Decalin-Containing Tetramic Acids and 4-Hydroxy-2-pyridones with

Article Cite This: J. Org. Chem. 2017, 82, 11474-11486

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Decalin-Containing Tetramic Acids and 4‑Hydroxy-2-pyridones with Antimicrobial and Cytotoxic Activity from the Fungus Coniochaeta cephalothecoides Collected in Tibetan Plateau (Medog) Junjie Han,†,‡,# Congcong Liu,†,§,# Li Li,⊥,# Hui Zhou,† Li Liu,†,‡ Li Bao,† Qian Chen,† Fuhang Song,∥ Lixin Zhang,∥ Erwei Li,† Ling Liu,† Yunfei Pei,¶ Cheng Jin,† Yanfen Xue,□ Wenbing Yin,† Yanhe Ma,□ and Hongwei Liu*,†,‡ Downloaded via UNIV OF LOUISIANA AT LAFAYETTE on January 14, 2019 at 06:08:56 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

†

State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China Savaid Medical School, University of Chinese Academy of Sciences, Beijing 100049, China § College of Life Sciences, Hebei University, Baoding 071002, China ⊥ Institute of Materia Medica, CAMS & PUMC, Beijing 100050, China ∥ CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China ¶ National Institutes for Food and Drug Control, Beijing 100050, China □ State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China ‡

S Supporting Information *

ABSTRACT: New tetramic acid derivatives, (±)-conipyridoins A−D (1−4), conipyridoins E (5) and F (6), and new 4-hydroxy-2-pyridone alkaloids (±)-didymellamide E (7), (+)-didymellamide B (8), (+)-N-hydroxyapiosporamide (9), and didymellamides F−H (10−12) were isolated and identified from the solid culture of the fungus Coniochaeta cephalothecoides. Chiral resolution of 1, 2, 3, 4, and 7 gave five pairs of enantiomers: 1a/1b, 2a/2b, 3a/3b, 4a/4b, and 7a/7b, respectively. Stereochemistry of 1a and 1b, and 2a and 2b was established and confirmed by the single-crystal X-ray diffraction and electronic circular dichroism (ECD) methods. Absolute configuration in 3a, 3b, 4a, 4b, 7a, and 7b was assigned by ECD calculations. Compounds 1−6 possess an unprecedented chemical skeleton featuring a decalin ring and a tetramic acid moiety. Compound 11 significantly inhibited the growth of Candida albicans and Aspergillus f umigatus with minimum inhibitory concentration (MIC) of 3.13 and 1.56 μM, respectively, and was further confirmed to be a new chitin synthesis inhibitor. Compound 5 exhibited the strongest activity against the growth of both Staphylococcus aureus and MRSA with MIC value of 0.97 μM. In the light of a co-occurrence of 3-acyl tetramic acids and biogenetically related pyridine alkaloids, the biosynthetic pathway for 1−12 was postulated.

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INTRODUCTION

Secondary metabolites from microorganisms are well-known for their ability to impede the growth of other nearby microorganisms. Fungi have been known as a valuable source of antifungal compounds.13−15 Fungi in the genus Coniochaeta (Coniochaetale) are composed of 101 species. They grow as saprotrophs, plant endophytes, and pathogens. Some Coniochaeta species have been reported to produce potent antibacterial and antifungal secondary metabolites, for example coniosetin from C. ellipsoidea, benzopyranone derivatives from C. saccardoi and C. tetraspora, and thiepinols and thienol from an endolichenic fungus Coniochaeta sp.16−19 In our search for new antifungal agents from fungi, the EtOAc extract of the fungus C. cephalothecoides was found to

Over the past 30 years, incidences of systemic fungal infections have soared as a result of increased populations with impaired immunity due to the occurrence of organ transplants, AIDS, diabetes, and various forms of cancer in clinics.1−5 According to statistics, the number of deaths from fungal infection reaches about 2 million each year.6 Pathogens belonging to four genera of fungi, Aspergillus, Candida, Cryptococcus, and Pneumocystis, have drawn most concern in clinics due to their mortality.7−12 The research and development of antifungal drugs is seriously lagging behind the increasing morbidity and mortality of fungal infection and drug resistance. Echinocadins are the only antifungal drug developed in the past 30 years. The overuse of antibiotics in the community and hospitals has accelerated the antibiotic-resistance crisis. © 2017 American Chemical Society

Received: August 10, 2017 Published: October 11, 2017 11474

DOI: 10.1021/acs.joc.7b02010 J. Org. Chem. 2017, 82, 11474−11486

Article

The Journal of Organic Chemistry

Figure 1. Structures of compounds 1−12.

show strong antifungal activity against Candida albicans and Aspergillus f umigates with minimum inhibitory concentration (MIC) values less than 100 μg/mL. Chemical investigation on its extract led to the identification of 12 secondary metabolites, including new tetramic acid derivatives (±)-conipyridoins A−D (1−4), conipyridoins E (5) and F (6), and new 4-hydroxy-2pyridone alkaloids (±)-didymellamide E (7), (+)-didymellamide B (8), (+)-N-hydroxyapiosporamide (9), and didymellamides F−H (10−12) (Figure 1). Details of the isolation, structure elucidation, and antimicrobial bioactivities of secondary metabolites isolated are presented here.

1 revealed the presence of 2 methyls [δH/δC 1.02 (d, J = 7.1 Hz)/18.4, 1.62 (s)/24.0], 3 methylenes [δH/δC 1.73 (m), 2.48 (d, J = 16.0 Hz)/31.2; 1.73 (m), 1.90 (m)/37.9 and 3.02 (dd, J = 7.8, 14.0 Hz), 3.39 (d, J = 3.8, 14.0 Hz)/38.3], 5 methines [δH/δC 4.40 (dd, J = 3.7, 7.8 Hz)/64.2, 4.17 (dd, J = 5.2, 10.8 Hz)/48.1, 2.93 (m)/33.5, 2.09 (m)/38.7, 1.99 (m)/32.2], 7 sp2 methines [δH/δC 5.38 (d, J = 3.7 Hz)/122.0, 5.54 (d, J = 9.8 Hz)/130.4, 5.74 (ddd, J = 2.4, 4.6, 9.8 Hz)/132.6, 7.29 (d, J = 8.4 Hz)/131.8 (×2), 7.11 (d, J = 8.4 Hz)/116.6 (×2)], and 7 sp2 quaternary carbons (δ 103.3, 127.8, 134.2, 158.3, 177.3, 192.5 and 195.9) in its structure. The 1H NMR spectrum of compound 1 showed signals of a typical AA′BB′ spin system attributable to H-21/25 [δ 7.29 (2H, d, J = 8.4 Hz)] and H-22/ 24 [δ 7.11 (2H, d, J = 8.4 Hz)], which indicated a 1,4substituted benzene. The 1H−1H COSY correlations of H-8-H9-H2-10-H-11, H2-13-H-14-H-15-H-16-H-17, and H-8-H-17H3-18 together with the HMBC correlations from H3-19 to C11, C-12, and C-13; from H3-18 to C-8, C-16, and C-17; and from H-9 to C-8, C-10, C-11, C-13, C-14, and C-17 confirmed the presence of the decalin ring moiety. Furthermore, the 1 H−1H COSY correlation of H-5-H2-6, the HMBC correlations of H-5 to C-2, C-4, C-6, and C-20; H2-6 to C-5, C-4, C-20, and C-21/25; H-8 to C-7 and C-3, and the chemical shifts of C-2 (δ 177.3), C-3 (δ 103.3), C-4 (δ 195.9), C-5 (δ 64.2), and C-7 (δ

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RESULTS AND DISCUSSION The fungus C. cephalothecoides was identified on the basis of the morphological features (Figure S1) and the ITS and LSU gene sequences. The strain was grown on rice medium, and the EtOAc extract of the solid culture was subjected to chromatographic separation using silica gel, ODS, Sephadex LH-20, and preparative HPLC to afford new secondary metabolites 1a, 1b, 2, 3a, 3b, 4a, 4b, 5, 6, 7a, 7b, and 8−12. (±)-Conipyrrolidone A (1) was isolated as a white needle crystal. A molecular formula of C24H27NO4 (12 degree of unsaturation) was assigned for 1 by the HRMS (ESI-TOF) and NMR data (Table 1). The 1H, 13C NMR, and HSQC spectra of 11475


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The Journal of Organic Chemistry Table 1. 1H and 13C NMR Data for Compounds 1−3 in Pyridine-d5a pos.

a

δC

1-NH 2 3 4 5 6

177.3 103.3 195.9 64.2 38.3

7 8 9 10

192.5 48.1 32.2 31.2

11 12 13

122.0 134.2 37.9

14 15 16 17 18 19 20 21 22 23 24 25

38.7 130.4 132.6 33.5 18.4 24.0 127.8 131.8 116.6 158.3 116.6 131.8

1 δH (J in Hz)

δC

2 δH (J in Hz)

9.97 brs

4.40 dd (3.7, 7.8) 3.02 dd (7.8, 14.0) 3.39 dd (3.7, 14.0) 4.17 1.99 1.73 2.48 5.38

dd (5.2, 10.8) m m d (16.0) d (3.7)

1.73 1.90 2.09 5.54 5.74 2.93 1.02 1.62

m m m d (9.8) ddd (2.4, 4.6, 9.8) m d (7.1) s

7.29 d (8.4) 7.11 d (8.4) 7.11 d (8.4) 7.29 d (8.4)

δC

9.96 brs 177.2 103.5 195.9 64.2 38.4 192.5 48.0 32.4 31.1 122.1 134.2 37.9 38.8 130.4 133.1 33.7 18.5 24.0 128.1 131.7 116.7 158.3 116.7 131.7

4.39 dd (3.8, 7.8) 3.08 dd (7.8, 14.0) 3.37 dd (3.8, 14.0) 4.18 2.00 1.75 2.40 5.37

m m m d (16.0) d (3.7)

1.70 1.90 2.11 5.53 5.73 2.94 1.08 1.61

m m m d (9.8) ddd (2.4, 4.6, 9.8) m d (7.1) s

7.29 d (8.4) 7.10 d (8.4) 7.10 d (8.4) 7.29 d (8.4)

3 δH (J in Hz) 10.60 brs

174.3 103.4 183.9 134.4 106.3 196.6 50.6 32.8 31.6 122.8 133.9 38.3 39.0 130.4 133.7 33.2 18.7 24.1 127.6 131.8 117.4 159.2 117.4 131.8

7.03 s

4.62 2.16 1.82 2.71 5.40

dd (5.5, 10.8) m m brd (16.0) d (2.7)

1.77 1.93 2.20 5.58 5.82 3.24 1.13 1.62

m m m d (9.8) m m d (7.1) s

7.86 d (8.2) 7.25 d (8.2) 7.25 d (8.2) 7.86 d (8.2)

“m” means multiplet or overlapped with other signals.

192.5) supported a tetramic acid moiety that was connected with decalin moiety through C-7 and linked with 1,4substituted benzene moiety through C-6 (Figure 2).

Figure 2. Selected key HMBC and 1H−1H COSY correlations of 1, 2, 9, and 10.

The partial relative configuration of 1 was confirmed by NOESY experiment (Figure 3). H-8, H-14, and H-17 were placed on the same face of the decalin ring, and H-9 and H3-18 were on the opposite face of the bicycle ring based on NOESY cross-peaks of H-8 with H-14 and H-17 and H3-18 with H-9. Fortunately, compound 1 was obtained in the crystalline form. Its structure was finally confirmed by single-crystal X-ray crystallographic analysis (Figure 4). An intramolecular hydrogen bond was formed between the carbonyl group at C-2 and the hydroxyl group at C-7 in 1. The single-crystal X-ray diffraction data showed the space group Pbca, indicating that conipyrrolidone A (1) was a racemic mixture, as further confirmed by an [α]25 D specific rotation of zero. The racemic 1 was resolved into (−)-conipyrrolidone A (1a) and (+)-con-

Figure 3. Selected key NOE correlations of 1 and 9.

ipyrrolidone A (1b) by a Kromasil 5-CelluCoat column using n-hexane/isopropanol (94/6, v/v) as eluent (Figure S4A). The electronic circular dichroism (ECD) spectra and [α]25 D values of 1a and 1b demonstrated their enantiomeric relationship. Absolute configurations of 1a and 1b were 11476


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Figure 4. X-ray crystallographic structures of 1 and 2.

and the almost zero value of the optical rotation confirmed the racemic feature of 2. An online HPLC using a Chiralpak stationary phase coupled with the CD detector was employed for the separation and the measurement of the ECD spectrum for 2. Two peaks corresponding to 2a and 2b were detected (Figure S4B). Following the approach outlined above, geometry optimization of 2a and 2b yielded three possible conformers (2−1−2−3, Figure S5B and Table S1). The respective absolute configurations of peak A and peak B were determined as 5R, 8S, 9S, 14R, 17S and 5S, 8R, 9R, 14S, 17R by comparison of their experimental and calculated ECD spectra (Figure 6).

established by comparison of the experimental ECD and theoretical ECD spectra calculated using time-dependent density functional theory (TDDFT)20,21 A conformational search using molecular mechanics yielded three possible conformers (1−1−1−3, Figure S5A and Table S1), which were reoptimized at B3LYP/6-31+G(d,p) level in methanol and used for the ECD calculations. A comparison of the observed ECD spectra for 1a and 1b with the calculated ECD spectra for the (5S, 8S, 9S, 14R, 17S)-1 and (5R, 8R, 9R, 14S, 17R)-1 enantiomers is shown in Figure 5. The overall ECD

Figure 5. Experimental CD spectra of (+)-conipyrrolidone A and (−)-conipyrrolidone A and the calculated ECD spectra of 1a and 1b (bandwidth σ = 0.20 eV).

Figure 6. Experimental CD spectra of two enantiomers (peak A and peak B) and the calculated ECD spectra of 2a and 2b (bandwidth σ = 0.40 eV).

spectra for (5S, 8S, 9S, 14R, 17S)-1 and (5R, 8R, 9R, 14S, 17R)1 enantiomers are in good accordance with the experimental ECD for 1a and 1b, respectively. Thus, compounds 1a and 1b were determined to be 5S, 8S, 9S, 14R, 17S and 5R, 8R, 9R, 14S, 17R, respectively. (±)-Conipyrrolidone B (2) was obtained as a white needle crystal. It was determined to have the same molecular formula of C24H27NO4 (12 degrees of unsaturation) as that of 1 by the HRMS (ESI-TOF) and NMR data (Table 1). The 1H, 13C NMR, IR, and UV spectra of 2 were quite similar to those of 1, suggesting an identical structural skeleton between 1 and 2. A comprehensive analysis of its 2D NMR spectra, including 1 H−1H COSY, HMQC, HMBC, and NOESY experiments, confirmed that 2 has the same planar structure and relative configuration as that of 1 (Figures 2 and S3). The proposed structure of 2 was finally confirmed by single-crystal X-ray crystallographic analysis (Figure 4). Between the carbonyl group at C-2 and the hydroxyl group at C-7, an intermolecular hydrogen bond was formed in 2 as in 1. The space group P21/n

(±)-Conipyrrolidones C (3) and D (4) were isolated as a yellow powder and determined to have the same molecular formula of C24H25NO4 (13 degrees of unsaturation) on the basis of HRMS (ESI-TOF) and NMR data. Compound 3 was deduced to be an analogue of 1 by the comparison of 1H and 13 C NMR data between 1 and 3 (Table 1). The structure of 3 was fully assigned as depicted in Figure 1 by the interpretation of its 2D NMR spectra. HMBC correlations from H-6 to C-20, C-4 and C-21/25 confirmed the presence of the Δ5,6 double bond [δH 7.03 (s), δC 106.3, 134.4] in 3. The NOESY correlations of HN-1 (δ 10.60) with H-21/25 (δ 7.86) were observed in the NOESY spectrum of 3, which supported the Zconfiguration for the Δ5,6 double bond in 3. Compound 4 was confirmed to possess the same planar structure as that of 3 by detailed analysis of NMR data. NOE correlations of H-8 (δ 4.41) with H3-18 (δ 1.24), H-9 (δ 2.60) with H-14 (δ 2.34) and H-17 (δ 2.95) in the NOESY spectrum of 4 indicated the α orientation of H-9, H-14, and H-17 and the β-orientation of 11477


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The Journal of Organic Chemistry H3-18 and H-8 (Figure S3), which is different from the relative configuration of 3. As found in compounds 1 and 2, (±)-conipyrrolidones C (3) and D (4) were confirmed to be a racemic mixture from their [α]25 D value of zero. Compounds 3 and 4 were separated into (+)-conipyrrolidone C (3a), (−)-conipyrrolidone C (3b), (−)-conipyrrolidone D (4a), and (+)-conipyrrolidone D (4b) by the same method used in 1, as shown in Figures S4C and S4D. The configuration of the Δ3,7 double bond of 3a was determined by the DP4 probability based on theoretical NMR calculation that has been proven to be a very powerful tool in natural products structure elucidation.22,23 Taking both 1H and 13 C chemical shift calculations for the DP4 probability analysis into consideration (Table S2), the relative configuration at Δ3,7 was defined to be Z. The relative configuration at Δ3,7 in 4 was also deduced to be Z due to the same biosynthetic origin. Following the approach outlined above, geometry optimization of 3a and 3b and 4a and 4b resulted in two possible conformers (3−1−3−2, Figure S5C, and 4−1−4−2, Figure S5D), respectively. The absolute configuration of 3a and 3b and 4a and 4b were determined as 8S, 9S, 14R, 17S and 8R, 9R, 14S, 17R and 8S, 9S, 14S, 17R and 8R, 9R, 14R, 17S, respectively, by the comparison of experimental and calculated ECD curves (Figures 7 and 8).

Figure 8. Experimental CD spectra of (+)-conipyrrolidone D and (−)-conipyrrolidone D and the calculated ECD spectra of 4a and 4b (bandwidth σ = 0.30 eV).

298 (−0.59); 6: CD (Δεmax) 213 (−0.54), 258 (+0.59), 290 (+0.58)] were consistent with those of 3a and 3b, respectively (Figure S6). Thus, the absolute configuration at C-8 in 5 and 6 were separately determined as 8S and 8R. In combination with the relative configuration determined, compounds 5 and 6 were determined to have the absolute configuration of 8S, 9S, 12S, 14R, 17S, and 8R, 9R, 12S, 14S, 17R, respectively. The molecular formula of (±)-didymellamide E (7) was determined to be C24H25NO4 (13 degrees of unsaturation) by the ion peak at m/z 392.1856 [M + H]+ (calcd for 392.1856). Comparison of its 1H and 13C NMR data with those of didymellamide B24 revealed a similar structure, including signals due to a 1,4-substituted benzene, the 4-hydroxypyridone moiety, and the decalin ring. A further comprehensive analysis of its 1H−1H COSY, HMQC, and HMBC spectra assigned the planar structure of 7. The HMBC correlations from H3-19 to C-11, C-12, and C-13 together with the 1H−1H COSY correlation of H-10-H-11 confirmed the presence of the Δ11,12 double bond (Figure S2). The relative configuration of 7 was assigned on the basis of NOESY data. NOE correlations of H318 with H-9, H-8 with H-14 and H-17 indicated α configuration for H-9 and H3-18, β-configuration for H-8, H14, and H-17. Compound 7 was deduced to be a mixture of enantiomers on the basis of the relatively smaller value of the specific rotation ([α]25 D = −7.57, c = 0.1, MeOH) and quite weak Cotton effects in the ECD spectrum. Two enantiomers (+)-didymellamide E (7a) and (−)-didymellamide E (7b) were separated (7a:7b = 55:45, Figure S4E) by chiral resolution as described for 1, 3, and 4. A conformational search using molecular mechanics calculations yielded two possible conformers (7−1−7−2, Figure S5E). Both conformers were reoptimized at the B3LYP/6-31+G(d,p) level in methanol and used for the ECD calculations. A comparison of the observed ECD spectra for 7a and 7b with the calculated ECD spectra for the (8S, 9S, 14R, 17S)-7 and (8R, 9R, 14S, 17R)-7 enantiomers is shown in Figure 9. The overall ECD spectra for (8S, 9S, 14R, 17S)-7 and (8R, 9R, 14S, 17R)-7 enantiomers are in accordance with the experimental ECD for 7a and 7b, respectively. Thus, compounds 7a and 7b were determined to be 8S, 9S, 14R, 17S and 8R, 9R, 14S, 17R, respectively. Compound 8 was isolated as light yellow powder, and its molecular formula was determined to be C24H27NO4 (12 degrees of unsaturation) by the HRESIMS and NMR data

Figure 7. Experimental CD spectra of (+)-conipyrrolidone C and (−)-conipyrrolidone C and the calculated ECD spectra of 3a and 3b (bandwidth σ = 0.45 eV).

The same molecular formula of C24H27NO4 (12 degrees of unsaturation) was determined for conipyridoins E (5) and F (6) by the HRMS (ESI-TOF) and NMR data. The 1H and 13C NMR spectroscopic data of 5 and 6 (Table 2) were similar to those of 3 (Table 1), except for the absence of the Δ11,12 double bond and the presence of an extra doublet methyl group in 5 and 6. Further analysis of the 2D NMR spectroscopic data, particularly the HMBC correlations from H3-19 to C-11, C-12 and C-13 confirmed the structural variations at C-11 and C-12 (Figure S2). NOE correlations of HN-1 with H-21/25 in the NOESY spectrum indicated the Z-orientation for the Δ5,6 double bond. The relative configuration in 5 and 6 was determined on the basis of NOESY data (Figure S3). Considering the same biosynthetic origins with those of 1−4, the relative configuration at Δ3,7 in 5 and 6 was deduced to be Z, respectively. The Cotton effects observed in the CD spectrum of 5 and 6 [5: CD (Δεmax) 213 (+0.56), 263 (−0.50), 11478


Article

The Journal of Organic Chemistry Table 2. 1H and 13C NMR Data for Compounds 4−6 in pyridine-d5a pos.

a

δC

4 δH (J in Hz)

1-NH 2 3 4 5 6 7 8 9 10

173.5 105.0 184.3 133.7 107.9 196.2 46.3. 34.7 29.1

11

119.7

4.41 2.60 2.22 2.30 5.35

12 13

132.3 35.7

2.08 m

14 15 16 17 18 19 20 21 22 23 24 25

34.4 132.4 132.5 36.9 21.0 24.3 126.9 132.0 117.4 159.6 117.4 132.0

δC

5 δH (J in Hz)

10.30 brs

6.95 s

2.34 5.93 5.63 2.95 1.24 1.68

t (8.8) m brd (17.0) m s

m m brd (9.8) m d (7.1) s

7.78 d (8.4) 7.21 d (8.4) 7.21 d (8.4) 7.78 d (8.4)

δC

10.49 brs 174.7 103.2 184.3 134.3 105.6 197.0 50.5 37.0 30.9

7.01 s 4.65 1.83 1.02 2.30 1.02 1.63 1.36 0.79 1.63 1.91 5.51 5.75 3.23 1.13 0.84

36.4 33.8 42.8 43.0 131.5 133.4 33.3 18.9 23.3 127.8 131.7 117.4 159.0 117.4 131.7

dd (5.8, 11.3) m m m m m m m m m brd (9.8) m m d (7.1) d (6.6)

7.84 d (8.1) 7.11 d (8.1) 7.11 d (8.1) 7.84 d (8.1)

6 δH (J in Hz) 10.66 brs

174.4 103.5 184.0 133.7 106.6 196.6 50.2 38.0 25.5 33.4 28.6 39.8 36.7 131.8 133.4 33.9 18.9 18.9 127.5 131.9 117.4 159.3 117.4 131.9

7.03 s 4.65 1.85 1.23 2.06 1.46 1.63 1.96 1.40 1.50 2.17 5.46 5.77 3.24 1.13 0.92

dd (5.5, 11.4) m m brd (11.5) m m m m m m brd (9.8) m m d (7.1) d (7.0)

7.86 d (8.2) 7.25 d (8.2) 7.25 d (8.2) 7.86 d (8.2)


the CD spectrum of 8 were almost consistent with those of 7a (Figure S7). Thus, compound 8 was assigned as 8S, 9S, 12S, 14R, 17S and named (+)-didymellamide B. Compounds 9 and 10 possessed the same molecular formula of C24H31NO7 (10 degrees of unsaturation), as determined by HRESIMS and NMR data. The 1H, 13C NMR, UV, and IR spectra of 9 were nearly identical with those of Nhydroxyapiosporamide,25,26 revealing the presence of the 2,3epoxycyclohexanol moiety, the 1,4-dihydroxy-2-pyridone moiety, and the decalin moiety in 9. The planar structure and relative configuration of 9 were determined to be the same as that of (−)-N-hydroxyapiosporamide by detailed interpretation of 2D NMR spectra recorded in CDCl3 and NMR data comparison between 9 and (−)-N-hydroxyapiosporamide (Figures 2 and 3). A long-range HMBC correlation from HO-4 to C-3, C-4, C-5, and C-7 and from H-8 to C-7 confirmed the connectivity between the 2-pyridone moiety and the C-7 ketone group (Table S3, Figures S40−42). The relative configuration of 9 was confirmed by NOESY experiment (Figure 3). NOE correlations of H-8 with H-10β, H-14, and H17; H-14 with H-11; and H-9 with H-10α and H3-18 indicated α-configuration for H-9 and H3-18 and β-configuration for H-8, H-11, H-14, and H-17. However, the experimental CD curve [CD (Δεmax) 222 (+2.33), 267 (−0.31), 315 (−1.40)] and the optical rotation ([α]25 D = +98.9) of 9 were completely contrary to those of N-hydroxyapiosporamide (CD (Δεmax) 215 (−2.14), 272 (+3.43), 314 (+4.86); [α]25 D = −56.3), supporting that compound 9 was the enantiomer of (−)-N-hydroxyapiosporamide. Hence, the absolute configuration of 9 was determined as 8S, 9S, 12S, 14R, 16S, 20R, 21S, 22S, 23S, and

Figure 9. Experimental CD spectra of (+)-didymellamide E and (−)-didymellamide E and the calculated ECD spectra of 7a and 7b (bandwidth σ = 0.35 eV).

(Table 3).The 1D NMR spectra of 8 were nearly identical with those of didymellamide B.24 Interpretation of its 2D NMR spectra, including 1H−1H COSY, HMQC, HMBC, and NOESY experiments, indicated the same planar structure and relative configuration as that of didymellamide B (Figures S2 and S3). The optical rotation data of 8 ([α]25 D = +95.9, c = 0.1, MeOH) were opposite those of didymellamide B ([α]25 D = −425, c = 0.14, MeOH), implying the enantiomeric relationship between them. The Cotton effects [CD (Δεmax) 246 (+0.77), 274 (−0.33), 318 (−0.35), 349 (+0.08)] detected in 11479


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The Journal of Organic Chemistry Table 3. 1H and 13C NMR Data for Compounds 7 and 8a

a

pos.

δC

1-NH 2 3 4 5 6 7 8 9 10

163.2 108.6 177.9 114.3 141.7 211.0 53.7 33.0 31.5

11

122.3

12 13

134.2 38.1

14 15 16 17 18 19 20 21 22 23 24 25 4-OH 23-OH

38.8 130.4 132.9 31.9 18.7 24.0 125.1 131.6 116.7 159.1 116.7 131.6

7b δH (J in Hz)

δC

8b δH (J in Hz)

13.31 brs

7.90 brs 5.04 2.18 1.77 2.60 5.43

dd (5.7, 10.8) m m m m

1.79 1.93 2.20 5.58 5.83 3.36 1.07 1.64

m m m brd (9.8) m m d (7.2) s

7.65 d (8.5) 7.32 d (8.5) 7.32 d (8.5) 7.65 d (8.5) 17.62 s 11.75 s

δC

13.30 brs 163.3 108.8 177.9 114.5 141.6 211.3 53.5 37.2 30.8 36.2 33.8 42.5 42.7 131.5 132.7 32.1 18.9 23.2 125.2 131.6 116.7 159.1 116.7 131.6

7.90 brs 5.04 1.85 1.02 2.20 1.02 1.69 1.38 0.79 1.63 1.89 5.51 5.76 3.35 1.08 0.85


7.64 d (8.5) 7.31 d (8.5) 7.31 d (8.5) 7.64 d (8.5) 17.70 s

8c δH (J in Hz) 11.69 brd (6.0)

161.3 106.8 175.9 112.5 140.9 209.7 51.8 35.8 29.4 35.0 32.5 41.4 41.3 130.4 131.6 30.6 17.8 22.4 123.3 130.2 115.0 156.8 115.0 130.2

7.55 d (6.2) 4.37 1.47 0.86 1.79 0.96 1.69 1.47 0.73 1.69 1.79 5.38 5.58 2.78 0.76 0.87


7.25 d (8.5) 6.78 d (8.5) 6.78 d (8.5) 7.25 d (8.5) 16.90 s 9.49 s

“m” means multiplet or overlapped with other signals. bNMR data were measured in pyridine-d5. cNMR data were measured in DMSO.

shifts of C-20 (δ 114.2), C-21 (δ 156.5), C-22 [δH/δC 5.10 (d, J = 9.8 Hz)/65.3], and C-23 [δH/δC 4.28 (m)/70.7], the 1H−1H COSY correlations of H-22-H-23-H2-24-H2-25, and the HMBC correlations from H-22 to C-20, C-21, C-23, and C-24, from H2-25 to C-20, C-21, C-23, and C-24, and from H-6 (δ 8.17, s) to C-3, C-5, C-20, and C-21 confirmed the presence of a 2,3epoxycyclohexenol moiety. The relative configurations in the decalin rings of 12 were deduced by interpretation of 3JHH values and NOESY correlations. The coupling constants for H-8/H-9 (10.4 Hz) and H-8/H-17 (5.5 Hz) and NOE correlations of H-8 with H10β, H-14, and H-17 and H3-18 with H-9 in the NOESY spectrum of 12 suggested the β-orientation of H-8, H-14, and H-17 and the α-orientation of H3-18 and H-9, as depicted in Figure S3. The CD spectrum of 12 was not obtained due to its poor solubility and lesser quantity isolated, which hampered further determination of its absolute configuration. The dihedral angles of the flexible C7−C8 single bonds in compounds 1−11 were analyzed on the basis of the D (C3− C7−C8-C9) angles of the lowest-energy conformations. From the relationship analysis between the stereochemical structures of compounds 1−11 and CD spectra, it was found that the dihedral angle D (C3−C7−C8−C9) affected the Cotton effects in different degrees. For compounds 1 and 2, the chiral center on the pyrrolidine-2,4-dione ring determined the tendency of CD spectra. Meanwhile, the dihedral angle D (C3−C7−C8− C9) in all the other compounds plays a determinant role on the sign of Cotton effects (see Table S4).

this compound was named (+)-N-hydroxyapiosporamide. The 1 H and 13C NMR spectra of 10 resembled those of 9. The difference in the structure between 10 and 9 was the orientation of the methyl group at C-12 [10: β CH3], which was supported by NOESY cross-peaks of H-8 with H-10β, H14, and H-17 and H-14 with H3-19 (without H-11) in the NOESY spectrum of 10. Thus, compound 10 was determined to be the 12-epimer of 9, and its absolute configuration was assigned as 8S, 9S, 12R, 14R, 16S, 20R, 21S, 22S, 23S by the comparison of ECD data between 9 and 10 (Figure S8). The structure of 11 was determined by a combination of HRESIMS and NMR data analysis (Figures S2 and S3). The ion at m/z 444.2019 [M + H]+ in the HRMS (ESI-TOF) spectrum of 11 determined the molecular formula of be C24H29NO7. The 1H and 13C NMR of 11 resembled those of 9/10, except for the presence of one more double bond (Table 4). Furthermore, the HMBC correlations from H3-19 to C-11, C-12, and C-13 confirmed the presence of the double bond between C-11 and C-12. Compound 11 showed similar Cotton effects in the experimental CD spectrum with those of 9 (Figure S9), which assigned the absolute configurations of 11 as 8S, 9S, 14R, 16S, 20R, 21S, 22S, 23S. It was designated as didymellamide G. Didymellamide H (12) had the molecular formula of C24H27NO5 (12 degrees of unsaturation), as determined by the ion peak at m/z 410.1965 [M + H]+ (calcd for 410.1962) in the HRESIMS spectrum. It was confirmed to be an analogue of 11 on the basis of NMR data analysis (Table 5). The chemical 11480


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The Journal of Organic Chemistry Table 4. 1H and 13C NMR Data for Compounds 9−11 in CD3ODa δC

pos.

a

2 3 4 5 6 7 8 9 10

159.6 108.6 175.6 114.6 140.0 211.7 54.5 37.6 30.9

11

36.6

12 13

34.3 43.1

14 15 16 17 18 19 20 21 22 23 24

43.2 131.7 132.5 32.4 18.4 22.9 70.4 60.5 57.7 67.1 25.7

25

31.7

9 δH (J in Hz)

δC 159.8 108.6 175.6 114.6 140.0 211.7 54.6 38.6 25.5

8.03 s 4.43 1.56 0.88 1.92 0.88 1.92 1.49 0.77 1.73 1.81 5.40 5.59 2.84 0.81 0.92

dd (5.2, 11.1) m m m m m m m m m brd (9.8) m s d (6.9) d (6.5)

3.63 3.41 4.12 1.36 1.80 1.67 2.24

brs brs m m m m m

33.2 29.1 40.1 36.8 132.0 132.9 32.3 18.4 18.8 70.5 60.6 57.7 67.2 25.8 31.8

δC

8.04 s

159.6 108.5 175.7 114.6 140.4 211.4 54.7 33.3 31.5

4.48 1.60 1.08 1.69 1.56 1.67 2.06 1.40 1.57 2.03 5.34 5.60 2.84 0.82 1.03

dd (5.2, 11.1) m m m m m m m m m brd (9.8) m s d (6.9) d (6.5)

3.64 3.41 4.12 1.35 1.83 1.70 2.25

d (2.5) brs m m m m dd (8.4, 13.0)

122.1 134.7 38.5 39.5 130.7 132.8 32.2 18.1 23.6 70.4 60.6 57.7 67.2 25.7 31.7

11 δH (J in Hz)

8.04 s 4.43 1.89 1.53 2.31 5.36

dd (5.2, 11.1) m m m s

1.78 2.01 2.07 5.50 5.68 2.86 0.83 1.67

m m m brd (9.8) ddd (2.3, 4.7, 9.8) m d (6.9) s

3.64 3.41 4.12 1.34 1.82 1.71 2.25

d (2.5) m m m m m dd (8.4, 13.0)


Table 5. 1H and 13C NMR Data for Compounds 12 in Pyridine-d5a pos.

δC

2 3 4 5 6 7 8 9 10

163.0 110.5 166.1 115.4 132.9 202.0 54.8 33.4 31.4

11 12

122.7 134.0

13

38.1

a

10 δH (J in Hz)

δH (J in Hz)

8.17 s 4.78 dd (5.5, 10.4) 2.25 m 2.05 m 2.89 m 5.42 s

1.79 m 1.93 m

pos.

δC

δH (J in Hz)

14 15 16 17 18 19 20 21 22

39.0 130.5 133.0 32.4 18.7 24.0 114.2 156.5 65.3

2.27 m 5.57 brd (9.8) 5.78 ddd (1.8, 4.7, 9.8) 3.17 m 1.15 d (7.2) 1.62 s

23 24

70.7 27.6

25

18.5

using L-tyrosine, acetyl CoA, and malonyl CoA as starting units.30−34 However, the intermediates related to tetramic acid derivatives have never been reported. In this study, we identified the key intermediate products 1−6 from fungi for the first time. Compounds 3 and 4 with a pyrrolidine-2,4-dione moiety were proposed as the key intermediates for the biosynthesis of 4-hydroxy-2-pryidone alkaloid. On the basis of our current work, the postulated biosynthetic pathway of 1−12 was revised as illustrated in Scheme 1. In brief, the polyene chain synthesized by a PKS gene is formed into the decalin ring by the Diels−Alder reaction. The decalin motif is further reacted with (Z)-2-amino-3-(4-hydroxyphenyl) acrylic acid derived from L-tyrosine to generate compounds 1/2. They are further oxidized to the corresponding 6-hydroxy derivatives 1/2 that transform into compounds 3/4 by the dehydration at C-5 and C-6. Compounds 5/6 are synthesized from 3/4 by reduction. Through an intramolecular rearrangement, compound 3 is converted to 7. Compound 7 undergoes further modifications to form 8−12. In the screening of antimicrobial activities (Table 6), compounds 9−11 showed strong antifungal activities against C. albicans or A. f umigatus with MIC in the range of 1.56−6.25 μM. Compounds 3a, 3b, 4a, 4b, 5, and 6 showed antibacterial activity against S. aureus and methicillin-resistant S. aureus (MRSA) with MIC in the range of 0.97−15.6 μM. More importantly, compound 5 exhibited strongest activity against the growth of both S. aureus and MRSA with MIC value of 0.97 μM. Among all isolates, compound 11 presented the strongest antifungal activity against A. f umigatus with MIC of 1.56 μM.

5.10 d (9.8) 4.28 2.05 2.38 2.44 2.68

m m m m m


Decalin-containing alkaloids have been isolated from Stagonosporopsis cucurbitacearum,24 Pogonomyrmex badius,25 Arthrinium arundinis,26 Apiospora montagnei,27,28 Phoma sp.,29 and Neosartorya f iscbri30 and were reported to possess antibacterial, antifungal, cytotoxic, and cholesteryl ester transfer protein (CETP) inhibitory activities. The biosynthesis of 4hydroxy-2-pryidone alkaloids with a decalin ring has been postulated via PKS and amino acid hybrid biogenetic pathway 11481


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The Journal of Organic Chemistry Scheme 1. Postulated Biogenetic Pathway of 1−12

Table 6. Bioactivities of Compounds 1−12a antimicrobial activity (MIC, μM)

cytotoxicityb (IC50, μM)

compound

SA

MRSA

EF

BS

CA

AF

1a 1b 2 3a 3b 4a 4b 5 6 7a 7b 8 9 10 11 12 Vanomycin Amphotericin B Taxol 5-Fluorouracil Cisplatin

31.3 31.3 31.3 7.80 7.80 7.80 15.6 0.97 3.90 >100 >100 15.6 >250 62.5 >250 62.5 0.43 NT NT NT NT

31.3 31.3 31.3 7.80 7.80 7.80 7.80 0.97 3.90 >100 >100 31.3 >250 125 250 62.5 0.43 NT NT NT NT

62.5 62.5 62.5 15.6 15.6 31.3 62.5 3.90 15.6 >100 >100 31.3 >250 125 250 250 0.22 NT NT NT NT

62.5 31.3 31.3 31.3 15.6 15.6 15.6 0.97 7.80 >100 >100 7.80 250 31.3 250 62.5 0.22 NT NT NT NT

100 >100 >100 100 100 >100 >100 >100 >100 >100 >100 >100 6.25 3.13 3.13 25.00 NT 1.11 NT NT NT

62.5 62.5 NT 15.6 25.0 >100 >100 >100 >100 >100 >100 12.5 12.5 6.25 1.56 >100 NT 0.84 NT NT NT

A549 96.8 ± 89.9 ± NT 40.3 ± 59.3 ± >100 >100 >100 74.3 ± >100 >100 21.9 ± 23.8 ± 99.8 ± 24.3 ± >100 NT NT 100 >100 NT >100 >100 >100 >100 >100 >100 >100 >100 20.8 ± 28.2 ± 19.0 ± 23.5 ± >100 NT NT 0.35 ± NT NT

8.26 1.72 3.65 7.53

0.02

H460 81.6 90.1 NT 45.4 74.4 NT NT NT NT NT NT 37.5 18.3 20.8 19.9 80.4 NT NT >10 1.13 >10

± 7.92 ± 2.33 ± 3.05 ± 6.16

± ± ± ± ±

3.88 4.36 1.71 2.80 4.03

± 0.17

a

SA: Staphylococcus aureus; MRSA: meticillin-resistant Staphylococcusaureus; EF: Enterococcus faecalis; BS: Bacillus subtilis; CA: Candida albicans; AF: Aspergilusfumigatus. NT: not tested. bThe cell growth was not influenced by compounds tested at 100 μM.

To detect the influence of 11 on the growth phenotype of A. f umigatus, the hyphae of A. f umigatus were treated with 11 at concentrations of 150 and 300 ng/mL. The biosynthesis of chitin in the cell wall was interrupted as supported by the brightness of the hyphae weaker than that in the control group. The distribution of chitin became uneven after 6 h of treatment of 11 by the staining of chitin with the calcofluor white (Figure 10). Compound 11 was supposed to be a new chitin synthesis inhibitor. As plants and vertebrates, including humans, do not contain chitin, chitin synthesis inhibitors have been thought to

be good candidates for the development of new drugs against fungal infections in humans and plants. Although not detected currently, we proposed that compounds 9 and 10 exerted their antifungal activity by inhibition of chitin synthesis. The exact action mechanism and further in vivo evaluation of these new alkaloids deserves investigation. In a cytotoxicity assay against A549, K562, and H460 cells lines (Table 6), compounds 8, 9, and 11 showed moderate antiproliferative activity against all tested cell lines with IC50 value less than 50 μM. A preliminary structure−activity 11482


Article


Figure 10. Effect of 11 on the growth phenotype of A. f umigatus. The hyphae obtained by culturing of 1.0 × 107 freshly harvested conidia was put on the coverslips to continue to culture with the tested compound at concentrations of 150 or 300 ng/mL for 2, 4, 6, and 8 h. After staining with Calcofluor white and DAPI, the hyphae without (column A) or with [columns B (150 ng/mL) and C (300 ng/mL)] compound 11 treatment were detected by fluorescence microscopy. Calcofluor white bound to the chitin in the cell walls of A. f umigatus and further shows chitin content by brightness of the hyphae, which was detected by fluorescence microscopy. respectively. Bruker Avance-500 spectrometer was used to acquire 1H and 13C NMR data using solvent signals (Pyridine-d5, δH 8.740/δC 150.35; CD3OD, δH 3.30/δC 49.9; DMSO-d6, δH 2.50/δC 39.5) as references. The HMQC and HMBC experiments were optimized for 145.0 and 8.0 Hz, respectively. TLC was carried out on Silica gel HSGF254 by spraying with 9% H2SO4 and heating. Silica gel (Qingdao Haiyang Chemical Co., Ltd., People’s Republic of China), LH-20 (Amersham Biosciences), and ODS (Lobar, 40−63 μm, Merck) were used for column chromatography. Semipreparative HPLC was performed using an Agilent 1200 HPLC system equipped with an Agilent DAD UV−vis spectrometric detector using a reversed-phase (RP) C8 column (Eclipse XDB-C8, 9.4 × 250 mm, Agilent, Inc.) with a flow rate of 2.0 mL/min and a Kromasil 5-CelluCoat column (4.6 × 250 mm, Akzo Nobel N.V.) with a flow rate of 0.8 mL/min. HPLCCD analysis was achieved using an Jasco LC2000 HPLC system coupled with a CD detector using a ChiralPak AD-H stationary phase with a flow rate of 0.8 mL/min. Fungal Material. Coniochaeta cephalothecoides Kamiya, Uchiy & Udagawa was isolated from the fruiting bodies of Trametes sp. collected in the Medog County of Tibet (29°18′32.27″ N, 95°16′08.35″ E, altitude: 1142 m), China, in May 2014.The fungus was identified mainly based on the morphological features.35 Sexual morph: Ascomata scattered, semi-immersed, subglobose to pyriform, black, harily, with a short neck; asci 8-spored, cylindrical; ascospores arranged uniseriately, hyaline to yellowish brown, variable in shape, often ovoid to pyriform, 8−13 × 5−7.5 × 3−4 μm, aseptate, smooth, with a longitudinal germ slit. Asexual morph: Mycelium hyaline, branched, septate. Conidiogenous cells phialidic, forming on branched hyphae, hyaline, ampuliform. Condia hyaline, aseptate, ovoid to ellipsoidal, 3.0−4.5 × 1.5−2.0 μm, smooth-walled. ITS (KY064029) and LSU (KY064030) sequences of this strain were also generated in the present paper and submitted to the GenBank. Fermentation and Extraction. C. cephalothecoides was cultured on slant of PDA at 26 °C for 7 days. Agar plugs were inoculated in a

relationship was obtained on the basis of the above bioactivities. In general, the formation of the tetramic acid moiety in the structures greatly increases the antibacterial activity. The oxidative dearomatization of the benzene ring attached at C-5 in the 4-dihydroxy-2-pyridone alkaloids facilitates the antifungal activity and cytotoxicity. In conclusion, 12 new alkaloids, including 5 pairs of racemic mixtures, were isolated from the solid culture of the fungus C. cephalothecoides collected in the Tibet Plateau. Compounds 1− 6 possess an unprecedented chemical skeleton featuring a decalin ring and a tetramic acid moiety. The resolution of the enantiomeric isomers was achieved on a chiral column to afford eight optical pure isomers. The ECD calculation method was used to determine the absolute configuration in new compounds. Tetramic acid derivatives (3a, 3b, 4a, 4b, 5, and 6) showed strong antibacterial activities against S. aureus, E. faecalis, and B. subtilis. 4-Hydroxy-2-pyridone alkaloids (8−12) exhibited antifungal activities against C. albicans or A. f umigatus. A preliminary investigation on the action mechanism of 11 indicates that it is a potent chitin synthesis inhibitor. The discovery of new tetramic acid derivatives enables us to elucidate the biosynthetic pathway for 4-hydroxy-2-pyridone alkaloids containing a decalin ring in this fungus.

■

EXPERIMENTAL SECTION

General Experimental Procedures. HRESIMS data were obtained on an Agilent Accurate-Mass-Q-TOF LC/MS 6520 instrument. UV, IR, optical rotation, and CD spectra were recorded on a Thermo Genesys-10S UV−vis spectrophotometer, a Nicolet IS5 FTIR spectrophotometer, an Anton Paar MCP 200 Automatic Polarimeter, and an Applied Photophysics Chirascan spectropolarimeter, 11483


Article

The Journal of Organic Chemistry 500 mL Erlenmeyer flask containing 200 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 before sterilization and incubation at 26 °C on a rotary shaker at 160 rpm for 5 d. Large scale cultivation was carried out in 30 500 mL Fernbach flasks each containing 80 g of rice and 120 mL of distilled H2O. Each flask was inoculated with 10.0 mL of the culture medium and incubated at 26 °C for 32 d. The fermented rice substrate was extracted with EtOAc by exhaustive maceration (3 × 4 L), and the organic solvent was evaporated to dryness under vacuum to afford the crude extract (16.8 g). Isolation and Characterization Data. The EtOAc fraction was subjected to a silica gel column chromatography (CC) eluted with a gradient of petroleum ether-ethyl acetate (v/v, 100:0, 100:1, 100:2, 100:5, 100:10) and dichloromethane-methanol (v/v, 100:1, 100:2, 100:4, 100:8, 100:15, 100:20, 0:100) to give 10 fractions (CC-1-CC10). Fractions CC-6 and CC-7 containing rich secondary metabolites were selected for further purification. Fraction CC-6 (5.6 g) was further separated on silica gel column by elution with increasing concentrations of acetone in dichloromethane to give 8 fractions (CC6-1-CC-6-8). Fraction CC-6-6 (2.3 g) was separated on ODS using a gradient of increasing methanol (30, 45, 60, 75, 85, and 100%) in acidic water (0.005% TFA) to afford 14 subfractions (CC-6-6-1- CC66-14). CC-6-6-6 (130 mg) was purified finally by RP-HPLC with acetonitrile:water (40:60) to give 1 (15.2 mg, tR 102.2 min) and 2 (1.6 mg, tR 113.5 min). Compounds 7 (25.1 mg, tR 81.9 min) and 8 (20.0 mg, tR 104.8 min) were obtained from CC-6-6-10 (280 mg) by RPHPLC using 71% methanol in acidic water (0.005% TFA). CC-6-6-12 (230 mg) was purified finally by RP-HPLC with 73% methanol in acidic water to afford 4 (13.5 mg, tR 80.2 min), 3 (25.2 mg, tR 89.5 min), 5 (4.2 mg, tR 101.3 min), and 6 (8.9 mg, tR 115.1 min). Compounds 9 (81 mg), 10 (28.8 mg), and 11 (438 mg) were obtained from fraction CC-6-7 (710 mg) by Sephadex LH-20 column separation with methanol as eluent. Fraction CC-7 (1.2 g) was chromatographed on ODS using a gradient elution of MeOH-H2O (25, 35, 40, 60, 70, and 100%) to afford 12 subfractions (CC-7-1-CC7-12). Compound 12 (1.2 mg) was obtained from subfractions CC-72 (13.2 mg) by RP-HPLC using 30% acetonitrile in acidic water (0.005% TFA). Enantioseparation of (±)-Conipyrrolidone A (1) and D (4) was carried out on a Kromasil 5-CelluCoat column (4.6 × 250 mm), respectively, using isopropanol/n-hexane, (6:94, +0.005% TFA) as mobile phase to afford 1b (tR 29.6 min, 2.2 mg), 1a (tR 39.4 min, 2.4 mg), 4a (tR 30.7 min, 1.0 mg), and 4b (tR 33.8 min, 0.8 mg). (±)-Conipyrrolidone C (3) was subjected to chiral HPLC using a Kromasil 5-CelluCoat column (isopropanol:n-hexane, 15:85, +0.005% TFA) to afford 3a (tR 24.2 min, 2.0 mg) and 3b (tR 34.2 min, 2.1 mg). The enantiomers 7a (tR 80.6 min, 0.6 mg) and 7b (tR 85.5 min, 0.7 mg) were obtained by chiral HPLC using a Kromasil 5-CelluCoat column eluting with isopropanol/n-hexane (3:97, +0.005% TFA). No racemization occurred when 1a was treated by the similar isolation procedure as described for 1 (Figure S54), which confirmed that the extraction and isolation procedure did not cause the racemization. HPLC-ECD chromatograms of racemic 2 were monitored at 285 nm (Chiralpak AD-H, n-hexane:isopropanol = 80:20, flow rate: 0.8 mL/min, Peak A: tR = 16.12 min, Peak B: tR = 26.83 min). (±)-Conipyrrolidone A (1). White needle crystal (MeOH); [α]25 D 0 (c 0.15, MeOH); UV (MeOH) λmax (log ε) 286 (4.30), 225 (3.87) nm; IR νmax 3342, 1653, 1604, 1513 cm−1. HRMS (ESI-TOF) m/z: [M + H]+ calcd for C24H28NO4 394.2013; found 394.2014. 1H NMR and 13C NMR, see Table 1. (−)-Conipyrrolidone A (1a). White needle crystal (MeOH); [α]25 D −10.2 (c 0.15, MeOH); CD (c 0.05, MeOH) λmax (Δε) 231 (−6.89), 259 (−1.89), 291 (−2.31). (+)-Conipyrrolidone A (1b). White needle crystal (MeOH); [α]25 D +10.0 (c 0.15, MeOH); CD (c 0.05, MeOH) λmax (Δε) 233 (+6.69), 260 (+1.85), 292 (+2.61). (±)-Conipyrrolidone B (2). White needle crystal (MeOH); [α]25 D 0 (c 0.15, MeOH); UV (MeOH) λmax (log ε) 286 (4.20), 225 (3.60) nm; IR νmax 3345, 1653, 1603, 1513 cm−1. HRMS (ESI-TOF) m/z:

[M + H]+ calcd for C24H28NO4 394.2013; found 394.2013. 1H NMR and 13C NMR, see Table 1. Peak A (2b). CD (i-PrOH/n-Hexane) λmax (mdeg) 232 (−1.02), 256 (−1.76), 290 (−1.10). Peak B (2a). CD (i-PrOH/n-Hexane) λmax (mdeg) 232 (+0.71), 256 (+1.65), 292 (+0.88). (±)-Conipyrrolidone C (3). Yellow powder (MeOH); [α]25 D 0 (c 0.15, MeOH); UV (MeOH) λmax (log ε) 379 (4.28), 295 (4.67), 245 (2.85) nm; IR νmax 3355, 1653, 1605), 1515 cm−1. HRMS (ESI-TOF) m/z: [M + H]+ calcd for C24H26NO4 392.1856; found 392.1860. 1H NMR and 13C NMR, see Table 1. (+)-Conipyrrolidone C (3a). [α]25 D +62.5 (c 0.1, MeOH); CD (c 0.02, MeOH) λmax (Δε) 208 (+6.38), 264 (−0.40), 305 (−0.66). (−)-Conipyrrolidone C (3b). [α]25 D −63.7 (c 0.1, MeOH); CD (c 0.02, MeOH) λmax (Δε) 209 (−5.95), 263 (+0.58), 301 (+1.04). (±)-Conipyrrolidone D (4). Yellow powder (MeOH); [α]25 D 0 (c 0.15, MeOH); UV (MeOH) λmax (log ε) 379 (4.28), 295 (4.67), 245 (2.85) nm; IR νmax 3355, 1655, 1603, 1518 cm−1. HRMS (ESI-TOF) m/z: [M + H]+ calcd for C24H26NO4 392.1856; found 392.1858. 1H NMR and 13C NMR, see Table 2. (−)-ConipyrrolidoneD (4a). [α]25 D −2.1 (c 0.04, MeOH); CD (c 0.02, MeOH) λmax (Δε)= 202 (+8.54), 236 (−0.06), 259 (+0.39), 305 (−0.62). (+)-ConipyrrolidoneD (4b). [α]25 D +2.0 (c 0.04, MeOH); CD (c 0.02, MeOH) λmax (Δε) 204 (−7.89), 237 (+0.16), 256 (−0.43), 305 (+0.61). Conipyridoin E (5). Yellow powder (MeOH); [α]25 D +22.3 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 380 (3.78), 295 (3.67), 244 (2.84) nm; CD (c 0.01, MeOH) (Δεmax) 213 (+0.56), 263 (−0.50), 298 (−0.59). IR νmax 3358, 1653, 1605, 1514 cm−1. HRMS (ESI-TOF) m/ z: [M + H]+ calcd for C24H28NO4 394.2013; found 394.2011. 1H NMR and 13C NMR, see Table 2. Conipyridoin F (6). Yellow powder (MeOH); [α]25 D −43.0 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 379 (4.28), 295 (4.67), 245 (2.85) nm; CD (c 0.01, MeOH) (Δεmax) 213 (−0.54), 258 (+0.59), 290 (+0.58). IR νmax 3359, 1653, 1604, 1519 cm−1. HRMS (ESI-TOF) m/ z: [M + H]+ calcd for C24H28NO4 394.2013; found 394.2015. 1H NMR and 13C NMR, see Table 2. (±)-Didymellamide E (7). Light yellow powder (MeOH); [α]25 D −7.57 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 348 (2.83) nm, 248 (4.02) nm, 213 (3.45) nm; IR: νmax 3338, 1652, 1607, 1541 cm−1. HRMS (ESI-TOF) m/z: [M + H]+ calcd for C24H26NO4 392.1856; found 392.1855. 1H NMR and 13C NMR, see Table 3. (+)-Didymellamide E (7a). [α]25 D +16.6 (c 0.08, MeOH); CD (c 0.01, MeOH) λmax (Δε)= 244 (+0.34), 274 (−0.14), 307 (−0.30), 349 (+0.11) (−)-Didymellamide E (7b). [α]25 D = −15.9 (c 0.08, MeOH); CD (c 0.01, MeOH) λmax (Δε)= 244 (−0.35), 277 (+0.11), 312 (+0.25), 354 (−0.08). (+)-Didymellamide B (8). Light yellow powder (MeOH); [α]25 D +95.9 (c 0.1, MeOH); UV (MeOH): λmax (log ε) 348 (2.85) nm, 247 (3.62) nm, 212 (3.53) nm; CD (c 0.01, MeOH) λmax (Δε) 246 (+0.77), 274 (−0.33), 318 (−0.35), 349 (+0.08); IR νmax 3368, 1651, 1602, 1540 cm−1. HRMS (ESI-TOF) m/z: [M + H]+ calcd for C24H28NO4 394.2013; found 394.2016. 1H NMR and 13C NMR, see Table 3. (+)-N-Hydroxyapiosporamide (9). Light yellow solid (MeOH); [α]25 D +98.9 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 338 (2.32) nm, 282 (2.17) nm, 224 (4.05) nm; CD (c 0.02, MeOH) λmax (Δε) 222 (+2.33), 267 (−0.31), 315 (−1.40), 347 (+0.60). IR νmax 3380, 1648, 1600 cm−1. HRMS (ESI-TOF) m/z: [M + H]+ calcd for C24H32NO7 446.2170; found 446.2173. 1H NMR and 13C NMR, see Table 4. Didymellamide F (10). Light yellow solid (MeOH); [α]25 D +87.9 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 338 (2.20) nm, 283 (2.07) nm, 225 (4.03) nm; CD (c 0.02, MeOH) λmax (Δε) 223 (+2.18), 272(−0.20), 314 (−1.16), 348 (+0.54). IR νmax 3383, 1645, 1602 cm−1. HRMS (ESI-TOF) m/z: [M + H]+ calcd for C24H32NO7 446.2170; found 446.2172. 1H NMR and 13C NMR, see Table 4. Didymellamide G (11). Light yellow solid (MeOH); [α]25 D +186.7 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 338 (2.12) nm, 281 (1.98) 11484


Article

The Journal of Organic Chemistry nm, 225 (3.92) nm; CD (c 0.02, MeOH) λmax (Δε) 263 (−0.44), 310 (−1.19), 346 (+0.44). IR νmax 3385, 1643, 1600 cm−1. HRMS (ESITOF) m/z: [M + H]+ calcd for C24H30NO7 444.2017; found 444.2019. 1H NMR and 13C NMR, see Table 4. Didymellamide H (12). Light yellow powder (MeOH); [α]D25 +106.9 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 368 (2.21) nm, 265 (2.30) nm, 231 (4.42) nm; IR νmax 3358, 1659, 1600 cm−1. HRMS (ESI-TOF) m/z: [M + H]+ calcd for C24H28NO5 410.1962; found 410.1965. 1H NMR and 13C NMR, see Table 5. X-ray Crystallographic Analysis of (±)-Conipyrrolidone A (1). Upon crystallization from pyridine using the vapor diffusion method, colorless needles of compound 1 were obtained. Data collection was performed on a Eos CCD using a graphitemonochromated Cu Kα radiation, λ = 1.54184 Å at 103.4 K. Crystal data: C25H31NO5, M = 425.51, space group orthorhombic, Pbca; unit cell dimensions were determined to be a = 14.3407(4) Å, b = 12.2598(4) Å, c = 25.8810(8) Å, α = β = γ = 90.00°, V = 4550.2(2) Å3, Z = 8, ρcalc = 1.242 mg/mm3, F (000) = 1824.0, μ (Cu Kα) = 0.696 mm−1. Unique reflections (12 859) were collected to θmax = 71.05°, in which 4339 reflections were observed [F2> 4σ(F2)]. The structure was solved by direct methods using the SHELXS-97 program and refined by the program SHELXL-97 and full-matrix least-squares calculations.36 In the structure refinements, nonhydrogen atoms were placed on the geometrically ideal positions by the “ride on” method. Hydrogen atoms bonded to oxygen were located by the structure factors with isotropic temperature factors. The final refinement gave R1 = 0.0389, wR2 = 0.0995 (w = 1/σ|F|2), and S = 1.038. CCDC 1501748 contains the supplementary crystallographic data for 1. These data can be obtained free of charge from the Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif. X-ray Crystallographic Analysis of (±)-Conipyrrolidone B (2). Colorless needles of compound 2 from pyridine were obtained. Data collection was performed on a Eos CCD using a graphitemonochromated Cu Kα radiation, λ = 1.54184 Å at 102.8 K. Crystal data: C24H27NO4, M = 393.47, space group monoclinic, P21/n; unit cell dimensions were determined to be a = 10.8380(2) Å, b = 10.16461(19) Å, c = 19.4057(4) Å, α = γ = 90.00°, β = 105.682(2)°, V = 2058.24(8) Å3, Z = 4, ρcalc = 1.270 mg/mm3, F (000) = 840, μ (Cu Kα) = 0.693 mm−1. Unique reflections (13 643) were collected to θmax = 71.14°, in which 3949 reflections were observed [F2> 4σ(F2)]. The structure refinements were conducted by the same method as that described for compound 1. The final refinement gave R1 = 0.0393, wR2 = 0.1009 (w = 1/σ|F|2), and S = 1.054. CCDC 1501749 contains the supplementary crystallographic data for 2. These data can be obtained from the Cambridge Crystallographic Data Centre via www.ccdc.cam. ac.uk/data_request/cif. Computation Section. Systematic conformational analyses for 1a/1b, 2a/2b, 3a/3b, 4a/4b, and 7a/7b were performed via the Molecular Operating Environment (MOE), ver. 2009.10. (Chemical Computing Group, Canada), software package using the MMFF94 molecular mechanics force field. The MMFF94 conformers were further optimized using TDDFT at B3LYP/6-31+G(d,p) basis set level in methanol with PCM model. The stationary points were checked as the true minima of the potential energy surface by verifying that they do not exhibit vibrational imaginary frequencies. ECD spectra were stimulated using a Gaussian function with various halfbandwidths indicated in text. 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 main conformers. The systematic errors in the prediction of the wavelength and excited-state energies are compensated for by employing UV correlation. 1H and 13C chemical shift calculations for the DP4 probability analysis were conducted according to a previously reported method.22 The shielding constants have been calculated with the GIAO method at the MPW1PW91/6-311++G(d,p) level. All quantum computations were performed using Gaussian 09 package37 on an IBM cluster machine located at the High Performance Computing Center of Peking Union Medical College.

Antimicrobial Bioassay. Assay for antibacterial activities including Staphylococcus aureus (ATCC 6538), meticillin-resistant S. aureus (MRSA, clinical isolates, Beijing Chao-yang Hospital), Enterococcus faecalis (clinical isolates, Beijing Chao-yang Hospital), Bacillus subtilis (ATCC 6051), and antifungal activities, including Candida albicans SC5314 and Aspergilus f umigatus (CGMCC 3.5835), were carried out as previously described method.38,39 The inhibition rate was calculated and plotted versus test concentrations to afford the MIC. MIC values were defined as the minimum concentration of compound that inhibited visible microbial growth. All of the experiments were performed in triplicate. Effect of 11 on the Growth Phenotype of A. f umigatus. According to the method described by Chen Jin (one of coauthors),40 first, 20 mL of complete liquid medium was inoculated with 1.0 × 107 freshly harvested conidia in a Petri dish at 37 °C. After cultured 16 h, the hyphae were collected, washed two times with ddH2O, and then put on the coverslips to continue to culture with the tested compound (DMSO as solvents and the final concentrations were at 150 and 300 ng/mL) for specified times: 2, 4, 6, and 8 h. The coverslips were kept in a humid environment, but the surfaces of the slips were kept dry. Fluorescence microscopy was done of the wild-type and compound 11 affected after staining with Calcofluor white and DAPI (4′, 6diamidino-2-phenylindole), and the chitin’s distribution and the abnormal conidiation was noted by fluorescence microscopy (Axiovert 200 M, Carl Zeiss). Cytotoxicity Assay. Cytotoxicity tests against A549, K562, and H460 cell lines were carried out as in the previously described method.41 Taxol, 5-Fluorouracil, and Cisplatin were used as the positive controls.

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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.joc.7b02010. NMR spectra for compounds 1−12, CD data, computational details, and Cartesian coordinates (PDF) X-ray crystallography data of 1 and 2 (CIF)

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AUTHOR INFORMATION

Corresponding Author

*Tel.: +86 10 64806074; E-mail: [email protected]. ORCID

Wenbing Yin: 0000-0002-9184-3198 Hongwei Liu: 0000-0001-6471-131X Author Contributions #

J. Han, C. Liu, and L. Li contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Financial support of the National Natural Science Foundation (Grants 81673334 and 21602247) and the Youth Innovation Promotion Association of Chinese Academy of Sciences (Grant 2014074) is gratefully acknowledged.

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REFERENCES

(1) Vallabhaneni, S.; Mody, R. K.; Walker, T.; Chiller, T. Infect. Dis. Clin. North Am. 2016, 30, 1−11. (2) Roemer, T.; Krysan, D. J. Cold Spring Harbor Perspect. Med. 2014, 4 (5), a019703. (3) Richardson, M. R. J. Antimicrob. Chemother. 2005, 56, S5−S11. (4) Brown, G. D.; Denning, D. W.; Levitz, S. M. Science 2012, 336 (6082), 647. (5) Zhai, B.; Lin, X. Curr. Pharm. Biotechnol. 2011, 12 (8), 1255− 1262.

11485


Article

The Journal of Organic Chemistry (6) Denning, D. W.; Bromley, M. J. Science 2015, 347 (6229), 1414− 1416. (7) Kullberg, B. J.; Arendrup, M. C. N. Engl. J. Med. 2015, 373, 1445−1456. (8) Butts, A.; Krysan, D. J. PLoS Pathog. 2012, 8 (9), 91−93. (9) Park, B. J.; Wannemuehler, K. A.; Marston, B. J.; Govender, N.; Pappas, P. G.; Chiller, T. M. AIDS 2009, 23, 525−530. (10) Lai, C. C.; Tan, C. K.; Huang, Y. T.; Shao, P. L.; Hsueh, P. R. J. Infect. Chemother. 2008, 14, 77−85. (11) Kołaczkowska, A.; Kołaczkowski, M. J. Antimicrob. Chemother. 2016, 71, 1438−1450. (12) Wong, S. S.; Samaranayake, L. P.; Seneviratne, C. J. Drug Discovery Today 2014, 19, 1721−1730. (13) Calugi, C.; Trabocchi, A.; Guarna, A. Expert Opin. Ther. Pat. 2011, 21 (3), 381−397. (14) Kamel, M. W.; Raizada, M. N. Front. Microbiol. 2013, 4, 65. (15) Di Santo, R. Nat. Prod. Rep. 2010, 27 (7), 1084−1098. (16) Segeth, M. P.; Bonnefoy, A.; Brönstrup, M.; Knauf, M.; Schummer, D.; Toti, L.; Vértesy, L.; Wetzel-Raynal, M. C.; Wink, J.; Seibert, G. J. Antibiot. 2003, 56 (2), 114−122. (17) Wang, H. J.; Gloer, J. B.; Scott, J. A.; Malloch, D. Tetrahedron Lett. 1995, 36 (33), 5847−5850. (18) Fujimoto, H.; Inagaki, M.; Satoh, Y.; Yoshida, E.; Yamazaki, M. Chem. Pharm. Bull. 1996, 44 (5), 1090−1092. (19) Wang, Y. C.; Niu, S. B.; Liu, S. C.; Guo, L. D.; Che, Y. S. Org. Lett. 2010, 12 (21), 5081−5083. (20) Bringmann, G.; Bruhn, T.; Maksimenka, K.; Hemberger, Y. Eur. J. Org. Chem. 2009, 2009, 2717−2727. (21) Wang, Q. X.; Qi, Q. Y.; Wang, K.; Li, L.; Bao, L.; Han, J. J.; Liu, M. M.; Zhang, L. X.; Cai, L.; Liu, H. W. Org. Lett. 2013, 15 (15), 3982−3985. (22) Smith, S. G.; Goodman, J. M. J. Am. Chem. Soc. 2010, 132 (37), 12946−12959. (23) Rodríguez, I.; Genta-Jouve, G.; Alfonso, C.; Calabro, K.; Alonso, E.; Sanchez, J. A.; Alfonso, A.; Thomas, O. P.; Botana, L. M. Org. Lett. 2015, 17, 2392−2395. (24) Haga, A.; Tamoto, H.; Ishino, M.; Kimura, E.; Sugita, T.; Kinoshita, K.; Takahashi, K.; Shiro, M.; Koyama, K. J. Nat. Prod. 2013, 76 (4), 750−754. (25) Lee, J. C.; Coval, S. J.; Clardy, J. J. Antibiot. 1996, 49, 693−696. (26) Wang, J.; Wei, X.; Qin, X.; Lin, X.; Zhou, X.; Liao, S.; Yang, B.; Liu, J.; Tu, Z.; Liu, Y. Org. Lett. 2015, 17 (3), 656−659. (27) Alfatafta, A. A.; Gloer, J. B.; Scott, J. A.; Malloch, D. J. Nat. Prod. 1994, 57, 1696−1702. (28) Williams, D. R.; Kammler, D. C.; Donnell, A. F.; Goundry, W. R. F. Angew. Chem., Int. Ed. 2005, 44, 6715−6718. (29) Shibazaki, M.; Taniguchi, M.; Yokoi, T.; Nagai, K.; Watanabe, M.; Suzuki, K.; Yamamoto, T. J. Antibiot. 2004, 57 (6), 379−382. (30) Fujimoto, H.; Ikeda, M.; Yamamoto, K.; Yamazaki, M. J. Nat. Prod. 1993, 56 (8), 1268−1275. (31) Schmidt, K.; Riese, U.; Li, Z.; Hamburger, M. J. Nat. Prod. 2003, 66 (3), 378−383. (32) Kemami Wangun, H. V.; Hertweck, C. Org. Biomol. Chem. 2007, 5 (11), 1702−1705. (33) Fujita, Y.; Oguri, H.; Oikawa, H. Tetrahedron Lett. 2005, 46 (35), 5885−5888. (34) Eley, K. L.; Halo, L. M.; Song, Z.; Powles, H.; Cox, R. J.; Bailey, A. M.; Lazarus, C. M.; Simpson, T. J. ChemBioChem 2007, 8 (3), 289− 297. (35) Kamiya, S.; Uchiyama, S.; Udagawa, S. I. Mycoscience 1995, 36, 377−383. (36) Sheldrick, G. M. SHELXS-97, A Program for X-Ray Crystal Structure Solution, and SHELXL-97, A Program for X-Ray Structure Refinement, University of Göttingen, Göttingen, Germany, 1997. (37) Frisch, M. J.; et al. Gaussian 09, revision C. 01. Gaussian, Inc.: Wallingford CT, 2010. (38) Ibraheim, Z. Z.; Abdel-Mageed, W. M.; Dai, H.; Guo, H.; Zhang, L.; Jaspars, M. Phytother. Res. 2012, 26 (4), 579−586.

(39) Song, F. H.; Liu, X. R.; Guo, H.; Ren, B.; Chen, C. C.; Piggott, A. M.; Yu, K.; Gao, H.; Wang, Q.; Liu, M.; Liu, X. T.; Dai, H. Q.; Zhang, L. X.; Capon, R. J. Org. Lett. 2012, 14 (18), 4770−4773. (40) Wang, J. Y.; Zhou, H.; Lu, H.; Du, T.; Luo, Y. M.; Wilsonc, I. B. H.; Jin, C. Fungal Genet. Biol. 2015, 76, 57−69. (41) Han, J. J.; Bao, L.; Tao, Q. Q.; Yao, Y. J.; Liu, X. Z.; Yin, W. B.; Liu, H. W. Org. Lett. 2015, 17 (10), 2538−2541.

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Decalin-Containing Tetramic Acids and 4-Hydroxy-2-pyridones with

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