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Guignardins A–F, spirodioxynaphthalenes from the endophytic fungus Guignardia sp. KcF8 as a new class of PTP1B and SIRT1 inhibitors
Tetrahedron 70 (2014) 5806e5814
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Guignardins AeF, spirodioxynaphthalenes from the endophytic fungus Guignardia sp. KcF8 as a new class of PTP1B and SIRT1 inhibitors Wen Ai a, b, Xiaoyi Wei c, Xiuping Lin a, Li Sheng d, Zhen Wang e, Zhengchao Tu e, Xianwen Yang a, Xuefeng Zhou a, Jia Li d, *, Yonghong Liu a, * a
Key Laboratory of Tropical Marine Bio-resources and Ecology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 510301, China University of Chinese Academy of Sciences, Beijing 100049, China c Key Laboratory of Plant Resources Conservation and Sustainable Utilization, South China Botanical Garden, Chinese Academy of Sciences, Guangzhou 510650, China d State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zu Chong Zhi Road, Zhang Jiang Hi-Tech Park, Shanghai 201203, China e Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 19 December 2013 Received in revised form 7 June 2014 Accepted 12 June 2014 Available online 17 June 2014
Six new guignardins AeF (1e6) were isolated from the cultures of endophytic fungus Guignardia sp. KcF8 derived of a mangrove plant Kandelia candel, along with three known analogues, palmarumycins C1 (7), BG1 (8), and JC1 (9). Compounds 2, 3, 7, and 8 showed antimicrobial activities. Compounds 5e7 exhibited significant cytotoxicities against 10 human tumor cell lines. Compound 3 also displayed significant inhibitory activity against human protein tyrosine phosphatase 1B and histone deacetylase silent information regulator T1enzymes, two key targets for the treatment of diabetes. This is the first report on the anti-PTP1B and anti-SIRT1 activities of spirodioxynaphthalenes. Ó 2014 Elsevier Ltd. All rights reserved.
Keywords: Spirodioxynaphthalene Kandelia candel Mangrove plant Endophytic fungus Guignardia sp.
1. Introduction Diabetes, a chronic metabolic disorder, affects about 5% of the population in the industrialized nations and accounts for over $200 billion in medical costs.1 In the year 2011, diabetes, directly and indirectly, accounted for about 5 million deaths worldwide. Type I diabetes often manifests in childhood and may result from autoimmune destruction of the b-cells. Type II diabetes, a more widespread metabolic disorder, generally manifests after the age of 40 and involves progressive development of insulin resistance leading to overt hyperglycemia. Silent information regulator T1 (SIRT1),2 an NADþ-dependent deacetylase, is a principal modulator of pathways downstream of calorie restriction that produce beneficial effects on glucose homeostasis and insulin sensitivity.3 The histone deacetylase SIRT1 plays an essential role in modulating several age-related diseases,4 such as type 1 diabetes.5 Protein tyrosine phosphatase 1B
* Corresponding authors. Tel./fax: þ86 020 8902 3244; e-mail addresses: jli@ mail.shcnc.ac.cn (J. Li), [email protected] (Y. Liu). http://dx.doi.org/10.1016/j.tet.2014.06.041 0040-4020/Ó 2014 Elsevier Ltd. All rights reserved.
(PTP1B), a negative regulator of insulin and leptin signal transduction, is one of the most promising therapeutic targets for treatment of type 2 diabetes and obesity.6 In our research toward the discovery of biologically active metabolites from endophytic fungus of Chinese mangrove plants, we obtained six new spirodioxynaphthalenes from the cultures of Guignardia sp. KcF8 isolated from the fruits of Kandelia candel, a mangrove tree used as a traditional medicine against rheumatoid arthritis, which were named guignardins AeF (1e6), and three known analogues, palmarumycins C1 (7),7 BG1 (8),9 and JC1 (9)8 (Fig. 1). In this paper, the isolation, structure elucidation, and biological activities of metabolites 1e9 were presented.
2. Results and discussion 2.1. Structure elucidation Guignardin A (1) was isolated as a white powder. The molecular formula of 1 was determined to be C23H18O6 (15 degrees of
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Fig. 1. The structures of the compounds 1e9.
unsaturation) by HRESIMS analysis [m/z 391.1166 (MþH)þ], which was also supported by the 1H and 13C NMR data (Tables 1 and 2). Apart from a set of NMR signal characteristic of the dioxynaphthalene moiety, the 1H and 13C NMR spectra showed signals for a 1,2,3-trisubstituted aromatic ring, a ketal carbon, two ketone moieties, two methylenes, and two hydroxyl groups [one of which is highly chelated (dH 11.92)]. The connectivities among these units were determined by the analysis of 1He1H COSY and HMBC spectra. Analysis of the COSY data led to the identification of three isolated proton spin systems corresponding to H-20 eH-30 eH-40 , H-50 eH60 eH-70, and H-5eH-6eH-7 subunits of structure 1. HMBC correlations of H-3 and H-110 with carbonyl C-1 and quaternary carbon C-2, H-3 with ketal carbon C-4, C-6, and C-110, H-110 with C-3 and C120 , H-130 with C-110 and C-120 led to the assignment of the nonaromatic ring. Correlation of H-5 to C-4, C-8, and C-10, H-6 to C-5, C-7, and C-9, H-7 to C-5, C-9, and C-10 enabled the connection of the C-9 and C-10 of the aromatic ring to C-1 and C-4, respectively, resulting in a tetralone moiety. The dioxynaphthalene unit must be linked to C-4 via two oxygen atoms of the ketal moiety, thereby completing the planar structure of 1 as shown in Fig. 1.
The absolute configuration was determined by ECD/TDDFT calculations using the truncated structure, which afforded the predicted gas phase and solution ECD spectra consistent with the experimental one (Fig. 2). MMFF conformational search generated eight lowest-energy conformers within a 2.5 kcal/mol relative free energies using DFT method at the B3LYP/6-31G (d) level (Fig. 3). The first five conformers were calculated for CD spectra in MeOH. Conformers (1a,cee) yielded solution CD curves closely similar to the experimental spectrum, while the simulated CD spectrum for 1b, displaying positive cotton effect around 220, was close to the mirror image of the experimental spectrum. Nevertheless, the Boltzmann-weighted spectra were highly consistent with the measured spectrum. Therefore, the absolute configuration was established as ()-2R. Guignardin B (2) was isolated as brown oil. The molecular formula of 2 was determined to be C20H14O5 (14 degrees of unsaturation) by HRESIMS analysis [m/z 333.0763 (MH)], 1H and 13C NMR data (Tables 1 and 2). The connectivities among these units were determined by the analysis of 1He1H COSY and HMBC spectra. Analysis of the COSY data led to the identification of two isolated
Table 1 1 H NMR data for (1, 3, 4; 500 MHz, CDCl3; 2; DMSO-d6) Position 1 2 3 5 6 7 20 30 40 50 60 70 11 13 OH-8 OH
1
2.58, d, 15.0 2.95, d, 15.0 7.44, dd, 8.0, 7.67, t, 8.0 7.15, dd, 8.0, 7.56, dd, 8.0, 7.46, t, 8.0 6.95, d, 7.0 6.94, d, 7.0 7.45, t, 7.5 7.56, dd, 8.0, 2.95, d, 17.5 3.11, d, 17.5 2.14, s 11.92, s
1.0 1.0 1.5
1.5
2
3
5.07, d, 4.0 4.74, dd, 4.0, 1.5 4.06, dd, 7.0, 1.5
6.28, s
7.41, 7.28, 6.94, 7.48, 7.44, 7.03, 6.78, 7.35, 7.46,
7.49, 7.68, 7.14, 7.55, 7.44, 6.94, 6.94, 7.44, 7.55,
d, 8.0 dd, 8.0, 7.5 d, 8.0 d, 8.0 t, 8.0 d, 7.5 d, 7.5 t, 8.0 d, 8.0
9.90, s 6.20, d, 7.0
4
d, 8.0 t, 8.0 d, 8.5 d, 8.0 t, 8.0 d, 7.5 d, 7.5 t, 8.0 d, 8.0
11.40, s 6.45, s
6.60, 7.15, 7.01, 7.59, 7.47, 6.91, 6.91, 7.47, 7.59, 2.37,
d, 7.5 t, 8.0 d, 8.0 d, 8.0 t, 8.0 d, 8.0 d, 8.0 t, 8.0 d, 8.0 s
12.27, s 6.55, s
5
6
5.31, d, 2.5 4.68, d, 2.5
4.08, d, 4.0 3.67, d, 4.0
7.46, 7.63, 7.14, 7.59, 7.49, 7.13, 6.91, 7.43, 7.56,
7.42, 7.62, 7.13, 7.58, 7.51, 7.17, 6.91, 7.45, 7.54,
d, 7.5 t, 8.0 d, 8.5 d, 8.5 t, 7.5 d, 7.5 d, 7.5 t, 7.5 d, 8.0
11.83, s
d, 8.0 t, 8.0 d, 8.5 d, 8.0 t, 7.5 d, 7.5 d, 7.5 t, 7.5 d, 8.5
11.35, s
5808
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Table 2 13 C NMR data for (1, 3, 4; 125 MHz, CDCl3; 2; DMSO-d6)
The relative configuration of 2 was deduced by analysis of He1H coupling constants. The large 3J1H,2H (4.0 Hz) and the small 3 J2H,3H (1.5 Hz) coupling constants indicated that H-1, H-2, and H-3 on the same side of the molecule. Conformational analysis using the method as described for compound 1 found two energy minima 2a and 2b within DG 1.5 kcal/mol. CD/TDDFT calculations for this compound were carried out using both low-energy conformers. As can be seen in Fig. 4, the Boltzmann-weighted CD spectrum in MeOH solution is in good agreement with the experimental spectrum (Fig. 4). Thus the absolute configuration of 2 could be determined as ()-(1R,2S,3R). Guignardin C (3) was isolated as white powder. The molecular formula of 3 was determined to be C20H12O5 (15 degrees of unsaturation) by HRESIMS analysis [m/z 333.0752 (MþH)þ], 1H and 13C NMR data (Tables 1 and 2). The connectivities among these units were determined by the analysis of HMBC spectra. HMBC correlations of H-3 with carbonyl C-1, C-3, ketal carbon C-4 and C-9, H-5 to C-4, C-7, C-8, and C-9, H-6 to C-5, C-7, C-8, C-9, and C-10, H-7 to C-5, C-8, and C-9 enabled the connection of the C-5 and C-9 of the aromatic ring to C-4 and C-10, respectively. Compared with 2, the completing the structure of 3 as shown in Fig. 1. Guignardin D (4) was isolated as red powder. The molecular formula of 4 was determined to be C23H14O8 (17 degrees of unsaturation) by HRESIMS analysis [m/z 441.2992 (MþNa)þ], 1H and 13C NMR data (Tables 1 and 2). The connectivities among these units were determined by the analysis of HMBC spectra. HMBC correlations of H-110 with carbonyl C-120 , correlation of H-5 to C-4, C-7, C9, and C-10, H-6 to C-5, C-7, C-8, C-9, and C-10, H-7 to C-6, C-8, and C-9 enabled the connection of the C-10 and C-8 of the aromatic ring to C-4 and C-9, respectively. Compared with 3, the completing the structure of 4 as shown in Fig. 1. Guignardin E (5) was isolated as brown oil. The molecular formula of 5 was determined to be C20H13ClO5 (14 degrees of unsaturation) by HRESIMS analysis m/z 369.0524 [MþH]þ, 1H and 13C NMR data (Tables 1 and 2). The connectivities among these units were determined by the analysis of 1He1H COSY and HMBC spectra. Analysis of the COSY NMR data led to the identification of two isolated proton spin systems corresponding to the H-2eH-3 and H5eH-6eH-7 subunits of structure 5. HMBC correlations of H-2 and 1
Position
1
2
3
4
5
6
1 2 3 4 5 6 7 8 9 10 10 20 30 40 50 60 70 80 90 100 110 120 130
201.2 s 73.5 s 39.9 t 97.9 s 118.2 d 137.8 d 119.9 d 162.8 s 113.4 s 139.9 s 146.9 s 121.3 d 127.7 d 109.8 d 109.5 d 127.5 d 121.2 d 147.1 s 113.2 s 134.3 s 49.1 t 206.7 s 31.3 q
65.6 d 57.9 d 71.1 d 100.4 s 118.9 d 130.3 d 116.9 d 154.9 s 123.6 s 132.2 s 147.0 s 120.7 d 127.4 d 109.5 d 108.6 d 127.2 d 120.6 d 146.8 s 113.0 s 133.9 s
184.1 s 109.3 d 147.3 s 95.1 s 119.6 d 137.7 d 120.0 d 161.9 s 112.3 s 140.1 s 146.2 s 121.2 d 127.6 d 109.9 d 109.9 d 127.6 d 121.2 d 146.2 s 113.3 s 134.2 s
177.5 s 123.6 s 133.2 s 95.2 s 116.2 d 136.7 d 120.1 d 163.3 s 112.9 s 142.0 s 145.5 s 121.0 d 127.6 d 109.3 d 109.3 d 127.6 d 121.0 d 145.5 s 111.3 s 133.8 s 20.8 q 165.7 s 165.5 s
194.2 s 62.5 d 72.3 d 98.7 s 120.4 d 137.9 d 118.8 d 162.1 s 114.3 s 137.5 s 146.7 s 121.8 d 127.9 d 110.1 d 109.2 d 127.8 d 121.6 d 146.1 s 113.0 s 134.3 s
196.5 s 53.2 d 53.3 d 96.0 s 119.0 d 137.6 d 120.0 d 161.9 s 112.2 s 136.9 s 147.0 s 121.4 d 127.8 d 110.1 d 109.3 d 127.6 d 121.3 d 146.7 s 112.8 s 134.2 s
Fig. 2. Key HMBC correlations and comparison between the calculated and the experimental ECD spectra of compound 1.
Fig. 3. Conformations of low-energy conformers of 1.
proton spin systems corresponding to the H-1eH-2eH-3 and H5eH-6eH-7 subunits of structure 2. HMBC correlations of H-1 with C-2, C-3, C-8, C-9, and C-10, H-3 with C-1, C-2, ketal carbon C-4 and C-10, OH-3 with C-2, C-3, and C-4 led to the assignment of the nonaromatic ring. Correlation of H-5 to C-4, C-8, C-9, and C-10, H-6 to C-5, C-7, C-8, and C-10, H-7 to C-6, C-8, and C-9 enabled the connection of the C-10 and C-8 of the aromatic ring to C-4 and C-9, respectively. Compared with 1, the completing the planar structure of 2 as shown in Fig. 1.
H-3 with carbonyl C-1 and ketal carbon C-4, H-3 with C-2 and C-10 led to the assignment of the nonaromatic ring. Correlation of H-5 to C-4, C-7, C-8, and C-9, H-6 to C-10, C-5, C-7, C-8, and C-9, H-7 to C-5, C-8, and C-9 enabled the connection of the C-5 and C-9 of the aromatic ring to C-4 and C-10, respectively. Compared with the known palmarumycins C1 (7),7 the completing the structure of 5 as shown in Fig. 1. The relative configuration of 5 was deduced by analysis of 1 He1H coupling constants. The small 3J2H,3H (2.5 Hz) coupling
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Fig. 4. Conformations of low-energy conformers and comparison of the calculated and the experimental ECD spectra of compound 2.
constants indicated that H-2 and H-3 having cis configuration. Conformational analysis using the method as described for compound 1 found two energy minima 5a and 5b within DG 3.5 kcal/ mol. CD/TDDFT calculations for this compound were carried out using both low-energy conformers. As can be seen in figure, the Boltzmann-weighted CD spectrum in MeOH solution is in good agreement with the experimental spectrum (Fig. 5). Thus the absolute configuration of 5 could be determined as ()-(2S,3S).
the (2R,3S) enantiomer. The CD spectra calculated for the (2R,3S) enantiomer of the lowest-energy conformer (100.0% population) with various functional (B3LYP, CAM-B3LYP) and the basis set (SVP, TZVP) reproduced well the experimental CD, with CAM-B3LYP/ TZVP giving the best agreement. Thus the absolute configuration of 6 could be determined as (2S,3R). Compounds 7e9 were identified as palmarumycins C1 (7),7 BG1 (8),9 and JC1 (9)8 by comparison the 1H and 13C NMR with those
Fig. 5. Conformations of low-energy conformers and comparison of the calculated and the experimental ECD spectra of compound 5.
Guignardin F (6) was isolated as colorless crystal. The molecular formula of 6 was determined to be C20H12O5 (14 degrees of unsaturation) by HRESIMS analysis m/z 331.0618 [MH], 1H and 13C NMR data (Tables 1 and 2). The connectivities among these units were determined by the analysis of 1He1H COSY and HMBC spectra. Analysis of the COSY NMR data led to the identification of two isolated proton spin systems corresponding to the H-2eH-3 and H5eH-6eH-7 subunits of structure 1. HMBC correlations of H-2 with C-3, ketal carbon C-4, and C-10, H-3 with C-1 and C-9 led to the assignment of the nonaromatic ring. Correlation of H-5 to C-4, C- 7, C-8, and C-9, H-6 to C-10, C-5, C-7, C-8, and C-9, H-7 to C-5, C-8, and C-9 enabled the connection of the C-5 and C-9 of the aromatic ring to C-4 and C-10, respectively. Compared with 5, the completing the structure of 6 as shown in Fig. 1. The relative configuration of 6 was deduced by analysis of 1 He1H coupling constants. The small 3J2H,3H (4.0 Hz) coupling constants indicated that H-2 and H-3 having cis configuration. The absolute configuration was determined by ECD/TDDFT calculations using the truncated structure, which afforded the predicted gas phase and solution ECD spectra consistent with the experimental one. The CD spectrum of 6 showed positive cotton effect at 210 nm and negative cotton effect around 220 nm (Fig. 6), which could be produced mirror image by the DFTCD calculations performed on
reported data. Palmarumycin C1 (7) was isolated as yellow crystal. The structure was confirmed by X-ray crystallographic analysis (Fig. 7), this is the first time to report the X-ray data. 2.2. Probably biosynthesis As compounds 1e9 were co-metabolites, we proposed that they would have the same biosynthetic pathway. The spirodioxynaphthalenes bear two naphthalenes, indicative of the fact that it is believed to be constructed from 1,8-dihydroxynaphalene or a suitable phenolic derivative, which could be generated from hexaketide or pentaketide.7 The major structure of 9 would be further modified by a number of addition, dehydration, reduction, chlorination, substitution, and hydrolysis reaction steps. The possible biosynthesis of 1e9 was presented as Fig. 8. 2.3. Biological activities The spirodioxynaphthalenes display a wide range of biological activities, including antibacterial, antifungal, algicidal, nematicidal, and antileishmanial effects, phospholipase D inhibitory, FPTase inhibitory, and antitumor activity.9
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Fig. 6. Conformations of low-energy conformers and comparison of the calculated and the experimental ECD spectra of compound 6.
In these tested antimicrobial activities, compounds 2, 3, 7, and 8 were active against gram positive and negative bacterial and several phytopathogenic fungi. The cytotoxicities of 1e9 were evaluated against a panel of 10 human tumor cell lines. The results indicated that compounds 5e7 exhibited significant cytotoxicity (Table 3). The chloride or epoxide and 1-position carbonyl group may be the important groups for the activities. All the isolated compounds were evaluated for their inhibitory activities against PTP1B and SIRT1 enzymes, compound 3 showed significant activities, IC50 25.7 and 43.9 mM (positive controls oleanolic acid and nicotinamide: IC50 1.90 and 50.81 mM). The 2position hydroxyl group may be the important group for the activities. To the best of our knowledge, this is the first paper reporting on the inhibitory activities of spirodioxynaphthalene against two drug target enzymes PTP1B and SIRT1. 3. Conclusion
Fig. 7. The X-ray structure of compound 7.
In standard antibacterial disc assays, compounds 2, 3, and 8 showed moderate activity against Staphylococcus aureus ATCC 29213 when tested at 50 mg/disc, the zones of inhibition 7, 8, and 8 mm, were noticed (positive control penicillin showed 12 mm at the same level). Compound 2 was active against Enterococcus faecalis ATCC 29212 at 50 mg/disc, causing zone of inhibition of 7 mm (penicillin: 20 mm). Compound 8 displayed significant activity against Aeromonas hydrophila ATCC 7966 at 50 mg/disc, causing inhibition zone of 12 mm (penicillin: 7 mm). None of the compounds showed significant activity against Acinetobacter baumannii ATCC 19606, Escherichia coli ATCC 25922, and Klebsiella pneumonia ATCC 13883 at this level. In tested antifungal activity, compounds 2, 7, and 8 showed marginal activity against Fusarium sp. at 25 mg/disc, the zones of inhibition 6, 2, and 2 mm, were noticed, where as positive control carbendazim tested at same level showed zone of inhibition 18 mm. Compounds 7 and 8 were active against the growth of Fusarium oxysporum f. sp. niveum, the zones of inhibition 6 and 2 mm (carbendazim: 12 mm). Compounds 2 and 8 displayed significant activity against Aspergillus niger, the zones of inhibition 7 and 4 mm (carbendazim: 10 mm). Compound 8 showed weak activity against Fusarium oxysporum f. sp. cucumeris and Rhizoctonia solani, the zones of inhibition 2 and 2 mm (carbendazim: 12 and 14 mm), respectively.
Six new spirodioxynaphthalenes, guignardins AeF (1e6), along with three known analogues, palmarumycins C1 (7), BG1 (8), and JC1 (9) were isolated from an endophytic fungus Guignardia sp. KcF8 of mangrove plant K. candel. Compound 8 displayed significant activity against A. hydrophila ATCC 7966, which a fish pathogens in aquiculture. Compounds 2, 7, and 8 showed antifungal activity against several plant pathogenic fungi. Compounds 5e7 exhibited cytotoxicities against a panel of 10 human tumor cell lines. Compound 3 displayed significant inhibitory activity (IC50¼25.7 and 43.9 mM) against human protein tyrosine phosphatase 1B (PTP1B) and silent information regulator T1 (SIRT1) enzymes. This is the first report on the anti-PTP1B and anti-SIRT1 activities of spirodioxynaphthalenes, which will become promising lead compound for the treatment of diabetes without toxic side effect. 4. Experimental section 4.1. General experimental procedures The NMR spectra were recorded on a Bruker AC 500 NMR spectrometer with TMS as an internal standard. HRESIMS data were measured on a Bruker micro TOF-QII mass spectrometer. Optical rotation values were measured with an Anton Paar MCP500 polarimeter. IR spectra were recorded on a Nicolet 6700 FT-IR spectrometer. UV spectra were recorded on a Shimadzu UV-2600 UVevis spectrophotometer. CD spectra were measured with a chirascan circular dichroism spectrometer (Applied Photophysics). The silica gel GF254 used for TLC was supplied by the
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Fig. 8. The possible biosynthesis of 1e9.
Table 3 The cytotoxicity of 5e7a K562 A549 Huh-7 H1975 MCF-7 U937 BGC823 HL60 HeLa MOLT-4 5 6 7 TSA
1.67 1.84 1.54 0.24
2.73 5.90 2.63 0.05
2.14 1.11 1.90 0.08
11.3 4.68 39.2 0.09
8.79 6.48 3.08 0.78
1.74 2.85 1.66 0.06
2.34 3.08 2.00 0.09
2.94 3.06 2.90 0.09
1.32 0.38 1.24 0.11
1.24 1.07 1.27 0.03
a Data as expressed in LD50 values (mM). K562: human erythromyeloblastoid leukemia cell line; A549: human lung cancer; Huh-7: human hepatocarcinoma; H1975: human erlotinib-resistant lung cancer; MCF-7: human breast cancer; U937: human leukemic monocyte lymphoma cancer; BGC823: human gastric cancer; HL60: human promyelocytic leukemia cancer; HeLa: human epithelial carcinoma; MOLT-4: human acute T lymphoblastic leukemia cancer.
Qingdao Marine Chemical Factory, Qingdao, China. Sephadex LH-20 gel (GE Healthcare, Sweden) was used. HPLC was carried on Hitachi L-2400 with YMC ODS column. Spots were detected on TLC under UV light or by heating after spraying with 5% H2SO4 in EtOH. Molecular Merck force field (MMFF) and DFT/TDDFT calculations were performed with Spartan’14 software package (Wavefunction Inc., Irvine, CA, USA) and Gaussian09 program package, respectively, using default grids and convergence criteria. MMFF conformational search generated low-energy conformers within a 10 kcal/mol energy window were subjected to geometry
optimization using DFT method at the B3LYP/6-31G (d) level. Frequency calculations were run at the same level to verify that each optimized conformer was a true minimum and to estimate their relative thermal free energies (DG) at 298.15 K. Solvent effects were taken into account by using polarizable continuum model (PCM). The TDDFT calculations were performed using the long-range corrected hybrid CAM-B3LYP and the hybrid B3LYP functionals, and Ahlrichs’ basis sets SVP (split valence plus polarization) and TZVP (triple zeta valence plus polarization).
4.2. Fungal material The fungus strain KcF8, identified as Guignardia sp., was isolated from fresh healthy fruits of mangrove plant K. candel. The plant material was collected at Daya Bay, Shenzhen city, Guangdong province, China, in April 2012. Following the surface sterilization of the fruits with 75% EtOH, small tissue samples from inside of the fruits were cut a septically and pressed onto MA agar (malt extract 15 g, sea salt 10 g, chloromycetin 0.2 g, agar 15 g, distilled water 1000 mL, pH 7.4e7.8) plates. From the growing cultures, pure strain of Guignardia sp. was isolated by repeated and reinoculation on MB agar (malt extract 15 g, sea salt 10 g, agar 15 g, distilled water 1000 mL, pH 7.4e7.8) plates.
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4.3. Identification of fungal culture The fungus (strain No. KcF8) was identified using a molecular biological protocol by DNA amplification and sequencing of the 18s rRNA gene. By comparison with the GenBank database, the most similar strains were Guignardia ardisiae, Guignardia mangiferae, Guignardia alliacea, all are 99% similarity. So the fungus strain KcF8 clearly belongs to the genus Guignardia, and was designated as Guignardia sp.
4.4. Extraction and isolation Rice (4 kg) in 20 1000 mL Erlenmeyer flasks (200 g each) was inoculated with liquid seed and incubated at 25 C under daylight for 2 months. The culture was broken up with a spatula and extracted three times with EtOAc (34 L). The combined EtOAc extract was filtered and evaporated to afford 74.7 g of crude extract. The crude EtOAc extract was partitioned between methanol and hexane. The resulting methanol fraction (27.7 g) was subjected to silica gel column chromatography (CC) eluted with petroleum ether/EtOAc in gradient eluant (80:20, 50:50, 30:70) followed by CHCl3/MeOH in gradient eluant (90:10, 80:20, 70:30, 60:40, 0:100) to obtain 16 fractions (fractions 1e16) on the monitor of TLC. Fraction 10 was purified by Sephadex LH-20 (CHCl3/MeOH, 1:1) first to give four subfractions (fractions 10.1e10.4). Fraction 10.2 (100.0 mg) was further purified by CC on silica gel (CHCl3/MeOH, 100:1) to give 4 (6.3 mg) and 8 (12.1 mg). Fraction 11 was purified by Sephadex LH-20 (CHCl3/MeOH, 1:1) first to give three subfractions (fractions 11.1e11.3). Fraction 11.2 (200.0 mg) was further purified by CC on silica gel (CHCl3/MeOH, gradient elution 100:1e10:1) to give 7 (11.2 mg), 9 (7.4 mg), and 6 (20.8 mg). Fraction 12 was purified by Sephadex LH-20 (CHCl3/MeOH, 1:1) first to give four subfractions (fractions 12.1e12.4). Fraction 12.2 (50.0 mg) was further purified by CC on silica gel (CHCl3/MeOH, 50:1) to give 1 (8.2 mg). Fraction 13 was purified by Sephadex LH-20 (CHCl3/ MeOH, 1:1) first to give two subfractions (fractions 13.1e13.2). Fraction 13.1 (80.0 mg) was further purified by CC on silica gel (CHCl3/MeOH, 50:1) to give 5 (15.4 mg). Fraction 13.2 (10.0 mg) was further purified by HPLC (MeOH/H2O, 75%) to give 3 (3.5 mg). Fraction 18 was purified by Sephadex LH-20 (MeOH) first to give three subfractions (fractions 18.1e18.3). Fraction 18.3 (20.0 mg) was further purified by CC on silica gel (CHCl3/MeOH, 20:1) to give 2 (4.6 mg). 4.4.1. Guignardin A (1). White powder; [a]25 D 16.7 (c 0.3, MeOH); UV (MeOH) lmax (log 3 ) 226 (4.63), 300 (3.71), 328 (3.70); IR nmax 3479, 1712, 1635, 1608, 1454, 1411, 1377, 1273, 956, 798, 744; 1H and 13 C NMR data see Tables 1 and 2; selected HMBC data, H-3/C-2, C4, C-10, C-11; H-5/C-4, C-7, C-9; H-6/C-5, C-8, C-10; H-7/C-5, C-8, C-9; H-11/C-3, C-2, C-10, C-12; H-13/C-11, C-12; HRESIMS 391.1166 [MþH]þ (calcd for 391.1176). 4.4.2. Guignardin B (2). Brown oil; [a]25 D 144 (c 0.3, MeOH); UV (MeOH) lmax (log 3 ) 226 (5.00), 287 (4.99), 296 (4.96), 299 (4.96), 310 (4.78), 313 (4.83), 327 (4.66); IR nmax 3267, 1608, 1411, 1377, 1273, 975, 794, 756; 1H and 13C NMR data see Tables 1 and 2; selected HMBC data, H-1/C-2, C-3, C-8, C-9, C-10; H-3/C-1, C-2, C4, C-10; H-5/C-4, C-7, C-8, C-9, C-10; H-6/C-4, C-7, C-8, C-9, C10; H-7/C-5, C-8, C-9; HRESIMS 333.0763 [MH]- (calcd for 333.0768). 4.4.3. Guignardin C (3). White powder; UV (MeOH) lmax (log 3 ) 224 (4.96), 258 (4.26), 299 (4.26), 328 (4.19); IR nmax 3394, 1635, 1608, 1458, 1411, 1377, 1265, 1168, 1060, 983, 891, 810, 756; 1H and 13C NMR data see Tables 1 and 2; selected HMBC data, H-3/C-2, C-1,
C-4, C-10; H-5/C-4, C-9, C-7; H-6/C-7, C-10, C-8; H-7/C-9, C-5, C-8; HRESIMS 333.0752 [MþH]þ (calcd for 333.0757). 4.4.4. Guignardin D (4). Red powder; UV (MeOH) lmax (log 3 ) 203 (4.68), 214 (4.46), 224 (4.51), 265 (3.82), 275 (3.84); IR nmax 1762, 1651, 1597, 1550, 1411, 1377, 1261, 1165, 1083, 1049, 956, 806, 740; 1H and 13C NMR data see Tables 1 and 2; selected HMBC data, H-5/C4, C-9, C-7; H-6/C-10, C-8; H-7/C-9, C-5; HRESIMS 441.2992 [MþNa]þ (calcd for 441.2999). 4.4.5. Guignardin E (5). Brown oil; UV (MeOH) lmax (log 3 ) 223 (4.98), 257 (4.36), 298 (4.38), 327 (4.36); IR nmax 3394, 1635, 1608, 1458, 1411, 1377, 1265, 1168, 1060, 983, 891, 810, 756; 1H and 13C NMR data see Tables 1 and 2; selected HMBC data, H-3/C-1, C-2, C-4, C-10; H-5/C-4, C-7, C-9; H-6/C-7, C-8, C-10; H-7/C-5, C-8, C-9; HRESIMS 369.0524 [MþH]þ (calcd for 369.0506). 4.4.6. Guignardin F (6). Colorless crystal; 1H and 13C NMR data see Tables 1 and 2; selected HMBC data, H-2/C-3, C-4, C-10; H-3/C1, C-9; H-5/C-4, C-7, C-8, C-9; H-6/C-5, C-7, C-8, C-9, C-10; H7/C-5, C-8, C-9; HRESIMS 331.0618 [MH] (calcd for 331.0612). 4.4.7. Palmarumycin C1 (7). Yellow crystal; 1H NMR (500 MHz, CDCl3): d 7.15 (1H, s), 7.46 (2H, d, J¼7.5 Hz, H-5), 7.69 (1H, t, J¼8.0 Hz, H-6), 7.18 (1H, d, J¼8.0 Hz, H-7), 6.99 (1H, d, J¼7.5 Hz, H20 , H-50 ), 7.49 (2H, t, J¼8.0 Hz, H-30 , H-60 ), 7.61 (2H, d, J¼8.0 Hz, H-40 , H-50 ), 11.87 (1H, s, HeOH); 13C NMR (125 MHz, CDCl3): d 181.9 (s, C1), 135.0 (s, C-2), 120.1 (d, C-3), 93.6 (s, C-4), 119.7 (d, C-5), 137.2 (d, C-6), 135.7 (d, C-7), 162.2 (s, C-8), 113.2 (s, C-9), 138.5 (s, C-10), 146.8 (s, C-10, C-90 ), 110.1 (d, C-20 , C-80 ), 127.7 (d, C-30 , C-70 ), 121.6 (d, C-40 , C-60 ), 112.8 (s, C-90 ), 134.2 (s, C-100 ). 4.4.8. Palmarumycin BG1 (8). White powder; 1H NMR (500 MHz, CDCl3): d 2.91 (1H, dd, J¼16.5, 4.0 Hz, H-2), 3.21 (1H, dd, J¼16.5, 4.0 Hz, H-2), 4.57 (1H, t, J¼4.0 Hz, H-3), 6.90 (1H, d, J¼7.5 Hz, H-50 ), 7.06 (1H, d, J¼7.5 Hz, H-7), 7.08 (1H, d, J¼7.5 Hz, H-40 ), 7.32 (1H, d, J¼7.5 Hz, H-20 ), 7.41 (1H, t, J¼7.5 Hz, H-60 ), 7.46 (1H, t, J¼7.5 Hz, H30 ), 7.52 (1H, d, J¼7.5 Hz, H-5), 7.54 (1H, d, J¼7.5 Hz, H-70 ), 7.55 (1H, t, J¼7.5 Hz, H-6), 12.34 (1H, eOH); 13C NMR (125 MHz, CDCl3): d 201.1 (s, C-1), 41.3 (t, C-2), 67.2 (d, C-3), 98.7 (s, C-4), 119.7 (d, C-5), 117.9 (d, C-6), 137.0 (d, C-7), 162.1 (s, C-8), 113.1 (s, C-9), 137.9 (s, C10), 147.1 (s, C-10 ), 121.3 (d, C-20 ), 127.7 (d, C-30 ), 109.5 (d, C-40 ), 108.8 (d, C-50 ), 127.6 (d, C-60 ), 121.0 (d, C-70 ), 146.3 (s, C-80 ), 115.3 (s, C-90 ), 134.1 (s, C-100 ). 4.4.9. Palmarumycin JC1 (9). White powder; 1H NMR (500 MHz, CDCl3): d 3.26 (1H, d, J¼11.0 Hz, HeOH), 3.74 (1H, dd, J¼4.5, 3.0 Hz, H-2), 3.87 (1H, d, J¼4.5 Hz, H-3), 5.44 (1H, dd, J¼11.0, 3.0 Hz, H-1), 6.92 (1H, d, J¼8.0 Hz, H-50 ), 7.05 (1H, dd, J¼8.0, 2.0 Hz, H-7), 7.14 (1H, d, J¼7.5 Hz, H-40 ), 7.37 (1H, t, J¼8.0 Hz, H-60 ), 7.40 (1H, dd, J¼8.0, 2.0 Hz, H-20 ), 7.44 (1H, t, J¼8.0 Hz, H-30 ), 7.51 (1H, t, J¼8.0 Hz, H-6), 7.55 (1H, d, J¼8.0 Hz, H-70 ), 7.57 (1H, d, J¼8.0 Hz, H-5), 8.31 (1H, s, HeOH); 13C NMR (125 MHz, CDCl3): d 66.6 (d, C-1), 52.8 (d, C-2), 54.2 (d, C-3), 96.7 (s, C-4), 119.3 (d, C-5), 130.6 (d, C-6), 118.9 (d, C-7), 156.5 (s, C-8), 118.5 (s, C-9), 132.0 (s, C-10), 147.3 (s, C-10 ), 121.1 (d, C-20 ), 127.7 (d, C-30 ), 109.9 (d, C-40 ), 109.1 (d, C-50 ), 127.5 (d, C-60 ), 121.0 (d, C-70 ), 147.2 (s, C-80 ), 112.8 (s, C-90 ), 134.1 (s, C-100 ). 4.5. X-ray crystallographic analysis of 7 Yellow crystal of C20H11ClO4 with M¼350.74. Unit cell dimensions a¼5.0690(2) A, b¼11.7367(6) A, c¼13.0641(8) A, V¼753.21(7) A3, Z¼2; crystal size 0.430.410.37 mm3. A total of 5025 unique reflections (q¼2.67e26.00 ) were collected using graphite monochromated Cu Ka radiation (l¼0.71073 A) on a Bruker Smart 1000 CCD diffractometer at 150(2) K. Absorption
W. Ai et al. / Tetrahedron 70 (2014) 5806e5814
corrections were done by semi-empirical from equivalents. The structure was solved by direct methods (SHELXS-97) and refined with Full-matrix least-squares on 2959 data, 0 restraints, and 229 variable parameters. Final R indicates R1¼0.0347, wR2¼0.0857 [I>2s (I)]. Crystallographic data for 7 has been deposited in the Cambridge Crystallographic Data Centre with the deposition number 963344. Copies of the data can be obtained, free of charge, on application to the director, CCDC, 12 Union Road, Cambridge CB21EZ, UK [fax: þ44 (0)1223 336033, or e-mail: [email protected]].
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Boston, MA). In a typical 100 mL assay mixture containing 50 mmol/ L 3-[N-morpholino]-propanesulfonic acid (MOPs), pH 6.5, 2 mmol/L pNPP, and 30 nmol/L recombinant PTP1B, activities were continuously monitored and the initial rate of the hydrolysis was determined using the early linear region of the enzymatic reaction kinetic curve. The IC50 was calculated with Prism 4 software (Graphpad, San Diego, CA) from the nonlinear curve fitting of the percentage of inhibition (% inhibition) versus the inhibitor concentration [I] by using the following equation: % Inhibition¼100/ (1þ[IC50/[I]]k), where k is the Hill coefficient. The positive control is oleanolic acid (IC50¼1.90 mM).
4.6. Antibacterial assay 4.10. SIRT1 inhibitory activity assay The ready made nutrient agar medium (38 g) was suspended in distilled water (1000 mL) and heated to boiling until it dissolved completely. The medium and Petridishes were autoclaved at temperature 121 C for 20 min. Disc diffusion assay was employed for testing antibacterial activity of the compounds. The medium was poured into sterile Petridishes under aseptic conditions in laminar flow chamber. Before the medium in the plate solidify, 2% cultured of test organism was inoculated and uniformly spread over the medium. Solutions were prepared by dissolving compound in MeOH and concentration (50 mg/mL) was made. After incubation, to each disc concentrations of test solutions were added (1 mL). The positive control penicillin was maintained with MeOH (50 mg/mL). The treated materials and the controls were kept in an incubator at 28 C for 12 h. Inhibition zones were measured and diameter was calculated in mm. Three replicates were maintained for each treatment. 4.7. Antifungal assay The method followed for antifungal bioassay is similar to that followed for antibacterial assay where in the medium is malt extract agar 40 g/L and the positive control is carbendazim. Concentration (25 mg/mL) of test solution was tested. Controls were maintained with acetone and carbendazim (25 mg/mL). The treated and the controls were kept in an incubator at 28 C for 3 days. Inhibition zones were measured and diameter was calculated in mm. Three replicates were maintained for each treatment. 4.8. Cytotoxicity Cytotoxicity was assayed with the CCK8 (DOjinDo, Japan) method.10 Cell lines, K562, A549, Huh-7, H1975, MCF-7, U937, BGC823, HL60, HeLa, and MOLT-4 were purchased from Shanghai Cell Bank, Chinese Academy of Sciences. Cells were routinely grown and maintained in mediums RPMI or DMEM with 10% FBS and with 1% penicillin/streptomycin. All cell lines were incubated in a Thermo/Forma Scientific CO2 Water Jacketed Incubator with 5% CO2 in air at 37 C. Cell viability assay was determined by the CCK8 (DOjinDo, Japan) assay. Cells were seeded at a density of 400e800 cells/well in 384 well plates and treated with various concentration of compounds or solvent control. After 72 h incubation, CCk8 reagent was added, and absorbance was measured at 450 nm using Envision 2104 multi-label Reader (PerkineElmer, USA). Dose response curves were plotted to determine the IC50 values using Prism 5.0 (GraphPad Software Inc., USA). 4.9. PTP1B inhibitory activity assay Recombinant PTP1B catalytic domain was expressed and purified according to a previous report.11 The enzymatic activities of the PTP1B catalytic domain were determined at 30 C by monitoring the hydrolysis of pNPP. Dephosphorylation of pNPP generates product pNP, which was monitored at an absorbance of 405 nm by the EnVision multi-label plate reader (PerkineElmer Life Sciences,
Compounds were dissolved in DMSO to prepare 10 mM stock solutions; a fluorescent-based assay was used to screen compounds that inhibit the deacetylase activity of SIRT1. The C-terminus of the acetyl substrate acetyl-Arg-His-Lys-Lys (3-acetyl) was labeled with 4-amino-7-methylcoumarin (AMC). When the substrate is deacetylated by SIRT1, AMC can be cleaved from the peptide by trypsin and detected by its fluorescence. The reaction conditions were as follows: 750 mMNADt, 31.25 mMac-RHKKac-AMC, and 1 mM enzyme in buffer containing 25 mM Tris (pH 8.0), 137 mM NaCl, 2.7 mM KCl, and 1 mM MgCl2. The reactions were carried out for 45 min at room temperature, stopped with 10 mM nicotinamide, and developed with 0.5 mg/mL trypsin. After incubation for 30 min, fluorescence intensities were measured with a microplate reader (POLARstar OPTIMA, BMG Labtech) at an excitation wavelength of 340 nm and an emission wavelength of 490 nm, the positive control is nicotinamide IC50¼50.81 mM.12 Acknowledgements This work was supported financially by the National Key Basic Research Program of China (973)’s Project (2010CB833800 and 2011CB915503), the National High Technology Research and Development Program (863 Program, 2013AA092901 and 2012AA092104), the National Natural Science Foundation of China (Nos. 31270402, 21172230, 20902094, 41176148, and 21002110), Guangdong Province-CAS Joint Research Program (2011B090300023 and 2012B091100264), and Guangdong Marine Economic Development and Innovation of Regional Demonstration Project (GD2012-D01-001 and GD2012-D01-002). Supplementary data NMR spectra of compounds 1e6. Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.tet.2014.06.041. References and notes 1. Nurul Islam, M.; Jung, H. A.; Sohn, H. S.; Kim, H. M.; Choi, J. S. Arch. Pharm. Res. 2013, 36, 542e552. 2. Canto, C.; Auwerx, J. Trends Endocrinol. Metab. 2009, 20, 325e331. 3. Milne, J. C.; Lambert, P. D.; Schenk, S.; Carney, D. P.; Smith, J. J.; Gagne, D. J.; Jin, L.; Boss, O.; Perni, R. B.; Vu, C. B.; Bemis, J. E.; Xie, R.; Disch, J. S.; Ng, P. Y.; Nunes, J. J.; Lynch, A. V.; Yang, H.; Galonek, H.; Israelian, K.; Choy, W.; Iffland, A.; Lavu, S.; Medvedik, O.; Sinclair, D. A.; Olefsky, J. M.; Jirousek, M. R.; Elliott, P. J.; Westphal, C. H. Nature 2007, 450, 712e716. 4. Kume, S.; Kitada, M.; Kanasaki, K.; Maegawa, H.; Koya, D. Arch. Pharm. Res. 2013, 36, 230e236. € ni-Schnetzler, M.; Hubbard, B. P.; Bouzakri, K.; Brunner, A.; 5. Biason-Lauber, A.; Bo €ni, M.; Meier, D. T.; Brorsson, C.; Timper, Cavelti-Weder, C.; Keller, C.; Meyer-Bo K.; Leibowitz, G.; Patrignani, A.; Bruggmann, R.; Boily, G.; Zulewski, H.; Geier, A.; Cermak, J. M.; Elliott, P.; Ellis, J. L.; Westphal, C.; Knobel, U.; Eloranta, J. J.; Kerr-Conte, J.; Pattou, F.; Konrad, D.; Matter, C. M.; Fontana, A.; Rogler, G.; Schlapbach, R.; Regairaz, C.; Carballido, J. M.; Glaser, B.; McBurney, M. W.; Pociot, F.; Sinclair, D. A.; Donath, M. Y. Cell Metab. 2013, 17, 448e455. 6. Owen, C.; Lees, E. K.; Grant, L.; Zimmer, D. J.; Mody, N.; Bence, K. K.; Delibegovic, M. Diabetologia 2013, 56, 2286e2296.
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7. Krohn, K.; Michel, A.; Florke, U.; Aust, H. J.; Draeger, S.; Schulz, B. Liebigs Ann. Chem. 1994, 11, 1099e1108. 8. Ravindranath, N.; Reddy, M. R.; Mahender, G.; Ramu, R.; Kumar, K. R.; Das, B. Phytochemistry 2004, 65, 2387e2390. n, T.; Miao, Z. H.; M romi, I. N.; Liu, H. L.; Ding, J.; 9. Cai, Y. S.; Kurta andi, A.; Koma Guo, Y. W. J. Org. Chem. 2011, 76, 1821e1830.
10. Wanka, L.; Iqbal, K.; Schreiner, P. R. Chem. Rev. 2013, 113, 3516e3604. 11. Yang, X.-N.; Li, J.-Y.; Zhou, Y.-Y.; Shen, Q.; Chen, J.-W.; Li, J. Biochim. Biophys. Acta Gen. Subj. 2005, 1726, 34e41. 12. Wu, J. H.; Zhang, D. Y.; Chen, L.; Li, J. N.; Wang, J. L.; Ning, C. Q.; Yu, N. F.; Zhao, F.; Chen, D. Y.; Chen, X. Y.; Chen, K. X.; Jiang, H. L.; Liu, H.; Liu, D. X. Eur. J. Med. Chem. 2013, 56, 761e780.
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Guignardins AeF, spirodioxynaphthalenes from the endophytic fungus Guignardia sp. KcF8 as a new class of PTP1B and SIRT1 inhibitors Wen Ai a, b, Xiaoyi Wei c, Xiuping Lin a, Li Sheng d, Zhen Wang e, Zhengchao Tu e, Xianwen Yang a, Xuefeng Zhou a, Jia Li d, *, Yonghong Liu a, * a
Key Laboratory of Tropical Marine Bio-resources and Ecology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 510301, China University of Chinese Academy of Sciences, Beijing 100049, China c Key Laboratory of Plant Resources Conservation and Sustainable Utilization, South China Botanical Garden, Chinese Academy of Sciences, Guangzhou 510650, China d State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zu Chong Zhi Road, Zhang Jiang Hi-Tech Park, Shanghai 201203, China e Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 19 December 2013 Received in revised form 7 June 2014 Accepted 12 June 2014 Available online 17 June 2014
Six new guignardins AeF (1e6) were isolated from the cultures of endophytic fungus Guignardia sp. KcF8 derived of a mangrove plant Kandelia candel, along with three known analogues, palmarumycins C1 (7), BG1 (8), and JC1 (9). Compounds 2, 3, 7, and 8 showed antimicrobial activities. Compounds 5e7 exhibited significant cytotoxicities against 10 human tumor cell lines. Compound 3 also displayed significant inhibitory activity against human protein tyrosine phosphatase 1B and histone deacetylase silent information regulator T1enzymes, two key targets for the treatment of diabetes. This is the first report on the anti-PTP1B and anti-SIRT1 activities of spirodioxynaphthalenes. Ó 2014 Elsevier Ltd. All rights reserved.
Keywords: Spirodioxynaphthalene Kandelia candel Mangrove plant Endophytic fungus Guignardia sp.
1. Introduction Diabetes, a chronic metabolic disorder, affects about 5% of the population in the industrialized nations and accounts for over $200 billion in medical costs.1 In the year 2011, diabetes, directly and indirectly, accounted for about 5 million deaths worldwide. Type I diabetes often manifests in childhood and may result from autoimmune destruction of the b-cells. Type II diabetes, a more widespread metabolic disorder, generally manifests after the age of 40 and involves progressive development of insulin resistance leading to overt hyperglycemia. Silent information regulator T1 (SIRT1),2 an NADþ-dependent deacetylase, is a principal modulator of pathways downstream of calorie restriction that produce beneficial effects on glucose homeostasis and insulin sensitivity.3 The histone deacetylase SIRT1 plays an essential role in modulating several age-related diseases,4 such as type 1 diabetes.5 Protein tyrosine phosphatase 1B
* Corresponding authors. Tel./fax: þ86 020 8902 3244; e-mail addresses: jli@ mail.shcnc.ac.cn (J. Li), [email protected] (Y. Liu). http://dx.doi.org/10.1016/j.tet.2014.06.041 0040-4020/Ó 2014 Elsevier Ltd. All rights reserved.
(PTP1B), a negative regulator of insulin and leptin signal transduction, is one of the most promising therapeutic targets for treatment of type 2 diabetes and obesity.6 In our research toward the discovery of biologically active metabolites from endophytic fungus of Chinese mangrove plants, we obtained six new spirodioxynaphthalenes from the cultures of Guignardia sp. KcF8 isolated from the fruits of Kandelia candel, a mangrove tree used as a traditional medicine against rheumatoid arthritis, which were named guignardins AeF (1e6), and three known analogues, palmarumycins C1 (7),7 BG1 (8),9 and JC1 (9)8 (Fig. 1). In this paper, the isolation, structure elucidation, and biological activities of metabolites 1e9 were presented.
2. Results and discussion 2.1. Structure elucidation Guignardin A (1) was isolated as a white powder. The molecular formula of 1 was determined to be C23H18O6 (15 degrees of
W. Ai et al. / Tetrahedron 70 (2014) 5806e5814
5807
Fig. 1. The structures of the compounds 1e9.
unsaturation) by HRESIMS analysis [m/z 391.1166 (MþH)þ], which was also supported by the 1H and 13C NMR data (Tables 1 and 2). Apart from a set of NMR signal characteristic of the dioxynaphthalene moiety, the 1H and 13C NMR spectra showed signals for a 1,2,3-trisubstituted aromatic ring, a ketal carbon, two ketone moieties, two methylenes, and two hydroxyl groups [one of which is highly chelated (dH 11.92)]. The connectivities among these units were determined by the analysis of 1He1H COSY and HMBC spectra. Analysis of the COSY data led to the identification of three isolated proton spin systems corresponding to H-20 eH-30 eH-40 , H-50 eH60 eH-70, and H-5eH-6eH-7 subunits of structure 1. HMBC correlations of H-3 and H-110 with carbonyl C-1 and quaternary carbon C-2, H-3 with ketal carbon C-4, C-6, and C-110, H-110 with C-3 and C120 , H-130 with C-110 and C-120 led to the assignment of the nonaromatic ring. Correlation of H-5 to C-4, C-8, and C-10, H-6 to C-5, C-7, and C-9, H-7 to C-5, C-9, and C-10 enabled the connection of the C-9 and C-10 of the aromatic ring to C-1 and C-4, respectively, resulting in a tetralone moiety. The dioxynaphthalene unit must be linked to C-4 via two oxygen atoms of the ketal moiety, thereby completing the planar structure of 1 as shown in Fig. 1.
The absolute configuration was determined by ECD/TDDFT calculations using the truncated structure, which afforded the predicted gas phase and solution ECD spectra consistent with the experimental one (Fig. 2). MMFF conformational search generated eight lowest-energy conformers within a 2.5 kcal/mol relative free energies using DFT method at the B3LYP/6-31G (d) level (Fig. 3). The first five conformers were calculated for CD spectra in MeOH. Conformers (1a,cee) yielded solution CD curves closely similar to the experimental spectrum, while the simulated CD spectrum for 1b, displaying positive cotton effect around 220, was close to the mirror image of the experimental spectrum. Nevertheless, the Boltzmann-weighted spectra were highly consistent with the measured spectrum. Therefore, the absolute configuration was established as ()-2R. Guignardin B (2) was isolated as brown oil. The molecular formula of 2 was determined to be C20H14O5 (14 degrees of unsaturation) by HRESIMS analysis [m/z 333.0763 (MH)], 1H and 13C NMR data (Tables 1 and 2). The connectivities among these units were determined by the analysis of 1He1H COSY and HMBC spectra. Analysis of the COSY data led to the identification of two isolated
Table 1 1 H NMR data for (1, 3, 4; 500 MHz, CDCl3; 2; DMSO-d6) Position 1 2 3 5 6 7 20 30 40 50 60 70 11 13 OH-8 OH
1
2.58, d, 15.0 2.95, d, 15.0 7.44, dd, 8.0, 7.67, t, 8.0 7.15, dd, 8.0, 7.56, dd, 8.0, 7.46, t, 8.0 6.95, d, 7.0 6.94, d, 7.0 7.45, t, 7.5 7.56, dd, 8.0, 2.95, d, 17.5 3.11, d, 17.5 2.14, s 11.92, s
1.0 1.0 1.5
1.5
2
3
5.07, d, 4.0 4.74, dd, 4.0, 1.5 4.06, dd, 7.0, 1.5
6.28, s
7.41, 7.28, 6.94, 7.48, 7.44, 7.03, 6.78, 7.35, 7.46,
7.49, 7.68, 7.14, 7.55, 7.44, 6.94, 6.94, 7.44, 7.55,
d, 8.0 dd, 8.0, 7.5 d, 8.0 d, 8.0 t, 8.0 d, 7.5 d, 7.5 t, 8.0 d, 8.0
9.90, s 6.20, d, 7.0
4
d, 8.0 t, 8.0 d, 8.5 d, 8.0 t, 8.0 d, 7.5 d, 7.5 t, 8.0 d, 8.0
11.40, s 6.45, s
6.60, 7.15, 7.01, 7.59, 7.47, 6.91, 6.91, 7.47, 7.59, 2.37,
d, 7.5 t, 8.0 d, 8.0 d, 8.0 t, 8.0 d, 8.0 d, 8.0 t, 8.0 d, 8.0 s
12.27, s 6.55, s
5
6
5.31, d, 2.5 4.68, d, 2.5
4.08, d, 4.0 3.67, d, 4.0
7.46, 7.63, 7.14, 7.59, 7.49, 7.13, 6.91, 7.43, 7.56,
7.42, 7.62, 7.13, 7.58, 7.51, 7.17, 6.91, 7.45, 7.54,
d, 7.5 t, 8.0 d, 8.5 d, 8.5 t, 7.5 d, 7.5 d, 7.5 t, 7.5 d, 8.0
11.83, s
d, 8.0 t, 8.0 d, 8.5 d, 8.0 t, 7.5 d, 7.5 d, 7.5 t, 7.5 d, 8.5
11.35, s
5808
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Table 2 13 C NMR data for (1, 3, 4; 125 MHz, CDCl3; 2; DMSO-d6)
The relative configuration of 2 was deduced by analysis of He1H coupling constants. The large 3J1H,2H (4.0 Hz) and the small 3 J2H,3H (1.5 Hz) coupling constants indicated that H-1, H-2, and H-3 on the same side of the molecule. Conformational analysis using the method as described for compound 1 found two energy minima 2a and 2b within DG 1.5 kcal/mol. CD/TDDFT calculations for this compound were carried out using both low-energy conformers. As can be seen in Fig. 4, the Boltzmann-weighted CD spectrum in MeOH solution is in good agreement with the experimental spectrum (Fig. 4). Thus the absolute configuration of 2 could be determined as ()-(1R,2S,3R). Guignardin C (3) was isolated as white powder. The molecular formula of 3 was determined to be C20H12O5 (15 degrees of unsaturation) by HRESIMS analysis [m/z 333.0752 (MþH)þ], 1H and 13C NMR data (Tables 1 and 2). The connectivities among these units were determined by the analysis of HMBC spectra. HMBC correlations of H-3 with carbonyl C-1, C-3, ketal carbon C-4 and C-9, H-5 to C-4, C-7, C-8, and C-9, H-6 to C-5, C-7, C-8, C-9, and C-10, H-7 to C-5, C-8, and C-9 enabled the connection of the C-5 and C-9 of the aromatic ring to C-4 and C-10, respectively. Compared with 2, the completing the structure of 3 as shown in Fig. 1. Guignardin D (4) was isolated as red powder. The molecular formula of 4 was determined to be C23H14O8 (17 degrees of unsaturation) by HRESIMS analysis [m/z 441.2992 (MþNa)þ], 1H and 13C NMR data (Tables 1 and 2). The connectivities among these units were determined by the analysis of HMBC spectra. HMBC correlations of H-110 with carbonyl C-120 , correlation of H-5 to C-4, C-7, C9, and C-10, H-6 to C-5, C-7, C-8, C-9, and C-10, H-7 to C-6, C-8, and C-9 enabled the connection of the C-10 and C-8 of the aromatic ring to C-4 and C-9, respectively. Compared with 3, the completing the structure of 4 as shown in Fig. 1. Guignardin E (5) was isolated as brown oil. The molecular formula of 5 was determined to be C20H13ClO5 (14 degrees of unsaturation) by HRESIMS analysis m/z 369.0524 [MþH]þ, 1H and 13C NMR data (Tables 1 and 2). The connectivities among these units were determined by the analysis of 1He1H COSY and HMBC spectra. Analysis of the COSY NMR data led to the identification of two isolated proton spin systems corresponding to the H-2eH-3 and H5eH-6eH-7 subunits of structure 5. HMBC correlations of H-2 and 1
Position
1
2
3
4
5
6
1 2 3 4 5 6 7 8 9 10 10 20 30 40 50 60 70 80 90 100 110 120 130
201.2 s 73.5 s 39.9 t 97.9 s 118.2 d 137.8 d 119.9 d 162.8 s 113.4 s 139.9 s 146.9 s 121.3 d 127.7 d 109.8 d 109.5 d 127.5 d 121.2 d 147.1 s 113.2 s 134.3 s 49.1 t 206.7 s 31.3 q
65.6 d 57.9 d 71.1 d 100.4 s 118.9 d 130.3 d 116.9 d 154.9 s 123.6 s 132.2 s 147.0 s 120.7 d 127.4 d 109.5 d 108.6 d 127.2 d 120.6 d 146.8 s 113.0 s 133.9 s
184.1 s 109.3 d 147.3 s 95.1 s 119.6 d 137.7 d 120.0 d 161.9 s 112.3 s 140.1 s 146.2 s 121.2 d 127.6 d 109.9 d 109.9 d 127.6 d 121.2 d 146.2 s 113.3 s 134.2 s
177.5 s 123.6 s 133.2 s 95.2 s 116.2 d 136.7 d 120.1 d 163.3 s 112.9 s 142.0 s 145.5 s 121.0 d 127.6 d 109.3 d 109.3 d 127.6 d 121.0 d 145.5 s 111.3 s 133.8 s 20.8 q 165.7 s 165.5 s
194.2 s 62.5 d 72.3 d 98.7 s 120.4 d 137.9 d 118.8 d 162.1 s 114.3 s 137.5 s 146.7 s 121.8 d 127.9 d 110.1 d 109.2 d 127.8 d 121.6 d 146.1 s 113.0 s 134.3 s
196.5 s 53.2 d 53.3 d 96.0 s 119.0 d 137.6 d 120.0 d 161.9 s 112.2 s 136.9 s 147.0 s 121.4 d 127.8 d 110.1 d 109.3 d 127.6 d 121.3 d 146.7 s 112.8 s 134.2 s
Fig. 2. Key HMBC correlations and comparison between the calculated and the experimental ECD spectra of compound 1.
Fig. 3. Conformations of low-energy conformers of 1.
proton spin systems corresponding to the H-1eH-2eH-3 and H5eH-6eH-7 subunits of structure 2. HMBC correlations of H-1 with C-2, C-3, C-8, C-9, and C-10, H-3 with C-1, C-2, ketal carbon C-4 and C-10, OH-3 with C-2, C-3, and C-4 led to the assignment of the nonaromatic ring. Correlation of H-5 to C-4, C-8, C-9, and C-10, H-6 to C-5, C-7, C-8, and C-10, H-7 to C-6, C-8, and C-9 enabled the connection of the C-10 and C-8 of the aromatic ring to C-4 and C-9, respectively. Compared with 1, the completing the planar structure of 2 as shown in Fig. 1.
H-3 with carbonyl C-1 and ketal carbon C-4, H-3 with C-2 and C-10 led to the assignment of the nonaromatic ring. Correlation of H-5 to C-4, C-7, C-8, and C-9, H-6 to C-10, C-5, C-7, C-8, and C-9, H-7 to C-5, C-8, and C-9 enabled the connection of the C-5 and C-9 of the aromatic ring to C-4 and C-10, respectively. Compared with the known palmarumycins C1 (7),7 the completing the structure of 5 as shown in Fig. 1. The relative configuration of 5 was deduced by analysis of 1 He1H coupling constants. The small 3J2H,3H (2.5 Hz) coupling
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Fig. 4. Conformations of low-energy conformers and comparison of the calculated and the experimental ECD spectra of compound 2.
constants indicated that H-2 and H-3 having cis configuration. Conformational analysis using the method as described for compound 1 found two energy minima 5a and 5b within DG 3.5 kcal/ mol. CD/TDDFT calculations for this compound were carried out using both low-energy conformers. As can be seen in figure, the Boltzmann-weighted CD spectrum in MeOH solution is in good agreement with the experimental spectrum (Fig. 5). Thus the absolute configuration of 5 could be determined as ()-(2S,3S).
the (2R,3S) enantiomer. The CD spectra calculated for the (2R,3S) enantiomer of the lowest-energy conformer (100.0% population) with various functional (B3LYP, CAM-B3LYP) and the basis set (SVP, TZVP) reproduced well the experimental CD, with CAM-B3LYP/ TZVP giving the best agreement. Thus the absolute configuration of 6 could be determined as (2S,3R). Compounds 7e9 were identified as palmarumycins C1 (7),7 BG1 (8),9 and JC1 (9)8 by comparison the 1H and 13C NMR with those
Fig. 5. Conformations of low-energy conformers and comparison of the calculated and the experimental ECD spectra of compound 5.
Guignardin F (6) was isolated as colorless crystal. The molecular formula of 6 was determined to be C20H12O5 (14 degrees of unsaturation) by HRESIMS analysis m/z 331.0618 [MH], 1H and 13C NMR data (Tables 1 and 2). The connectivities among these units were determined by the analysis of 1He1H COSY and HMBC spectra. Analysis of the COSY NMR data led to the identification of two isolated proton spin systems corresponding to the H-2eH-3 and H5eH-6eH-7 subunits of structure 1. HMBC correlations of H-2 with C-3, ketal carbon C-4, and C-10, H-3 with C-1 and C-9 led to the assignment of the nonaromatic ring. Correlation of H-5 to C-4, C- 7, C-8, and C-9, H-6 to C-10, C-5, C-7, C-8, and C-9, H-7 to C-5, C-8, and C-9 enabled the connection of the C-5 and C-9 of the aromatic ring to C-4 and C-10, respectively. Compared with 5, the completing the structure of 6 as shown in Fig. 1. The relative configuration of 6 was deduced by analysis of 1 He1H coupling constants. The small 3J2H,3H (4.0 Hz) coupling constants indicated that H-2 and H-3 having cis configuration. The absolute configuration was determined by ECD/TDDFT calculations using the truncated structure, which afforded the predicted gas phase and solution ECD spectra consistent with the experimental one. The CD spectrum of 6 showed positive cotton effect at 210 nm and negative cotton effect around 220 nm (Fig. 6), which could be produced mirror image by the DFTCD calculations performed on
reported data. Palmarumycin C1 (7) was isolated as yellow crystal. The structure was confirmed by X-ray crystallographic analysis (Fig. 7), this is the first time to report the X-ray data. 2.2. Probably biosynthesis As compounds 1e9 were co-metabolites, we proposed that they would have the same biosynthetic pathway. The spirodioxynaphthalenes bear two naphthalenes, indicative of the fact that it is believed to be constructed from 1,8-dihydroxynaphalene or a suitable phenolic derivative, which could be generated from hexaketide or pentaketide.7 The major structure of 9 would be further modified by a number of addition, dehydration, reduction, chlorination, substitution, and hydrolysis reaction steps. The possible biosynthesis of 1e9 was presented as Fig. 8. 2.3. Biological activities The spirodioxynaphthalenes display a wide range of biological activities, including antibacterial, antifungal, algicidal, nematicidal, and antileishmanial effects, phospholipase D inhibitory, FPTase inhibitory, and antitumor activity.9
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Fig. 6. Conformations of low-energy conformers and comparison of the calculated and the experimental ECD spectra of compound 6.
In these tested antimicrobial activities, compounds 2, 3, 7, and 8 were active against gram positive and negative bacterial and several phytopathogenic fungi. The cytotoxicities of 1e9 were evaluated against a panel of 10 human tumor cell lines. The results indicated that compounds 5e7 exhibited significant cytotoxicity (Table 3). The chloride or epoxide and 1-position carbonyl group may be the important groups for the activities. All the isolated compounds were evaluated for their inhibitory activities against PTP1B and SIRT1 enzymes, compound 3 showed significant activities, IC50 25.7 and 43.9 mM (positive controls oleanolic acid and nicotinamide: IC50 1.90 and 50.81 mM). The 2position hydroxyl group may be the important group for the activities. To the best of our knowledge, this is the first paper reporting on the inhibitory activities of spirodioxynaphthalene against two drug target enzymes PTP1B and SIRT1. 3. Conclusion
Fig. 7. The X-ray structure of compound 7.
In standard antibacterial disc assays, compounds 2, 3, and 8 showed moderate activity against Staphylococcus aureus ATCC 29213 when tested at 50 mg/disc, the zones of inhibition 7, 8, and 8 mm, were noticed (positive control penicillin showed 12 mm at the same level). Compound 2 was active against Enterococcus faecalis ATCC 29212 at 50 mg/disc, causing zone of inhibition of 7 mm (penicillin: 20 mm). Compound 8 displayed significant activity against Aeromonas hydrophila ATCC 7966 at 50 mg/disc, causing inhibition zone of 12 mm (penicillin: 7 mm). None of the compounds showed significant activity against Acinetobacter baumannii ATCC 19606, Escherichia coli ATCC 25922, and Klebsiella pneumonia ATCC 13883 at this level. In tested antifungal activity, compounds 2, 7, and 8 showed marginal activity against Fusarium sp. at 25 mg/disc, the zones of inhibition 6, 2, and 2 mm, were noticed, where as positive control carbendazim tested at same level showed zone of inhibition 18 mm. Compounds 7 and 8 were active against the growth of Fusarium oxysporum f. sp. niveum, the zones of inhibition 6 and 2 mm (carbendazim: 12 mm). Compounds 2 and 8 displayed significant activity against Aspergillus niger, the zones of inhibition 7 and 4 mm (carbendazim: 10 mm). Compound 8 showed weak activity against Fusarium oxysporum f. sp. cucumeris and Rhizoctonia solani, the zones of inhibition 2 and 2 mm (carbendazim: 12 and 14 mm), respectively.
Six new spirodioxynaphthalenes, guignardins AeF (1e6), along with three known analogues, palmarumycins C1 (7), BG1 (8), and JC1 (9) were isolated from an endophytic fungus Guignardia sp. KcF8 of mangrove plant K. candel. Compound 8 displayed significant activity against A. hydrophila ATCC 7966, which a fish pathogens in aquiculture. Compounds 2, 7, and 8 showed antifungal activity against several plant pathogenic fungi. Compounds 5e7 exhibited cytotoxicities against a panel of 10 human tumor cell lines. Compound 3 displayed significant inhibitory activity (IC50¼25.7 and 43.9 mM) against human protein tyrosine phosphatase 1B (PTP1B) and silent information regulator T1 (SIRT1) enzymes. This is the first report on the anti-PTP1B and anti-SIRT1 activities of spirodioxynaphthalenes, which will become promising lead compound for the treatment of diabetes without toxic side effect. 4. Experimental section 4.1. General experimental procedures The NMR spectra were recorded on a Bruker AC 500 NMR spectrometer with TMS as an internal standard. HRESIMS data were measured on a Bruker micro TOF-QII mass spectrometer. Optical rotation values were measured with an Anton Paar MCP500 polarimeter. IR spectra were recorded on a Nicolet 6700 FT-IR spectrometer. UV spectra were recorded on a Shimadzu UV-2600 UVevis spectrophotometer. CD spectra were measured with a chirascan circular dichroism spectrometer (Applied Photophysics). The silica gel GF254 used for TLC was supplied by the
W. Ai et al. / Tetrahedron 70 (2014) 5806e5814
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Fig. 8. The possible biosynthesis of 1e9.
Table 3 The cytotoxicity of 5e7a K562 A549 Huh-7 H1975 MCF-7 U937 BGC823 HL60 HeLa MOLT-4 5 6 7 TSA
1.67 1.84 1.54 0.24
2.73 5.90 2.63 0.05
2.14 1.11 1.90 0.08
11.3 4.68 39.2 0.09
8.79 6.48 3.08 0.78
1.74 2.85 1.66 0.06
2.34 3.08 2.00 0.09
2.94 3.06 2.90 0.09
1.32 0.38 1.24 0.11
1.24 1.07 1.27 0.03
a Data as expressed in LD50 values (mM). K562: human erythromyeloblastoid leukemia cell line; A549: human lung cancer; Huh-7: human hepatocarcinoma; H1975: human erlotinib-resistant lung cancer; MCF-7: human breast cancer; U937: human leukemic monocyte lymphoma cancer; BGC823: human gastric cancer; HL60: human promyelocytic leukemia cancer; HeLa: human epithelial carcinoma; MOLT-4: human acute T lymphoblastic leukemia cancer.
Qingdao Marine Chemical Factory, Qingdao, China. Sephadex LH-20 gel (GE Healthcare, Sweden) was used. HPLC was carried on Hitachi L-2400 with YMC ODS column. Spots were detected on TLC under UV light or by heating after spraying with 5% H2SO4 in EtOH. Molecular Merck force field (MMFF) and DFT/TDDFT calculations were performed with Spartan’14 software package (Wavefunction Inc., Irvine, CA, USA) and Gaussian09 program package, respectively, using default grids and convergence criteria. MMFF conformational search generated low-energy conformers within a 10 kcal/mol energy window were subjected to geometry
optimization using DFT method at the B3LYP/6-31G (d) level. Frequency calculations were run at the same level to verify that each optimized conformer was a true minimum and to estimate their relative thermal free energies (DG) at 298.15 K. Solvent effects were taken into account by using polarizable continuum model (PCM). The TDDFT calculations were performed using the long-range corrected hybrid CAM-B3LYP and the hybrid B3LYP functionals, and Ahlrichs’ basis sets SVP (split valence plus polarization) and TZVP (triple zeta valence plus polarization).
4.2. Fungal material The fungus strain KcF8, identified as Guignardia sp., was isolated from fresh healthy fruits of mangrove plant K. candel. The plant material was collected at Daya Bay, Shenzhen city, Guangdong province, China, in April 2012. Following the surface sterilization of the fruits with 75% EtOH, small tissue samples from inside of the fruits were cut a septically and pressed onto MA agar (malt extract 15 g, sea salt 10 g, chloromycetin 0.2 g, agar 15 g, distilled water 1000 mL, pH 7.4e7.8) plates. From the growing cultures, pure strain of Guignardia sp. was isolated by repeated and reinoculation on MB agar (malt extract 15 g, sea salt 10 g, agar 15 g, distilled water 1000 mL, pH 7.4e7.8) plates.
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4.3. Identification of fungal culture The fungus (strain No. KcF8) was identified using a molecular biological protocol by DNA amplification and sequencing of the 18s rRNA gene. By comparison with the GenBank database, the most similar strains were Guignardia ardisiae, Guignardia mangiferae, Guignardia alliacea, all are 99% similarity. So the fungus strain KcF8 clearly belongs to the genus Guignardia, and was designated as Guignardia sp.
4.4. Extraction and isolation Rice (4 kg) in 20 1000 mL Erlenmeyer flasks (200 g each) was inoculated with liquid seed and incubated at 25 C under daylight for 2 months. The culture was broken up with a spatula and extracted three times with EtOAc (34 L). The combined EtOAc extract was filtered and evaporated to afford 74.7 g of crude extract. The crude EtOAc extract was partitioned between methanol and hexane. The resulting methanol fraction (27.7 g) was subjected to silica gel column chromatography (CC) eluted with petroleum ether/EtOAc in gradient eluant (80:20, 50:50, 30:70) followed by CHCl3/MeOH in gradient eluant (90:10, 80:20, 70:30, 60:40, 0:100) to obtain 16 fractions (fractions 1e16) on the monitor of TLC. Fraction 10 was purified by Sephadex LH-20 (CHCl3/MeOH, 1:1) first to give four subfractions (fractions 10.1e10.4). Fraction 10.2 (100.0 mg) was further purified by CC on silica gel (CHCl3/MeOH, 100:1) to give 4 (6.3 mg) and 8 (12.1 mg). Fraction 11 was purified by Sephadex LH-20 (CHCl3/MeOH, 1:1) first to give three subfractions (fractions 11.1e11.3). Fraction 11.2 (200.0 mg) was further purified by CC on silica gel (CHCl3/MeOH, gradient elution 100:1e10:1) to give 7 (11.2 mg), 9 (7.4 mg), and 6 (20.8 mg). Fraction 12 was purified by Sephadex LH-20 (CHCl3/MeOH, 1:1) first to give four subfractions (fractions 12.1e12.4). Fraction 12.2 (50.0 mg) was further purified by CC on silica gel (CHCl3/MeOH, 50:1) to give 1 (8.2 mg). Fraction 13 was purified by Sephadex LH-20 (CHCl3/ MeOH, 1:1) first to give two subfractions (fractions 13.1e13.2). Fraction 13.1 (80.0 mg) was further purified by CC on silica gel (CHCl3/MeOH, 50:1) to give 5 (15.4 mg). Fraction 13.2 (10.0 mg) was further purified by HPLC (MeOH/H2O, 75%) to give 3 (3.5 mg). Fraction 18 was purified by Sephadex LH-20 (MeOH) first to give three subfractions (fractions 18.1e18.3). Fraction 18.3 (20.0 mg) was further purified by CC on silica gel (CHCl3/MeOH, 20:1) to give 2 (4.6 mg). 4.4.1. Guignardin A (1). White powder; [a]25 D 16.7 (c 0.3, MeOH); UV (MeOH) lmax (log 3 ) 226 (4.63), 300 (3.71), 328 (3.70); IR nmax 3479, 1712, 1635, 1608, 1454, 1411, 1377, 1273, 956, 798, 744; 1H and 13 C NMR data see Tables 1 and 2; selected HMBC data, H-3/C-2, C4, C-10, C-11; H-5/C-4, C-7, C-9; H-6/C-5, C-8, C-10; H-7/C-5, C-8, C-9; H-11/C-3, C-2, C-10, C-12; H-13/C-11, C-12; HRESIMS 391.1166 [MþH]þ (calcd for 391.1176). 4.4.2. Guignardin B (2). Brown oil; [a]25 D 144 (c 0.3, MeOH); UV (MeOH) lmax (log 3 ) 226 (5.00), 287 (4.99), 296 (4.96), 299 (4.96), 310 (4.78), 313 (4.83), 327 (4.66); IR nmax 3267, 1608, 1411, 1377, 1273, 975, 794, 756; 1H and 13C NMR data see Tables 1 and 2; selected HMBC data, H-1/C-2, C-3, C-8, C-9, C-10; H-3/C-1, C-2, C4, C-10; H-5/C-4, C-7, C-8, C-9, C-10; H-6/C-4, C-7, C-8, C-9, C10; H-7/C-5, C-8, C-9; HRESIMS 333.0763 [MH]- (calcd for 333.0768). 4.4.3. Guignardin C (3). White powder; UV (MeOH) lmax (log 3 ) 224 (4.96), 258 (4.26), 299 (4.26), 328 (4.19); IR nmax 3394, 1635, 1608, 1458, 1411, 1377, 1265, 1168, 1060, 983, 891, 810, 756; 1H and 13C NMR data see Tables 1 and 2; selected HMBC data, H-3/C-2, C-1,
C-4, C-10; H-5/C-4, C-9, C-7; H-6/C-7, C-10, C-8; H-7/C-9, C-5, C-8; HRESIMS 333.0752 [MþH]þ (calcd for 333.0757). 4.4.4. Guignardin D (4). Red powder; UV (MeOH) lmax (log 3 ) 203 (4.68), 214 (4.46), 224 (4.51), 265 (3.82), 275 (3.84); IR nmax 1762, 1651, 1597, 1550, 1411, 1377, 1261, 1165, 1083, 1049, 956, 806, 740; 1H and 13C NMR data see Tables 1 and 2; selected HMBC data, H-5/C4, C-9, C-7; H-6/C-10, C-8; H-7/C-9, C-5; HRESIMS 441.2992 [MþNa]þ (calcd for 441.2999). 4.4.5. Guignardin E (5). Brown oil; UV (MeOH) lmax (log 3 ) 223 (4.98), 257 (4.36), 298 (4.38), 327 (4.36); IR nmax 3394, 1635, 1608, 1458, 1411, 1377, 1265, 1168, 1060, 983, 891, 810, 756; 1H and 13C NMR data see Tables 1 and 2; selected HMBC data, H-3/C-1, C-2, C-4, C-10; H-5/C-4, C-7, C-9; H-6/C-7, C-8, C-10; H-7/C-5, C-8, C-9; HRESIMS 369.0524 [MþH]þ (calcd for 369.0506). 4.4.6. Guignardin F (6). Colorless crystal; 1H and 13C NMR data see Tables 1 and 2; selected HMBC data, H-2/C-3, C-4, C-10; H-3/C1, C-9; H-5/C-4, C-7, C-8, C-9; H-6/C-5, C-7, C-8, C-9, C-10; H7/C-5, C-8, C-9; HRESIMS 331.0618 [MH] (calcd for 331.0612). 4.4.7. Palmarumycin C1 (7). Yellow crystal; 1H NMR (500 MHz, CDCl3): d 7.15 (1H, s), 7.46 (2H, d, J¼7.5 Hz, H-5), 7.69 (1H, t, J¼8.0 Hz, H-6), 7.18 (1H, d, J¼8.0 Hz, H-7), 6.99 (1H, d, J¼7.5 Hz, H20 , H-50 ), 7.49 (2H, t, J¼8.0 Hz, H-30 , H-60 ), 7.61 (2H, d, J¼8.0 Hz, H-40 , H-50 ), 11.87 (1H, s, HeOH); 13C NMR (125 MHz, CDCl3): d 181.9 (s, C1), 135.0 (s, C-2), 120.1 (d, C-3), 93.6 (s, C-4), 119.7 (d, C-5), 137.2 (d, C-6), 135.7 (d, C-7), 162.2 (s, C-8), 113.2 (s, C-9), 138.5 (s, C-10), 146.8 (s, C-10, C-90 ), 110.1 (d, C-20 , C-80 ), 127.7 (d, C-30 , C-70 ), 121.6 (d, C-40 , C-60 ), 112.8 (s, C-90 ), 134.2 (s, C-100 ). 4.4.8. Palmarumycin BG1 (8). White powder; 1H NMR (500 MHz, CDCl3): d 2.91 (1H, dd, J¼16.5, 4.0 Hz, H-2), 3.21 (1H, dd, J¼16.5, 4.0 Hz, H-2), 4.57 (1H, t, J¼4.0 Hz, H-3), 6.90 (1H, d, J¼7.5 Hz, H-50 ), 7.06 (1H, d, J¼7.5 Hz, H-7), 7.08 (1H, d, J¼7.5 Hz, H-40 ), 7.32 (1H, d, J¼7.5 Hz, H-20 ), 7.41 (1H, t, J¼7.5 Hz, H-60 ), 7.46 (1H, t, J¼7.5 Hz, H30 ), 7.52 (1H, d, J¼7.5 Hz, H-5), 7.54 (1H, d, J¼7.5 Hz, H-70 ), 7.55 (1H, t, J¼7.5 Hz, H-6), 12.34 (1H, eOH); 13C NMR (125 MHz, CDCl3): d 201.1 (s, C-1), 41.3 (t, C-2), 67.2 (d, C-3), 98.7 (s, C-4), 119.7 (d, C-5), 117.9 (d, C-6), 137.0 (d, C-7), 162.1 (s, C-8), 113.1 (s, C-9), 137.9 (s, C10), 147.1 (s, C-10 ), 121.3 (d, C-20 ), 127.7 (d, C-30 ), 109.5 (d, C-40 ), 108.8 (d, C-50 ), 127.6 (d, C-60 ), 121.0 (d, C-70 ), 146.3 (s, C-80 ), 115.3 (s, C-90 ), 134.1 (s, C-100 ). 4.4.9. Palmarumycin JC1 (9). White powder; 1H NMR (500 MHz, CDCl3): d 3.26 (1H, d, J¼11.0 Hz, HeOH), 3.74 (1H, dd, J¼4.5, 3.0 Hz, H-2), 3.87 (1H, d, J¼4.5 Hz, H-3), 5.44 (1H, dd, J¼11.0, 3.0 Hz, H-1), 6.92 (1H, d, J¼8.0 Hz, H-50 ), 7.05 (1H, dd, J¼8.0, 2.0 Hz, H-7), 7.14 (1H, d, J¼7.5 Hz, H-40 ), 7.37 (1H, t, J¼8.0 Hz, H-60 ), 7.40 (1H, dd, J¼8.0, 2.0 Hz, H-20 ), 7.44 (1H, t, J¼8.0 Hz, H-30 ), 7.51 (1H, t, J¼8.0 Hz, H-6), 7.55 (1H, d, J¼8.0 Hz, H-70 ), 7.57 (1H, d, J¼8.0 Hz, H-5), 8.31 (1H, s, HeOH); 13C NMR (125 MHz, CDCl3): d 66.6 (d, C-1), 52.8 (d, C-2), 54.2 (d, C-3), 96.7 (s, C-4), 119.3 (d, C-5), 130.6 (d, C-6), 118.9 (d, C-7), 156.5 (s, C-8), 118.5 (s, C-9), 132.0 (s, C-10), 147.3 (s, C-10 ), 121.1 (d, C-20 ), 127.7 (d, C-30 ), 109.9 (d, C-40 ), 109.1 (d, C-50 ), 127.5 (d, C-60 ), 121.0 (d, C-70 ), 147.2 (s, C-80 ), 112.8 (s, C-90 ), 134.1 (s, C-100 ). 4.5. X-ray crystallographic analysis of 7 Yellow crystal of C20H11ClO4 with M¼350.74. Unit cell dimensions a¼5.0690(2) A, b¼11.7367(6) A, c¼13.0641(8) A, V¼753.21(7) A3, Z¼2; crystal size 0.430.410.37 mm3. A total of 5025 unique reflections (q¼2.67e26.00 ) were collected using graphite monochromated Cu Ka radiation (l¼0.71073 A) on a Bruker Smart 1000 CCD diffractometer at 150(2) K. Absorption
W. Ai et al. / Tetrahedron 70 (2014) 5806e5814
corrections were done by semi-empirical from equivalents. The structure was solved by direct methods (SHELXS-97) and refined with Full-matrix least-squares on 2959 data, 0 restraints, and 229 variable parameters. Final R indicates R1¼0.0347, wR2¼0.0857 [I>2s (I)]. Crystallographic data for 7 has been deposited in the Cambridge Crystallographic Data Centre with the deposition number 963344. Copies of the data can be obtained, free of charge, on application to the director, CCDC, 12 Union Road, Cambridge CB21EZ, UK [fax: þ44 (0)1223 336033, or e-mail: [email protected]].
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Boston, MA). In a typical 100 mL assay mixture containing 50 mmol/ L 3-[N-morpholino]-propanesulfonic acid (MOPs), pH 6.5, 2 mmol/L pNPP, and 30 nmol/L recombinant PTP1B, activities were continuously monitored and the initial rate of the hydrolysis was determined using the early linear region of the enzymatic reaction kinetic curve. The IC50 was calculated with Prism 4 software (Graphpad, San Diego, CA) from the nonlinear curve fitting of the percentage of inhibition (% inhibition) versus the inhibitor concentration [I] by using the following equation: % Inhibition¼100/ (1þ[IC50/[I]]k), where k is the Hill coefficient. The positive control is oleanolic acid (IC50¼1.90 mM).
4.6. Antibacterial assay 4.10. SIRT1 inhibitory activity assay The ready made nutrient agar medium (38 g) was suspended in distilled water (1000 mL) and heated to boiling until it dissolved completely. The medium and Petridishes were autoclaved at temperature 121 C for 20 min. Disc diffusion assay was employed for testing antibacterial activity of the compounds. The medium was poured into sterile Petridishes under aseptic conditions in laminar flow chamber. Before the medium in the plate solidify, 2% cultured of test organism was inoculated and uniformly spread over the medium. Solutions were prepared by dissolving compound in MeOH and concentration (50 mg/mL) was made. After incubation, to each disc concentrations of test solutions were added (1 mL). The positive control penicillin was maintained with MeOH (50 mg/mL). The treated materials and the controls were kept in an incubator at 28 C for 12 h. Inhibition zones were measured and diameter was calculated in mm. Three replicates were maintained for each treatment. 4.7. Antifungal assay The method followed for antifungal bioassay is similar to that followed for antibacterial assay where in the medium is malt extract agar 40 g/L and the positive control is carbendazim. Concentration (25 mg/mL) of test solution was tested. Controls were maintained with acetone and carbendazim (25 mg/mL). The treated and the controls were kept in an incubator at 28 C for 3 days. Inhibition zones were measured and diameter was calculated in mm. Three replicates were maintained for each treatment. 4.8. Cytotoxicity Cytotoxicity was assayed with the CCK8 (DOjinDo, Japan) method.10 Cell lines, K562, A549, Huh-7, H1975, MCF-7, U937, BGC823, HL60, HeLa, and MOLT-4 were purchased from Shanghai Cell Bank, Chinese Academy of Sciences. Cells were routinely grown and maintained in mediums RPMI or DMEM with 10% FBS and with 1% penicillin/streptomycin. All cell lines were incubated in a Thermo/Forma Scientific CO2 Water Jacketed Incubator with 5% CO2 in air at 37 C. Cell viability assay was determined by the CCK8 (DOjinDo, Japan) assay. Cells were seeded at a density of 400e800 cells/well in 384 well plates and treated with various concentration of compounds or solvent control. After 72 h incubation, CCk8 reagent was added, and absorbance was measured at 450 nm using Envision 2104 multi-label Reader (PerkineElmer, USA). Dose response curves were plotted to determine the IC50 values using Prism 5.0 (GraphPad Software Inc., USA). 4.9. PTP1B inhibitory activity assay Recombinant PTP1B catalytic domain was expressed and purified according to a previous report.11 The enzymatic activities of the PTP1B catalytic domain were determined at 30 C by monitoring the hydrolysis of pNPP. Dephosphorylation of pNPP generates product pNP, which was monitored at an absorbance of 405 nm by the EnVision multi-label plate reader (PerkineElmer Life Sciences,
Compounds were dissolved in DMSO to prepare 10 mM stock solutions; a fluorescent-based assay was used to screen compounds that inhibit the deacetylase activity of SIRT1. The C-terminus of the acetyl substrate acetyl-Arg-His-Lys-Lys (3-acetyl) was labeled with 4-amino-7-methylcoumarin (AMC). When the substrate is deacetylated by SIRT1, AMC can be cleaved from the peptide by trypsin and detected by its fluorescence. The reaction conditions were as follows: 750 mMNADt, 31.25 mMac-RHKKac-AMC, and 1 mM enzyme in buffer containing 25 mM Tris (pH 8.0), 137 mM NaCl, 2.7 mM KCl, and 1 mM MgCl2. The reactions were carried out for 45 min at room temperature, stopped with 10 mM nicotinamide, and developed with 0.5 mg/mL trypsin. After incubation for 30 min, fluorescence intensities were measured with a microplate reader (POLARstar OPTIMA, BMG Labtech) at an excitation wavelength of 340 nm and an emission wavelength of 490 nm, the positive control is nicotinamide IC50¼50.81 mM.12 Acknowledgements This work was supported financially by the National Key Basic Research Program of China (973)’s Project (2010CB833800 and 2011CB915503), the National High Technology Research and Development Program (863 Program, 2013AA092901 and 2012AA092104), the National Natural Science Foundation of China (Nos. 31270402, 21172230, 20902094, 41176148, and 21002110), Guangdong Province-CAS Joint Research Program (2011B090300023 and 2012B091100264), and Guangdong Marine Economic Development and Innovation of Regional Demonstration Project (GD2012-D01-001 and GD2012-D01-002). Supplementary data NMR spectra of compounds 1e6. Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.tet.2014.06.041. References and notes 1. Nurul Islam, M.; Jung, H. A.; Sohn, H. S.; Kim, H. M.; Choi, J. S. Arch. Pharm. Res. 2013, 36, 542e552. 2. Canto, C.; Auwerx, J. Trends Endocrinol. Metab. 2009, 20, 325e331. 3. Milne, J. C.; Lambert, P. D.; Schenk, S.; Carney, D. P.; Smith, J. J.; Gagne, D. J.; Jin, L.; Boss, O.; Perni, R. B.; Vu, C. B.; Bemis, J. E.; Xie, R.; Disch, J. S.; Ng, P. Y.; Nunes, J. J.; Lynch, A. V.; Yang, H.; Galonek, H.; Israelian, K.; Choy, W.; Iffland, A.; Lavu, S.; Medvedik, O.; Sinclair, D. A.; Olefsky, J. M.; Jirousek, M. R.; Elliott, P. J.; Westphal, C. H. Nature 2007, 450, 712e716. 4. Kume, S.; Kitada, M.; Kanasaki, K.; Maegawa, H.; Koya, D. Arch. Pharm. Res. 2013, 36, 230e236. € ni-Schnetzler, M.; Hubbard, B. P.; Bouzakri, K.; Brunner, A.; 5. Biason-Lauber, A.; Bo €ni, M.; Meier, D. T.; Brorsson, C.; Timper, Cavelti-Weder, C.; Keller, C.; Meyer-Bo K.; Leibowitz, G.; Patrignani, A.; Bruggmann, R.; Boily, G.; Zulewski, H.; Geier, A.; Cermak, J. M.; Elliott, P.; Ellis, J. L.; Westphal, C.; Knobel, U.; Eloranta, J. J.; Kerr-Conte, J.; Pattou, F.; Konrad, D.; Matter, C. M.; Fontana, A.; Rogler, G.; Schlapbach, R.; Regairaz, C.; Carballido, J. M.; Glaser, B.; McBurney, M. W.; Pociot, F.; Sinclair, D. A.; Donath, M. Y. Cell Metab. 2013, 17, 448e455. 6. Owen, C.; Lees, E. K.; Grant, L.; Zimmer, D. J.; Mody, N.; Bence, K. K.; Delibegovic, M. Diabetologia 2013, 56, 2286e2296.
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7. Krohn, K.; Michel, A.; Florke, U.; Aust, H. J.; Draeger, S.; Schulz, B. Liebigs Ann. Chem. 1994, 11, 1099e1108. 8. Ravindranath, N.; Reddy, M. R.; Mahender, G.; Ramu, R.; Kumar, K. R.; Das, B. Phytochemistry 2004, 65, 2387e2390. n, T.; Miao, Z. H.; M romi, I. N.; Liu, H. L.; Ding, J.; 9. Cai, Y. S.; Kurta andi, A.; Koma Guo, Y. W. J. Org. Chem. 2011, 76, 1821e1830.
10. Wanka, L.; Iqbal, K.; Schreiner, P. R. Chem. Rev. 2013, 113, 3516e3604. 11. Yang, X.-N.; Li, J.-Y.; Zhou, Y.-Y.; Shen, Q.; Chen, J.-W.; Li, J. Biochim. Biophys. Acta Gen. Subj. 2005, 1726, 34e41. 12. Wu, J. H.; Zhang, D. Y.; Chen, L.; Li, J. N.; Wang, J. L.; Ning, C. Q.; Yu, N. F.; Zhao, F.; Chen, D. Y.; Chen, X. Y.; Chen, K. X.; Jiang, H. L.; Liu, H.; Liu, D. X. Eur. J. Med. Chem. 2013, 56, 761e780.