Mar. Drugs 2014, 12(1), 477-490; doi:10.3390/md12010477

Communication
Alkaloids from the Mangrove-Derived Actinomycete Jishengella endophytica 161111
Pei Wang 1, Fandong Kong 1, Jingjing Wei 2, Yi Wang 1, Wei Wang 1, Kui Hong 2,* and Weiming Zhu 1,*
1
Key Laboratory of Marine Drugs, Ministry of Education of China, School of Medicine and Pharmacy, Ocean University of China, Qingdao 266003, China; E-Mails: wangpei850212@163.com (P.W.); kongfandong501@126.com (F.K.); wangyi0213@ouc.edu.cn (Y.W.); wwwakin@ouc.edu.cn (W.W.)
2
Key Laboratory of Combinatorial Biosynthesis and Drug Discovery, Ministry of Education, School of Pharmaceutical Sciences, Wuhan University, Wuhan 430071, China; E-Mail: rxwzq@163.com
*
Authors to whom correspondence should be addressed; E-Mails: kuihong31@whu.edu.cn (K.H.); weimingzhu@ouc.edu.cn (W.Z.); Tel./Fax: +86-532-8203-1268 (W.Z.).
Received: 28 November 2013; in revised form: 24 December 2013 / Accepted: 7 January 2014 /
Published: 21 January 2014

Abstract

: A new alkaloid, 2-(furan-2-yl)-6-(2S,3S,4-trihydroxybutyl)pyrazine (1), along with 12 known compounds, 2-(furan-2-yl)-5-(2S,3S,4-trihydroxybutyl)pyrazine (2), (S)-4-isobutyl-3-oxo-3,4-dihydro-1H-pyrrolo[2,1-c][1,4]oxazine-6-carbaldehyde (3), (S)-4-isopropyl-3-oxo-3,4-dihydro-1H-pyrrolo[2,1-c][1,4]oxazine-6-carbaldehyde (4), (4S)-4-(2-methylbutyl)-3-oxo-3,4-dihydro-1H-pyrrolo[2,1-c][1,4]oxazine-6-carbaldehyde (5), (S)-4-benzyl-3-oxo-3,4-dihydro-1H-pyrrolo[2,1-c][1,4]oxazine-6-carbaldehyde (6), flazin (7), perlolyrine (8), 1-hydroxy-β-carboline (9), lumichrome (10), 1H-indole-3-carboxaldehyde (11), 2-hydroxy-1-(1H-indol-3-yl)ethanone (12), and 5-(methoxymethyl)-1H-pyrrole-2-carbaldehyde (13), were isolated and identified from the fermentation broth of an endophytic actinomycetes, Jishengella endophytica 161111. The new structure 1 and the absolute configurations of 26 were determined by spectroscopic methods, J-based configuration analysis (JBCA) method, lactone sector rule, and electronic circular dichroism (ECD) calculations. Compounds 811 were active against the influenza A virus subtype H1N1 with IC50 and selectivity index (SI) values of 38.3(±1.2)/25.0(±3.6)/39.7(±5.6)/45.9(±2.1) μg/mL and 3.0/16.1/3.1/11.4, respectively. The IC50 and SI values of positive control, ribavirin, were 23.1(±1.7) μg/mL and 32.2, respectively. The results showed that compound 9 could be a promising new hit for anti-H1N1 drugs. The absolute configurations of 25, 13C nuclear magnetic resonance (NMR) data and the specific rotations of 36 were also reported here for the first time.
Keywords:
mangrove; actinomycete; Jishengella endophytica 161111; pyrazine derivative; anti-H1N1 virus activity

1. Introduction

Mangroves, unique forest ecosystems found mainly in the tropical and subtropical intertidal regions, represent a rich biological diversity and a high population of actinomycetes [1,2]. From these actinomycetes, many bioactive compounds such as cytotoxic streptocarbazoles A and B have been obtained [3]. In addition, there is also evidence that the mangrove ecosystem is a largely unexplored source for novel actinomycetes with the potential to produce biologically active secondary metabolites [4]. In order to pursue bioactive products from mangrove actinomycetes, a novel endophytic actinomycetes, identified as Jishengella endophytica 161111, had been isolated from the root of the mangrove plant, Xylocarpus granatum (Meliaceae) [5]. J. endophytica 161111 was found to produce alkaloids in a saline culture medium by thin layer chromatography (TLC) visualizing with Dragendorff’s reagent. Chemical investigations on the ethyl acetate (EtOAc) extract of fermentation broth of strain 161111 resulted in the isolation and identification of three pyrazine derivatives, 2-(furan-2-yl)-6-(2S,3S,4-trihydroxybutyl)pyrazine (1) and 2-(furan-2-yl)-5-(2S,3S,4-trihydroxybutyl) pyrazine (2) [6] and lumichrome (10) [7]; four pyrrololactones, (S)-4-isobutyl-3-oxo-3,4-dihydro-1H-pyrrolo[2,1-c][1,4]oxazine-6-carbaldehyde (3) [8], (S)-4-isopropyl-3-oxo-3,4-dihydro-1H-pyrrolo[2,1-c][1,4]oxazine-6-carbaldehyde (4) [8], (4S)-4-(2-methylbutyl)-3-oxo-3,4-dihydro-1H-pyrrolo[2,1-c][1,4]oxazine-6-carbaldehyde (5) [8,9] and (S)-4-benzyl-3-oxo-3,4-dihydro-1H-pyrrolo[2,1-c][1,4]oxazine-6-carbaldehyde (6) [10]; three β-carbolines, flazin (7) [11], perlolyrine (8) [12] and 1-hydroxy-β-carboline (9) [13]; along with 1H-indole-3-carboxaldehyde (11) [14,15], 2-hydroxy-1-(1H-indol-3-yl) ethanone (12) [14,15], and 5-(methoxymethyl)-1H-pyrrole-2-carbaldehyde (13) [16] (Figure 1). Compounds 811 showed anti-influenza A (H1N1) virus activity with the half maximal inhibitory concentration (IC50) and selectivity index (SI) values of 38.3(±1.2)/25.0(±3.6)/39.7(±5.6)/45.9(±2.1) μg/mL and 3.0/16.1/3.1/11.4, respectively. Pyrazine alkaloids from marine organisms exhibited cytotoxic and antimicrobial activities [17,18,19,20,21], three of which were identified from the mangrove plant [22] and fungi [23,24]. β-Carbolines, as a type of natural indol alkaloids, displayed cytotoxic [25], antiviral [26,27], antimicrobial [28], antiparasitic [29], and antithrombotic activities [30]. Apart from the terrestrial organisms, marine organisms, including mangrove fungi [24,31], are also a major source of β-carbolines [32,33,34,35,36,37,38,39,40]. Pyrrololactones that had been reported as the volatile components of the roasted roots of Cichorium intrybus [8] were not identified from the marine organisms.

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Figure 1. Chemical structures of compounds 113 from J. endophytica 161111.

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Figure 1. Chemical structures of compounds 113 from J. endophytica 161111.
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2. Results and Discussion

2.1. Structure Elucidation

The EtOAc extract of the fermentation broth of J. endophytica 161111 was subjected to extensive chromatographic separations over silica gel, RP-18, Sephadex LH-20 and high performance liquid chromotography (HPLC) to yield the new compound 1 and the known compounds 213.

Compound 1 was obtained as brown oil. Its molecular formula was determined as C12H14N2O4 on the basis of high resolution electrospray ionization mass spectrum (HRESIMS) peak at m/z 273.0849 [M + Na]+ (calcd. for C12H14N2O4Na 273.0846). The similarity of 1D- and 2D-NMR data (Table S2 in Supplementary Information) between 1 and the known crotonine (2) [6] indicated the similar planar structure. The signals at δH 7.23 (1H, d, J = 4.0 Hz), 6.63 (1H, dd, J = 2.1, 4.0 Hz) and 7.72 (1H, d, J = 2.1 Hz) revealed the presence of a α-substituted furan moiety, which was further identified by the 1H-1H correlation spectroscopy (COSY) of H-3′/H-4′/H-5′ and the key heteronuclear multiple bond correlations (HMBC) of H-3′ (δ 7.23) to C-2′ (δ 151.1), C-4′ (δ 112.0) and C-5′ (δ 144.6), of H-4′ (δH 6.63) to C-2′, C-3′ (δ 110.5) and C-5′, and of H-5′ (δ 7.72) to C-2′ and C-3′. The existence of 2,3,4-trihydroxybutyl moiety was deduced from the 1H-1H COSY correlations of H-1″/H-2″/H-3″/H-4″ along with the key HMBC correlations of H-1″ (δ 3.24/2.93) to C-2″ (δ 71.5) and C-3″ (δ 74.8), H-2″ (δ 4.03) to C-3″ and C-4″ (δ 63.2), H-3″ (δ 3.58) to C-1″ (δ 38.4), C-2″ and C-4″, and H-4″ (δ 3.80/3.65) to C-2″ and C-3″. The remainder C4H2N2 displayed two sp2 methine signals at δH/C 8.78/136.7 and 8.38/142.7, and two sp2 quaternary carbon signals at δC 144.2 and 155.3, which were ascribable to a disubstituted pyrazine nucleus [41]. The obvious HMBC correlations from H-3 (δ 8.78) to C-2 (δ 144.2) and C-2′, H-5 (δ 8.38) to C-3 (δ 136.7) and C-6 (δ 155.3), and from H-2″ to C-5 (δ 142.7) and C-6 indicated that the α-substituted furan moiety and the 2,3,4-trihydroxybutyl moiety were linked onto C-2 and C-6 of the pyrazine nucleus, respectively. The relative configuration of 1 was determined by J-based configuration analysis (JBCA) method [42]. The low temperature NMR (−4 °C) of 1 revealed a large coupling constant between H-2″ and H-3″ (J = 7.2 Hz), indicating an anti-relationship between the two protons. In addition, low temperature NMR (−4 °C) of 1 also displayed nuclear Overhauser effect spectroscopy (NOESY) of H-1″/H-3″, H-1″/H-4″, and H-4″/H-2″. These data indicated threo-configuration between C-2″ and C-3″ (Figure 2). The absolute configuration of 1 was determined by use of quantum chemical ECD calculation [43]. The preliminary conformational distribution search was performed by HyperChem 7.5 software. The corresponding minimum geometries were further fully optimized by using density functional theory (DFT) at the B3LYP/6-31G(d) level as implemented in the Gaussian 03 program package. The stable conformers obtained were submitted to ECD calculation by the time-dependent DFT (TDDFT) (B3LYP/6-31G(d)) method. The overall predicted ECD spectrum of 1 was subsequently compared with the measured one [43]. The measured circular dichroism (CD) curve of 1 showed Cotton effect at λmax (Δε) 310 (−0.14), 284 (+0.12) and 237 (−1.3) nm, matching well with the calculated ECD curve of (2″S,3″S)-1 (Figure 3). Thus, the new structure, 1, was established as 2-(furan-2-yl)-6-(2S,3S,4-trihydroxybutyl)pyrazine.

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Figure 2. Selected 2D NMR correlations for 1 and Newman projections showing NOESY correlations and 3 JH-2″,H-3″ values of 1 and 2.

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Figure 2. Selected 2D NMR correlations for 1 and Newman projections showing NOESY correlations and 3 JH-2″,H-3″ values of 1 and 2.
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Figure 3. CD and calculated ECD spectra for 1 and 2.

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Figure 3. CD and calculated ECD spectra for 1 and 2.
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Although the planar structures of 25 had reported in the literatures [6,8,9], their absolute configurations have not been resolved yet. The planar structures of 2 and 6 here were elucidated by comparison of their NMR data with those reported [6,10]. Then, the same methods as 1 were used to resolve the relative and absolute configurations of 2. The large 3 JH-2″,H-3″ value (7.1 Hz) and the NOESY correlations of H-1″/H-3″ and H-1″/H-4″/H-2″ in the low temperature NMR (−4 °C) of 2 revealed the same threo-configuration of two hydroxy groups at C-2″ and C-3″ (Figure 2). Compound 2 showed a CD Cotton effect at λmax (Δε) 316 (−0.54), 267 (+0.35) and 235 (−1.0) nm, matching with the calculated ECD curve of (2″S,3″S)-2 (Figure 3). Thus, compound 2 was determined as 2-(furan-2-yl)-5-(2S,3S,4-trihydroxybutyl)pyrazine.

Though absolute structure 6 had been presented in the literature [10], no reference data on absolute configuration, such as CD and specific rotation of 6, could be used. Lactone sector rule [44] could be used to determine the absolute configuration of 6. The molecule was viewed from the line on the plane of the ester group along the bisectrix of the O–C=O angle, i.e., the line from C-2 to C-3 as shown in Figure 4 for (3S). The functional group at C-3 lying in the back upper left sector was responsible for the positive CD Cotton effect resulted from n–π* transition of lactone, which was well in accordance with the positive Cotton effect at λmax 293 nm of 6 (Figure 4). The deduction was further confirmed by ECD quantum chemical calculations using the TDDFT method at the B3LYP/6-31G(d) level in Gaussian 03 [43]. The measured CD curve of 6 showed Cotton effect at λmax (Δε) 293 (+13.1), 249 (–4.7), 211 (+6.8) nm, matching with the calculated ECD curve of (3S)-6 and opposite to that of (3R)-6 (Figure 5). Thus, the absolute configuration of 6 was unambiguous determined as S-, that is (S)-4-benzyl-3-oxo-3,4-dihydro-1H-pyrrolo[2,1-c][1,4]oxazine-6-carbaldehyde.

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Figure 4. Application of lactone sector rule to 36.

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Figure 4. Application of lactone sector rule to 36.
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Figure 5. CD and calculated ECD spectra of 36.

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Figure 5. CD and calculated ECD spectra of 36.
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Compounds 35 were found to have the molecular formulas of C12H15NO3, C11H13NO3, and C12H15NO3 by HRESI-MS peaks at m/z 222.1131 [M + H]+, 208.0974 [M + H]+, and 222.1129 [M + H]+(calcd. for C12H16NO3 222.1125, C11H14NO3 208.0968, C12H16NO3 222.1125), respectively. The similarity of ultraviolet (UV) and NMR spectra between 35 and 6 indicates compounds 35 contain the same3-oxo-3,4-dihydro-1H-pyrrolo[2,1-c][1,4]oxazine-6-carbaldehyde nucleus. The remaining moieties of C4H9, C3H7 and C4H9 for 35 could be deduced as isobutyl, isopropyl, and sec-butyl, respectively, from their 1D NMR data (Table 1). Thus, the planar structures of 35 were 4-isobutyl-3-oxo-3,4-dihydro-1H-pyrrolo[2,1-c][1,4]oxazine-6-carbaldehyde [8], 4-isopropyl-3-oxo-3,4-dihydro-1H-pyrrolo[2,1-c][1,4]oxazine-6-carbaldehyde [8], and 4-(2-methylbutyl)-3-oxo-3,4-dihydro-1H-pyrrolo [2,1-c] [1,4]oxazine-6-carbaldehyde [8,9], respectively. The planar structure 3 was further supported by 1H-1H COSY correlations of H-14/H-13/H-12/H-11/H-3 and H-6 (δH 7.14) to H-7 (δH 6.31) combined with the key HMBC correlations from H-11 (δH 2.00, 1.60) to C-2 (δC 167.5), C-3 (δC 56.4), C-13 (δC 21.6), and C-14 (δC 23.3), from H-9 (δH 5.70, 5.51) to C-2, C-7 (δC 107.2), and C-8 (δC 132.5), from H-3 (δH 5.65) to C-2, C-8, C-12 (δC 24.7), of H-7 (δH 6.31) to C-5 (δC 130.7), C-6 (δC 125.2), of H-6 to C-7, C-8, and C-10 (δC 180.1). The same lactone sector rule and ECD quantum chemical calculation used in 6 were also applied to elucidate the absolute configurations of 35. Thus, the same (3S)-configurations of 35 could be deduced from the same negative sign of the specific rotations and the positive CD Cotton effects at the long wave length along with concordance of CD with the calculated ECD of (3S)-isomer and opposition to ECD of (3R)-isomer (Figure 5).

Table 1. 1H and 13C NMR data for 36 (δ in ppm).

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Table 1. 1H and 13C NMR data for 36 (δ in ppm).
Position3456
δC, TypeδH, Mult. (J in Hz)δC, TypeδH, Mult. (J in Hz)δC, TypeδH, Mult. (J in Hz)δC, TypeδH, Mult. (J in Hz)
2167.5, qC 166.7, qC 166.5, qC 167.2, qC
356.4, CH5.65, dd, (5.7, 10.2)63.1, CH5.36, d, (8.0)62.0, CH5.46, d, (6.9)59.0, CH5.84, t, (5.6)
5130.7, qC 131.4, qC 131.2, qC 130.7, qC
6125.2, CH7.14, d, (4.0)125.4, CH7.17, d, (4.0)125.6, CH7.2, d, (3.8)125.4, CH7.19, d, (4.0)
7107.2, CH6.31, d, (4.0)107.2, CH6.34, d, (4.0)107.1, CH6.34, d, (3.8)106.4, CH6.17, d, (4.0)
8132.5, qC 132.7, qC 132.8, qC 132.4, qC
963.7, CH25.70, d, (15.2)64.2, CH25.64, d, (15.5)64.4, CH25.64, d, (15.6)63.6, CH25.24, d, (15.1)
5.51, d, (15.2)5.53, d, (15.5)5.54, d, (15.6)4.06, d, (15.1)
10180.1, CH9.49, s180.2, CH9.51, s180.0, CH9.5, s179.9, CH9.51, s
1141.3, CH22.00, ddd, (4.3, 10.2, 13.4)32.4, CH2.33, m39.3, CH2.08, m39.5, CH23.33, m
1.60, ddd, (5.7, 9.3, 13.4)
1224.7, CH1.66, m19.3, CH30.97, d, (6.8)25.8, CH21.39, m; 1.25, m135.1, qC
1321.6, CH31.00, d, (6.4)18.8, CH30.92, d, (6.8)15.5, CH30.90, d, (7.0)129.8, CH6.85, d, (7.3)
1423.3, CH30.89, d, (6.4) 11.8, CH30.91, t, (7.1)129.1, CH7.25, t, (7.3)
15 128.2, CH7.29, t, (7.3)
16 129.1, CH7.25, t, (7.3)
17 129.8, CH6.85, d, (7.3)

2.2. The Bioactivities of Compounds 113 from J. endophytica 161111

Compounds 113 were tested for antivirus effects on H1N1 by the cytopathic effect (CPE) inhibition assay [45,46] and ribavirin was used as the positive control with an IC50 value of 23.1 ± 1.7 μg/mL. The results (Table S1 in Supplementary Information) showed that compounds 811 showed moderate anti-H1N1 activity with IC50 values of 38.3 ± 1.2, 25.0 ± 3.6, 39.7 ± 5.6, and 45.9 ± 2.1 μg/mL, respectively. In addition, the cytotoxic effects of 811 on Madin-Daby canine kidney (MDCK) normal cells were also evaluated by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) [47]. The results showed that compounds 811 and ribavirin exhibited very weak cytotoxicities against MDCK normal cell with the half maximal cytotoxic concentration (CC50) values of 116.3 ± 12.1, 403.2 ± 31.4, 124.1 ± 10.5, 522.5 ± 24.5, and 744.2 ± 18.5 μg/mL, respectively. The SI values of compounds 811 and ribavirin were 3.0, 16.1, 3.1, 11.4, and 32.2, respectively.

Both naturally occurring and chemically synthesized β-carbolines exhibited good activities against a set of virus, such as human immunodeficiency virus (HIV) [26,48], herpes simplex virus (HSV) [27,49], vaccinia virus [50], vesicular stomatitis virus [50,51], poliovirus [49], and influenza A and B virus [51]. It was reported that the derivatives with the oxathiazepine 7-membered ring showed potential activities against influenza A and B [51]. This study showed that the simple β-carboline alkaloids were active against H1N1 virus and the unsubstituted H-3 is essential for the anti-H1N1 activity of this kind of compounds. In addition, the compound 9 could be a promising new hit for anti-H1N1 drugs. In addition, further chemical modifications are necessary to improve the anti-H1N1 virus activity.

3. Experimental Section

3.1. General Experimental Procedures

Specific rotations were obtained on a JASCO P-1020 digital polarimeter. UV spectra were measured on a Beckman DU 640 spectrophotometer. IR spectra were recorded on a Nicolet Nexus 470 spectrophotometer as KBr disks. CD spectra were collected using a JASCO J-715 spectropolarimeter. NMR data of 7, 8, 11, and 13 were measured on a JEOL JNM-ECP 600 spectrometer, while NMR of 1, 2, 36, 9, 10, and 12, and all NOESY spectra were recorded on a Bruker Avance 600 spectrometer. Electrospray ionization mass spectra (ESIMS) and HRESIMS measurements were taken on a Q-TOFULTIMA GLOBAL GAA076 LC mass spectrometer. Semipreparative HPLC was performed using an octadecylsilyl (ODS) column (YMC-pak ODS-A, Allentown, PA, USA, 10 × 250 mm, 5 μm, 4.0 mL/min) and C3 column (Agilent Zorbax StableBond C3, Palo Alto, CA, USA, 4.6 × 150 mm, 5 μm, 1.0 mL/min). TLC and column chromatography (CC, 2.5 × 103 cm) were performed on plates pre-coated with silica gel GF254 (10–40 μm, Qingdao Marine Chemical Factory, Qingdao, China), and over Sephadex LH-20 (Amersham Biosciences, Uppsala, Sweden), respectively. Vacuum-liquid chromatography (VLC, 6 × 24 cm and 3.6 × 30 cm) utilized silica gel (200–300 mesh, Qingdao Marine Chemical Factory, Qingdao, China) and RP-18 (40–63 μm, Merck, Darmstadt, Germany). Glucose (Shanghai Huixing Biochemical Reagent Co., Ltd., Shanghai, China); yeast extract and peptone (Beinjing Shuangxuan Microbe Culture Medium Products Factory, Beijing, China); CaCO3 (Tianjijn Bodi Chemical Co., Ltd., Tianjin, China); KNO3 (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China); NaCl (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China).

3.2. Actinomycetes Material

Jishengella endophytica 161111 was isolated and identified from the healthy roots of the mangrove plant, Xylocarpus granatum, collected from the mangrove reserve zone in Hainan Province, China [5]. The producing strain was prepared on oatmeal agar (ISP3) medium and stored in Hong’s Lab. at 4 °C.

3.3. Fermentation and Extraction

The spores of J. endophytica 161111 were directly cultured in 1000 mL Erlenmeyer flasks containing 200 mL fermentation media consisted of glucose 2%, yeast extract 0.5%, peptone 0.5%, KNO3 1.5%, CaCO3 0.4%, and NaCl 0.4% (pH 7.2). The cultures were incubated on a rotatory shaker at 220 rpm at 28 °C for 30 days. The whole fermentation broth (100 L) was divided into three equal parts and extracted three times with equal volumes of EtOAc separately. The whole EtOAc solutions were combined and evaporated under reduced pressure to give a dark brown gum (10 g).

3.4. Purification and Identification

The EtOAc extract (10 g) was subjected to SiO2 VLC, eluting with CH2Cl2–petroleum ether (0%~100%) and then with MeOH–CH2Cl2 (0%~50%), to give nine fractions (Fr.1–Fr.9). Fraction 3 (1.31 g) was separated into three subfractions by CC over Sephadex LH-20, eluted with MeOH/CH2Cl2 (1:1). Fraction 3-3 (351 mg) was further subjected to HPLC separation eluteding with 50% MeCN/H2O to yield 3 (2 mg, tR 9.93 min) and 6 (1 mg, tR 10.74 min). Compound 7 (25 mg) was purified from the precipitate of fraction 5 (512 mg) after washing with MeOH, and the filtrate of fraction 5 was separated into five parts (Fr.5-1–Fr.5-4) by Sephadex LH-20 with CH3OH. Compound 11 (3 mg, tR 6.46 min) was obtained from Fr.5-4 (71 mg) by HPLC purification eluting with 60% MeOH. Fraction 4 (413 mg) was separated by CC over RP-18 to afford four subfractions (Fr.4-1–Fr.4-5), and compounds 9 (3 mg, tR 7.3 min) and 10 (3 mg, tR 9.7 min) were obtained from Fr.4-4 (93 mg) by HPLC purifications eluted with 60% MeOH and 50% MeOH, respectively. Fr.4-2 (87 mg) and Fr.4-3 (77 mg) were both further separated into four parts (Fr.4-2-1–Fr.4-2-4 and Fr.4-3-1–Fr.4-3-4) by Sephadex LH-20 with MeOH. Compounds 12 (4 mg, tR 8.97 min) and 13 (5 mg, tR 7.6 min) were purified from Fr.4-2-2 (21 mg) and Fr.4-2-3 (17 mg) by HPLC eluting with 40% and 60% MeOH, respectively. And Fr.4-3-2 (16 mg) and Fr.4-3-3 (10 mg) were purified by HPLC eluting with 70% MeOH to give 4 (2 mg, tR 5.3 min) and 5 (2 mg, tR 5.0 min), respectively. Fraction 7 (414 mg) gave four parts (Fr.7-1–Fr.7-4) after subjection to CC over Sephadex LH-20 with MeOH. Fr.7-2 (92 mg) was further purified by HPLC, with 30% MeOH, to yield the mixture (3 mg, tR 12.33 min) of 1 and 2 by ODS column. The mixture was further purified by HPLC over C3 column with 25% MeOH to give pure 1 (2 mg, tR 10.08 min) and 2 (1 mg, tR 11.98 min). Compound 8 (7 mg, tR 8.68 min) was obtained from Fr.7-3 (133 mg) by HPLC with 50% MeOH.

2-(Furan-2-yl)-6-(2S,3S,4-trihydroxybutyl)pyrazine (1): A brown oil. [α]D20 −11.5 (c 0.05, MeOH); UV (MeOH) λmax (log ε) 240 (3.4), 273 (3.6), 328 (3.4) nm; CD (c 0.05, MeOH) λext (Δε) 310 (−0.14), 284 (+0.12), 237 (−1.3) nm; 1H (600 MHz, MeOH-d4) and 13C NMR (150 MHz, MeOH-d4), see Table 2; HRESIMS m/z 273.0849 [M + Na]+ (calcd for C12H14N2O4Na 273.0846).

Table 2. 1H and 13C NMR data for 1 and 2 (δ in ppm).

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Table 2. 1H and 13C NMR data for 1 and 2 (δ in ppm).
Position12
δC, TypeδH, Mult. (J in Hz)δH, Mult. (J in Hz, −4 °C)δC, TypeδH, Mult. (J in Hz)δH, Mult. (J in Hz, −4 °C)
2144.2, qC 142.5, qC
3136.7, CH8.78, s8.79, s138.7, CH8.90, d, (0.7)8.91, d, (1.0)
5142.7, CH8.38, s8.38, s153.4, qC
6155.3, qC 144.3, CH8.50, d, (0.7)8.50, d, (1.0)
2′151.1, qC 151.0, qC
3′110.5, CH7.23, d, (4.0)7.25, d, 3.4109.9, CH7.18, d, (3.4)7.19,d, (3.4)
4′112.0, CH6.63, dd, (2.1, 4.0)6.64, dd, (1.7, 3.4)111.9, CH6.63, d, (1.7, 3.4)6.64, dd, (1.7, 3.4)
5′144.6, CH7.72, d, (2.1)7.74, d, (1.7)144.6, CH7.71, d, (1.7)7.73, d, (1.7)
1″38.4, CH23.24, dd, (3.1, 14.0)3.24, dd, (2.9, 14.0)38.3, CH23.22, dd, (2.8, 14.0)3.22, dd, (2.8, 14.0)
2.93, dd, (9.4, 14.0)2.91, dd, (9.7, 14.0)2.93, dd, (9.4, 14.0)2.91, dd, (9.5, 14.1)
2″71.5, CH4.03, ddd, (3.1, 7.0, 9.4)4.01, ddd, (2.9, 7.2, 9.7)71.6, CH3.97, ddd, (2.8, 7.0, 9.4)3.95, ddd, (2.8, 7.1, 9.5)
3″74.8, CH3.58, ddd, (3.8, 7.0, 6.2)3.57, ddd, (3.8, 7.2, 6.5)74.8, CH3.57, (3.7, 7.0, 6.5)3.56, ddd, (3.7, 7.1, 6.5)
4″63.2, CH23.80, dd, (3.8, 11.4)3.80, dd, (3.8, 11.2) 63.2, CH23.79, dd, (3.7, 11.3)3.78, dd, (3.7, 11.3)
3.65, dd, (6.2, 11.4)3.63, dd, (6.5, 11.2)3.64, dd, (6.2, 11.3)3.62, dd, (6.5, 11.3)

2-(Furan-2-yl)-5-(2S,3S,4-trihydroxybutyl)pyrazine (2): Brown oil. [α]D20 −11.3 (c 0.03, MeOH); UV (MeOH) λmax (log ε) 240 (3.2), 273 (3.4), and 328 (3.1) nm; CD (c 0.05, MeOH) λext (Δε) 316 (−0.54), 267 (+0.35), 235 (−1.0) nm; 1H (600 MHz, MeOH-d4) and 13C NMR (150 MHz, MeOH-d4), see Table 2; HRESIMS m/z 273.0849 [M + Na]+ (calcd for C12H14N2O4Na 273.0846).

(S)-4-Isobutyl-3-oxo-3,4-dihydro-1H-pyrrolo[2,1-c][1,4]oxazine-6-carbaldehyde (3): A brown oil. [α]D20 −32 (c 0.05, acetone); UV (MeOH) λmax (log ε) 254 (3.3), and 291 (3.9) nm; CD (c 0.08, MeOH) λmax (Δε) 292 (+2.9), 248 (−1.0), 207 (+2.5) nm; 1H (600 MHz, DMSO-d6) and 13C NMR (150 MHz, DMSO-d6), see Table 1; HRESIMS m/z 222.1131 [M + H]+ (calcd for C12H14N2O4Na 222.1125).

(S)-4-Isopropyl-3-oxo-3,4-dihydro-1H-pyrrolo[2,1-c][1,4]oxazine-6-carbaldehyde (4): A brown oil. [α]D20 −88 (c 0.12, acetone); UV (MeOH) λmax (log ε) 254 (3.4) and 291 (4.03) nm; CD (c 0.02, MeOH) λmax (Δε) 298 (+16.1), 290 (−1.9), 280 (+6.6), 253 (−8.9), 208 (+8.8) nm; 1H (600 MHz, DMSO-d6) and 13C NMR (150 MHz, DMSO-d6), see Table 1; HRESIMS m/z 208.0974 [M + H]+ (calcd for C12H14N2O4Na 208.0968).

(4S)-4-(2-Methylbutyl)-3-oxo-3,4-dihydro-1H-pyrrolo[2,1-c][1,4]oxazine-6-carbaldehyde (5): A brown oil; [α]D20 −57 (c 0.06, acetone); UV (MeOH) λmax (log ε) 254 (3.6) and 291 (4.2) nm; CD (c 0.08, MeOH) λmax (Δε) 293 (+5.8), 252 (−2.7), 208 (+3.1) nm; 1H (600 MHz, DMSO-d6) and 13C NMR (150 MHz, DMSO-d6), see Table 1; HRESIMS m/z 222.1129 [M + H]+ (calcd for C12H14N2O4Na 222.1125).

(S)-4-Benzyl-3-oxo-3,4-dihydro-1H-pyrrolo[2,1-c][1,4]oxazine-6-carbaldehyde (6): A brown oil. [α]D20 −46 (c 0.1, acetone); UV (MeOH) λmax (log ε) 257 (3.4) and 295 (3.8) nm; CD (c 0.02, MeOH) λmax (Δε) 293 (+13.1), 249 (−4.7), 211 (+6.8) nm; 1H (600 MHz, DMSO-d6) and 13C NMR (150 MHz, DMSO-d6) see Table 1; HRESIMS m/z 256.0976 [M + H]+ (calcd for C12H14N2O4Na 256.0968).

3.5. Bioassays

The antiviral activity against H1N1 virus was evaluated by the CPE inhibition assay [45,46]. Confluent MDCK cell monolayers were firstly incubated with influenza virus (A/Puerto Rico/8/34 (H1N1), PR/8) at 37 °C for 1 h. After removing the virus dilution, cells were maintained in infecting media (RPMI 1640, 4 μg/mL of trypsin) containing different concentrations of test compounds and ribavirin (positive control) at 37 °C. After 48 h incubation at 37 °C, the cells were fixed with 100 μL of 4% formaldehyde for 20 min at room temperature. After removal of the formaldehyde, the cells were stained with 0.1% crystal violet for 30 min. The plates were washed and dried, and the intensity of crystal violet staining for each well was measured in a microplate reader (Bio-Rad, Hercules, CA, USA) at 570 nm. The IC50 was calculated as the compound concentration required inhibiting influenza virus yield at 48 h post-infection by 50%. Ribavirin was used as the positive control with an IC50 value of 23.1 ± 1.7 μg/mL.

Cytotoxicity was assayed by the MTT [47]. In the MTT assay, MDCK normal cell line were cultured in RPMI-1640, supplemented with 10% FBS, under a humidified atmosphere of 5% CO2 and 95% air at 37 °C, cell suspensions with a density of 4.6 × 104 cells/mL (198 μL) was plated in 96-well microtiter plates and incubated for 24 h. Then, the test solutions in MeOH (2 μL) were added to each well and further incubated for 36 h. The MTT solution (20 µL, 5 mg/mL in RPMI-1640 medium) was then added to each well and incubated for 4 h. Old medium containing MTT (150 μL) was then gently replaced by dimethylsulfoxide (DMSO) and pipetted to dissolve any formazan crystals formed. Absorbance was then determined on a Spectra Max Plus plate reader at 570 nm. Ribavirin was used as positive control (CC50 744.2 ± 18.5 μg/mL for MDCK normal cell line).

4. Conclusions

A new pyrazine derivative, 2-(furan-2-yl)-6-(2S,3S,4-trihydroxybutyl)pyrazine (1), was isolated and identified from the fermentation products of Jishengella endophytica 161111 endophytic with mangrove plant, Xylocarpus granatum (Meliaceae). The absolute configurations and the key reference data of [α]D, CD and 13C NMR of 2-(furan-2-yl)-5-(2S,3S,4-trihydroxybutyl)pyrazine (2), (S)-4-isobutyl-3-oxo-3,4-dihydro-1H-pyrrolo[2,1-c][1,4]oxazine-6-carbaldehyde (3), (S)-4-isopropyl-3-oxo-3,4-dihydro-1H-pyrrolo[2,1-c][1,4]oxazine-6-carbaldehyde (4), and (4S)-4-(2-methylbutyl)-3-oxo-3,4-dihydro-1H-pyrrolo[2,1-c][1,4]oxazine-6-carbaldehyde (5) were reported here for the first time. In addition, β-carboline alkaloid 9 exhibited moderate anti-H1N1 virus activity and weak cytotoxicity.

Acknowledgments

This work was financially supported by the grants from 973 Program of China (No. 2010CB833804), from NSFC (Nos. 41376148, 21172204, 81373298 & 31170467), from 863 Program of China (Nos. 2013AA092901, 2012AA092104 and 2011AA09070106), and from the Special Fund for Marine Scientific Research in the Public Interest of China (No. 2010418022-3). The authors are grateful to Haipeng Lin (Institute of Tropical Biosiences and Biotechnology) for his help on preparing the crude extracts from the 100 L fermentation broth.

Conflicts of Interest

The authors declare no conflict of interest.

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