Absolute Configuration of Bioactive Azaphilones from the Marine

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Absolute Configuration of Bioactive Azaphilones from the MarineDerived Fungus Pleosporales sp. CF09‑1 Fei Cao,*,† Zhi-Hui Meng,† Xing Mu,‡ Yu-Fei Yue,† and Hua-Jie Zhu*,† †

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College of Pharmaceutical Sciences, Key Laboratory of Pharmaceutical Quality Control of Hebei Province, Key Laboratory of Medicinal Chemistry and Molecular Diagnostics of Education Ministry of China, Hebei University, Baoding 071002, People’s Republic of China ‡ College of Life Sciences, Hebei University, Baoding 071002, People’s Republic of China S Supporting Information *

ABSTRACT: Investigation of the marine-derived fungus Pleosporales sp. CF09-1 cultured in modified PDB medium led to the isolation of six new azaphilone derivatives, pleosporalones B and C (1 and 2) and pleosporalones E−H (4−7), and one known analogue (3). The absolute configurations of C-2′ and C-3′ in 3 were assigned by a vibrational circular dichroism method. The C-11 relative configurations for the pair of C-11 epimers (4 and 5) were established by comparing the magnitude of the computed 13C NMR chemical shifts (Δδcalcd) with the experimental 13C NMR values (Δδexp) for the epimers. Antiphytopathogenic and anti-Vibrio activities were evaluated for 1−7. Pleosporalone B (1) exhibited potent antifungal activities against the fungi Alternaria brassicicola and Fusarium oxysporum with the same MIC value of 1.6 μg/mL, which were stronger than the positive control ketoconazole among these compounds. Additionally, pleosporalone C (2) displayed significant activity against the fungus Botryosphaeria dothidea (MIC, 3.1 μg/mL). Compounds 6 and 7 displayed moderate anti-Vibrio activities against Vibrio anguillarum and Vibrio parahemolyticus, with MIC values of 13 and 6.3 μg/mL for 6 and 6.3 and 25 μg/mL for 7, respectively.

I

activities against three marine-derived Vibrio spp. were tested for 1−7. Herein, we report the details of the isolation, structure elucidation, absolute configuration determination, and antimicrobial activities of 1−7.

n recent years, fungi inhabiting marine environments have attracted widespread attention for their ability to produce bioactive natural products with diverse structures.1 Additionally, it has been proven by genomic analyses that many secondary metabolite biosynthetic gene clusters in marinederived fungi can be transcriptionally suppressed under certain cultivation conditions.2−4 Using different conditions, including varying the period of cultivation, the composition of the culture medium, and temperature, is a popular approach to activate these gene clusters to generate new bioactive structures.2 In the course of our previous efforts to discover bioactive natural products from marine sources, an azaphilone derivative with an aromatic A-ring, was isolated from the marine-derived fungus Pleosporales sp. CF09-1 grown on solid rice medium.5 In order to explore the ability of this fungus to generate other bioactive azaphilone derivatives, changes in the composition of the culture medium were employed. Investigation of the fungus Pleosporales sp. CF09-1 cultured in modified potato dextrose broth (PDB) medium led to the isolation of seven azaphilone derivatives, pleosporalones B−H (1−7). Electronic circular dichroism (ECD), vibrational circular dichroism (VCD), or gauge-independent atomic orbital (GIAO) 13C NMR calculation methods were used to determine the absolute configurations of 1−7. The antifungal activities against three plant pathogens and anti-Vibrio © XXXX American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION Pleosporalone B (1) was isolated as a yellow oil. Its positive HRESIMS spectrum gave the molecular formula of C27H29ClO8, indicating 13 degrees of unsaturation. In the NMR spectra of 1 (Tables 1 and 2), the signals corresponding to the three keto groups [δC 196.0 (C-15), 191.3 (C-8), and 186.5 (C-6)], two ester carbonyls [δC 173.8 (C-1′) and 171.4 (C-1″)], three trisubstituted double bonds [δH 7.91 (1H, s, H1); δC 153.6 (C-1) and 114.9 (C-8a), δH 6.66 (1H, s, H-4); δC 166.5 (C-3) and 111.4 (C-4), and [δH 5.69 (1H, q, J = 6.5 Hz, H-5′); δC 130.5 (C-4′) and 127.7 (C-5′)], and two tetrasubstituted double bonds [δC 138.4 (C-4a) and 111.4 (C-5) and δC 155.9 (C-11) and 130.3 (C-10)] were observed. These functional groups accounted for 10 of the 13 degrees of unsaturation, and the remaining three thus required a tricyclic system (A-, B-, and C-rings) in 1. Moreover, one oxygenbearing nonprotonated carbon [δC 84.9 (C-7)] and one singlet Received: December 7, 2018

A

DOI: 10.1021/acs.jnatprod.8b01030 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Table 2. 13C NMR Data (δ) of 1−3 (125 MHz)

Table 1. 1H NMR Data (δ) of 1−3 (500 MHz, J in Hz) no.

1a

2b

3a

1 4 5 9 12 13 14 16 2′ 3′ 5′ 6′ 7′ 8′ 2″

7.91, s 6.66, s

8.21, s 6.61, s 5.49, s 1.57, s 6.78, d (7.5) 7.19, dd (8.0, 7.5) 6.74, d (8.0) 2.29, s 2.95, dq (11.0, 6.5) 5.25, d (11.0) 5.67, q (6.5) 1.65, d (6.9) 1.05, d (6.9) 1.62, s 2.10, s

7.86, s 6.17, s 5.53, s 1.55, s 2.52, m 2.04, m 2.49, m 2.06, s 2.94, dq (11.0, 6.9) 5.29, d (11.0) 5.68, q (6.9) 1.62, d (6.9) 1.07, d (6.9) 1.57, s 2.13, s

1.57, s 2.54, m 2.01, m 2.52, m 2.09, s 2.95, dq (11.0, 6.5) 5.35, d (11.0) 5.69, q (6.5) 1.63, d (6.9) 1.10, d (6.9) 1.58, s 2.12, s

a

no.

1a

2b

3a

1 3 4 4a 5 6 7 8 8a 9 10 11 12 13 14 15 16 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 1″ 2″

153.6, CH 166.5, C 111.4, CH 138.4, C 111.4, C 186.5, C 84.9, C 191.3, C 114.9, C 22.3, CH3 130.3, C 155.9, C 32.8, CH2 21.6, CH2 37.3, CH2 196.0, C 23.1, CH3 173.8, C 41.4, CH 81.0, CH 130.5, C 127.7, CH 13.3, CH3 14.0, CH3 10.7, CH3 171.4, C 21.4, CH3

156.8, CH 158.3, C 115.0, CH 145.9, C 106.8, CH 194.8, C 86.1, C 194.1, C 116.4, C 22.8, CH3 120.4, C 139.9, C 122.4, CH 132.5, CH 114.3, CH 157.3, C 19.9, CH3 175.0, C 42.4, CH 82.4, CH 132.1, C 128.3, CH 13.2, CH3 14.2, CH3 10.7, CH3 172.0, C 21.3, CH3

154.2, CH 165.4, C 114.1, CH 142.3, C 107.7, CH 192.6, C 84.5, C 192.6, C 115.1, C 22.1, CH3 130.1, C 153.8, C 33.8, CH2 21.5, CH2 38.4, CH2 195.4, C 22.9, CH3 173.7, C 41.1, CH 80.7, CH 130.7, C 127.1, CH 13.1, CH3 13.9, CH3 10.6, CH3 170.3, C 21.2, CH3

Recorded in CDCl3. bRecorded in CD3OD.

a

Recorded in CDCl3. bRecorded in CD3OD.

methyl [δH 1.57 (H3-9); δC 22.3 (C-9)] attached at C-7 [HMBC correlation from H-9 to C-7] were observed for 1. These NMR data indicated 1 belongs to the family of azaphilone derivatives, which always contain an oxygenated bicyclic nucleus (A- and B-rings) and a stereogenic nonprotonated center (C-7).6,7 Careful comparison of the NMR data of 1 with those of the known azaphilone cohaerin B, previously isolated from the fungus Hypoxylon cohaerens,8 revealed that 1 and cohaerin B share the same azaphilone nucleus structure. In fact, the notable difference between 1 and cohaerin B lay within the respective side chains connected at C-7. The 2D NMR data of 1 (Figure 1) including the COSY cross-peaks of H-2′/H-3′/H3-7′ and H-5′/H3-6′ and the key HMBC correlations from H-3′ to C-1″, from H3-7′ to C-1′/C3′, and from H3-8′ to C-3′/C-4′/C-5′ suggested the presence of a 3-acetoxy-2,4-dimethylhex-4-enoic acid moiety (side chain) in 1. The Cl atom attached to C-5 was established by the lack of another site to place the Cl in 1 and by comparing the chemical shift of C-5 of 1 with that of the known azaphilones with a 5-Cl.9 Furthermore, compared to cohaerin B, 1 lacks the 13-hydroxy group, which was verified by the COSY cross-peaks of H2-12/H2-13/H2-14. On the basis of the above NMR data analyses, the planar structure of 1 was identified.

Figure 1. COSY and key HMBC correlations of 1 and 4.

Pleosporalone C (2) had the molecular formula C27H28O8 (14 degrees of unsaturation) by position ion HRESIMS. The 1 H and 13C NMR data of 2 (Tables 1 and 2) indicated an azaphilone derivative and were closely related to those of 1. The main differences were the presence of trisubstituted aromatic ring signals [δH 6.78 (1H, d, J = 7.5 Hz, H-12), 6.74 (1H, d, J = 8.0 Hz, H-14), and 7.19 (1H, dd, J = 8.0, 7.5 Hz, H-13)] in the 1H NMR spectrum of 2, instead of the −CH2CH2CH2− signals [δH 2.54 (2H, m, H2-12), 2.52 (2H, m, H2-14), and 2.01 (2H, m, H2-13)] for 1. The above NMR data suggested that a cyclohexanone ring as in 1 was hydroxylated, followed by an elimination of water, then a tautomerization to form a 3-methylphenol ring (C-ring) in 2, which was confirmed by the HMBC correlations from H-4 and H2-12 to C-10 (δC 120.4) and COSY cross-peaks of H2-12/H213/H2-14 in 2. The other difference between 2 and 1 was that the 5-Cl group in 1 was absent in 2, which was indicated by the key HMBC correlations from H-5 to C-7 and C-8a of 2. Pleosporalone D (3) was also obtained as an azaphilone derivative structurally resembling 1. Its molecular formula, C27H30O8, was determined from the HRESIMS spectrum, B

DOI: 10.1021/acs.jnatprod.8b01030 J. Nat. Prod. XXXX, XXX, XXX−XXX

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at C-5). The same phenomenon for a chlorinated azaphilone (opposite C-7 configuration for a chlorinated versus a hydrogen-substituted metabolite) was also previously reported from a different fungus.9 The relative configurations of C-2′ and C-3′ of 1−3 were established by J-based configuration analyses. The large coupling constant (J2′,3′ = 11.0 Hz) indicated a erythro configuration at C-2′ and C-3′ in 1−3.11,12 Thus, two possible absolute configurations, (2′S,3′R)-1/2/3 and (2′R,3′S)-1/2/3, were present for 1−3, respectively. Because of the low yields of 1 and 2, compound 3 was chosen to determine the absolute configurations of C-2′ and C-3′ by studying its ECD and VCD spectra. First, the predicted ECD spectra of two possible structures [(2′S,3′R) and (2′R,3′S)] of 3 were calculated using the time-dependent density functional theory (TD-DFT) method. The results showed that both of the predicted ECD spectra for (2′S,3′R)-3 and (2′R,3′S)-3 matched well with the measured ECD spectrum of 3 (Figure S1), suggesting that it was not possible to determine the absolute configurations of C-2′ and C-3′ in the side chains of 1−3 by the ECD method. Recently, a VCD approach has become a robust and reliable alternative for the absolute configuration characterizations of natural products, but only when multiple milligram amounts are available.13−15 Thus, the experimental IR and VCD spectra of 3 (10.0 mg) were measured in 120 μL of CDCl3. The IR and VCD frequencies of (2′S,3′R)-3 and (2′R,3′S)-3 were calculated at the B3LYP/6311+G(d)//B3LYP/6-311+G(d) level in the gas phase, and the spectra were used to compare with the experimental VCD spectra of 3. As shown in Figure 3, the calculated VCD signals of (2′S,3′R)-3 had better agreement with the experimental VCD signals of 3, indicating the (2′S,3′R) configuration for 3. Based on a shared biogenesis, the absolute configurations could be proposed as (2′S,3′R,7S) for 1−3. Pleosporalone E (4) was obtained as a yellow oil with a molecular formula of C22H26O9 determined by positive HRESIMS. The characterized NMR data of 4 (Table 3), including one ketone carbonyl signal [δC 198.4 (C-6)], one trisubstituted double bond signal [δH 5.86 (1H, s, H-5); δC 158.9 (C-4a) and 124.3 (C-5)], and one singlet methyl signal with a singlet [δH 1.22 (H3-9); δC 20.4 (C-9)], suggested that 4 was also an azaphilone derivative. Two aromatic proton signals at δH 6.15 (1H, d, J = 2.0 Hz) and 6.13 (1H, d, J = 2.0 Hz) and six aromatic carbon signals indicated that an oorsellinic acid moiety was connected at C-8 in 4,16 which was

indicating the loss of a Cl atom compared with that of 1. The planar structure of 3 was the same as the known azaphilone FA200B, which has been previously published in the patent literature.10 However, the relative and absolute configurations of the known azaphilone FA200B were not defined due to the high conformational flexibility of the molecule. By detailed comparison of the [α]D, UV, IR, 1H NMR, and 13C NMR data of 3 with those of FA200B (Table S1), the highly similar data revealed that 3 was the known metabolite FA200B with unassigned configurations. Defining the configurations of pleosporalones B−D (1−3) was an interesting and difficult exercise in the present work. The configuration of the double bond between C-4′ and C-5′ in 1−3 could be assigned as the E-configuration by the NOESY experiments of 1−3, based on the correlations between H3-6′ and H3-8′ and between H-3′ and H-5′ in 1− 3. The absolute configurations of C-7 in 1−3 were easily assigned as S, S, and S, respectively, by the negative ECD Cotton effect for 1 and the positive ECD Cotton effects for 2 and 3 around 360 nm (Figure 2), according to the earlier

Figure 2. Experimental ECD spectra of 1−3.

references.8,9 It was interesting that visual inspection of compound 1 versus compounds 2 and 3 showed that the C7 configuration was opposite spatially, even though all were formally 7S (due to the change in priority with Cl substitution

Figure 3. Comparison of the calculated VCD spectra of (2′S,3′R)-3 and (2′R,3′S)-3 and the experimental VCD spectra of 3. C

DOI: 10.1021/acs.jnatprod.8b01030 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Table 3. 1H and 13C NMR NMR Data for 4 and 5 (1H 600 MHz, 13C 150 MHz, CD3OD) 4

5

no.

δC, type

δH (J in Hz)

δC, type

1α

63.9, CH2

3.66, dd (10.0, 10.0) 3.75, dd (10.0, 7.2)

63.6, CH2

1β 3 4α 4β 4a 5 6 7 8 8a 9 10α 10β 11 12α 12β 13 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′

108.0, C 43.0, CH2 158.9, C 124.3, CH 198.4, C 75.1, C 76.8, CH 40.9, CH 20.4, CH3 45.6, CH2 81.8, CH 72.7, CH2 57.4, CH3 172.0, C 105.3, C 166.4, C 102.1, CH 164.1, C 113.1, CH 145.2, C 25.2, CH3

2.59, d (14.4) 2.74, d (14.4) 5.86, s

5.18, 3.08, 1.22, 1.85, 2.29, 4.03, 3.86, 3.78, 3.20,

configuration (Figures S23 and S31). Thus, the absolute configurations of C-3, C-7, C-8, and C-8a in the bicyclic nucleus of 4 and 5 could be defined as 3S, 7S, 8S, and 8aS, respectively, based on the unambiguous relative configurations. However, the experimental ECD spectra of 4 and 5 were almost identical, indicating that the ECD method had limitations in the assignment of the C-11 absolute configurations for 4 and 5. VCD spectra and optical rotations (OR) were computed for determining the C-11 absolute configuration of 4 and 5. However, both approaches were unsuccessful due to the similar VCD spectra and OR values of 4 and 5. Recently, computational methods for atomic chemical shift calculations have been developed and used for the relative configuration identifications of complex natural compounds.18−21 Compounds 4 and 5 are a pair of epimers with more than one stereogenic carbon. The carbons near C11 in 4 should have different chemical shifts from those of the corresponding carbons in 5. If the chemical shifts for 4 and 5 and the chemical shift differences between those carbons could be accurately computed, the configurations at C-11 of 4 and 5 could be established by comparing the magnitude of the computed chemical shifts (Δδcalcd.) for the (11R)-epimer and (11S)-epimer. The theoretical differences (Δδcalcd) could be compared with the experimental values (Δδexp) for determining the relative configurations for 4 and 5. Thus, four GIAO methods, B3LYP/6-311+G(d,p)//B3LYP/6-31+G(d) in the gas phase (method 1), B3LYP/6-311+G(2d,p)//B3LYP/631+G(d) in the gas phase (method 2), B3LYP/6-311+G(d,p)//B3LYP/6-31+G(d) in MeOH using the PCM model (method 3), and B3LYP/6-311+G(2d,p)//B3LYP/6-31+G(d) in MeOH using the PCM model (method 4) were used to calculate the 13C NMR values of the (11R)-epimer and the (11S)-epimer. The relative errors (Δδcalcd) between the computed 13C chemical shifts of the (11R)-epimer and (11S)-epimer and the relative errors (Δδexp) between the experimental 13C NMR data of 4 and 5 are summarized in Table 4. Based on the relative error magnitudes (Δδcalcd and

d (7.6) m s dd (14.2, 2.7) dd (14.2, 6.8) m d (10.2) dd (10.2, 4.8) s

6.13, d (2.0) 6.15, d (2.0) 2.44, s

δH (J in Hz) 3.65, m 3.77, dd (10.9, 6.6)

107.5, C 42.7, CH2 159.1, C 124.3, CH 198.4, C 75.1, C 76.8, CH 40.9, CH 20.4, CH3 44.3, CH2 81.1, CH 73.9, CH2 58.0, CH3 172.0, C 105.3, C 166.4, C 102.1, CH 164.1,C 113.1, CH 145.2, C 25.2, CH3

2.44, d (14.5) 2.71, d (14.5) 5.81, s

5.16, d (7.6) 3.06, m 1.20, s 1.99, dd (14.2, 7.7) 2.05, dd (14.2, 1.6) 4.01, m 4.06, dd (9.4, 6.4) 3.67, m 3.19, s

6.08, d (2.0) 6.14, d (2.0) 2.42, s

confirmed by the HMBC correlation from H-8 and C-1′ (Figure 1). The above NMR indicated that 4 was a structural analogue of pinophilin E, which was previously obtained from a gorgonian-derived strain of the fungus Penicillium pinophilum.17 The differences between them were the trisubstituted double bond between C-3 and C-4 in pinophilin E was absent in 4, the 12-Me in pinophilin E was replaced by an oxygenbearing methylene [δH 3.86 (1H, d, J = 10.2 Hz, H-12α), 3.78 (1H, dd, J = 10.2, 4.8 Hz, H-12β); δC 72.7 (C-12)] in 4, and an additional methoxy group [δH 3.20 (3H, s, H-13); δC 57.4 (C-13)] was present in 4. More importantly, the key HMBC correlation from H-12 to C-3 for 4 was observed, declaring that 4 was a C-3-spiroketal azaphilone. The relative configuration of 4 was assigned by a NOESY experiment, which showed NOE correlations from H-1β/H-9 to H-8, from H-8a to H-4α, and from H-10 to H-1α/4α. Pleosporalone F (5) had the same molecular formula C22H26O9 as 4. The 1H and 13C NMR data (Table 3) of 5 and 4 showed striking similarity, suggesting the same structural nucleus. In fact, only differences in the 13C NMR signals for C10 and C-12 were observed [δC 44.3 (C-10) and 73.9 (C-12) in 5 versus 45.6 (C-10) and 72.7 (C-12) in 4], indicating that the structural difference between 5 and 4 should be located at C-11. This was confirmed by the divergence of their 1H NMR signals attributed to H2-10 and H2-12. Moreover, a NOESY experiment indicated that 5 had the same relative configurations of C-3, C-7, C-8, and C-8a in 4. Thus, 5 was the 11epimer of 4. According to previous references,16,17 negative ECD Cotton effects for 4 and 5 around 340 nm indicated the 7S-

Table 4. Chemical Shift Differences of Selected Carbons in 4 and 5 experimental Δδexp

calculated Δδcalcd for (11R)-epimer − (11S)epimer

no.

4−5

method 1

C-1 C-3 C-4 C-4a C-10 C-11 C-12 C-13

0.3 0.5 0.3 −0.2 1.3 0.7 −1.2 −0.6

0.7 1.5 −0.5 1.4 2.3 1.8 0.2 −0.9

method 2 method 3 method 4 0.2 0.8 −0.6 0.1 2.1 1.2 −0.5 −1.4

0.3 0.5 0.7 0.1 2.6 1.0 −2.8 −1.1

0.4 0.6 0.8 −0.1 2.4 1.0 −2.6 −1.1

Δδexp), the configurations of C-11 for 4 and 5 were suggested to be R and S, respectively. Furthermore, this conclusion was confirmed by calculating the relative errors (Δδcalcd) between the computed 1H NMR chemical shifts of the (11R)-epimer and (11S)-epimer (Table S2). Pleosporalones G and H (6 and 7) were also isolated as a pair of epimeric azaphilone derivatives with highly conserved NMR data (Table 5) and the same molecular formula, C22H26O9. Compared with the known compound pinophilin B,22 an azaphilone obtained from the fungus P. pinophilum, the D

DOI: 10.1021/acs.jnatprod.8b01030 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Table 5. 1H and 13C NMR NMR Data for 6 and 7 (1H 300 MHz, 13C 75 MHz, Acetone-d6) 6

7

no.

δC, type

δH (J in Hz)

δC, type

1

69.1, CH2

3.85, dd (12.6, 10.8)

69.0, CH2

4.49, dd (10.8, 5.1) 3 4 4a 5 6 7 8 8a 9 10 11 12 13 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′

165.6, C 101.8, CH 151.0, C 116.0, CH 195.4, C 74.6, C 76.5, CH 35.5, CH 19.9, CH3 37.4, CH2 80.3, CH 63.8, CH2 57.8, CH3 171.6, C 104.8, C 164.0, C 103.1, CH 166.3,C 112.8, CH 145.1, C 24.8, CH3

6.26, s 5.66, s

5.27, 3.46, 1.29, 2.42, 3.52, 3.54, 3.46,

powerful physicochemical approach for the structural investigation of azaphilone derivatives (1−7). Fungal pathogens cause many of the most serious crop diseases. Searching for new anti-phytopathogenic agents from marine-derived fungi for controlling fungal pathogens has become a research trend.28,29 In previous literature, azaphilones have also been reported with a wide range of biological activities, such as antifungal,30,31 antibacterial,9,32 cytotoxic,8,16 and inhibition against several enzyme activities.7,16 Therefore, the inhibitory activities of 1−7 against three plant pathogenic fungi, Alternaria brassicicola, Botryosphaeria dothidea, and Fusarium oxysporum, were tested. Among these compounds, 1−3 were active at various levels against the three tested plant pathogens, with the MIC values ranging from 1.6 to 25 μg/mL (Table 6). Compound 1 exhibited potent antifungal activities

d (9.9) m s m m m s

5.70, brs 6.33, brs 2.60, s

165.6, C 101.7, CH 151.2, C 116.0, CH 195.6, C 74.6, C 76.4, CH 35.4, CH 19.8, CH3 37.1, CH2 80.3, CH 63.7, CH2 57.6, CH3 171.5, C 104.7, C 164.0, C 103.1, CH 166.1, C 112.7, CH 144.9, C 24.7, CH3

δH (J in Hz) 3.84, dd (12.6, 10.8) 4.47, dd (10.8, 5.1) 6.26, s 5.66, s

5.24, 3.42, 1.29, 2.43, 3.50, 3.53, 3.35,

d (9.9) m s d (4.2) m m s

Table 6. Anti-Phytopathogenic Activities of Compounds 1− 7 compound MIC (μg/mL)

5.71, brs

strain

1

2

3

4−7

ketoconazole

A. brassicicola B. dothidea F. oxysporum

1.6 13 1.6

6.3 3.1 25

13 13 6.3

50 50 50

3.1 3.1 6.3

against the fungi A. brassicicola and F. oxysporum with the same MIC value of 1.6 μg/mL, which were stronger than the positive control ketoconazole. Additionally, compound 2 displayed significant activity against the fungus B. dothidea (MIC, 3.1 μg/mL). However, compounds 4−7 displayed no activity against the selected strains (MIC > 50 μg/mL). The results indicate the potential value of these new azaphilones as agricultural fungicides. Vibriosis is one of the bacterial diseases that causes serious damage and great losses to mariculture.33,34 Thus, the antiVibrio activities against Vibrio anguillarum, V. parahemolyticus, and V. alginolyticus of the new compounds 1−7 were explored. Compounds 6 and 7 displayed moderate anti-Vibrio activities against V. anguillarum and V. parahemolyticus, with MIC values of 13 and 6.3 μg/mL for 6 and 6.3 and 25 μg/mL for 7, respectively. Compounds 4 and 5 showed weak anti-Vibrio activities against Vibrio alginolyticus with the same MIC values of 25 μg/mL.

6.32, brs 2.58, s

C-10/C-11 double bond in pinophilin B was hydrogenated in 6 and 7, and an additional methoxy group was connected to C11 in 6 and 7, which were confirmed by the 2D NMR data of 6 and 7. Thus, the planar structures of 6 and 7 were determined. The configurations of the bicyclic nucleus for 6 and 7 were also defined by comparing their NOESY and ECD (Figures S39 and S47) spectra with those of the known compound talaraculone A.16 The determination of the C-11 configurations in 6 and 7 was a challenging task. An attempt for the GIAO 13 C NMR calculations of 6 and 7 was also made (Table S3). However, the relative errors (Δδexp) between experimental 13C NMR data of 6 and 7 were much smaller than the relative errors (Δδcalcd) between the computed 13C chemical shifts of the (11R)-epimer and (11S)-epimer, indicating that the calculated NMR shift differences for assigning the 6 and 7 epimers did not provide definitive evidence for assigning the configurations. Thus, the configurations at C-11 in 6 and 7 remain unassigned. Azaphilones, which exhibit diverse biological activities, are a class of polyketides containing a pyrone-quinone bicyclic core and a stereogenic nonprotonated center.6,7 Due to the intriguing structural features and biological properties, the research focus on azaphilones has become a hot-spot in the field of natural product medicinal chemistry.22−27 These interesting azaphilones usually possess multiple stereogenic centers, leading to difficulties in determining their absolute configurations. Particularly, when the stereogenic carbons exist in the fatty chains of azaphilone derivatives, there are few methods to address this problem due to the high conformational flexibility of fatty chains.8,9,27 In the present research, VCD and GIAO 13C NMR calculation methods were used as a

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EXPERIMENTED SECTION

General Experimental Procedures. NMR data were recorded on Bruker Avance III 600 MHz NMR (600 MHz for 1H and 150 MHz for 13C), Agilent DD2 500 MHz NMR (500 MHz for 1H and 125 MHz for 13C), and Bruker AV300 300 MHz NMR (300 MHz for 1 H and 75 MHz for 13C) spectrometers, using tetramethylsilane as an internal standard. The other experimental procedures were performed as reported previously.35 Isolation of the Fungal Material. The marine-derived fungus Pleosporales sp. CF09-1 with GenBank accession number MK409359 has been previously described.5 Liquid fermentation of the fungus Pleosporales sp. CF09-1 was carried out in 200 Erlenmeyer flasks [each containing 400 mL of PDB culture broth and 13 g MgCl2 (guided by the UV and MS characteristics, it was found that the strain CF09-1 produced previously unrecognized compounds exhibiting the typical azaphilone UV spectrum when 3.3% MgCl2 was added)] and cultivated at 28 °C without shaking for 45 days. The mycelia was separated from the broth, the cell walls were broken by ultrasonication, and the cell material was extracted three times with EtOAc. The broth was also extracted using EtOAc two times. The combined organic phases were evaporated to dryness to provide an E

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conformers were performed by Gaussian 09 (Gaussian Inc.) software. TD-DFT at the B3LYP/6-311++G(2d,p) level in the gas phase was used for ECD calculations with a total of 60 excited states for (2′S,3′R)-3 and (2′R,3′S)-3, respectively. A standard deviation of 0.5 eV was applied for ECD simulations for both (2′S,3′R)-3 and (2′R,3′S)-3. VCD calculations for (2′S,3′R)-3 and (2′R,3′S)-3 were carried out at the B3LYP/6-311+G(d) level in the gas phase. For molecules (11R)-4, (11S)-5, (11R)-6, and (11S)-7, totally 73 stable conformers for 4 with relative energy within 10.0 kcal/mol, 76 conformers for 5, 78 conformers for 6, and 80 conformers for 7 were obtained, respectively. B3LYP theory at the basis sets of 6311+G(d,p) and 6-311+G(2d,p) in the gas phase and in MeOH solution using the PCM model, respectively, were applied for 13C NMR calculations for both 4 and 5. Two approaches, B3LYP/6311+G(d,p) and B3LYP/6-311+G(2d,p) in MeOH solvent using the PCM model, were used to calculate the 13C NMR values of 6 and 7. Boltzmann statistics were used for final simulations of the ECD, VCD, and 13C NMR for these molecules. Anti-phytopathogenic Assay. Three plant pathogenic fungi, Alternaria brassicicola CICC 2646, Botryosphaeria dothidea CICC 2678, and Fusarium oxysporum CICC 41029, were used for activity testing by the conventional broth dilution assay.5 The fungi were cultivated on potato dextrose agar (PDA) medium and incubated at 28 °C for 72 h. The seed culture was diluted in PYG medium (peptone 1.0%, glucose 2.0%, and yeast extract 1.0%) and delivered into presterilized 96-well plates. Stock solutions of 10 mg/mL prepared by dissolving each metabolite in DMSO were serial diluted in PDA medium to achieve in-test concentrations ranging from 0.78 to 100 μg/mL. The plates were incubated for 48 h in a humidified incubator at 28 °C. Ketoconazole was used as the positive control, and DMSO was used as the negative control. Anti-Vibrio Assay. Anti-Vibrio activities against Vibrio anguillarum CICC 10475, Vibrio parahemolyticus CICC 10552, and Vibrio alginolyticus CICC 21664 for 1−7 were also evaluated by the conventional broth dilution assay.5 LB medium (peptone 10 g/L, yeast extract 5 g/L, and NaCl 10 g/L) was used for microcultures, and ciprofloxacin was used as a positive control. The MICs of ciprofloxacin are 6.3 μg/mL against V. anguillarum, 3.1 μg/mL against V. parahemolyticus, and 13 μg/mL against V. alginolyticus.

EtOAc extract (106.0 g). The extract was subjected to silica gel column chromatography (CC) with a CH2Cl2/MeOH gradient system (100:0, 80:1, 60:1, 40:1, 20:1, 10:1, 1:1, and 0:100) to yield eight fractions, Fr.1−Fr.8. Fr. 4 (20.0 g) was separated by silica gel CC with a petroleum ether (PE)/EtOAc gradient system (from 4:1 to 1:1) to offer four subfractions, Fr.4.1−Fr.4.4. Subfraction Fr.4.3 was further purified on Sephadex LH-20 and ODS silica gel and by semipreparative HPLC using a Waters RP-18 column (5 μm, 10 × 250 mm) at a flow rate of 2.0 mL/min (MeOH/H2O, 65:35) to afford 1 (5.6 mg), 2 (6.2 mg), and 3 (20.0 mg). Subfraction Fr.4.4 was further purified by silica gel CC and semipreparative HPLC using a Waters Silica 2-ethylpyridine column (5 μm, 10 × 250 mm) at a flow rate of 2.0 mL/min (PE/EtOH, 90:10) to give 4 (9.5 mg) and 5 (9.0 mg). Fr. 5 (3.2 g) was divided into five parts, Fr.5.1−Fr.5.5, by ODS silica gel eluting with MeOH/H2O (30−80%). Subfraction Fr.5.4 was directly separated by semipreparative HPLC using a Daicel Chiralpak IA column (5 μm, 10 × 250 mm) at a flow rate of 2.0 mL/ min (PE/EtOH, 75:25) to yield 6 (8.4 mg) and 7 (8.0 mg). Pleosporalone B (1): yellow oil; [α]20D −96.0 (c 0.50, MeOH); UV (MeOH), λmax (log ε) 224 (4.40), 346 (3.90) nm; ECD (MeOH), λmax (Δε) 215 (5.31), 296 (1.22), 366 (−5.00) nm; IR (KBr), νmax 2926, 1742, 1676, 1643, 1450, 1238, 1126, 1022 cm−1; 1H and 13C NMR data, Tables 1 and 2; HRESIMS m/z 517.1630 [M + H]+ (calcd for C27H3035ClO8, 517.1624). Pleosporalone C (2): yellow oil; [α]20D +120 (c 0.50, MeOH); UV (MeOH), λmax (log ε) 219 (4.05), 337 (3.53) nm; ECD (MeOH), λmax (Δε) 275 (−4.21), 291 (6.45) nm; IR (KBr), νmax 3608, 2931, 1728, 1672, 1453, 1376, 1237, 1149, 1034 cm−1; 1H and 13C NMR data, Tables 1 and 2; HRESIMS m/z 481.1847 [M + H]+ (calcd for C27H29O8, 481.1857). Pleosporalone D (3): yellow oil; [α]20D +146 (c 0.50, MeOH); UV (MeOH), λmax (log ε) 221 (4.12), 335 (3.61) nm; ECD (MeOH), λmax (Δε) 275 (−4.09), 291 (6.27) nm; IR (KBr), νmax 2926, 1741, 1675, 1642, 1455, 1235, 1122, 1026 cm−1; 1H and 13C NMR data, Tables 1 and 2; HRESIMS m/z 483.2011 [M + H]+ (calcd for C27H31O8, 483.2013). Pleosporalone E (4): yellow oil; [α]20D +13.6 (c 0.20, MeOH); UV (MeOH), λmax (log ε) 218 (5.11), 263 (4.23), 308 (3.60) nm; ECD (MeOH), λmax (Δε) 226 (3.94), 236 (2.30), 255 (4.97), 337 (−2.70) nm; IR (KBr), νmax 3392, 2923, 1718, 1640, 1447, 1249, 1153, 989 cm−1; 1H and 13C NMR data, Table 3; HRESIMS m/z 435.1646 [M + H]+ (calcd for C22H27O9, 435.1650). Pleosporalone F (5): yellow oil. [α]20D +10.8 (c 0.20, MeOH); UV (MeOH) λmax (log ε) 218 (4.98), 263 (4.01), 308 (3.47) nm; ECD (MeOH), λmax (Δε) 227 (3.55), 235 (2.96), 252 (5.35), 338 (−2.68) nm; IR (KBr), νmax 3390, 2925, 1722, 1645, 1449, 1252, 1160, 982 cm−1; 1H and 13C NMR data, Table 3; HRESIMS m/z 435.1643 [M + H]+ (calcd for C22H27O9, 435.1650). Pleosporalone G (6): yellow oil. [α]20D +75.2 (c 1.00, MeOH); UV (MeOH), λmax (log ε) 219 (4.06), 270 (3.78), 316 (3.98) nm; ECD (MeOH), λmax (Δε) 215 (2.76), 241 (−1.70), 261 (2.65), 326 (−1.00), 364 (4.96) nm; IR (KBr), νmax 3529, 2923, 1612, 1456, 1261, 1051 cm−1; 1H and 13C NMR data, Table 4; HRESIMS m/z 457.1454 [M + H]+ (calcd for C22H26O9Na, 457.1469). Pleosporalone H (7): yellow oil. [α]20D +91.0 (c 1.00, MeOH); UV (MeOH), λmax (log ε) 219 (4.05), 271 (3.76), 317 (4.00) nm; ECD (MeOH), λmax (Δε) 215 (2.61), 241 (−1.62), 261 (2.52), 325 (−0.96), 365 (4.71) nm; IR (KBr), νmax 3530, 2923, 1610, 1455, 1263, 1049 cm−1; 1H and 13C NMR data, Table 4; HRESIMS m/z 457.1454 [M + H]+ (calcd for C22H26O9Na, 457.1469). Computational Section. Conformational searches for the molecules (2′S,3′R)-3, (2′R,3′S)-3, (11R)-4, (11S)-5, (11R)-6, and (11S)-7 were carried out using the MMFF94S force field by the BARISTA software (CONFLEX Corporation). For molecules (2′S,3′R)-3 and (2′R,3′S)-3, totally 51 stable conformers for (2′S,3′R)-3 with relative energy within a 10.0 kcal/mol energy window and 50 conformers for (2′R,3′S)-3 were recorded, respectively. DFT calculations were used to optimize the conformers at the B3LYP/6-31G(d) and B3LYP/6-311+G(d) levels, respectively. The ECD, VCD, and GIAO 13C NMR calculations for the stable

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.8b01030.

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1D and 2D NMR and HRESIMS spectra of the new compounds (1, 2, and 4−7); ECD spectra of 4−7; and quantum mechanical calculation data for 3 and 4−7 (PDF)

AUTHOR INFORMATION

Corresponding Authors

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

Fei Cao: 0000-0002-5676-3176 Hua-Jie Zhu: 0000-0002-7263-2360 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (Nos. 41606174; 21877025), the Natural Science Foundation of Hebei Province of China (No. F

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B2017201059), and the High Performance Computer Center of Hebei University.

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