C 112.8, CH 162.8, C 173.4, C 125.7, CH 151.5, C 27.5, CH3

d

2.53, 2.38, 5.63, 5.74,

HMBC

dd (13.4, 4.4) dd (13.4, 10.9) ddd (16.0, 10.9, 4.4) d (16.0)

3, 1, 2, 2,

4 3, 4, 11, 20 4, 5 3, 5, 21, 22

1.72, d (14.6) 0.74, dd (14.6, 4.3) 1.78, br s

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

4.09, 2.32, 1.24, 2.32, 2.57, 2.07,

8, 1, 1, 1, 1, 1,

d (11.6) m dd (13.1, 11.6) m dd (14.9, 13.3) dd (14.9, 2.2)

10, 11, 23 9 9 9, 13, 20 11 10, 11, 13, 14

2.93, d (17.2) 2.88, d (17.2)

14, 16 13, 14, 16

5.91, d (0.8)

13, 17, 24

1.56, 1.05, 1.08, 1.08, 2.09, 2.84, 2.35,

1, 2, 11 4, 5, 6, 22 4, 5, 6, 21 7, 8, 9 13, 18, 19 6, 7, 26, 27 6, 7, 8, 26, 27

s s s s s dd (17.0, 5.3) d (17.0)

6.91, s

26, 27, 29, 30

7.15, s

26, 29, 30, 32, 33

2.37, s

26, 31, 32

a

Recorded at 150 MHz in acetone-d6. bRecorded at 600 MHz in acetone-d6. cRecorded at 150 MHz in CDCl3. dRecorded at 600 MHz in CDCl3. 1679

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was proposed by comparison of the experimental and simulated electronic circular dichroism (ECD) spectra calculated using the time-dependent density functional theory (TDDFT)17 for two isomers, (1S,7R,8S,9S,11R,13S,14S)-1 (1a) and (1R,7S,8R,9R,11S,13R,14R)-1 (1b). A systematic conformational analysis was performed for 1a and 1b with the Molecular Operating Environment (MOE) software package using the MMFF94 molecular mechanics force field calculation. The selected conformers were then reoptimized using TDDFT at the B3LYP/6-311G(d,p) level to afford the lowestenergy conformers. The conformers were further filtered based on the Boltzmann-population rule, resulting in one significant conformer for each configuration (Figure S25). The overall calculated ECD spectra of 1a and 1b were then generated by Gaussian broadening (Figure 2). The experimental ECD curve of 1 was nearly identical to that calculated for 1a, suggesting the 1S,7R,8S,9S,11R,13S,14S absolute configuration for 1.

COSY NMR data revealed the connectivity of C-12 to C-11, which was supported by the HMBC correlations from H2-12 to C-1, C-10, and C-11 (Figure 1). HMBC cross-peaks from H2-

Figure 1. 1H−1H COSY, key HMBC, and NOESY correlations of phomanolide C (1).

12 to C-13, C-14, and C-19 and from H2-15 to C-13, C-14, and C-16 indicated that the C-13−C-15 fragment is connected to both C-12 and the C-19 olefinic carbon via C-13 and to the C-16 carboxylic carbon at C-15. A weak but distinctive fourbond HMBC correlation was observed from H2-15 to C-1,6,13 combined with the chemical shift values for C-1 (δC 86.2) and C-14 (δC 104.0), suggesting that C-1 and C-14 were all connected to the same oxygen atom to form a tetrahydro-2Hpyran ring (C), identical to that found in 5 and 6 (Figure 1). The HMBC cross-peaks from H3-24 to C-13, C-18, and C-19 and from H-18 to C-13 and C-17 revealed that the C-17 carboxylic carbon and C-24 were both connected to the C-18− C-19 olefin at C-18 and C-19, respectively. Considering the upfield chemical shift value for C-17 (δC 160.4 in 1 vs 170.5 in 5) and the doubly oxygenated nature of C-14 (δC 104.0), C-17 was connected to C-14 via an ester linkage to form a 5,6dihydro-2H-pyranone moiety (A), rather than a furanone via connecting to C-13 as that found in 5. Considering the unsaturation requirement of 1, the C-16 carboxylic carbon has to acylate the oxygen atom attached to C-13 (δC 81.1) to form a tetrahydrofuranone ring (B), which was supported by a fourbond HMBC correlation from H2-12 to C-16, similar to those observed in the tropolonic sesquiterpenoids neosetophomone A6 and 6.13 Collectively, the gross structure of 1 was established as shown. The relative configuration of rings D−F in 1 was deduced to be the same as that of 5 by analysis of the 1H−1H coupling constants (Table 1) and NOESY data (Figure 1). The C-3/C4 olefin was assigned the E-geometry based on the large (15.8 Hz) coupling constant observed for H-3/H-4. The large J values observed for H-9/H-10b (11.8 Hz) and H-11/H-12a (14.2 Hz) revealed their trans-diaxial orientations. The NOESY correlation of H3-20 with H-9 placed these protons on the same side of the ring system, whereas those of H3-23 with H-7 and H-10b indicated that H-7, H-10b, and H3-23 were on the opposite side of the molecule. The C-18/C-19 olefin was assigned the Z-geometry based on the NOESY correlation of H3-24 with H-18. NOESY correlations of H-12a with H3-20 and H3-24 placed these protons on the same side of rings A and C. Therefore, the relative configuration of 1 was deduced as shown (Figure 1). The absolute configuration of 1

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

Phomanolide D (2) was determined to have a molecular formula of C33H40O9 (14 degrees of unsaturation) based on HRESIMS and the NMR data (Table 1), which is 28 mass units higher than that of 1. Analysis of its NMR data revealed the presence of the same partial structure (rings A−E) as that found in 1, except that those corresponding to the tetrasubstituted aryl ring in 1 were significantly different in 2. Considering the downfield chemical shift values for the aromatic protons (δH 6.91 and δH 7.15 in 2 vs 6.11 and 6.27 in 1) and the tropolonic carbonyl signal (δC 173.4) in 2, the aryl moiety in 1 was replaced by a tropolone unit. HMBC correlations from H2-25 to C-26 and C-27, from H-28 to C-26, C-27, C-29, and C-30, from H-31 to C-26, C-29, C-30, C-32, and C-33, and from H3-33 to C-26, C-31, and C-32 confirmed this assignment. Therefore, the planar structure of 2 was established as shown. The relative configuration of 2 was deduced to be the same as that of 1 by comparison of their 1 H−1H coupling constants (Table 1) and NOESY data (Figure 3). The absolute configuration for 2 was also proposed by comparison of the experimental and calculated ECD spectra for the enantiomers (1S,7R,8S,9S,11R,13S,14S)-2 (2a) and 1680

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Figure 3. 1H−1H COSY and key HMBC and NOESY correlations for 2−4.

rings A−D as found in 1, but the benzopyran unit (rings E and F) in 1 was absent. Specifically, the C-7 methine unit (δH/δC 1.87/33.0) and the C-8 oxygenated tertiary carbon (δC 81.0) in 1 were replaced by an olefin moiety in 3, which was confirmed by HMBC correlations from H-7 to C-9 and C-23 and from H3-23 to C-7, C-8, and C-9, thereby completing the planar structure of 3. Compound 3 was deduced to have the same relative configuration as 1 and 2 by comparison of their 1 H−1H coupling constants (Tables 1 and 2) and NOESY data (Figure 3). The C-7/C-8 olefin was also assigned the Zgeometry based on a NOESY correlation of H-7 with H3-23. The absolute configuration for 3 was deduced by comparison of the experimental and calculated ECD spectra for the enantiomers (1S,9S,11R,13S,14S)-3 (3a) and (1R,9R,11S,13R,14R)-3 (3b). The MMFF94 conformational search followed by B3LYP/6-311G(d,p) TDDFT reoptimization afforded the lowest energy conformers, which were further filtered based on the Boltzmann-population rule, resulting in two significant conformers with 46.8% and 53.16% populations, respectively, for each configuration (Figure S27). The overall calculated ECD spectra were then generated according to Boltzmann weighting of the two conformers. The experimental ECD spectrum of 3 matched the calculated ECD curve of 3a (Figure S28), suggesting the 1S,9S,11R,13S,14S absolute configuration for 3. Phomanolide F (4) was assigned a molecular formula of C32H40O8 (13 degrees of unsaturation) by HRESIMS. Interpretation of the 1H and 13C NMR data of 4 (Table 2) revealed the presence of the same cyclo-undeca[b]pyran unit and furo[3,2-b]pyran-2,5-dione unit (rings A−D) as those found in 6 and the same benzopyran moiety (rings E and F) as that found in 1 and 5, which were confirmed by relevant 1 H− 1 H COSY and HMBC correlations. The relative configuration of 4 was also deduced by analysis of 1H−1H coupling constants (Table 2) and NOESY correlations (Figure 3) and was further confirmed by X-ray crystallography using Cu Kα radiation (Figure 5), from which the absolute configuration was determined to be 1S,7R,8S,9S,11R,13S,19R based on the value of the Flack parameter, 0.00(16).18

(1R,7S,8R,9R,11S,13R,14R)-2 (2b). The MMFF94 conformational search followed by TDDFT reoptimization at the B3LYP/6-311G(d,p) basis set level afforded the lowest energy conformers for 2a and 2b, which were further filtered based on the Boltzmann-population rule, resulting in one significant conformer for each configuration (Figure S26). The calculated ECD spectra of enantiomers 2a and 2b were then generated by Gaussian broadening (Figure 4). The experimental ECD spectrum of 2 matched reasonably well with the calculated one for 2a, suggesting that 2 has the 1S,7R,8S,9S,11R,13S,14S absolute configuration. The molecular formula of phomanolide E (3) was determined to be C24H32O6 (nine degrees of unsaturation) on the basis of HRESIMS data. Interpretation of its NMR data (Table 2) revealed the presence of the same partial structure of

Figure 4. Experimental ECD spectrum of 2 in MeOH and the calculated ECD spectra of 2a and 2b. 1681

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

81.4, C 46.6, CH2 120.8, CH 145.7, CH 39.0, C 41.4, CH2 125.7, CH 140.4, C 67.3, CH 36.9, CH2 31.2, CH 34.6, CH2 80.7, C 103.5, C 43.7, CH2

4

δHb (J in Hz) 2.52, 2.31, 5.07, 5.33,

ddd (14.8, 2.9, 1.6) dd (14.8, 10.1) ddd (15.8, 10.1, 2.9) dd (15.8, 1.6)

δC,c mult.

HMBC 1, 3, 2, 2,

2.18, dd (13.2, 10.2) 1.75, dd (13.2, 7.3) 5.34, dd (10.2, 7.3)

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

4.72, 1.89, 1.21, 2.04, 2.45, 2.04,

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

ddd (9.1, 6.4, 3.7) ddd (14.9, 6.4, 5.4) ddd (14.9, 9.1, 2.8) m d (12.4) t (12.4)

3.25, d (16.9) 2.72, d (16.9)

124.6, CH 145.5, CH 35.2, C 46.5, CH2 32.4, 80.3, 70.8, 32.0,

5.92, q (1.4)

13, 17, 24

156.8, C 21.2, CH3 30.2, CH3 22.1, CH3 19.2, CH3 16.5, CH3

1.33, 1.02, 0.94, 1.71, 2.07,

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

CH C CH CH2

39.7, CH 35.8, CH2 79.9, C 162.0, C 101.9, CH

14, 16 13, 14, 16

170.5, C 160.6, C 117.6, CH

s s s s d (1.4)

86.2, C 46.1, CH2

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

163.2, C 171.9, C 41.9, CH2 85.6, 21.4, 29.6, 27.2, 15.6, 20.4, 29.7,

C CH3 CH3 CH3 CH3 CH3 CH2

111.1, C 153.6, C 101.5, CH 154.7, C 110.1, CH 139.8, C 19.5, CH3 3.54, d (3.7)

8, 9, 10

δHd (J in Hz) 2.65, 2.59, 5.58, 5.82,

ddd (13.4, 4.6, 1.6) dd (13.4, 10.9) ddd (16.0, 10.9, 4.6) dd (16.0, 1.6)

HMBC 1, 1, 2, 2,

3, 4, 11 3, 4, 11, 20 5 5, 6, 22

1.73, d (14.7) 0.81, dd (14.7, 4.5) 1.71, dd (5.3, 4.5)

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

4.08, 2.38, 1.27, 2.74, 2.79, 1.59,

7, 8, 11, 23 12 1, 8, 9, 11, 12 1, 12 1, 11, 24 1, 10, 11, 13, 14, 24

d (11.5) dd (14.6, 5.2) ddd (14.6, 11.5, 2.0) ddd (3.8, 5.2, 2.0) t (13.8) d (13.8)

5.59, s

13, 14

2.98, d (17.8) 2.82, d (17.8)

13, 17, 19 17, 24

1.49, 1.04, 1.10, 1.03, 1.66, 2.69, 2.29,

1, 2, 11 4, 5, 6, 22 4, 5, 6, 21 7, 8, 9 13, 18, 19 6, 7, 26 6, 7, 8, 26, 27, 31

s s s s s dd (16.0, 5.3) d (16.0)

6.12, d (2.4)

26, 27, 30

6.30, d (2.4) 2.16, s 4.69, br s

26, 28, 29, 32 26, 30, 31

a

Recorded at 150 MHz in acetone-d6. bRecorded at 600 MHz in acetone-d6. cRecorded at 150 MHz in CDCl3. dRecorded at 600 MHz in CDCl3.

subunit in 1−3 has not been documented. Biogenetically, compounds 1−4 could be the hetero-Diels−Alder adducts of tropolone orthoquinone methides (b′) and 10-hydroxyhumulene (humulenol, 3′) via a hypothetical key intermediate c (Scheme 1), and this intermediate is now identified as the recently published tropolonic sesquiterpenoid neosetophomone B.6 The discovery of these unique secondary metabolites provided evidence for previously proposed hetero-Diels−Alder reaction cascades leading to the generation of this class of tropolonic meroterpenoids.

Compounds 1−4 were tested for cytotoxicity against three human glioma cell lines (SH-SY5Y, U251, and H4) and two other tumor cell lines (MCF-7 and HeLa). Compounds 2 and 4 showed weak cytotoxic effects toward these tumor cells (Table 3), whereas 1 and 3 did not show detectable activity at 50 μM. Phomanolides C−F (1−4) are new tropolonic meroterpenoids with unique structural features. Although they share some common structural fragments with those reported tropolone-incoporating and tropolone-derived meroterpenoids, compounds 1−3 incorporate an unprecedented trioxa[4.4.3]propellane subunit in their skeletons. Oxygen-containing propellane units have been encountered in natural products and synthetic compounds,6,19−24 and examples of natural products include the dioxa[4.3.3]propellane-incorporating neosetophomone A6 and the dioxa[4.4.3]propellane-containing cephalotanins A and B.19 The trioxa[4.4.3]propellane

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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

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MeOH−H2O gradient, and the resulting subfraction (85 mg) eluted with 65:35 MeOH−H2O was further purified by semipreparative RP HPLC (Agilent Zorbax SB-C18 column; 5 μm; 9.4 × 250 mm; 75% MeOH in H2O for 55 min; 2 mL/min) to afford 1 (6.4 mg, tR 52.45 min), 4 (4.3 mg, tR 46.50 min), 5 (5.5 mg, tR 36.54 min), and 6 (1.5 mg, tR 32.16 min). The fraction (58 mg) eluted with 70:30 MeOH− H2O was further separated by Sephadex LH-20 CC eluting with MeOH, and the resulting subfractions were combined and purified by semipreparative RP HPLC (Agilent Zorbax SB-C18 column; 5 μm; 9.4 × 250 mm; 55% CH3CN in H2O for 10 min, followed by 55−58% over 40 min; 2 mL/min) to afford 2 (1.8 mg, tR 36.78 min). The fraction (2.8 g) eluted with 400:7 CH2Cl2−MeOH was separated by silica gel CC using petroleum ether−acetone−MeOH gradient elution, and the resulting subfraction (76 mg) eluted with 37:13 petroleum ether−acetone was further separated by Sephadex LH-20 CC eluting with MeOH. The subfractions were combined and further purified by semipreparative RP HPLC (Agilent Zorbax SB-C18 column; 5 μm; 9.4 × 250 mm; 67% CH3CN in H2O for 45 min; 2 mL/min) to afford 3 (1.6 mg, tR 40.55 min). Phomanolide C (1): white powder; [α]25D +86.0 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 211 (3.07), 226 (2.84), 282 (2.18) nm; CD (c 2.3 × 10−4 M, MeOH) λmax (Δε) 200 (+41.3), 237 (−3.8) nm; IR (neat) νmax 3466 (br), 2867, 2157, 1805, 1732, 1619, 1596, 1461, 1303, 1137, 1022 cm−1; 1H, 13C NMR and HMBC data see Table 1; NOESY correlations (acetone-d6, 600 MHz) H-3 ↔ H3-21; H-4 ↔ H3-22; H-6a ↔ H-9; H-6b ↔ H3-22; H-7 ↔ H3-22, H3-23; H-9 ↔ H-6a, H3-20; H-10b ↔ H-12b, H3-23; H-12a ↔ H3-20, H3-24; H-12b ↔ H-10b; H-18 ↔ H3-24; H3-20 ↔ H-9, H-12a; H3-21 ↔ H-3; H322 ↔ H-4, H-7; H3-23 ↔ H-7, H-10b; H3-24 ↔ H-12a, H-18; HRESIMS m/z 553.2801 [M + H]+ (calcd for C32H41O8, 553.2796). Phomanolide D (2): white powder; [α]25D +160.7 (c 0.03, MeOH); UV (MeOH) λmax (log ε) 206 (4.37), 262 (4.30), 271 (4.35) nm; CD (c 1.7 × 10−3 M, MeOH) λmax (Δε) 218 (+2.8), 237 (−0.8), 267 (+4.9), 302 (+0.2), 338 (+0.4), 371 (+1.0) nm; IR (neat) νmax 3355 (br), 2927, 1799, 1728, 1662, 1589, 1440, 1386, 1296, 1086, 1037 cm−1; 1H, 13C NMR and HMBC data see Table 1; NOESY correlations (CDCl3, 600 MHz) H-3 ↔ H3-21; H-4 ↔ H322; H-6a ↔ H-9; H-6b ↔ H3-22; H-7 ↔ H3-22, H3-23; H-9 ↔ H-6a, H3-20; H-10b ↔ H-12b, H3-23; H-12a ↔ H3-20, H3-24; H-12b ↔ H10b; H-18 ↔ 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-18; HRESIMS m/z 581.2741 [M + H]+ (calcd for C33H41O9, 581.2745). Phomanolide E (3): white powder; [α]25D +72.4 (c 0.06, MeOH); UV (MeOH) λmax (log ε) 206 (4.01), 333 (1.80) nm; CD (c 1.2 × 10−3 M, MeOH) λmax (Δε) 204(+44.4), 247 (−5.5) nm; IR (neat) νmax 3448 (br), 2926, 2856, 1802, 1732, 1651, 1441, 1383, 1287, 1258, 1128, 1026, 993 cm−1; 1H, 13C NMR and HMBC data see Table 2; NOESY correlations (acetone-d6, 600 MHz) H-3 ↔ H3-21; H-4 ↔ H3-22; H-6a ↔ H-9; H-7 ↔ H3-21, H3-23; H-9 ↔ H-6a, H10a; H-10a ↔ H-9, H3-20; H-12a ↔ H3-24; H-12b ↔ H3-20; H-18 ↔ H3-24; H3-20 ↔ H-10a, H-12b; H3-21 ↔ H-3, H-7; H3-22 ↔ H-4; H3-23 ↔ H-7; H3-24 ↔ H-12a, H-18; HRESIMS m/z 417.2276 [M + H]+ (calcd for C24H33O6, 417.2272). Phomanolide F (4): colorless needles (MeOH−acetone); mp 262−264 °C (dec); [α]25D +66.0 (c 0.05, MeOH); UV (MeOH) λmax (log ε) 208 (4.42), 230 (4.09), 246 (4.05) nm; CD (c 1.8 × 10−4 M, MeOH) λmax (Δε) 206 (+40.30), 254 (−15.11) nm; IR (neat) νmax

Figure 5. Thermal ellipsoid representation of 4. (Note: A different numbering system is used for the structural data deposited with the CCDC.) 750 spectrophotometer. 1H and 13C NMR data were acquired with Bruker Avance III-600 spectrometer using solvent residual signals (CDCl3: δH 7.26/δC 77.2; acetone-d6: δH 2.05/δC 29.8, 206.1) as references. The HSQC and HMBC experiments were optimized for 145.0 and 8.0 Hz, respectively. ESIMS and HRESIMS data were obtained using an Agilent Accurate-Mass-Q-TOF LC/MS G6550 instrument equipped with an electrospray ionization (ESI) source. All MS experiments were performed in the positive ion mode. HPLC separations were performed on an Agilent 1260 instrument equipped with a variable-wavelength 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 morphology and sequence (Genbank Accession No. KP896486) analysis of the ITS region of the rDNA and assigned the accession number 10481 in the China General Microbial Culture Collection (CGMCC) at the Institute of Microbiology, Chinese Academy of Sciences, Beijing. 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, 40 pieces were used to inoculatein eight 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, eight 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. Fermentation was carried out in 80 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 30 days. Extraction and Isolation. The fermented material was extracted repeatedly with EtOAc (4 × 12.0 L), and the organic solvent was evaporated to dryness under vacuum to afford the crude extract (80.0 g), which was fractionated by silica gel vacuum liquid chromatography (VLC) using petroleum ether−CH2Cl2−MeOH gradient elution. The fraction (2.1 g) eluted with 400:8 CH2Cl2−MeOH was separated by reversed-phase silica gel column chromatography (CC) eluting with a

Table 3. Cytotoxicity of Compounds 2 and 4a IC50 (μM) glioma cell lines

tumor cell lines

compound

SH-SY5Y

U251

H4

MCF-7

2 4 cisplatin

131.9 ± 2.3 83.5 ± 4.6 18.3 ± 3.6

38.8 ± 3.8

44.6 ± 3.6

68.4 ± 5.3

29.4 ± 3.2

29.6 ± 4.8

16.2 ± 2.6

HeLa 50.0 ± 6.8 14.8 ± 3.9

No activity was detected for compounds 1 and 3 at 50 μM.

a

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Scheme 1. Hypothetical Biosynthetic Pathways for 1−6

3435 (br), 2928, 2867, 2360, 1797, 1712, 1618, 1462, 1224, 1091 cm−1; 1H, 13C NMR and HMBC data see Table 2; NOESY correlations (CDCl3, 600 MHz) H-3 ↔ H3-20, H3-21; H-4 ↔ H3-22; H-6a ↔ H-9; H-6b ↔ H3-22; H-7 ↔ H3-22, H3-23; H-9 ↔ H-6a, H320; H-10b ↔ H3-23; H-12a ↔ H3-20, H3-24; H-18a ↔ H3-24; H-18b ↔ H-12b, 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, H2-18; HRESIMS m/z 575.2619 [M + Na]+ (calcd for C32H40O8Na, 575.2615). X-ray Crystallographic Analysis of 4.25 Upon crystallization from MeOH−acetone (2:1) using the vapor diffusion method, colorless crystals were obtained for 4. A crystal (0.55 × 0.04 × 0.03 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 = 1.54184 Å at 107.0(10) K. Crystal data: C33H44O9, M = 584.68,26 space group orthorhombic, P2(1)2(1)2; unit cell dimensions a = 22.2561(9) Å, b = 16.0137(5) Å, c = 8.7122(3) Å, V = 3105.05(19) Å3, Z = 4, Dcalcd = 1.251 mg/m3, μ = 0.738 mm−1, F(000) = 1256. The structure was solved by direct methods using SHELXL-9727 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.28 The 11 640 measurements yielded 5899 independent reflections after equivalent data were averaged and Lorentz and polarization corrections were applied. The final refinement gave R1 = 0.0495 and wR2 = 0.1192 [I > 2σ(I)]. Computational Details. Conformational analyses for 1−3 were performed via the MOE version 2009.10 (Chemical Computing Group, Canada) software package with LowModeMD at the MMFF94 force field. The MMFF94 conformational analyses were further optimized using TDDFT at the B3LYP/6-311G(d,p) basis set level in MeOH with the IEFPCM model. 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 and 150 lowest electronic transitions were calculated for compounds 1 and 3, and 2, respectively. The rotational strengths of each electronic excitation were given using both dipole length and dipole velocity representations. The ECD spectrum was simulated in SpecDis29 using a Gaussian function with half-bandwidths of 0.30 and 0.42 eV for compounds 1 and 3, and 2, 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 correlations. All quantum computations were performed using the Gaussian 09 package.30 MTS Assay.31 Cells were seeded in 96-well plates at a density of 1.0 × 104 cells/well in 100 μL of complete culture medium. After cell attachment overnight, the medium was removed, and each well was treated with 100 μL of medium containing 0.1% DMSO or appropriate concentrations of the test compounds and the positive control cisplatin and incubated with cells at 37 °C for 48 h in a 5% CO2-containing incubator. Proliferation was assessed by adding 20 μL of MTS (Promega) to each well in the dark, after 90 min of incubation at 37 °C. The optical density was recorded on a microplate reader at 490 nm. Three duplicate wells were used for each concentration, and all the tests were repeated three times.

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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.9b00281. NMR spectra of 1−4, ECD calculations for 1−3 (PDF)

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X-ray data of 4 (CIF)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Yongsheng Che: 0000-0003-1126-2754 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS J.Z., Y.L., F.R., and Y.Z. were supported in part by grant 81402832 from the National Natural Science Foundation of China; Y.C. was supported by the CAMS Innovation Fund for Medical Sciences (2018-I2M-3-005). 1684

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Article

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