Thiazolo[5,4-b]pyridine Alkaloid and Seven ar-Bisabol Sesquiterpenes Produced by the Endophytic Fungus Penicillium janthinellum.

We investigated the secondary metabolites present in Penicillium janthinellum MPT-25, an endophytic fungus isolated from Taxus wallichiana var. chinensis (Pilger) Florin. Chemical characterization of the solid cultured extract resulted in the isolation of 11 compounds, including eight previously undescribed metabolites: a thiazolo[5,4-b]pyridine alkaloid, janthinedine A (1), and seven ar-bisabol sesquiterpenes, janthinepenes A-G (2-8). Their structures were elucidated by a combination of extensive spectroscopic methods, including single-crystal X-ray diffraction and ECD spectra. The antimicrobial activities of these compounds were evaluated against seven agricultural pathogenic fungi and eight clinically drug-resistant bacteria.

Introduction

Plant endophytic fungi can inhabit various types of healthy tissues in host plants without causing disease symptoms.[1] The mutually beneficial symbiotic relationship between host plants and their endophytic fungi has been established due to a long period of co-evolution and, consequently, endowed with the ability of fungi to encode biologically active metabolites with distinctive scaffolds.[2−6] Since Strobel discovered the “gold” bioactive compound taxol from an endophytic fungus acquired from the phloem of Taxus brevifolia, endophytic fungi have received increasing attention as potential producers of peculiar and bioactive metabolites.[7] Furthermore, the endophytic fungus Fusarium proliferatum (MTCC 9690), isolated from Dysoxylum binectariferum, can produce the same chromane alkaloid, rohitukine, as the host plant, which suggests that the metabolites generated by endophytic fungi are potential candidates for natural medicines that can be directly isolated from plant tissues.[8] Additionally, chemical investigations on endophytic fungi have provided new methods to solve the problem of shortage of some natural plants.[9]

Owing to our interest in antimicrobial compounds extracted from endophytic fungi, the strain MPT-25, which was obtained from the stems of Taxus wallichiana var. chinensis (Pilger) Florin and identified as Penicillium janthinellum, has attracted our attention. P. janthinellum produces diverse metabolites, including brefeldin A (BFA) derivatives,[10−12] alkaloids,[13−20] azaphilones,[21,22] terpenoids,[23,24] meroterpenoids,[25] restricticin derivatives,[26] and heterocyclic dipeptides,[27,28] and exhibits extensive antitumoral,[10,12,19] antibacterial,[15,16,18,21] and cytoprotective activities.[27,28] A phytochemical investigation on the EtOAc extract of this fungus led to the isolation of 11 metabolites: janthinedine A (1), janthinepenes A–G (2–8), and three known compounds (9–11). Janthinedine A possesses a thiazolo[5,4-b]pyridine skeleton[29] and janthinepenes A–G belong to a medicinally interesting class of sesquiterpenes—ar-bisabol sesquiterpenes.[30,31] Herein, the detailed isolation and structural elucidation, in addition to the antimicrobial activities, are described.

Results and Discussion

Janthinedine A (1) was isolated as yellow crystals. The molecular formula of C14H10N2O3S with 11 degrees of unsaturation was deduced by positive high-resolution electrospray ionization mass spectrometry (HRESIMS), with an ion peak at m/z 287.04841. Data analysis of 1H, 13C, and heteronuclear single-quantum correlation (HSQC) NMR spectra (Table ) revealed the presence of a para-substituted benzene moiety, with significant signals at δH 8.53 (2H, d, J = 8.9 Hz) and 6.93 (2H, d, J = 8.9 Hz); δC 165.4 (C-5′), 135.3 (C-3′), 135.3 (C-7′), 127.1 (C-2′), 116.5 (C-4′), and 116.5 (C-6′). The other signals displayed one ketone carbonyl carbon at δC 183.8 (C-1′); four sp2 quaternary carbons at δC 169.4 (C-2), 163.7 (C-6), 159.2 (C-7a), and 147.5 (C-3a); two sp2 methines [δH 8.57 (1H, d, J = 8.5 Hz), δC 134.6 (C-4); 7.80 (1H, d, J = 8.5 Hz), δC 120.9 (C-5)]; and one oxygenated methylene at δH 4.85 (2H, s) and δC 65.8 (C-8). The heteronuclear multiple-bond correlations (HMBC) from H-3′ (H-7′) to C-1′ and C-5′ and from H-4′ (H-6′) to C-2′, demonstrated the presence of the 4-substituted benzoyl group. Furthermore, the thiazolopyridine moiety, similar with phenyl(thiazolo[5,4-b]pyridin-2-yl)methanone,[32] was supported by the correlation spectroscopy (COSY) cross-signal of H-4/H-5; HMBC correlations of H-4 with C-6 and C-7a and H-5 with C-3a, C-6, and C-7a (Figure ); and the molecular formula. Correlations of H-8 with C-5 and C-6 in the HMBC spectrum indicated that the oxygenated methyl unit was located at C-6. Therefore, the skeleton structure of compound 1 was composed of a 4-substituted benzoyl group and a 2-substituted thiazolo[5,4-b]pyridin-5-ylmethanol. According to the similar chemical shifts of 1 [δC 183.8 (C-1′), 169.4 (C-2)] and phenyl(thiazolo[5,4-b]pyridin-2-yl)methanone [δC 185.3 (C-1′), 167.8 (C-2)], the benzoyl group was assigned to C-2. Then, a hydroxyl group was attached to the benzoyl group based on the molecular formula. Successful crystallization and subsequent X-ray diffraction (Figure ) allowed for unambiguous determination of the entire structure of 1.

Table 1

1H (500 MHz) and 13C (125 MHz) NMR Data of Compound 1 in CD3OD

no.	δH (J in Hz)	δC	no.	δH (J in Hz)	δC
2		169.4	1′		183.8
3a		147.5	2′		127.1
4	8.57 d (8.5)	134.6	3′	8.53 d (8.9)	135.3
5	7.80 d (8.5)	120.9	4′	6.93 d (8.9)	116.5
6		163.7	5′		165.4
7a		159.2	6′	6.93 d (8.9)	116.5
8	4.85 s	65.8	7′	8.53 d (8.9)	135.3

Figure 1

Key 1H–1H COSY and HMBC correlations of compounds 1–8.

Figure 2

X-ray ORTEP drawing of compounds 1 and 2.

Janthinepene A (2) was obtained as colorless crystals. The negative-mode HRESIMS spectrum of 2 displayed an [M – H]− ion peak at m/z 245.11912, corresponding to a molecular formula of C15H18O3 with 13 indices of hydrogen deficiency. The 1H NMR resonance signals (Table ) indicated one 1,4-disubstituted benzene ring with a coupling pattern of [δH 7.98 (2H, d, J = 8.4 Hz) and 7.51 (2H, d, J = 8.4 Hz)] and one terminal double bond [δH 5.07 (1H, s) and 4.85 (1H, s)]. In combination with the HSQC data, signals were observed corresponding to one carbonyl carbon [δC 170.1 (C-15)], two olefinic carbons [δC 147.2 (C-11) and 110.0 (C-12)], one oxygenated methine [δH 4.46 (1H, t, J = 7.1 Hz); δC 83.7 (C-10)], one oxygenated tetrasubstituted carbon [δC 86.3 (C-7)], two sp3 methylenes [δH 2.23 (1H, m), 2.14 (1H, m), 1.94 (1H, m), and 1.85 (1H, m); δC 40.2 (C-8), 31.8 (C-9)], and two methyl groups [δH 1.78 (3H, s) and 1.56 (3H, s); δC 30.3 (C-14), 18.3 (C-13)] in 13C NMR (Table ) and DEPT spectra. The aforementioned data suggested that 2 was an ar-bisabol sesquiterpene similar to (+)-sielboldianin B.[30,31] Further analysis of the 2D NMR spectra indicated the planar structure. 1H–1H COSY correlations between H-8/H-9/H-10 of the spin system and HMBC correlations of H-12 with C-10/C-13, H-13 with C-10/C-12, H-10 with C-12/C-13, and H-9 with C-11 demonstrated the location of the isopropenyl fragment at C-10 (Figure ). Moreover, a carboxyl unit was connected to the benzene ring based on the key HMBC correlation of H-3 (H-5) with C-15. Thus, the planar structure of 2 was determined, as shown. To understand the relative configuration, we acquired the nuclear Overhauser effect spectroscopy (NOESY) spectrum of 1. The observed NOESY correlations between H-10 and H-8α/H-12/H-2 and between H3-14 and H-9β indicated that H-10, H-8α, and the benzene ring were found in a co-facial orientation on the tetrahydrofuran ring, while H3-14 and H-9β were on the opposite side (Figure ). The X-ray diffraction experiment using Ga Kα radiation with a Flack parameter of 0.01(4) confirmed the 7S,10S absolute configuration of compound 2 (Figure ).

Table 2

1H NMR Data for Compounds 2–8 (500 MHz, CD3OD)

no.	2	3	4	5	6	7	8
2	7.51 d (8.4)	7.52 d (8.5)	7.49 d (8.4)	7.48 d (8.4)	7.56 d (8.4)	7.50 d (8.4)	7.49 d (8.5)
3	7.98 d (8.4)	7.98 d (8.5)	7.96 d (8.4)	7.96 d (8.4)	7.95 d (8.4)	7.96 d (8.4)	7.95 d (8.5)
4
5	7.98 d (8.4)	7.98 d (8.5)	7.96 d (8.4)	7.96 d (8.4)	7.95 d (8.4)	7.96 d (8.4)	7.95 d (8.4)
6	7.51 d (8.4)	7.52 d (8.5)	7.49 d (8.4)	7.48 d (8.4)	7.56 d (8.4)	7.50 d (8.4)	7.49 d (8.5)
8α	2.23 m	2.24 dt (12.3, 7.1)	2.21 dt (12.4, 7.5)	2.19 dt (12.4, 7.7)	2.19 m	2.23 m	2.22 dt (12.4, 7.4)
8β	2.14 m	2.16 m	2.11 ddd (12.4, 8.2, 6.2)	2.14 m	2.07 m	2.09 m	2.15 m
9α	1.94 m	1.92 m	1.85 m	1.88 m	2.04 m	1.93 m	1.91 m
9β	1.85 m	2.00 m	1.82 m	1.80 m	1.87 m	1.73 m	1.80 m
10	4.46 t (7.1)	4.58 t (7.1)	3.98 dd (13.7, 6.3)	3.87 dd (14.9, 7.4)	4.00 t (7.1)	3.82 t (7.2)	4.17 dd (15.0, 7.2)
11			1.79 m	1.84 m			2.57 m
12	5.07 s	4.16 s	3.65 dd (10.7, 5.8)	3.77 dd (10.7, 5.2)	1.19 s	1.22 s	1.16 d (7.0)
	4.85 s		3.48 dd (10.7, 6.7)	3.54 dd (10.7, 6.4)
13	1.78 s	5.25 s	1.06 d (6.8)	0.95 d (6.9)	1.21 s	1.25 s
		5.17 s
14	1.56 s	1.56 s	1.49 s	1.50 s	1.49 s	1.52 s	1.50 s

Table 3

13C (125 MHz) NMR Data of Compounds 2–8 in CD3OD

no.	2	3	4	5	6	7	8
1	154.9	155.0	154.8	155.0	154.3	154.5	154.7
2	125.8	125.8	125.6	125.7	125.7	125.7	125.7
3	130.8	130.8	130.7	130.7	130.4	130.7	130.7
4	125.8	125.8	125.6	125.7	125.7	125.7	125.7
5	130.8	130.8	130.7	130.7	130.4	130.7	130.7
6	125.8	125.8	125.6	125.7	125.7	125.7	125.7
7	86.3	86.1	85.6	85.3	85.8	86.0	86.0
8	40.2	40.1	40.4	40.6	40.3	40.4	40.3
9	31.8	32.4	30.3	30.2	27.4	27.5	29.9
10	83.7	81.1	82.8	81.8	86.5	86.9	82.0
11	147.2	151.2	42.5	41.9	72.8	72.5	47.2
12	110.0	63.3	66.2	65.9	25.6	25.8	14.0
13	18.3	110.0	13.4	12.9	26.3	25.9	179.7
14	30.3	30.3	30.8	30.6	28.9	30.4	30.7
15	170.0	169.8	171.0	170.4	172.8	171.4	171.1

Figure 3

Key NOESY correlations of compounds 2–8.

Janthinepene B (3) was a colorless oil with a molecular formula of C15H18O4, which was confirmed by the (−)-HRESIMS ion peak at m/z 261.11433 [M – H]− [calcd for C15H17O4 (261.11268)], indicating seven degrees of unsaturation. The 1H and 13C NMR data (Tables and 3) of 3 closely resemble those of 2, indicating their structural similarity. However, these 1D NMR data, in conjunction with the HSQC spectrum, revealed that compound 3 has one less methyl group but one additional oxygenated methylene signal [δH 4.16 (2H, s); δC 63.3 (C-12)] compared with 2. We further constructed the planar structure of 3 by 2D NMR spectroscopic analysis (Figure ). Key HMBC correlations of H-12 (δH 4.16) with C-10/C-13 and of H-13 (δH 5.25, 5.17) with C-10/C-12, indicated that the C-12 position was oxygenated and the terminal double bond was between C-11 and C-13. Thereby, we established the planar structure of 3, as depicted. In addition, the obvious NOESY correlations between H-10 and H-8α/H-13/H-2 and between H3-14 and H-12/H-9β revealed that 3 and 2 share the same relative configurations (Figure ). Finally, the absolute stereochemistry was deduced based on the similar ECD (electronic circular dichroism) curves of 2 and 3 (Figure ).

Figure 4

Experimental ECD spectra of janthinepenes A–G (2–8) in MeOH.

Janthinepenes C (4) and D (5) shared the same molecular formula of C15H20O4, shown by the (+)-HRESIMS ion peaks at m/z 265.14340 [M + H]+ [calcd for C15H21O4 (265.14398)]. The 1H and 13C NMR data (Tables and 3) of 4 and 5 closely resembled those of 2, evidencing their structural similarity. 1H–1H COSY spectra displayed the spin systems of H-12/H-11/H-10/H-9/H-8 and H3-13/H-11 in combination with the molecular formula, and the location of the isopropanol group was determined as C-10 in compounds 4 and 5 (Figure ). Further interpretation of the 2D NMR spectra indicated they possessed the same planar structure. The relative stereochemistry of the tetrahydrofuran ring was determined to be the same as in the aforementioned ar-bisabol sesquiterpenes based on key NOESY correlations between H-10 and H-2 and between H-9β and H3-14 in 4 and 5. The NOESY correlations between H-12 and H-9 and between H3-13 and H3-14 in 4, while NOESY correlations of H-12/H-9 and H-11/H3-14 in 5, suggested that the relative configurations of C-11 was as shown. Additionally, we ultimately determined the absolute stereochemistry of 4 and 5 based on the similarities in their ECD curves with those of 2 (Figure ).

The molecular formula of janthinepenes E (6) and F (7) was determined to be C15H20O4 based on their HRESIMS data—the same as for 4 and 5. Subsequent comparison of the 1D NMR data (Tables and 3) confirmed that 6 and 7 contain an additional methyl and an oxygenated tetrasubstituted carbon (6: δC 72.8; 7: δC 72.5), replaying the methine and oxygenated methylene groups in 4 and 5. In HMBC spectra, cross-peaks from H3-12/H3-13 to C-10 and H-9 to C-11 indicated that a propane-2,2-diyl-2-ol unit was located at C-10. Thus, we determined the planar structures of 6 and 7. As shown in Figure , the NOESY cross-peaks from H3-14 to H-10/H-9β and H3-12 to H-9α/H-9β in 6 and from H-10 to H-2 and H3-14 to H3-12/H-9β in 7 indicated the relative configurations. Finally, the similar ECD curves of these ar-bisabol sesquiterpenes allowed us to determine the absolute configurations as 7S,10R in 6 and 7S,10S in 7.

Janthinepene G (8) has a molecular formula of C15H18O5, with seven degrees of unsaturation. The 1D NMR data revealed that 8 was an ar-bisabol sesquiterpene equipped with the same 4-(tetrahydrofuran-2-yl)benzoic acid unit. Meanwhile, extensive analysis of the 2D spectra indicated isopropionic acid connected to C-10 based on 1H–1H COSY correlations of H-8/H-9/H-10/H-11/H3-12 and HMBC cross-peaks from H-10/H-11/H3-12 to C-13. Hence, the planar structure of 8 was established. The relative configuration of the p-furanbenzoic acid skeleton was similar to that of 2–5 based on the NOESY cross-peaks of H-10 to H-2 and H-9β to H3-14. Furthermore, the NOESY correlations between H3-12 and H-9 and between H-11 and H-9β/H3-14 indicated the relative stereochemistry of C-11. As these ar-bisabol sesquiterpenes should share the same biosynthetic pathway in these fungi and the similar optical rotations and ECD curves, we presumed that the absolute configuration of the tetrahydrofuran ring was 7S,10S.

In addition to the new structures described above, we confirmed the presence of three known compounds (9–11) as 7-dehydrobrefeldin A (9),[11] BFA (10),[33] and campyrone B (11)[34] through a comparison of their spectroscopic data with values in the literature.

The antimicrobial activities of compounds 1–11 were evaluated against eight clinically drug-resistant bacteria and seven agricultural pathogenic fungi. Compound 9 showed weak antifungal activity against Alternaria fragriae, with an MIC value of 25 μg/mL, and compound 10 exhibited moderate antifungal activity against A. fragriae, with an MIC value of 12.5 μg/mL (Table S1). None of the evaluated compounds were active against the selected drug-resistance bacteria (Table S2).

To summarize, we isolated 11 metabolites including eight previously uncharacterized structures, janthinedine A (1) and janthinepenes A–G (2–8), from the endophytic fungus P. janthinellum. Janthinedine A represents the first example of a natural product featuring the novel thiazolo[5,4-b]pyridine skeleton, and the ar-bisabol sesquiterpenes were first obtained from P. janthinellum.[35−40] The evaluation of the antimicrobial activities revealed that compounds 9 and 10 possess weak antifungal capabilities. Our findings indicate that plant endophytic fungi are an important source of new natural products with potential antimicrobrial applications.

Experimental Section

General Experimental Procedures

A WRX-4 micromelting point apparatus was used to record the melting points (Changzhou Dedu Inc., Changzhou, China). An Autopol IV automatic polarimeter equipped with a 1.0 mL cell was arranged to measure optical rotations (Rudolph Research Analytical, Hackettstown, NJ, U.S.A.). An IRTracer-100 spectrophotometer was used to record IR spectra (Shimadzu, Kyoto, Japan). UV spectra were acquired using a UH5300 spectrophotometer (Hitachi Limited, Tokyo, Japan). A Chirascan plus circular dichroism spectrophotometer was applied to obtain the ECD data (Applied Photophysics Ltd, Leatherhead, Surrey, U.K.). An APEX-II CCD/BRUKER D8 QUEST diffractometer equipped with graphite-monochromatized Ga Kα radiation was arranged to measure the X-ray crystallography data (Bruker, Karlsruhe, Germany). HRESIMS data were obtained using a SolariX spectrometer (Bruker, Karlsruhe, Germany). 1D and 2D NMR spectra were recorded using a DRX-500 MHz spectrometer (Bruker, Karlsruhe, Germany). Chemical shifts are expressed in parts per million, with reference to the CD3OD (δH 3.31/δC 49.0) signals. Compounds were purified on a Hanbon newstyle semi-preparative HPLC system equipped with a UV detector (Hanbon Sci. and Tech., Jiangsu, China). Silica gel (80–100, 100–200, and 200–300 mesh; Anhui Liangchen Inc., Anhui, China), ODS (50 μm; YMC, Kyoto, Japan), and Sephadex LH-20 (Pharmacia Biotech, Uppsala, Sweden) were used as the packing materials for column chromatography (CC). Medium-pressure liquid chromatography (MPLC) was applied using a Sanotac MP0502C system (Shanghai Sanwei Scientific Instrument Co., Ltd., Shanghai, China).

Fungal Material

The strain MPT-25 was isolated from the stems of T. wallichiana var. chinensis (Pilger) Florin, collected in Shijiazhuang, Hebei province, PR China, in September 2015, and then identified as P. janthinellum based on analysis and comparison of the ITS region of the rDNA sequence (GenBank accession no. MZ048774). The voucher strain is maintained at the School of Pharmaceutical Sciences, South-Central Minzu University.

Fermentation and Isolation

To prepare the seed culture, the strain P. janthinellum MPT-25 was cultivated on potato dextrose agar (PDA) for 7 days under 28 °C. We first filled each tissue culture flasks (500 mL) with 80 mL of distilled water and 100 g of rice and then sterilized by autoclaving. The PDA plates were cut into small pieces (approximately 0.5 × 0.5 × 0.5 cm3) and inoculated into tissue culture flasks. A total of 400 flasks for fungal fermentation were used. After inoculation, all tissue cultures were statically incubated at 28 °C. After 38 days of fermentation, cultures were harvested from the flasks for chemical characterization. Rice medium was ultrasonically extracted with the same volume of CH3CH2OH five times to give a brown syrup, followed by suspending in H2O and extracting with EtOAc to yield an crude extract (200 g).

The EtOAc extract (200 g) was separated by silica gel CC (100–200 mesh) and eluted with CH2Cl2–MeOH (100:1–0:1, v/v) to give six fractions (Fr. A–F).

Fr. C (20.6 g) was chromatographed on reversed-phase ODS MPLC (MeOH–H2O, 20:80–100:0) to afford six fractions (C1–C6). Fr. C3 was chromatographed on Sephadex LH-20 (CH2Cl2–MeOH 1:1) and followed by semi-preparative HPLC (MeOH–H2O–HCOOH, 72:28:0.05, v/v) to yield 2 (12.7 mg, tR = 19.0 min, 3.0 mL/min).

Fr. D (48.4 g) was chromatographed on reversed-phase ODS MPLC (MeOH–H2O, 20:80–100:0) to obtain seven fractions (D1–D7). Fr. D2 was chromatographed on Sephadex LH-20 (MeOH) to obtain five subfractions (Fr. D2.1–Fr. D2.5). Fr. D2.1 was further purified by semi-preparative HPLC (MeCN–H2O, 30:70, v/v) to yield 11 (20.5 mg, tR = 21.0 min, 2.0 mL/min). Chromatography of Fr. D2.2 on a silica gel column was carried out, eluting with a gradient system of CH2Cl2–MeOH (100:1–70:1, v/v), followed by semi-preparative HPLC (MeOH–H2O, 65:35, v/v) to yield 9 (2 mg, tR = 17.0 min, 2.0 mL/min). Chromatography of Fr. D2.3 on a silica gel column was carried out, eluting with CH2Cl2–MeOH (70:1, v/v) to yield 3 (25.7 mg). Fr. D2.4 was further purified on semi-preparative HPLC (MeCN–H2O, 32:68, v/v) to yield 1 (11.8 mg, tR = 26.0 min, 2.0 mL/min).

Fr. E (39.2 g) was separated by MPLC (MeOH–H2O, 20:80–100:0) to obtain eight fractions (E1–E8). Fr. E4 was chromatographed on Sephadex LH-20 (MeOH) to give three subfractions (Fr. E4.1–Fr. E4.3). Chromatography of Fr. E4.2 on a silica gel column was carried out, eluting with a gradient of PE–EA (4:1–1:1, v/v) to give two subfractions (Fr. E4.2.1–Fr. E4.2.2). Fr. E4.2.1 was further purified by semi-preparative HPLC (MeCN–H2O–HCOOH, 33:67:0.05, v/v) to yield 6 (1.1 mg, tR = 18.0 min, 3.0 mL/min) and 7 (1.4 mg, tR = 19.0 min, 3.0 mL/min). Chromatography of Fr. E4.3 on a silica gel column was carried out, eluting with a gradient of PE–EA–HAc (5:1:0.006, v/v) to offer four subfractions. Fr. E4.3.2 was further purified by semi-preparative HPLC (MeCN–H2O–HCOOH, 35:65:0.05, v/v) to yield 4 (3.9 mg, tR = 20.0 min, 2.0 mL/min) and 5 (3.0 mg, tR = 23.0 min, 2.0 mL/min). Fr. E4.3.3 was further purified by semi-preparative HPLC (MeCN–H2O–HCOOH, 30:70:0.05, v/v) to yield 8 (2.6 mg, tR = 28.0 min, 3.0 mL/min). Fr. E4.3.4 was further purified using semi-preparative HPLC (MeCN–H2O–HCOOH, 27:73:0.05, v/v) to yield 10 (3.4 mg, tR = 37.0 min, 4.0 mL/min).

Janthinedine A (1)

Yellow crystals, mp 189.4–226.4 °C, UV (MeOH) λmax (log ε): 215 (4.11), 335 (4.04) nm; IR (KBr) νmax: 1080, 1126, 1174, 1282, 1319, 1577, 1600, 2850, 2920 cm–1; 1H and 13C NMR data, see Table ; HRESIMS m/z: 287.04841 [M + H]+ (calcd for C14H11N2O3S+, 287.04904).

Janthinepene A (2)

White crystals, mp 121.4–122.6 °C, [α]D20 −46.6 (c 1.0, MeOH); UV (MeOH) λmax (log ε): 235 (4.03) nm; CD (MeOH) λmax (Δε): 205 (4.31), 236 (−3.24) nm; IR (KBr) νmax: 1541, 1558, 2360, 2924 cm–1; 1H and 13C NMR data, see Tables and 3; HRESIMS m/z: 245.11912 [M – H]− (calcd for C15H17O3–, 245.11777).

Janthinepene B (3)

Colorless oil, [α]D20 −24.4 (c 0.1, MeOH); UV (MeOH) λmax (log ε): 235 (4.60) nm; CD (MeOH) λmax (Δε): 205 (6.09), 236 (−6.60) nm; IR (KBr) νmax: 1016, 1080, 1267, 1701, 2972 cm–1; 1H and 13C NMR data, see Tables and 3; HRESIMS m/z: 261.11433 [M – H]− (calcd for C15H17O4–, 261.11268).

Janthinepene C (4)

Colorless oil, [α]D20 −17.1 (c 1.0, MeOH); UV (MeOH) λmax (log ε): 235 (4.83) nm; CD (MeOH) λmax (Δε): 204 (5.31), 236 (−8.27) nm; IR (KBr) νmax: 1016, 1261, 1406, 1701, 2970 cm–1; 1H and 13C NMR data, see Tables and 3; HRESIMS m/z: 265.14340 [M + H]+ (calcd for C15H21O4+, 265.14398).

Janthinepene D (5)

Colorless oil, [α]D20 −21.3 (c 1.0, MeOH); UV (MeOH) λmax (log ε): 235 (4.89) nm; CD (MeOH) λmax (Δε): 205 (−6.63), 233 (−11.36) nm; IR (KBr) νmax: 1016, 1261, 1406, 1701, 2970 cm–1; 1H and 13C NMR data, see Tables and 3; HRESIMS m/z: 265.14340 [M + H]+ (calcd for C15H21O4+, 265.14398).

Janthinepene E (6)

Colorless oil, [α]D20 −20.0 (c 0.1, MeOH); UV (MeOH) λmax (log ε): 235 (4.12) nm; CD (MeOH) λmax (Δε): 201 (7.17), 236 (−0.90) nm; IR (KBr) νmax: 1083, 1409, 1610, 1697, 2920 cm–1; 1H and 13C NMR data, see Tables and 3; HRESIMS m/z: 265.14378 [M + H]+ (calcd for C15H21O4+, 265.14398).

Janthinepene F (7)

Colorless oil, [α]D20 −22.0 (c 0.5, MeOH); UV (MeOH) λmax (log ε): 235 (4.73) nm; CD (MeOH) λmax (Δε): 204 (3.18), 236 (−5.97) nm; IR (KBr) νmax: 1261, 1406, 1610, 1697, 2974 cm–1; 1H and 13C NMR data, see Tables and 3; HRESIMS m/z: 263.12998 [M – H]− (calcd for C15H19O4–, 263.12833).

Janthinepene G (8)

Colorless oil, [α]D20 −27.6 (c 1.0, MeOH); UV (MeOH) λmax (log ε): 235 (4.38) nm; CD (MeOH) λmax (Δε): 205 (3.00), 237 (−9.33) nm; IR (KBr) νmax: 1078, 1290, 1417, 1691, 2980 cm–1; 1H and 13C NMR data, see Tables and 3; HRESIMS m/z: 277.10941 [M – H]− (calcd for C15H17O5–, 277.10760).

X-ray Crystal Structure Analysis

Crystals of 1 and 2 were obtained from MeOH–H2O. Intensity data were collected on a Bruker D8 QUEST diffractometer equipped with an APEX II CCD using Ga Kα radiation. Cell refinement and data reduction were conducted using a Bruker SAINT, and data were corrected for absorption effects using the multi-scan method (SADABS). The structures were solved using the Bruker SHELXTL software package.[41] Crystallographic data (excluding structure factor tables) for compounds 1 and 2 were deposited at the Cambridge Crystallographic Data Center (CCDC) as supplementary publications no. CCDC 2156926 for 1 and CCDC 2156925 for 2.

Crystal Data for 1

C14H10N2O4S, M = 302.30, a = 11.883(8) Å, b = 53.61(4) Å, c = 4.243(4) Å, α = 90°, β = 90.06(4)°, γ = 90°, V = 2703(4) Å3, T = 200.15 K, space group Cc, Z = 8, Dcalc = 1.486 g/cm3, F(000) = 1248.0, μ (Ga Kα) = 1.507 mm–1, 12 612 reflections measured, 5454 independent reflections (Rint = 0.1066, Rσ = 0.1064). The final R1 value was 0.2390 [I > 2σ(I)]. The final wR2 value was 0.5007 [I > 2σ(I)]. The final R1 value was 0.2429 (all data). The final wR2 value was 0.5170 (all data). The goodness of fit on F2 was 2.345.

Crystal Data for 2

C30H36O6, M = 492.59, a = 5.8678(9) Å, b = 11.0202(16) Å, c = 41.904(6) Å, α = 90°, β = 90°, γ = 90°, V = 2709.7(7) Å3, T = 200(2) K, space group P212121, Z = 4, Dcalc = 1.207 g/cm3, F(000) = 1056.0, μ (Ga Kα) = 0.427 mm–1, 47 437 reflections measured, 6366 independent reflections (Rint = 0.0477, Rσ = 0.0278). The final R1 value was 0.0423 [I > 2σ(I)]. The final wR2 value was 0.1167 [I > 2σ(I)]. The final R1 value was 0.0437 (all data). The final wR2 value was 0.1179 (all data). The goodness of fit on F2 was 1.048. Flack parameter = 0.01(4).

Biological Assays

Antibacterial Assay

Methicillin-resistant Staphylococcus aureus, carbapenem-resistant Klebsiella neumonia, carbapenems-resistant Acinetobacter baumannii, carbapenems-resistant Escherichia coli, carbapenems-resistant Pseudomonas aeruginosa, multidrug-resistant Enterococcus faecium, multidrug-resistant Staphylococcus epidermidis, and multidrug-resistant Enterococcus faecalis were cultivated to evaluate the antibacterial activities of compounds 1–11in vitro. According to the previous report,[42] samples were assayed in sterile 96-well plates based on the modified broth dilution test method with DMSO and ciprofloxacin as the negative and positive control, respectively.

Antifungal Assay

The isolated compounds were also evaluated for their antifungal activity against agricultural pathogenic fungi (Verticillium dahliae Kleb, Sclerotinia sclerotiorum, Rhizoctonia solani, A. fragriae, Fusarium oxysporum f. sp. vasinfectum, Botryospuaeria berengeriana, and Helminthosporium maydis) previously described with DMSO and ketoconazole as the negative and positive control, respectively.[42]