- Email: [email protected]
Proangiogenic penibishexahydroxanthone A from the marine-derived fungus Penicillium sp. ZZ486A
Tetrahedron Letters 60 (2019) 1393–1396
Contents lists available at ScienceDirect
Tetrahedron Letters journal homepage: www.elsevier.com/locate/tetlet
Proangiogenic penibishexahydroxanthone A from the marine-derived fungus Penicillium sp. ZZ486A Mengxuan Chen a, Yuhan Gui b, Hongrui Zhu b, Zhizhen Zhang a,⇑, Hou-Wen Lin b,⇑ a
Ocean College, Zhoushan Campus, Zhejiang University, Zhoushan 316021, PR China Research Center for Marine Drugs, State Key Laboratory of Oncogenes and Related Genes, Department of Pharmacy, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200127, PR China b
a r t i c l e
i n f o
a b s t r a c t
Article history: Received 28 March 2019 Revised 16 April 2019 Accepted 23 April 2019 Available online 23 April 2019
Penibishexahydroxanthone A, a new hexahydroxanthone homodimer with a rare highly oxidized hexacyclic framework, was identified from the marine-derived Penicillium sp. ZZ486A. Its structure was determined by a combination of extensive NMR spectroscopical analyses, HRESIMS data, ECD calculation, and single crystal X-ray diffraction analysis. Penibishexahydroxanthone A showed in vivo activity in promoting angiogenesis in a dose-dependent manner by rescuing VRI-induced vascular insufficiency in zebrafish. This study is the first report of the proangiogenic activity of hexahydroxanthones. Ó 2019 Elsevier Ltd. All rights reserved.
Keywords: Penicillium sp. ZZ486A Marine-derived fungus Penibishexahydroxanthone A Proangiogenesis
Introduction Angiogenesis refers to the establishment of the mature blood vessel network through expansion and remodeling of the preexisting vascular primordium [1]. Pathological angiogenesis is a hallmark of cancers and various ischaemic and inflammatory diseases [2]. Antiangiogenic therapies provide new hope for the successful treatment of cancer, whereas proangiogenic molecules can repair the tissue damages to cure ischaemic diseases [2,3]. Various angiogenic approaches for ischaemic diseases have been tested in clinical trials and most interventions involved the delivery of vascular endothelial growth factor (VEGF) to the ischaemic tissue to stimulate growth of new vessels [2]. VEGF is a key regulator of physiological angiogenesis and its biological effects are mediated by two receptor tyrosine kinases VEGFR-1 and VEGFR-2 [4]. VEGFR tyrosine kinase inhibitor (VRI) displays antiangiogenic properties and is used to chemically induce vascular insufficiency in zebrafish to study the mechanisms of vascular morphogenesis in pathological conditions [5] and also to discover novel proangiogenic compounds. Hexahydroxanthone derivatives have been isolated from fungal, bacterial, or lichenoid sources [6]. They included applanatins [7], monodictysins [8], and isocochlioquinones [9]. So far, only two
dimers of hexahydroxanthones (phomoxanthones C and D) have been reported [10]. Monodictysin B had inhibitory activity against cytochrome P450 1A [8] and isocochlioquinones A and C showed cytotoxicity against human cervical carcinoma Hela cells [9]. Isocochlioquinone A also had activity in inhibiting the growth of Plasmodium falciparum [11]. In the course of our research for discovering novel bioactive compounds from marine microorganism [12–16], a fungus strain ZZ486A was isolated from the gut of an octopus. Chemical investigation on the culture of this strain in BMPM liquid medium resulted in the isolation of a novel homodimer of hexahydroxanthone, named penibishexahydroxanthone A (1, Fig. 1). Herein, we describe the isolation and culture of strain ZZ486A as well as the structural elucidation and bioactive evaluation of penibishexahydroxanthone A.
⇑ Corresponding authors. E-mail addresses: (H.-W. Lin).
[email protected]
(Z.
https://doi.org/10.1016/j.tetlet.2019.04.034 0040-4039/Ó 2019 Elsevier Ltd. All rights reserved.
Zhang),
[email protected]
Fig. 1. Structures of penibishexahydroxanthone A (1) and phomoxanthone C (1a).
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M. Chen et al. / Tetrahedron Letters 60 (2019) 1393–1396
Table 1 C (150 MHz) and 1H (600 MHz) NMR data for 1 (in DMSO d6).
13
No.
13
1, 1a 2, 2a 3, 3a 4, 4a 5, 5a 6, 6a 7, 7a 8, 8a 9, 9a 10, 10a
158.2, C 106.9, CH 140.7, CH 116.9, C 159.4, C 106.3, C 192.3, C 71.8, C 198.6, C 43.1, CH2
C, type
1
H (J in Hz)
– 6.66, 1H, d (8.5) 7.53, 1H, d (8.5) – – – – – – bH: 2.26, 1H, dd (14.5, 5.2); aH: 2.81, 1H, dd (14.5, 13.2)
Results and discussion The strain ZZ486A was assigned as Penicillium sp. ZZ486A (Supplementary Data, Fig. S1 and Table S1) based on the result of ITS rDNA sequence (Fig. S2). A crude extract prepared from the culture of strain ZZ486A in BMPM liquid medium was partitioned by EtOAc. The EtOAc partition was separated by repeated column chromatography, following by HPLC purification, to give compound 1. Penibishexahydroxanthone A (1) was obtained as a yellow needle crystal (in CH3OH). Its molecular formula C32H30O16 was determined by HRESIMS at m/z 671.1612 [M+H]+ (calcd for C32H31O16, 671.1612). The IR spectrum of 1 exhibited absorptions at 3382, 1672, and 1621 cm 1 for hydroxyl, carbonyl, and benzene groups, respectively. Its symmetric dimer structure through C-4 and C-4a connection was indicated by its only sixteen 13C NMR signals for three carbonyls (dC 198.6, 192.3, and 168.3), six aromatic carbons
Fig. 2. Key HMBC and 1H-1H COSY correlations of penibishexahydroxanthone A (1).
No.
13
1
11, 11a 12, 12a 13, 13a 14, 14a 15, 15a 16, 16a OH-5, 5a OH-8, 8a OH-12, 12a
32.2, CH 73.0, CH 90.0, C 168.3, C 52.9, CH3 18.6, CH3 – – –
1.93, 1H, m 4.22, 1H, dd (10.5, 6.0) – – 3.55, 3H, s 1.07, 3H, d (6.3) 11.81, 1H, s 8.18, 1H, s 5.95, 1H, d (6.0)
C, type
H (J in Hz)
(dC 159.4, 158.2, 140.7, 116.9, 106.9, and 106.3), two nonprotonated carbons linked to oxygen (dC 90.0 and 71.8), one oxymethine (dC 73.0), one methoxyl (dC 52.9), one methylene (dC 43.1), one methine (dC 32.2), and one methyl (dC 18.6) (Table 1). Analysis of 1 H NMR spectrum suggested the presence of three hydroxyls (dH 11.81, 1H, s; 8.18, 1H, s; 5.95, 1H, d, 6.0 Hz), one 1,2,3,4-substituted aromatic ring (dH 6.66 and 7.53, each 1H, d, 8.5 Hz), one oxymethine (dH 4.22, 1H, dd, J = 10.5, 6.0 Hz), one methoxyl (dH 3.55, 3H, s), one methylene (dH 2.81, 1H, dd, 14.5, 13.2 Hz; 2.26, 1H, dd, 14.5, 5.2 Hz), one methine (dH 1.93, 1H, m), and one methyl (dH 1.07, 3H, d, 6.3 Hz) (Table 1). The three carbonyls and ring A accounted for seven out of nine degrees of unsaturation, which is half of the 18 degrees required by the molecular formula and the remaining two were from rings B and C. The positions of OH-8, OH-12, CH3-15, and CH3-16 were established by the following HMBC correlations (Fig. 2): OH-8 (dH 8.18) with C-7 (dC 192.3), C8 (dC 71.8), C-9 (dC 198.6), and C-13 (dC 90.0); OH-12 (dH 5.95) with C-11 (dC 32.2), C-12 (dC 73.0), and C-13; CH3-15 (dH 3.55) with C-14 (dC 168.3); and CH3-16 (dH 1.07) with C-10 (dC 43.1), C-11, and C12. HMBC correlations of OH-8 with C-9 and H-10 (dH 2.26, 2.81) with C-9 also indicated a ketone group at C-9. The relative configuration of 1 was assigned by NOE information. As shown in Fig. 3, key NOE correlations of OH-8 with H-10a (dH 2.81) and H-12, H10a with H-12 and H3-16, and H-12 with H3-16 indicated an a-orientation for these protons, whereas NOE cross-peaks of H-11 with H-10b (dH 2.26), OH-12, and H3-15 were suggestive of a b-orientation for these protons. The result (Fig. 4) from single crystal X-ray diffraction analysis further confirmed these assignments. The Ga Ka data of crystal X-ray diffraction analysis may be not good enough in assigning the absolute configuration because the Flack parameter is 0.1(2). As a result, a computational method
Fig. 3. Key NOE correlations of penibishexahydroxanthone A (1).
M. Chen et al. / Tetrahedron Letters 60 (2019) 1393–1396
1395
Fig. 4. X-ray crystal structure of penibishexahydroxanthone A (1, Ga Ka radiation).
Fig. 5. Experimental and calculated ECD spectra of penibishexahydroxanthone A (1).
was applied to clarify the absolute configuration of 1 by comparing the experimental ECD spectrum with the time-dependent densityfunctional theory (TDDFT) calculated ECD spectra [17,18]. The CIF profile of X-ray crystals was chosen for ECD calculation. As shown in Fig. 5, the calculated ECD curve of penibishexahydroxanthone A (1) was in good agreement with the experimental ECD spectrum at 205–400 nm. Therefore, the absolute configurations of penibishexahydroxanthone A (1) were assigned to be 8S, 11S, 12R, 13S, 8aS, 11aS, 12aR, 13aS (Fig. 4). Based on the foregoing evidence, the structure of penibishexahydroxanthone A (1) was elucidated as shown in Fig. 1. The full assignments of 13C and 1H NMR data (Table 1) of penibishexahydroxanthone A (1) were made based
on a combination of 13C, 1H, HSQC, 1H-1H COSY, HMBC, and NOE spectroscopical analyses. The closest structural analogue of penibishexahydroxanthone A is phomoxanthone C (1a, Fig. 1) that was isolated from the Thai mangrove endophytic fungus Phomopsis sp. xy21. Their structural differences were the different substitutes at C-9 and C-13 as well as the different configurations at C-12. To the best of our knowledge, penibishexahydroxanthone A is the first homodimer of a highly oxidized hexahydroxanthone with a ketone group at C-9. A model [5] of vascular insufficiency in zebrafish induced by vatalanib (PTK787), a VEGFR tyrosine kinase inhibitor [19], was used to evaluate the effect of penibishexahydroxanthone A on angiogenesis. Intersomitic vessels (ISVs) sprouted and elongated from the dorsal aorta and posterior cardinal vein were considered as intact vessels [5]. Salvianic acid A sodium (SAAS) [20] was used as a positive control. As shown in Fig. 6A, ISVs’ growth was significantly suppressed by pretreatment with PTK787 (0.15 lg/mL) for 3 h in Tg(flk1:EGFP) zebrafish embryos. ISVs’ growth was rescued after incubation with penibishexahydroxanthone A for 24 h in concentrations of 10, 20, and 40 lM. Quantitative analysis showed that penibishexahydroxanthone A rescued the PTK787-induced blood vessel loss in a dose-dependent manner (Fig. 6B). In addition, penibishexahydroxanthone A showed moderate antimicrobial activity with MIC values of 15 lg/mL for Candida albicans, 20 lg/mL for Escherichia coli, and 25 lg/mL for Methicillin-resistant Staphylococcus aureus.
Fig. 6. A: Typical images on inflammatory sites in PTK787-induced blood vessel loss in zebrafish embryos. B: Quantitative analysis showed that penibishexahydroxanthone A (1) rescued PTK787-induced blood vessel loss in a dose-dependent manner. Data was represented as the mean ± SEM (n = 5). ##p < 0.01 vs. the Control group; **p < 0.01 vs. PTK787-induced group. Bar = 1 mm.
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Conclusion
References
Marine fungus Penicillium sp. ZZ486A was isolated from the gut of an octopus. Large culture of strain ZZ486A in BMPM liquid medium produced penibishexahydroxanthone A, the first homodimer of a highly oxidized hexahydroxanthone with a ketone group at C-9. Its structure was elucidated based on the NMR and HRESIMS data, ECD calculation, and X-ray diffraction analysis. Penibishexahydroxanthone A showed in vivo activity in rescuing VRI-induced blood vessel loss in the model of vascular insufficiency in zebrafish.
[1] C.A. Franco, S. Liebner, H. Gerhardt, Curr. Opin. Genet. Dev. 19 (2009) 476–483. [2] P. Carmeliet, R.K. Jain, Nature 407 (2000) 249–257. [3] J. Tímár, B. Döme, K. Fazekas, Á. Janovics, S. Paku, Pathol. Oncol. Res. 7 (2001) 85–94. [4] N. Ferrara, H.P. Gerber, J. LeCouter, Nat. Med. 9 (2003) 669–676. [5] S. Li, Y.Y. Dang, G.O.L. Che, Y.W. Kwan, S.W. Chan, G.P.H. Leung, S.M.Y. Lee, M.P. M. Hoi, Toxicol. Appl. Pharm. 280 (2014) 408–420. [6] M. Kye, B. Stefan, Chem. Rev. 112 (2012) 3717–3776. [7] F. Wang, Z.J. Dong, J.K. Liu, Naturforsch 62 (2007) 1329–1332. [8] A. Krick, S. Kehraus, C. Gerhauser, K. Klimo, M. Nieger, A. Maier, H.H. Fiebig, I. Atobdiresei, G. Raabe, J. Fleischhauer, G.M. König, J. Nat. Prod. 70 (2007) 353– 360. [9] P. Phuwapraisirisan, K. Sawang, P. Siripong, S. Tip-pyang, Tetrahedron Lett. 48 (2007) 5193–5195. [10] P. Wang, Y.F. Luo, M. Zhang, J.G. Dai, W.J. Wang, J. Wu, J. Asian Nat. Prod. Res. 20 (2017) 217–226. [11] C. Osterhage, G.M. König, U. Höller, A.D. Wright, J. Nat. Prod. 65 (2002) 306– 313. [12] X. Ye, K. Anjum, T. Song, W. Wang, Y. Liang, M. Chen, H. Huang, X.Y. Lian, Z. Zhang, Phytochemistry 135 (2017) 151–159. [13] L. Chen, W. Chai, W. Wang, T. Song, X.Y. Lian, Z. Zhang, J. Nat. Prod. 80 (2017) 1450–1456. [14] M. Chen, W. Chai, R. Zhu, T. Song, Z. Zhang, X.Y. Lian, Tetrahedron 74 (2018) 2100–2106. [15] M. Chen, W. Chai, T. Song, M. Ma, X.Y. Lian, Z. Zhang, Sci. Rep. 8 (2018) 72. [16] T. Song, M. Chen, Z. Ge, W. Chai, X.C. Li, Z. Zhang, X.Y. Lian, J. Org. Chem. 83 (2018) 13395–13401. [17] K. Matsumura, T. Taniguchi, J.D. Reimer, S. Noguchi, S.J. Fujita, R. Sakai, Org. Lett. 20 (2018) 3039–3043. [18] W.H. Jiao, B.H. Cheng, G.D. Chen, G.H. Shi, J. Li, T.Y. Hu, H.W. Lin, Org. Lett. 20 (2018) 3092–3095. [19] A.K. Gaumann, H.C. Drexler, S.A. Lang, O. Stoeltzing, S. Diermeier-Daucher, E. Buchdunger, J. Wood, G. Bold, G. Breier, Int. J. Oncol. 45 (2014) 2267–2277. [20] N. Zhang, H. Zou, L. Jin, J. Wang, M. Zhong, P. Huang, B. Gu, S. Mao, C. Zhang, H. Chen, Acta Pharmacol. Sin. 31 (2010) 421–428.
Conflict of interest The authors declare no conflict of interest.
Acknowledgments This research work was supported by the National Natural Science Foundation of China (No. 81773587). We thank Dr. Huping Zhu (Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences) for performing the NMR spectrometry and Dr. Xuebing Leng for the test and analysis of the X-ray diffraction.
Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.tetlet.2019.04.034.
Contents lists available at ScienceDirect
Tetrahedron Letters journal homepage: www.elsevier.com/locate/tetlet
Proangiogenic penibishexahydroxanthone A from the marine-derived fungus Penicillium sp. ZZ486A Mengxuan Chen a, Yuhan Gui b, Hongrui Zhu b, Zhizhen Zhang a,⇑, Hou-Wen Lin b,⇑ a
Ocean College, Zhoushan Campus, Zhejiang University, Zhoushan 316021, PR China Research Center for Marine Drugs, State Key Laboratory of Oncogenes and Related Genes, Department of Pharmacy, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200127, PR China b
a r t i c l e
i n f o
a b s t r a c t
Article history: Received 28 March 2019 Revised 16 April 2019 Accepted 23 April 2019 Available online 23 April 2019
Penibishexahydroxanthone A, a new hexahydroxanthone homodimer with a rare highly oxidized hexacyclic framework, was identified from the marine-derived Penicillium sp. ZZ486A. Its structure was determined by a combination of extensive NMR spectroscopical analyses, HRESIMS data, ECD calculation, and single crystal X-ray diffraction analysis. Penibishexahydroxanthone A showed in vivo activity in promoting angiogenesis in a dose-dependent manner by rescuing VRI-induced vascular insufficiency in zebrafish. This study is the first report of the proangiogenic activity of hexahydroxanthones. Ó 2019 Elsevier Ltd. All rights reserved.
Keywords: Penicillium sp. ZZ486A Marine-derived fungus Penibishexahydroxanthone A Proangiogenesis
Introduction Angiogenesis refers to the establishment of the mature blood vessel network through expansion and remodeling of the preexisting vascular primordium [1]. Pathological angiogenesis is a hallmark of cancers and various ischaemic and inflammatory diseases [2]. Antiangiogenic therapies provide new hope for the successful treatment of cancer, whereas proangiogenic molecules can repair the tissue damages to cure ischaemic diseases [2,3]. Various angiogenic approaches for ischaemic diseases have been tested in clinical trials and most interventions involved the delivery of vascular endothelial growth factor (VEGF) to the ischaemic tissue to stimulate growth of new vessels [2]. VEGF is a key regulator of physiological angiogenesis and its biological effects are mediated by two receptor tyrosine kinases VEGFR-1 and VEGFR-2 [4]. VEGFR tyrosine kinase inhibitor (VRI) displays antiangiogenic properties and is used to chemically induce vascular insufficiency in zebrafish to study the mechanisms of vascular morphogenesis in pathological conditions [5] and also to discover novel proangiogenic compounds. Hexahydroxanthone derivatives have been isolated from fungal, bacterial, or lichenoid sources [6]. They included applanatins [7], monodictysins [8], and isocochlioquinones [9]. So far, only two
dimers of hexahydroxanthones (phomoxanthones C and D) have been reported [10]. Monodictysin B had inhibitory activity against cytochrome P450 1A [8] and isocochlioquinones A and C showed cytotoxicity against human cervical carcinoma Hela cells [9]. Isocochlioquinone A also had activity in inhibiting the growth of Plasmodium falciparum [11]. In the course of our research for discovering novel bioactive compounds from marine microorganism [12–16], a fungus strain ZZ486A was isolated from the gut of an octopus. Chemical investigation on the culture of this strain in BMPM liquid medium resulted in the isolation of a novel homodimer of hexahydroxanthone, named penibishexahydroxanthone A (1, Fig. 1). Herein, we describe the isolation and culture of strain ZZ486A as well as the structural elucidation and bioactive evaluation of penibishexahydroxanthone A.
⇑ Corresponding authors. E-mail addresses: (H.-W. Lin).
[email protected]
(Z.
https://doi.org/10.1016/j.tetlet.2019.04.034 0040-4039/Ó 2019 Elsevier Ltd. All rights reserved.
Zhang),
[email protected]
Fig. 1. Structures of penibishexahydroxanthone A (1) and phomoxanthone C (1a).
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M. Chen et al. / Tetrahedron Letters 60 (2019) 1393–1396
Table 1 C (150 MHz) and 1H (600 MHz) NMR data for 1 (in DMSO d6).
13
No.
13
1, 1a 2, 2a 3, 3a 4, 4a 5, 5a 6, 6a 7, 7a 8, 8a 9, 9a 10, 10a
158.2, C 106.9, CH 140.7, CH 116.9, C 159.4, C 106.3, C 192.3, C 71.8, C 198.6, C 43.1, CH2
C, type
1
H (J in Hz)
– 6.66, 1H, d (8.5) 7.53, 1H, d (8.5) – – – – – – bH: 2.26, 1H, dd (14.5, 5.2); aH: 2.81, 1H, dd (14.5, 13.2)
Results and discussion The strain ZZ486A was assigned as Penicillium sp. ZZ486A (Supplementary Data, Fig. S1 and Table S1) based on the result of ITS rDNA sequence (Fig. S2). A crude extract prepared from the culture of strain ZZ486A in BMPM liquid medium was partitioned by EtOAc. The EtOAc partition was separated by repeated column chromatography, following by HPLC purification, to give compound 1. Penibishexahydroxanthone A (1) was obtained as a yellow needle crystal (in CH3OH). Its molecular formula C32H30O16 was determined by HRESIMS at m/z 671.1612 [M+H]+ (calcd for C32H31O16, 671.1612). The IR spectrum of 1 exhibited absorptions at 3382, 1672, and 1621 cm 1 for hydroxyl, carbonyl, and benzene groups, respectively. Its symmetric dimer structure through C-4 and C-4a connection was indicated by its only sixteen 13C NMR signals for three carbonyls (dC 198.6, 192.3, and 168.3), six aromatic carbons
Fig. 2. Key HMBC and 1H-1H COSY correlations of penibishexahydroxanthone A (1).
No.
13
1
11, 11a 12, 12a 13, 13a 14, 14a 15, 15a 16, 16a OH-5, 5a OH-8, 8a OH-12, 12a
32.2, CH 73.0, CH 90.0, C 168.3, C 52.9, CH3 18.6, CH3 – – –
1.93, 1H, m 4.22, 1H, dd (10.5, 6.0) – – 3.55, 3H, s 1.07, 3H, d (6.3) 11.81, 1H, s 8.18, 1H, s 5.95, 1H, d (6.0)
C, type
H (J in Hz)
(dC 159.4, 158.2, 140.7, 116.9, 106.9, and 106.3), two nonprotonated carbons linked to oxygen (dC 90.0 and 71.8), one oxymethine (dC 73.0), one methoxyl (dC 52.9), one methylene (dC 43.1), one methine (dC 32.2), and one methyl (dC 18.6) (Table 1). Analysis of 1 H NMR spectrum suggested the presence of three hydroxyls (dH 11.81, 1H, s; 8.18, 1H, s; 5.95, 1H, d, 6.0 Hz), one 1,2,3,4-substituted aromatic ring (dH 6.66 and 7.53, each 1H, d, 8.5 Hz), one oxymethine (dH 4.22, 1H, dd, J = 10.5, 6.0 Hz), one methoxyl (dH 3.55, 3H, s), one methylene (dH 2.81, 1H, dd, 14.5, 13.2 Hz; 2.26, 1H, dd, 14.5, 5.2 Hz), one methine (dH 1.93, 1H, m), and one methyl (dH 1.07, 3H, d, 6.3 Hz) (Table 1). The three carbonyls and ring A accounted for seven out of nine degrees of unsaturation, which is half of the 18 degrees required by the molecular formula and the remaining two were from rings B and C. The positions of OH-8, OH-12, CH3-15, and CH3-16 were established by the following HMBC correlations (Fig. 2): OH-8 (dH 8.18) with C-7 (dC 192.3), C8 (dC 71.8), C-9 (dC 198.6), and C-13 (dC 90.0); OH-12 (dH 5.95) with C-11 (dC 32.2), C-12 (dC 73.0), and C-13; CH3-15 (dH 3.55) with C-14 (dC 168.3); and CH3-16 (dH 1.07) with C-10 (dC 43.1), C-11, and C12. HMBC correlations of OH-8 with C-9 and H-10 (dH 2.26, 2.81) with C-9 also indicated a ketone group at C-9. The relative configuration of 1 was assigned by NOE information. As shown in Fig. 3, key NOE correlations of OH-8 with H-10a (dH 2.81) and H-12, H10a with H-12 and H3-16, and H-12 with H3-16 indicated an a-orientation for these protons, whereas NOE cross-peaks of H-11 with H-10b (dH 2.26), OH-12, and H3-15 were suggestive of a b-orientation for these protons. The result (Fig. 4) from single crystal X-ray diffraction analysis further confirmed these assignments. The Ga Ka data of crystal X-ray diffraction analysis may be not good enough in assigning the absolute configuration because the Flack parameter is 0.1(2). As a result, a computational method
Fig. 3. Key NOE correlations of penibishexahydroxanthone A (1).
M. Chen et al. / Tetrahedron Letters 60 (2019) 1393–1396
1395
Fig. 4. X-ray crystal structure of penibishexahydroxanthone A (1, Ga Ka radiation).
Fig. 5. Experimental and calculated ECD spectra of penibishexahydroxanthone A (1).
was applied to clarify the absolute configuration of 1 by comparing the experimental ECD spectrum with the time-dependent densityfunctional theory (TDDFT) calculated ECD spectra [17,18]. The CIF profile of X-ray crystals was chosen for ECD calculation. As shown in Fig. 5, the calculated ECD curve of penibishexahydroxanthone A (1) was in good agreement with the experimental ECD spectrum at 205–400 nm. Therefore, the absolute configurations of penibishexahydroxanthone A (1) were assigned to be 8S, 11S, 12R, 13S, 8aS, 11aS, 12aR, 13aS (Fig. 4). Based on the foregoing evidence, the structure of penibishexahydroxanthone A (1) was elucidated as shown in Fig. 1. The full assignments of 13C and 1H NMR data (Table 1) of penibishexahydroxanthone A (1) were made based
on a combination of 13C, 1H, HSQC, 1H-1H COSY, HMBC, and NOE spectroscopical analyses. The closest structural analogue of penibishexahydroxanthone A is phomoxanthone C (1a, Fig. 1) that was isolated from the Thai mangrove endophytic fungus Phomopsis sp. xy21. Their structural differences were the different substitutes at C-9 and C-13 as well as the different configurations at C-12. To the best of our knowledge, penibishexahydroxanthone A is the first homodimer of a highly oxidized hexahydroxanthone with a ketone group at C-9. A model [5] of vascular insufficiency in zebrafish induced by vatalanib (PTK787), a VEGFR tyrosine kinase inhibitor [19], was used to evaluate the effect of penibishexahydroxanthone A on angiogenesis. Intersomitic vessels (ISVs) sprouted and elongated from the dorsal aorta and posterior cardinal vein were considered as intact vessels [5]. Salvianic acid A sodium (SAAS) [20] was used as a positive control. As shown in Fig. 6A, ISVs’ growth was significantly suppressed by pretreatment with PTK787 (0.15 lg/mL) for 3 h in Tg(flk1:EGFP) zebrafish embryos. ISVs’ growth was rescued after incubation with penibishexahydroxanthone A for 24 h in concentrations of 10, 20, and 40 lM. Quantitative analysis showed that penibishexahydroxanthone A rescued the PTK787-induced blood vessel loss in a dose-dependent manner (Fig. 6B). In addition, penibishexahydroxanthone A showed moderate antimicrobial activity with MIC values of 15 lg/mL for Candida albicans, 20 lg/mL for Escherichia coli, and 25 lg/mL for Methicillin-resistant Staphylococcus aureus.
Fig. 6. A: Typical images on inflammatory sites in PTK787-induced blood vessel loss in zebrafish embryos. B: Quantitative analysis showed that penibishexahydroxanthone A (1) rescued PTK787-induced blood vessel loss in a dose-dependent manner. Data was represented as the mean ± SEM (n = 5). ##p < 0.01 vs. the Control group; **p < 0.01 vs. PTK787-induced group. Bar = 1 mm.
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Conclusion
References
Marine fungus Penicillium sp. ZZ486A was isolated from the gut of an octopus. Large culture of strain ZZ486A in BMPM liquid medium produced penibishexahydroxanthone A, the first homodimer of a highly oxidized hexahydroxanthone with a ketone group at C-9. Its structure was elucidated based on the NMR and HRESIMS data, ECD calculation, and X-ray diffraction analysis. Penibishexahydroxanthone A showed in vivo activity in rescuing VRI-induced blood vessel loss in the model of vascular insufficiency in zebrafish.
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Conflict of interest The authors declare no conflict of interest.
Acknowledgments This research work was supported by the National Natural Science Foundation of China (No. 81773587). We thank Dr. Huping Zhu (Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences) for performing the NMR spectrometry and Dr. Xuebing Leng for the test and analysis of the X-ray diffraction.
Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.tetlet.2019.04.034.