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New bioactive pyrrospirones C−I from a marine-derived fungus Penicillium sp. ZZ380
Tetrahedron 74 (2018) 884e891
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New bioactive pyrrospirones CI from a marine-derived fungus Penicillium sp. ZZ380 Tengfei Song a, Mengxuan Chen a, Weiyun Chai a, Zhizhen Zhang a, *, Xiao-Yuan Lian b, ** a b
Ocean College, Zhoushan Campus, Zhejiang University, Zhoushan 316021, PR China College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 17 November 2017 Received in revised form 1 January 2018 Accepted 9 January 2018 Available online 11 January 2018
Seven rare pyrrospirones CI (1e7) as well as 18 known compounds were isolated from a marinederived fungus Penicillium sp. ZZ380. Structures of the new pyrrospirones were elucidated by extensive NMR spectroscopic analyses, HRESIMS data, and Mosher's method. Pyrrospirone D (2) was also confirmed by X-ray diffraction analysis. Pyrrospirone G (5) showed potent activity in inhibiting the proliferation of different glioma cells with IC50 values of 1.06e8.52 mM and pyrrospirones C (1), F (4), and I (7) had antimicrobial activity against the growth of both methicillin-resistant Staphylococcus aureus and Escherichia coli with MIC values of 2.0e5.0 mg/mL. © 2018 Elsevier Ltd. All rights reserved.
Keywords: Penicillium sp. ZZ380 Marine fungus Pyrrospirones Antiproliferative and antibacterial activities Glioma cells Methicillin-resistant Staphylococcus aureus Escherichia coli
1. Introduction Glioma is the most common type of primary malignant brain tumor1 and surgery following by adjuvant radiotherapy and chemotherapy are always considered in all patients.2 Because glioma usually locates at important brain function areas, which makes surgical resection extremely difficult, so chemotherapy has played a more important role in the treatment and prevention of glioma. So far, very few drugs, including temozolomide, carmustine, and lomustine, have been approved for treating glioma. Moreover, most of the current anti-glioma drugs are alkylating agents with limited efficacy and serious side effects.3,4 Therefore, there is an urgent need to discover lead compounds for the development of novel anti-glioma drugs. Marine-derived natural products are important sources for the discovery of new anticancer agents.5e7 As a part of our ongoing project for the discovery of novel antiglioma agents from marine microorganisms,8e15 a marine fungus strain ZZ380 was isolated from a sample of wild crab. This marine
* Corresponding author. ** Corresponding author.. E-mail addresses: [email protected] (X.-Y. Lian).
(Z.
https://doi.org/10.1016/j.tet.2018.01.015 0040-4020/© 2018 Elsevier Ltd. All rights reserved.
Zhang),
[email protected]
fungus was assigned as Penicillium sp. ZZ380 (Fig. S1 and Table S1) based on its ITS rDNA sequence result (Fig. S2). A crude extract prepared from the culture of this isolated fungus in BMPM medium showed activity in inhibiting the proliferation of human glioma U87MG cells with an inhibition rate of 55.1%. Chemical investigation of this active extract resulted in the isolation of seven new pyrrospirones CI (1e7) and 18 known compounds (8e25) (Fig. 1 and Supplementary Data Fig. S3). Herein, we described the isolation and culture of strain ZZ380, the isolation and structural elucidation of new pyrrospirones, and their activities in inhibiting the proliferation of glioma cells and the growth of methicillinresistant Staphylococcus aureus (MRSA), Escherichia coli, and Candia albicans.
2. Results and discussion A large culture of Penicillium sp. ZZ380 was conducted in BMPM liquid medium and a crude extract prepared from the culture was separated by column chromatography, followed by HPLC purification, to afford compounds 1e25. Based on the NMR and HRESIMS data and the comparison to the reported data, 18 known compounds were identified as GKK1032B (8),16 chrysophanol (9), emodin (10),17 citreorosein-3-O-sulphate (11), emodin-3-O-
T. Song et al. / Tetrahedron 74 (2018) 884e891
885
Fig. 1. Structures of compounds 1e8 isolated from Penicillium sp. ZZ380.
sulphate (12),18 methyl 8-hydroxy-6-methyl-9-oxo-9H-xanthene1-carboxylate (13),19 yicathin C (14),20 coniochaetone B (15),21 sclerotinin C (16),22 dihydrocitrinone (17),23 1,9-dihydroxy-3(hydroxymethyl)-10-methoxydibenz[b,e]oxepin-6,11-dione (18),24 moniliphenone (19),25 phenol A (20),26 neocyclocitrinol A (21), neocyclocitrinol C (22), neocyclocitrinol D (23),27 citrinin H1 (24),28 and penicitrinone A (25).29 Compounds 1e7 were identified as new pyrrospirone alkaloids, named as pyrrospirones CI, respectively. The detailed structural elucidations of these new compounds are described as the following. Compounds 1 and 2 have the same molecular formula of C33H43NO5 deduced from their HRESIMS and 13C NMR data. The UV spectra of 1 and 2 showed similar absorption at around 278 nm for a phenyl group. Two exchangeable protons (d 6.49 or 6.60 for a hydroxyl group and d 9.72 or 9.73 for an amide group) were observed in the 1H NMR spectra of 1 and 2. Detailed 1H, 13C, and HSQC NMR spectroscopical interpretation indicated that both 1 and 2 have two carbonyls, six aromatic carbons, two olefin carbons, four quaternary carbons, two oxymethines, one methoxyl, six methines, five methylenes, and five methyls (Tables 1 and 2). The 1H NMR spectra of the benzene rings in 1 and 2 did not display a simple A2B2 type coupling pattern but rather complex splitting patterns due to the inequality of H-23/H-27 and H-24/H-26 (Table 2). Further NMR analyses including HMBC correlations (Fig. 2) revealed that 1 and 2 have a spiro ring system and are analogues of pyrrospirones A (1a) and B (2a)30 with a methyl at C-12 and a methoxyl at C-19. The relative stereochemistry of 1 was confirmed by a combination of NOE correlations and coupling constants.30 The b-orientation of H-5, H-7, H-9, H-13, H-17, and H3-32 was indicated by NOE correlations: bH-17 (d 5.26) with H-13 (d 3.88), H-16b (d 2.66), and H3-32 (d 1.83); H-7 (d 2.25) with H-5 (d 1.13), H-9 (d 4.86), H-13, and H3-32; H-5 with H-9; H-9 with H-13; H-13 with H3-32 (Fig. 3). NOE correlations of OH-17 (d 6.49) with H-16a (d 1.86), H3-30 (d 1.36) with aH-1(d 1.90), H-2 (d 1.78), H-4 (d 2.02), and H-8 (d 3.53) suggested a a-orientation for these protons. The large coupling constants of 3J4,5 (11.2 Hz) and 3J7,8 (13.6 Hz), together with above the NOE information, supported the trans-juncture for A/B and B/C rings. The NOESY spectrum of 1 also showed NOE correlations (Fig. 3) from H-21b (d 3.29) to NH (d 9.72) and H-27 (d 7.33), from H-
Table 1 13 C NMR data of pyrrospirones CI (1e7, 125 MHz, in pyridine-d5). C
1
2
3
4
3a
4a
5
6
7
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 OAc-17
49.3 28.5 46.1 27.9 61.6 42.3 53.4 46.5 86.6 138.8 126.8 51.6 51.7 201.2 59.9 49.9 71.6 176.8 93.2 36.4 46.0 128.8 133.4 124.9 158.9 120.3 133.7 23.4 20.3 17.1 21.5 27.4 49.4 e
48.9 28.5 46.1 28.0 60.8 42.4 52.3 48.5 89.9 136.7 132.3 48.2 51.6 200.1 59.5 46.3 70.8 177.9 93.6 38.8 46.0 128.6 133.4 124.8 160.0 118.6 135.0 23.3 20.3 16.7 21.5 21.0 49.4 e e
49.4 28.5 46.1 27.9 61.7 42.3 53.4 46.4 86.7 138.8 126.8 51.5 51.9 201.3 60.4 50.5 71.7 176.2 88.5 43.9 47.7 129.6 133.3 124.9 158.8 120.4 133.7 23.3 20.3 17.1 21.4 27.3 e e e
49.0 28.5 46.1 27.9 60.7 42.3 52.4 48.5 90.0 136.7 132.3 48.0 51.7 200.2 60.0 47.0 70.8 177.2 88.7 46.3 47.6 129.4 133.2 124.7 159.8 118.7 134.9 23.2 20.2 16.6 21.3 20.7 e e e
49.2 28.5 46.0 27.9 61.5 42.3 53.1 46.0 86.5 140.2 125.6 49.9 51.7 200.1 59.7 47.7 74.9 175.5 88.6 43.5 45.1 129.6 133.3 124.9 158.8 120.4 133.9 23.4 20.3 17.0 21.3 26.6 e 170.6 21.3
48.8 28.5 46.1 28.0 60.7 42.4 52.1 48.2 89.8 138.1 130.1 46.5 51.8 199.5 59.7 42.2 73.1 176.5 88.8 46.0 47.7 129.4 133.3 124.8 159.8 118.6 135.1 23.3 20.2 16.6 21.5 21.2 e 170.6 21.3
48.7 28.5 46.1 28.0 61.0 42.3 52.0 45.4 86.8 141.6 126.9 60.6 51.9 202.1 59.7 56.5 208.9 175.8 88.9 42.4 47.4 129.6 133.1 124.6 158.8 120.1 133.7 23.3 20.4 17.0 21.0 23.6 e e e
49.2 28.6 46.2 28.1 61.3 42.4 52.9 47.0 88.3 138.4 133.1 46.4 52.8 202.6 60.4 36.6 41.6 176.7 88.5 45.1 47.9 129.7 133.3 124.8 159.2 119.8 134.3 23.4 20.3 16.9 21.1 28.0 e e e
49.0 28.6 46.2 28.0 61.2 42.5 52.6 46.7 88.3 138.5 129.5 51.4 51.1 200.2 62.2 132.4 140.7 174.0 88.6 43.4 47.8 129.5 133.3 124.8 159.2 119.3 134.3 23.3 20.4 16.9 21.1 28.6 e e e
e
23 (d 7.23) to H-20a (d 3.13) and H-21a (d 2.97), from H-24 (d 7.11) to H-8, and from H-20b (d 1.90) to H3-33 (d 3.36), confirming that the relative configuration of the spiro ring system is as shown.30 Similarly, the relative stereochemistry of 2 was also established
886
T. Song et al. / Tetrahedron 74 (2018) 884e891
Table 2 1 H NMR data of pyrrospirones CF (1e4, 500 MHz, in pyridine-d5, J ¼ Hz). H
1
1a 1b 2 3a 3b 4 5 7 8 9 11 13 16a 16b 17 20
1.90, 0.76, 1.78, 1.72, 0.54, 2.02, 1.13, 2.25, 3.53, 4.86, 6.03, 3.88, 1.86, 2.66, 5.26, 1.89, 3.13, 2.97, 3.29, 7.23, 7.11, 7.04, 7.33, 0.91, 1.21, 1.36, 1.91, 1.83, 3.36, 6.49, e 9.72, e
21 23 24 26 27 28 29 30 31 32 33 OH-17 OH-19 NH OAc-17
2 m; t (12.0) m m; q (11.9) m dd (11.2, 7.5) d (13.6) m dd (7.5, 6.1) br s d (6.0) t (12.8, 10.9); dd (12.8, 5.5) dd (10.9, 5.5) d (14.6); d (14.6) d (13.2); d (13.2) dd (8.1, 2.2) dd (8.1, 2.5) dd (8.5, 2.5) dd (8.5, 2.2) d (6.3) d (6.3) s s s s br s s
3
1.90, 0.71, 1.74, 1.70, 0.54, 1.96, 1.04, 1.99, 3.09, 4.59, 5.87, 4.08, 2.08, 3.41, 3.50, 1.90, 3.43, 3.03, 3.31, 7.34, 7.16, 7.13, 7.36, 0.89, 1.17, 1.22, 1.88, 1.80, 3.34, 6.60, e 9.73, e
m; t (12.1) m m; q (12.2) m dd (11.3, 8.1) d (13.5) m dd (8.1, 5.3) br s d (9.5) dd (12.5, 4.1); t (12.5, 12.5) m d (14.7); d (14.7) d (13.2); d (13.2) dd (8.0, 2.2) dd (8.0, 2.6) dd (8.6, 2.6) dd (8.6, 2.2) d (6.3) d (6.3) s s s s d (5.8) s
Fig. 2. 1H-1H COSY and key HMBC correlations of pyrrospirones C (1) and D (2).
by the coupling constants and NOE correlations as depicted in Fig. 3. Since compound 2 developed suitable crystals from MeOH, an X-ray diffraction analysis was carried out (Fig. 4), which confirmed the proposed structure based on the analysis of spectroscopic data. Compounds 1 and 2 have different configurations at C-17, which were assigned by the Mosher ester NMR method. Treatment of 1
4
1.90, 0.75, 1.77, 1.70, 0.54, 2.03, 1.13, 2.26, 3.54, 4.88, 5.99, 3.89, 1.84, 2.48, 5.26, 2.08, 3.56, 3.20, 3.49, 7.29, 7.13, 7.07, 7.44, 0.90, 1.22, 1.36, 1.90, 1.82, e 6.40, 7.95, 9.79, e
m; t (12.1) m m; q (12.2) m dd (11.2, 7.7) d (13.6) m dd (7.7, 5.8) br s d (5.9) t (12.1, 11.0); dd (12.7, 5.4) m d (14.0); d (14.0) d (13.2); d (13.2) dd (8.2, 2.0) dd (8.2, 2.3) dd (8.6, 2.3) dd (8.6, 2.0) d (6.5) d (6.1) s s s br s s s
1.86, 0.69, 1.70, 1.67, 0.54, 1.92, 1.02, 1.96, 3.04, 4.57, 5.78, 4.02, 1.81, 3.32, 3.39, 2.06, 3.81, 3.21, 3.46, 7.37, 7.15, 7.13, 7.44, 0.87, 1.15, 1.20, 1.84, 1.72, e 6.40, 7.97, 9.72, e
3a m; t (12.1) m m; q (12.0) m dd (11.3, 8.1) d (13.1) m dd (8.1, 5.8) br s d (9.5) dd (12.1, 4.1); t (12.1, 12.1) m d (14.5); d (14.5) d (13.3); d (13.3) dd (8.2, 2.0) dd (8.2, 2.5) dd (8.6, 2.5) dd (8.6, 2.0) d (6.3) d (6.3) s s s d (5.7) s s
1.88, m; 0.74, t (12.0) 1.77, m 1.70, m; 0.55, q (12.1) 2.01, m 1.10, dd (11.3, 7.5) 2.15, d (13.6) 3.45, m 4.85, dd (7.5, 6.0) 5.59, br s 3.91, d (6.0) 1.49, t (12.6, 11.0); 2.40, dd (12.6, 5.2) 6.35, dd (11.0, 5.2) 2.00, d (14.1); 3.49, d (14.1) 3.22, d (13.3); 3.52, d (13.3) 7.29, dd (8.0, 2.0) 7.12, dd (8.0, 2.5) 7.05, dd (8.6, 2.5) 7.42, dd (8.6, 2.0) 0.91, d (6.3) 1.22, d (6.3) 1.33, s 1.91, s 1.56, s e e 8.05, s 10.00, s 1.99, s
with (R)-a-methoxy-a-(trifluoromethyl) phenylacetyl chloride (RMTPA-Cl) or S-MTPA-Cl gave S-MTPA ester (1c) or R-MTPA ester (1d). The 1H NMR chemical shift differences (DdS-R, Fig. 5) between 1c and 1d in negative value for H-32 and positive values for H-16 and H-20 were observed, indicating a 17R configuration for 1.30 In the same way, treatment of 2 with R-MTPA-Cl or S-MTPA-Cl gave SMTPA ester 2c or R-MTPA ester 2d. The positiveDdS-R value for H-11 and negativeDdS-R values for H-16 and H-20 were suggestive of a 17S configuration for 2. The different configurations at C-17 in 1 and 2 were also differentiated by the significantly different 13C NMR chemical shifts for C-8 to C-12, C-16, C-20, C-32 (Table 1) and the different 1H NMR chemical shifts and coupling patterns for H-16 and H-17 (Table 2). Based on the foregoing evidence, compounds 1 and 2 were identified as new members of pyrrospirones A (1a) and B (2a), named as pyrrospirones C (1) and D (2). The assignments of 13 C and 1H NMR data (Tables 1 and 2) were made based on the analysis of HSQC, 1H-1H COSY, HMBC (Fig. 2), and NOE correlations (Fig. 3).
Fig. 3. Key NOE correlations of pyrrospirones C (1) and D (2).
T. Song et al. / Tetrahedron 74 (2018) 884e891
Fig. 4. X-ray crystal structure of pyrrospirone D (2).
Fig. 5. DdS-R values for the MTPA esters (1c, 1d, 2c, and 2d).
Compounds 3 and 4 were originally isolated as a mixture, which was not able to be separated by HPLC using different HPLC columns. In order to separate 3 and 4, the mixture was acetylated and then separated by HPLC to give 3a and 4a, which were in return hydrolyzed using 3N LiOH to produce 3 and 4. The HRESIMS and 13C NMR data showed that 3 and 4 also have the same molecular formula of C32H41NO5, a CH2 unit less than 1 and 2. The 13C NMR data (Table 1) of 3 and 4 bore a close resemblance to those of 1 and 2, respectively, except for the different chemical shifts for C-19 to C-21 due to the absence of a methoxy at C-19 in 3 and 4, implying that 3 and 4 were the analogues of 1 and 2 without the methoxy at C-19. Just like 1 and 2, the structural difference between 3 and 4 is also due to the different configurations at C-17, which was indicated by the different 13C NMR chemical shifts for C-8 to C-12, C-16, C-20, C32 and the different 1H NMR chemical shifts and the coupling patterns for H-16 and H-17 (Tables 1 and 2). Compounds 3 and 4 were thus elucidated as new alkaloids, named as pyrrospirones E (3) and F (4). The UV, 1H and 13C spectra of 5 suggested that 5 is also an analogue of pyrrospirone alkaloids. Its HRESIMS spectrum showed [MþH]þ ion at m/z 518.2896 and [MþNa]þ ion at m/z 540.2722, corresponding to a molecular formula C32H39NO5, two protons less than that of 3. Detailed comparison of the NMR data of 5 with those of 3 suggested that the structural difference between 5 and 3 was that the oxymethine (dC 71.7; dH 5.26) at C-17 in 3 was replaced by a carbonyl group (dC 208.9) in 5. The significant divergence in 13C NMR chemical shifts (Table 1) for C-10, C-12, C-16, C-17, and C-32 between 5 and 3 were observed because of the different substitutes at C-17 in 5 and 3. Compound 5 was thus identified as a new compound, named as pyrrospirone G. The 13C and 1H NMR data of 5 were listed in Tables 1 and 3, respectively. Compound 6 is also a pyrrospirone alkaloid based on the UV and NMR spectroscopic analyses. Its molecular formula C32H41NO4 was deduced from its HRESIMS ions at m/z 504.3118 for [MþH]þ and 526.2927 for [MþNa]þ, 16 mass units less than that of 3. Further NMR comparison indicated that 6 differs from 3 only in the substitute at C-17. In the NMR spectra of 3, the signals for an oxymethine (dC 71.7; dH 5.26) was replaced by the signals for a methylene (dC 41.6; dH 1.45, 2.47) in 6. Therefore, 6 was assigned as
887
an analogue of pyrrospirone E (3) with a methylene at C-17. Compound 6 is a new alkaloid, named as pyrrospirone H, and its 13C and 1 H NMR data were showed in Tables 1 and 3. The molecular formula C32H39NO4 for 7, established by its HRESIMS ions at m/z 502.2952 of [MþH]þ and 524.2767 of [MþNa]þ, is two protons less than that of 6. Compared to 6, the NMR spectra of 7 showed the existence of an additional pair of double bond (dC 132.4, 140.7; dH 5.26, d, 9.2 Hz, 5.47, d, 9.2 Hz) without the two methylenes (dC 36.6, 41.6) at C-16 and C-17 in 6. Accordingly, the structure of 7 was elucidated as an analogue of pyrrospirone H (6) with a double bond at C-16 and C-17. Compound 7 is also a new pyrrospirone alkaloid, named as pyrrospirone I. The 13 C and 1H NMR data of 7 see Tables 1 and 3. Pyrrospirones A (1a) and B (1b), as unique alkaloids with a spiro ring system and a new type of pyrrocidines,30 were previously isolated from an endophytic fungus Neonectria ramulariae Wollenw KS-246. Although pyrrospirones CI (1e7) were related to pyrrospirones A (1a) and B (1b), their structures are different in the substituent at C-12 with a methyl for 1e7 and a hydrogen for 1a and 1b. It was reported that pyrrospirones A (1a) and B (1b) could biosynthetically originate from pyrrocidine B (26).31 Therefore, it is suggested herein that pyrrospirones CI (1e7) might derive from GKK1032A1 (27) or GKK1032A2 (28). The possible biosynthetic origin of pyrrospirones CI (1e7) has been proposed in Fig. 6. Pyrrospirones CI (1e7) could be the results from the unusual 6endo-tet electrophilic cyclization of the epoxide derivatives (29, 30) of GKK1032A1 (27) or GKK1032A2 (28) after oxidation of the vinyl group. To the best of our knowledge, this study is the first report of this type of pyrrospirones CI (1e7). Pyrrospirones CI (1e7) were assayed for their activity in inhibiting the proliferation of glioma U87MG, U251, SHG44, and C6 cells by sulforhodamine B (SRB) assay. Doxorubicin (DOX, a chemotherapeutic drug)32 was used as a positive control. The results (Table 4) indicated that pyrrospirone G (5) had good activity with IC50 values of 1.06e8.52 mM, as compared to the activity of the positive control DOX with IC50 values of 0.47e8.03 mM. Pyrrospirones CF (1e4), H (6), and I (7) showed moderate activity with IC50 values of 7.44e26.64 mM. Pyrrospirones CI (1e7) were also evaluated for their activity in suppressing the growth of MRSA, E. coli, and C. albicans by using micro broth dilution method. Gentamicin (an antibiotic against both Gram-positive and Gram-negative bacteria) and amphotericin B (an antifungal drug) were used as positive controls. It has been found (Table 4) that pyrrospirones CF (1e4), H (6), and I (7) showed activity against both MRSA and E. coli (MIC: 2.0e19.0 mg/ mL) with pyrrospirone F (4) as the most active compound with MIC values of 2.0e3.0 mg/mL. None of the seven tested alkaloids showed antifungal activity. 3. Conclusion Marine-derived fungi of the genus Penicillium are able to synthesize both previously known and novel compounds with diverse structures and bioactivities.33 In this study, a marine crabassociated fungus Penicillium sp. ZZ380 was found to produce new pyrrospirones CI (1e7) as well as 18 known compounds with different structural classes. The structures of the new pyrrospirones were determined based on their NMR and HRESIMS data as well as the Mosher and X-ray diffraction methods. To the best of our knowledge, this study is the first report of this type of pyrrospirone alkaloids with both a spiro junction and a methyl at C-12. Bioactive assay demonstrated that pyrrospirone G (5) showed potent activity against the proliferation of four different glioma cells with IC50 values of 1.06e8.52 mM and that pyrrospirones C (1), F (4), and I (7) had antimicrobial activity in suppressing the growth of both MRSA
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T. Song et al. / Tetrahedron 74 (2018) 884e891
Table 3 1 H NMR data of pyrrospirones GI (5e7, 500 MHz, in pyridine-d5, J ¼ Hz). H
4a
1a; 1b 2 3a; 3b 4 5 7 8 9 11 13 16a 16b 17 20
1.84, 1.70, 1.67, 1.93, 1.02, 1.91, 3.02, 4.57, 5.26, 4.04, 1.75, 3.12, 4.51, 2.11, 3.81, 3.24, 3.49, 7.39, 7.17, 7.15, 7.44, 0.89, 1.16, 1.19, 1.78, 1.64, 8.05, 9.93, 1.96,
21 23 24 26 27 28 29 30 31 32 OH-19 NH OAc-17
5 m; 0.67, t (12.3) m m; 0.53, q (12.0) m dd (11.3, 8.1) d (13.0) m dd (8.1, 5.2) br s d (9.6) dd (12.9, 4.7); t (12.9, 12.9) dd (12.9, 4.7) d (14.3); d (14.3) d (13.1); d (13.1) dd (8.2, 2.2) dd (8.2, 2.5) dd (8.5, 2.5) dd (8.5, 2.2) d (6.3) d (6.3) s s s br s s s
1.83, 1.75, 1.72, 1.99, 1.16, 2.11, 3.53, 4.92, 5.09, 4.00, 2.59, 2.71, e 2.04, 3.48, 3.23, 3.48, 7.32, 7.17, 7.11, 7.40, 0.90, 1.24, 1.31, 1.77, 1.89, 8.19, 10.3, e
6 m; 0.71, t (11.9) m m; 0.54, q (12.1) m dd (11.5, 8.1) d (13.2) m dd (8.1, 6.4) br s d (6.3) d (16.3); d (16.3) d (14.8); d (14.8) d (13.4); d (13.4) dd (8.1, 2.2) dd (8.1, 2.5) dd (8.6, 2.5) dd (8.6, 2.2) d (6.3) d (6.3) s s s br s s
7
1.88, 1.74, 1.70, 1.98, 1.05, 2.01, 3.25, 4.70, 4.98, 3.81, 1.16,
m; 0.71, t (12.0) m m; 0.53, q (12.2) m dd (11.4, 7.9) d (13.9) m dd (7.9, 5.6) br s d (7.6) m; 2.03, m
1.85, 1.72, 1.69, 1.97, 1.12, 2.05, 3.31, 4.79, 5.10, 4.13, 5.26,
m; 0.70, t (12.3) m m; 0.54, q (12.0) m dd (11.3, 8.1) d (13.4) m dd (8.1, 5.9) br s d (7.7) d (9.2)
1.45, 2.01, 3.62, 3.23, 3.50, 7.32, 7.13, 7.09, 7.46, 0.90, 1.19, 1.28, 1.83, 1.46, 8.01, 9.72, e
m; 2.47, m d (14.1); d (14.1) d (13.3); d (13.1) dd (8.0, 2.2) dd (8.0, 2.5) dd (8.6, 2.5) dd (8.6, 2.2) d (6.3) d (6.3) s s s br s s
5.47, 2.22, 3.63, 3.27, 3.52, 7.34, 7.16, 7.14, 7.45, 0.90, 1.22, 1.27, 1.81, 1.65, 8.13, 9.92, e
d (9.2) d (14.3); d (14.3) d (13.2); d (13.2) dd (8.0, 2.0) dd (8.0, 2.3) dd (8.3, 2.3) dd (8.3, 2.0) d (6.3) d (6.3) s s s br s s
Fig. 6. Possible biosynthetic origin of pyrrospirones CI (1e7).
Table 4 Activities of pyrrospirones CI (1e7) against the proliferation of glioma cells and the growth of MRSA and E. coli. Compounds
1 2 3 4 5 6 7 DOX Gentamicin
Glioma cells (IC50: mM)
Bacteria (MIC: mg/mL)
U87MG
U251
SHG44
C6
MRSA
E. coli
12.15 ± 0.61 9.95 ± 0.50 16.24 ± 0.68 12.44 ± 0.81 1.06 ± 0.05 12.89 ± 0.64 13.67 ± 0.53 1.20 ± 0.06 e
22.12 ± 1.11 23.39 ± 1.27 26.64 ± 2.14 22.82 ± 1.15 1.28 ± 0.06 23.92 ± 1.20 13.46 ± 0.76 8.03 ± 1.20 e
10.03 ± 0.10 13.74 ± 0.55 15.76 ± 1.20 8.93 ± 0.76 2.14 ± 0.11 13.02 ± 0.79 7.44 ± 0.97 0.67 ± 0.10 e
13.87 ± 1.26 14.56 ± 0.95 21.03 ± 3.15 14.87 ± 1.66 8.52 ± 1.01 23.24 ± 2.24 19.18 ± 2.11 0.47 ± 0.10 e
4.0 12.0 10.0 2.0 >50 19.0 4.0 e 0.5
5.0 3.0 11.0 3.0 >50 4.0 2.0 e 1.0
T. Song et al. / Tetrahedron 74 (2018) 884e891
and E. coli with MIC values of 2.0e5.0 mg/mL. 4. Experimental section 4.1. General experimental procedures UV spectra were recorded on a METASH UV-8000 (Shanghai METASH Instruments Co. Ltd., China). Optical rotation and ECD spectra were measured on a JASCO DIP-370 digital polarimeter and a JASCO J715 spectropolarimeter (JASCO, Japan), respectively. HRESIMS data were acquired on an Agilent 6230 TOF LC/MS spectrometer. NMR spectra were acquired on a Bruker 500 spectrometer using standard pulse programs and acquisition parameters. Chemical shifts were expressed in d (ppm) and referred to the NMR solvent used. Octadecyl-functionalized silica gel (ODS, Cosmosil 75C18-Prep, Nacalai Tesque Inc., Japan) was used for column chromatography. HPLC separation was performed on Agilent 1260 HPLC system (HPLC A), or CXTH 3000 prepared HPLC system (HPLC B, Beijing Chuangxintongheng Science & Technology Co. Ltd., China), or SHIMADZU 20A prepared HPLC system (HPLC C). All solvents used for this study were purchased from the Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). Human glioma U87MG (JDS2568), U251 (XB-0439), SHG44 (RX-J150) cells and rat glioma C6 (XB-003) cells were obtained from the Cell Bank of the Chinese Academy of Sciences. Methicillin-resistant Staphylococcus aureus (MRSA) ATCC 43300, Escherichia coli ATCC 25922, and Candida albicans were gifts from Drs. Zhongjun Ma, Pinmei Wang, and Bin Wu, respectively. Doxorubicin (DOX, >98.0%) was ordered from Sigma-Aldrich, and gentamicin (99.6%) and amphotericin B (>95.0%) from Meilune Biotechnology Co. Ltd. (Dalian, China). 4.2. Isolation and taxonomic identity of strain ZZ380 Strain ZZ380 was isolated from a sample of wild crab (Pachygrapsus crassipes), which had been living on the seaside rocks of Putuo Mountain (Zhoushan, China). A voucher sample (PCPT201603) was authenticated by one of the authors (T. Song) and deposited in the Laboratory of Institute of Marine Biology, Ocean College, Zhoushan Campus, Zhejiang University, China. Briefly, a crab was dipped in 75% ethyl alcohol for 10 s and then mashed in sterile water. The mash was centrifugated (5000 r/min, 5 min) to produce a supernatant. The supernatant (1 mL) was diluted into 10%, 1%, and 0.1% (V/V) with sterile water. The 0.1% diluted suspension of 200 mL was covered on the surface of PDA (Potato Dextrose Agar) solid medium in a petri dish and then incubated at 28 C for seven days. The single colony was picked and then transferred to another PDA dish. After several days of growth at 28 , the single colony (strain ZZ380, Fig. S1) that grew well was transferred onto PDA slants, which were stored at 4 C for further use. The strain ZZ380 was identified using ITS rDNA sequence analysis by Legenomics (Hangzhou, China) and its DNA sequence using BLAST (nucleotide sequence comparison) was compared to the GenBank database. The ITS rDNA sequence of strain ZZ380 has been deposited in GenBank (accession number: MF503479). A voucher strain (Penicillium sp. ZZ380) was preserved at the Laboratory of Institute of Marine Biology, Ocean College, Zhoushan Campus, Zhejiang University, China.
889
which contains 250 mL of BMPM medium (glucose 20 g, glycerol 20 g, soy flour 10 g, cotton seed embryo meal 10 g, (NH4)2SO4 1 g, CaCO3 10 g, tap water 1000 mL). All flasks were incubated at 28 C for 30 days in stationary state. A total of 40.0 L culture was made for this study. 4.4. Extraction and isolation of compounds 125 The 40.0 L culture was centrifuged to give fermentation broth and mycelia. The mycelia were extracted with MeOH three times to afford a mycelia extract after the removal of MeOH. The fermentation broth was partitioned with EtOAc three times to give an EtOAc extract after the removal of EtOAc. A mixture of the mycelia extract and the EtOAc extract was fractionated on a ODS column eluted successively with 80% MeOH, 90% MeOH, and 100% MeOH to give six fractions (Fr. 1 to Fr. 6) based on the results of TLC analysis. Each fraction was further separated by ODS column, following by HPLC purification, to give compounds 1e25. The isolation procedure and separation conditions were depicted in Fig. S4. 4.4.1. Pyrrospirone C (1) Colorless amorphous powder; molecular formula C33H43NO5; [a]25 D þ5.5 (c 0.50, MeOH); ECD (10 mg/L, MeOH) lmax (Dε) 211 (þ150.1), 233 (23.0), 288 (26.4) nm; UV (MeOH) lmax (log ε) 205 (4.49), 228 (4.01), 279 (3.39) nm; 13C NMR data (125 MHz, in pyridine-d5), see Table 1, 1H NMR data (500 MHz, in pyridine-d5), see Table 2; HRESIMS m/z [MþH]þ 534.3217 (calcd for C33H44NO5, 534.3219), [MþNa]þ 556.3030 (calcd for C33H43NNaO5, 556.3039), and [MþK]þ 572.2777 (calcd for C33H43KNO5, 572.2778). 4.4.2. Pyrrospirone D (2) Colorless amorphous powder; molecular formula C33H43NO5; [a]25 D þ13.6 (c 0.38, MeOH); ECD (10 mg/L, MeOH) lmax (Dε) 212 (þ36.2), 233 (þ0.86), 280 (4.4) nm; UV (MeOH) lmax (log ε) 207 (4.40), 227 (4.08), 277 (3.38) nm; 13C NMR data (125 MHz, in pyridine-d5), see Table 1, 1H NMR data (500 MHz, in pyridine-d5), see Table 2; HRESIMS m/z [MþH]þ 534.3216 (calcd for C33H44NO5, 534.3219), [MþNa]þ 556.3030 (calcd for C33H43NNaO5, 556.3039), and [MþK]þ 572.2781 (calcd for C33H43KNO5, 572.2778). 4.4.3. Pyrrospirone E (3) Colorless amorphous powder; molecular formula C32H41NO5; [a]25 D þ80.3 (c 0.15, MeOH); ECD (10 mg/L, MeOH) lmax (Dε) 212 (þ73.5), 235 (11.5), 288 (12.6) nm; UV (MeOH) lmax (log ε) 205 (4.19), 230 (3.68), 279 (3.05) nm; 13C NMR data (125 MHz, in pyridine-d5), see Table 1, 1H NMR data (500 MHz, in pyridine-d5), see Table 2; HRESIMS m/z [MþH]þ 520.3064 (calcd for C32H42NO5, 520.3063), [MþNa]þ 542.2879 (calcd for C32H41NNaO5, 542.2882), and [MþK]þ 558.2610 (calcd for C32H41KNO5, 558.2622).
4.3. Large culture of strain ZZ380
4.4.4. Pyrrospirone F (4) Colorless amorphous powder; molecular formula C32H41NO5; [a]25 D þ99.9 (c 0.20, MeOH), ECD (10 mg/L, MeOH) lmax (Dε) 213 (þ138.2), 237 (þ5.7), 281 (16.4) nm; UV (MeOH) lmax (log ε) 205 (4.19), 228 (3.77), 280 (2.97) nm; 13C NMR data (125 MHz, in pyridine-d5), see Table 1, 1H NMR data (500 MHz, in pyridine-d5), see Table 2; HRESIMS m/z [MþH]þ 520.3057 (calcd for C32H42NO5, 520.3063), [MþNa]þ 542.2875 (calcd for C32H41NNaO5, 542.2882), and [MþK]þ 558.2622 (calcd for C32H41KNO5, 558.2622).
Colonies of the strain ZZ380 from the PDA slants were inoculated into a 500 mL Erlenmeyer flask containing 250 mL of PDB (Potato Dextrose Broth) medium and then incubated at 28 C for three days on a rotary shaker (180 rpm) to produce seed broth. The seed broth (5 mL) was inoculated into a 500 mL Erlenmeyer flask,
4.4.5. Pyrrospirone G (5) Colorless amorphous powder; molecular formula C32H39NO5; [a]25 D þ24.6 (c 0.50, MeOH); ECD (10 mg/L, MeOH) lmax (Dε) 212 (þ164.5), 235 (94.7), 280 (þ3.6) nm; UV (MeOH) lmax (log ε) 205 (4.23), 227 (3.95), 277 (2.87) nm; 13C NMR data (125 MHz, in
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pyridine-d5), see Table 1, 1H NMR data (500 MHz, in pyridine-d5), see Table 3; HRESIMS m/z [MþH]þ 518.2896 (calcd for C32H40NO5, 518.2906), [MþNa]þ 540.2722 (calcd for C32H39NNaO5, 540.2726), and [MþK]þ 556.2455 (calcd for C32H39KNO5, 556.2465). 4.4.6. Pyrrospirone H (6) Colorless amorphous powder; molecular formula C32H41NO4; [a]25 D þ18.5 (c 0.50, MeOH); ECD (10 mg/L, MeOH) lmax (Dε) 211 (þ190.4), 235 (24.7), 286 (26.6) nm; UV (MeOH) lmax (log ε) 207 (4.36), 227 (3.86), 276 (2.90) nm; 13C NMR data (125 MHz, in pyridine-d5), see Table 1, 1H NMR data (500 MHz, in pyridine-d5), see Table 3; HRESIMS m/z [MþH]þ 504.3118 (calcd for C32H42NO4, 504.3114), [MþNa]þ 526.2927 (calcd for C32H41NNaO4, 526.2933), and [MþK]þ 542.2672 (calcd for C32H41KNO4, 542.2673). 4.4.7. Pyrrospirone I (7) Colorless amorphous powder; molecular formula C32H39NO4; [a]25 D þ118.2 (c 0.10, MeOH); ECD (10 mg/L, MeOH) lmax (Dε) 211 (þ87.9), 232(32.7), 283 (9.7) nm; UV (MeOH) lmax (log ε) 206 (4.22), 278 (3.22) nm; 13C NMR data (125 MHz, in pyridine-d5), see Table 1, 1H NMR data (500 MHz, in pyridine-d5), see Table 3; HRESIMS m/z [MþH]þ 502.2952 (calcd for C32H40NO4, 502.2957) and [MþNa]þ 524.2767 (calcd for C32H39NNaO4, 524.2777). 4.5. Acetylation of 3 and 4
HPLC (column: Zorbax SB-C18, 250 4.6 mm, 5 mm; mobile phase: MeOH/H2O, 92/8; flow rate: 1.3 mL/min). Two compounds of SMTPA ester of pyrrospirone C (1c, 5.6 mg, tR 16.1 min) and R-MTPA ester of pyrrospirone C (1d, 5.8 mg, tR 15.8 min) were obtained for NMR analysis. Through the same process, MTPA esterification of pyrrospirone D (2, 5.0 mg) gave S-MTPA ester of pyrrospirone D (2c, 2.7 mg, tR 17.0 min) and R-MTPA ester of pyrrospirone D (2d, 2.4 mg, tR 16.7 min). 4.8. X-ray crystallographic analysis of pyrrospirone D (2) X-ray diffraction analysis was carried out on an Xcalibur Atlas Gemini Ultra diffractometer (Agilent Technologies) with Cu Ka radiation (l ¼ 1.541 84 Å) at 293 K. The structures were solved by direct methods (SHELXS-2008) and refined with full-matrix leastsquares on F2 (SHELXL-2015). All non-hydrogen atoms were refined anisotropically, and all hydrogen atoms were placed in idealized positions and refined as riding atoms with the relative isotropic parameters. Crystallographic data for 2 have been deposited at the Cambridge Crystallographic Data Centre (CCDC Number: 1580841). Copies of the data can be obtained, free of charge, through application to the Director, CCDC, 12 Union Road, Cambridge CB2 1EZ, UK (fax: þ 44 (0)1223-336033, or e-mail: http://[email protected]. uk/[email protected]).
The mixture (25 mg) of 3 and 4 dissolved in Ac2O/pyridine (1: 2, 3 mL) was allowed to stand at room temperature for 24 h. After addition of 1 mL of water, the resulting mixture was dried in vacuo to give a residue. This residue was applied to an ODS column eluted successively with water and MeOH. The MeOH fraction was further separated by Agilent 1260 HPLC (column: Agilent Zorbax SB-C18, 250 9.2 mm, 5 mm; mobile phase: ACN/H2O, 87/13; flow rate: 2.0 mL/min) to give acetylation products (3a and 4a) of 3 and 4.
4.8.1. Crystallographic data of pyrrospirone D (2) Colorless needle crystals, C33H43NO5, Mr 533.70, monoclinic, space group P 1 21 1, a ¼ 6.74932 (14) Å, b ¼ 13.8982(2) Å, c ¼ 16.8690(3) Å, a ¼ 90.00 , b ¼ 100.4959(18) , g ¼ 90.00 , V ¼ 1555.90 (5) Å3, Z ¼ 2, D ¼ 1.208 g/cm3, crystal size 0.49 0.16 0.13 mm3, F(000) ¼ 612.0, T ¼ 293 K, The final R1 value is 0.0459 (wR2 ¼ 0.1179) for 5090 reflections [I 2s (I)]. CCDC Number: 1580841.
4.5.1. Compound 3a Colorless amorphous powder; molecular formula C34H43NO6; 13 C NMR data (125 MHz, in pyridine-d5), see Table 1, 1H NMR data (500 MHz, in pyridine-d5), see Table 2; HRESIMS m/z [MþH]þ 562.3166 (calcd for C34H44NO6, 562.3169) and [MþNa]þ 584.2984 (calcd for C34H43NNaO6, 584.2988).
4.9. Culture of glioma cells
4.5.2. Compound 4a Colorless amorphous powder; molecular formula C34H43NO6; 13 C NMR data (125 MHz, pyridine-d5), see Table 1, 1H NMR data (500 MHz, pyridine-d5), see Table 3; HRESIMS m/z [MþH]þ 562.3162 (calcd for C34H44NO6, 562.3169) and [MþNa]þ 584.2980 (calcd for C34H43NNaO6, 584.2988). 4.6. Alkaline hydrolysis of 3a and 4a Each (5 mg) of 3a and 4a dissolved in a mixture of MeOH/H2O (3:1, 2 mL) was hydrolyzed by using 1 mL 3 N LiOH in MeOH/H2O (3:1) for 12 h at room temperature. The reaction mixture was neutralized with 3N HCl to pH 6e8 and then concentrated under reduced pressure to give deacetylation products 3 and 4. 4.7. MTPA esterification of pyrrospirones C (1) and D (2) Pyrrospirone C (1, 11 mg) in 2 mL anhydrous pyridine was divided into 2 equal portions. Mosher regent R-MTPA-Cl or SMTPA-Cl (each 50 mL) was added to each portion. Each mixture was stirred at room temperature for 2 h and the reaction was terminated by an addition of 1 mL MeOH. Each resulting mixture was dried in vacuo to give a residue, which was purified by Agilent 1260
Human glioma U87MG cells were cultured in MEM (Minimum Essential Medium, Gibco) with 10% FBS (Fetal Bovine Serum, PAA Laboratories Inc.), human glioma U251 and rat glioma C6 cells in DMEM (Dulbecco's Modified Eagle Medium, Gibco), and human glioma SHG44 in RPMI-1640 Medium (Roswell Park Memorial Institution 1640 Medium, Gibco). All cells were incubated at 37 C in a humidified incubator with 5% CO2. Cells after the third generation were used for the experiment. 4.10. Sulforhodamine B (SRB) assay The SRB assay8,13 was used to evaluate the activities of compounds 1e7 against the proliferation of glioma U87MG, U251, SHG44, and C6 cells. Doxorubicin (DOX) was used as a positive control. 4.11. Antimicrobial assay Micro broth dilution method11 was applied to determine the antimicrobial activities of compounds 1e7 against the growth of MRSA, E. coli, and C. albicans. Gentamicin (an antibiotic against both Gram-positive and Gram-negative bacteria) and amphotericin B (an antifungal drug) were used as positive controls. Conflicts of interest The authors declare no conflict of interest.
T. Song et al. / Tetrahedron 74 (2018) 884e891
Acknowledgments This study was supported by the National Natural Science Foundation of China (81273428, 81773587, and 81773769). We appreciate Mrs. Jianyang Pan (Pharmaceutical Informatics Institute of Zhejiang University) for performing the NMR spectrometry and Dr. Jiyong Liu (Department of Chemistry of Zhejiang University) for performing the X-ray diffraction analysis. Appendix A. Supplementary data Supplementary data related to this article can be found at https://doi.org/10.1016/j.tet.2018.01.015. References 1. 2. 3. 4. 5. 6. 7. 8. 9.
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New bioactive pyrrospirones CI from a marine-derived fungus Penicillium sp. ZZ380 Tengfei Song a, Mengxuan Chen a, Weiyun Chai a, Zhizhen Zhang a, *, Xiao-Yuan Lian b, ** a b
Ocean College, Zhoushan Campus, Zhejiang University, Zhoushan 316021, PR China College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 17 November 2017 Received in revised form 1 January 2018 Accepted 9 January 2018 Available online 11 January 2018
Seven rare pyrrospirones CI (1e7) as well as 18 known compounds were isolated from a marinederived fungus Penicillium sp. ZZ380. Structures of the new pyrrospirones were elucidated by extensive NMR spectroscopic analyses, HRESIMS data, and Mosher's method. Pyrrospirone D (2) was also confirmed by X-ray diffraction analysis. Pyrrospirone G (5) showed potent activity in inhibiting the proliferation of different glioma cells with IC50 values of 1.06e8.52 mM and pyrrospirones C (1), F (4), and I (7) had antimicrobial activity against the growth of both methicillin-resistant Staphylococcus aureus and Escherichia coli with MIC values of 2.0e5.0 mg/mL. © 2018 Elsevier Ltd. All rights reserved.
Keywords: Penicillium sp. ZZ380 Marine fungus Pyrrospirones Antiproliferative and antibacterial activities Glioma cells Methicillin-resistant Staphylococcus aureus Escherichia coli
1. Introduction Glioma is the most common type of primary malignant brain tumor1 and surgery following by adjuvant radiotherapy and chemotherapy are always considered in all patients.2 Because glioma usually locates at important brain function areas, which makes surgical resection extremely difficult, so chemotherapy has played a more important role in the treatment and prevention of glioma. So far, very few drugs, including temozolomide, carmustine, and lomustine, have been approved for treating glioma. Moreover, most of the current anti-glioma drugs are alkylating agents with limited efficacy and serious side effects.3,4 Therefore, there is an urgent need to discover lead compounds for the development of novel anti-glioma drugs. Marine-derived natural products are important sources for the discovery of new anticancer agents.5e7 As a part of our ongoing project for the discovery of novel antiglioma agents from marine microorganisms,8e15 a marine fungus strain ZZ380 was isolated from a sample of wild crab. This marine
* Corresponding author. ** Corresponding author.. E-mail addresses: [email protected] (X.-Y. Lian).
(Z.
https://doi.org/10.1016/j.tet.2018.01.015 0040-4020/© 2018 Elsevier Ltd. All rights reserved.
Zhang),
[email protected]
fungus was assigned as Penicillium sp. ZZ380 (Fig. S1 and Table S1) based on its ITS rDNA sequence result (Fig. S2). A crude extract prepared from the culture of this isolated fungus in BMPM medium showed activity in inhibiting the proliferation of human glioma U87MG cells with an inhibition rate of 55.1%. Chemical investigation of this active extract resulted in the isolation of seven new pyrrospirones CI (1e7) and 18 known compounds (8e25) (Fig. 1 and Supplementary Data Fig. S3). Herein, we described the isolation and culture of strain ZZ380, the isolation and structural elucidation of new pyrrospirones, and their activities in inhibiting the proliferation of glioma cells and the growth of methicillinresistant Staphylococcus aureus (MRSA), Escherichia coli, and Candia albicans.
2. Results and discussion A large culture of Penicillium sp. ZZ380 was conducted in BMPM liquid medium and a crude extract prepared from the culture was separated by column chromatography, followed by HPLC purification, to afford compounds 1e25. Based on the NMR and HRESIMS data and the comparison to the reported data, 18 known compounds were identified as GKK1032B (8),16 chrysophanol (9), emodin (10),17 citreorosein-3-O-sulphate (11), emodin-3-O-
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885
Fig. 1. Structures of compounds 1e8 isolated from Penicillium sp. ZZ380.
sulphate (12),18 methyl 8-hydroxy-6-methyl-9-oxo-9H-xanthene1-carboxylate (13),19 yicathin C (14),20 coniochaetone B (15),21 sclerotinin C (16),22 dihydrocitrinone (17),23 1,9-dihydroxy-3(hydroxymethyl)-10-methoxydibenz[b,e]oxepin-6,11-dione (18),24 moniliphenone (19),25 phenol A (20),26 neocyclocitrinol A (21), neocyclocitrinol C (22), neocyclocitrinol D (23),27 citrinin H1 (24),28 and penicitrinone A (25).29 Compounds 1e7 were identified as new pyrrospirone alkaloids, named as pyrrospirones CI, respectively. The detailed structural elucidations of these new compounds are described as the following. Compounds 1 and 2 have the same molecular formula of C33H43NO5 deduced from their HRESIMS and 13C NMR data. The UV spectra of 1 and 2 showed similar absorption at around 278 nm for a phenyl group. Two exchangeable protons (d 6.49 or 6.60 for a hydroxyl group and d 9.72 or 9.73 for an amide group) were observed in the 1H NMR spectra of 1 and 2. Detailed 1H, 13C, and HSQC NMR spectroscopical interpretation indicated that both 1 and 2 have two carbonyls, six aromatic carbons, two olefin carbons, four quaternary carbons, two oxymethines, one methoxyl, six methines, five methylenes, and five methyls (Tables 1 and 2). The 1H NMR spectra of the benzene rings in 1 and 2 did not display a simple A2B2 type coupling pattern but rather complex splitting patterns due to the inequality of H-23/H-27 and H-24/H-26 (Table 2). Further NMR analyses including HMBC correlations (Fig. 2) revealed that 1 and 2 have a spiro ring system and are analogues of pyrrospirones A (1a) and B (2a)30 with a methyl at C-12 and a methoxyl at C-19. The relative stereochemistry of 1 was confirmed by a combination of NOE correlations and coupling constants.30 The b-orientation of H-5, H-7, H-9, H-13, H-17, and H3-32 was indicated by NOE correlations: bH-17 (d 5.26) with H-13 (d 3.88), H-16b (d 2.66), and H3-32 (d 1.83); H-7 (d 2.25) with H-5 (d 1.13), H-9 (d 4.86), H-13, and H3-32; H-5 with H-9; H-9 with H-13; H-13 with H3-32 (Fig. 3). NOE correlations of OH-17 (d 6.49) with H-16a (d 1.86), H3-30 (d 1.36) with aH-1(d 1.90), H-2 (d 1.78), H-4 (d 2.02), and H-8 (d 3.53) suggested a a-orientation for these protons. The large coupling constants of 3J4,5 (11.2 Hz) and 3J7,8 (13.6 Hz), together with above the NOE information, supported the trans-juncture for A/B and B/C rings. The NOESY spectrum of 1 also showed NOE correlations (Fig. 3) from H-21b (d 3.29) to NH (d 9.72) and H-27 (d 7.33), from H-
Table 1 13 C NMR data of pyrrospirones CI (1e7, 125 MHz, in pyridine-d5). C
1
2
3
4
3a
4a
5
6
7
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 OAc-17
49.3 28.5 46.1 27.9 61.6 42.3 53.4 46.5 86.6 138.8 126.8 51.6 51.7 201.2 59.9 49.9 71.6 176.8 93.2 36.4 46.0 128.8 133.4 124.9 158.9 120.3 133.7 23.4 20.3 17.1 21.5 27.4 49.4 e
48.9 28.5 46.1 28.0 60.8 42.4 52.3 48.5 89.9 136.7 132.3 48.2 51.6 200.1 59.5 46.3 70.8 177.9 93.6 38.8 46.0 128.6 133.4 124.8 160.0 118.6 135.0 23.3 20.3 16.7 21.5 21.0 49.4 e e
49.4 28.5 46.1 27.9 61.7 42.3 53.4 46.4 86.7 138.8 126.8 51.5 51.9 201.3 60.4 50.5 71.7 176.2 88.5 43.9 47.7 129.6 133.3 124.9 158.8 120.4 133.7 23.3 20.3 17.1 21.4 27.3 e e e
49.0 28.5 46.1 27.9 60.7 42.3 52.4 48.5 90.0 136.7 132.3 48.0 51.7 200.2 60.0 47.0 70.8 177.2 88.7 46.3 47.6 129.4 133.2 124.7 159.8 118.7 134.9 23.2 20.2 16.6 21.3 20.7 e e e
49.2 28.5 46.0 27.9 61.5 42.3 53.1 46.0 86.5 140.2 125.6 49.9 51.7 200.1 59.7 47.7 74.9 175.5 88.6 43.5 45.1 129.6 133.3 124.9 158.8 120.4 133.9 23.4 20.3 17.0 21.3 26.6 e 170.6 21.3
48.8 28.5 46.1 28.0 60.7 42.4 52.1 48.2 89.8 138.1 130.1 46.5 51.8 199.5 59.7 42.2 73.1 176.5 88.8 46.0 47.7 129.4 133.3 124.8 159.8 118.6 135.1 23.3 20.2 16.6 21.5 21.2 e 170.6 21.3
48.7 28.5 46.1 28.0 61.0 42.3 52.0 45.4 86.8 141.6 126.9 60.6 51.9 202.1 59.7 56.5 208.9 175.8 88.9 42.4 47.4 129.6 133.1 124.6 158.8 120.1 133.7 23.3 20.4 17.0 21.0 23.6 e e e
49.2 28.6 46.2 28.1 61.3 42.4 52.9 47.0 88.3 138.4 133.1 46.4 52.8 202.6 60.4 36.6 41.6 176.7 88.5 45.1 47.9 129.7 133.3 124.8 159.2 119.8 134.3 23.4 20.3 16.9 21.1 28.0 e e e
49.0 28.6 46.2 28.0 61.2 42.5 52.6 46.7 88.3 138.5 129.5 51.4 51.1 200.2 62.2 132.4 140.7 174.0 88.6 43.4 47.8 129.5 133.3 124.8 159.2 119.3 134.3 23.3 20.4 16.9 21.1 28.6 e e e
e
23 (d 7.23) to H-20a (d 3.13) and H-21a (d 2.97), from H-24 (d 7.11) to H-8, and from H-20b (d 1.90) to H3-33 (d 3.36), confirming that the relative configuration of the spiro ring system is as shown.30 Similarly, the relative stereochemistry of 2 was also established
886
T. Song et al. / Tetrahedron 74 (2018) 884e891
Table 2 1 H NMR data of pyrrospirones CF (1e4, 500 MHz, in pyridine-d5, J ¼ Hz). H
1
1a 1b 2 3a 3b 4 5 7 8 9 11 13 16a 16b 17 20
1.90, 0.76, 1.78, 1.72, 0.54, 2.02, 1.13, 2.25, 3.53, 4.86, 6.03, 3.88, 1.86, 2.66, 5.26, 1.89, 3.13, 2.97, 3.29, 7.23, 7.11, 7.04, 7.33, 0.91, 1.21, 1.36, 1.91, 1.83, 3.36, 6.49, e 9.72, e
21 23 24 26 27 28 29 30 31 32 33 OH-17 OH-19 NH OAc-17
2 m; t (12.0) m m; q (11.9) m dd (11.2, 7.5) d (13.6) m dd (7.5, 6.1) br s d (6.0) t (12.8, 10.9); dd (12.8, 5.5) dd (10.9, 5.5) d (14.6); d (14.6) d (13.2); d (13.2) dd (8.1, 2.2) dd (8.1, 2.5) dd (8.5, 2.5) dd (8.5, 2.2) d (6.3) d (6.3) s s s s br s s
3
1.90, 0.71, 1.74, 1.70, 0.54, 1.96, 1.04, 1.99, 3.09, 4.59, 5.87, 4.08, 2.08, 3.41, 3.50, 1.90, 3.43, 3.03, 3.31, 7.34, 7.16, 7.13, 7.36, 0.89, 1.17, 1.22, 1.88, 1.80, 3.34, 6.60, e 9.73, e
m; t (12.1) m m; q (12.2) m dd (11.3, 8.1) d (13.5) m dd (8.1, 5.3) br s d (9.5) dd (12.5, 4.1); t (12.5, 12.5) m d (14.7); d (14.7) d (13.2); d (13.2) dd (8.0, 2.2) dd (8.0, 2.6) dd (8.6, 2.6) dd (8.6, 2.2) d (6.3) d (6.3) s s s s d (5.8) s
Fig. 2. 1H-1H COSY and key HMBC correlations of pyrrospirones C (1) and D (2).
by the coupling constants and NOE correlations as depicted in Fig. 3. Since compound 2 developed suitable crystals from MeOH, an X-ray diffraction analysis was carried out (Fig. 4), which confirmed the proposed structure based on the analysis of spectroscopic data. Compounds 1 and 2 have different configurations at C-17, which were assigned by the Mosher ester NMR method. Treatment of 1
4
1.90, 0.75, 1.77, 1.70, 0.54, 2.03, 1.13, 2.26, 3.54, 4.88, 5.99, 3.89, 1.84, 2.48, 5.26, 2.08, 3.56, 3.20, 3.49, 7.29, 7.13, 7.07, 7.44, 0.90, 1.22, 1.36, 1.90, 1.82, e 6.40, 7.95, 9.79, e
m; t (12.1) m m; q (12.2) m dd (11.2, 7.7) d (13.6) m dd (7.7, 5.8) br s d (5.9) t (12.1, 11.0); dd (12.7, 5.4) m d (14.0); d (14.0) d (13.2); d (13.2) dd (8.2, 2.0) dd (8.2, 2.3) dd (8.6, 2.3) dd (8.6, 2.0) d (6.5) d (6.1) s s s br s s s
1.86, 0.69, 1.70, 1.67, 0.54, 1.92, 1.02, 1.96, 3.04, 4.57, 5.78, 4.02, 1.81, 3.32, 3.39, 2.06, 3.81, 3.21, 3.46, 7.37, 7.15, 7.13, 7.44, 0.87, 1.15, 1.20, 1.84, 1.72, e 6.40, 7.97, 9.72, e
3a m; t (12.1) m m; q (12.0) m dd (11.3, 8.1) d (13.1) m dd (8.1, 5.8) br s d (9.5) dd (12.1, 4.1); t (12.1, 12.1) m d (14.5); d (14.5) d (13.3); d (13.3) dd (8.2, 2.0) dd (8.2, 2.5) dd (8.6, 2.5) dd (8.6, 2.0) d (6.3) d (6.3) s s s d (5.7) s s
1.88, m; 0.74, t (12.0) 1.77, m 1.70, m; 0.55, q (12.1) 2.01, m 1.10, dd (11.3, 7.5) 2.15, d (13.6) 3.45, m 4.85, dd (7.5, 6.0) 5.59, br s 3.91, d (6.0) 1.49, t (12.6, 11.0); 2.40, dd (12.6, 5.2) 6.35, dd (11.0, 5.2) 2.00, d (14.1); 3.49, d (14.1) 3.22, d (13.3); 3.52, d (13.3) 7.29, dd (8.0, 2.0) 7.12, dd (8.0, 2.5) 7.05, dd (8.6, 2.5) 7.42, dd (8.6, 2.0) 0.91, d (6.3) 1.22, d (6.3) 1.33, s 1.91, s 1.56, s e e 8.05, s 10.00, s 1.99, s
with (R)-a-methoxy-a-(trifluoromethyl) phenylacetyl chloride (RMTPA-Cl) or S-MTPA-Cl gave S-MTPA ester (1c) or R-MTPA ester (1d). The 1H NMR chemical shift differences (DdS-R, Fig. 5) between 1c and 1d in negative value for H-32 and positive values for H-16 and H-20 were observed, indicating a 17R configuration for 1.30 In the same way, treatment of 2 with R-MTPA-Cl or S-MTPA-Cl gave SMTPA ester 2c or R-MTPA ester 2d. The positiveDdS-R value for H-11 and negativeDdS-R values for H-16 and H-20 were suggestive of a 17S configuration for 2. The different configurations at C-17 in 1 and 2 were also differentiated by the significantly different 13C NMR chemical shifts for C-8 to C-12, C-16, C-20, C-32 (Table 1) and the different 1H NMR chemical shifts and coupling patterns for H-16 and H-17 (Table 2). Based on the foregoing evidence, compounds 1 and 2 were identified as new members of pyrrospirones A (1a) and B (2a), named as pyrrospirones C (1) and D (2). The assignments of 13 C and 1H NMR data (Tables 1 and 2) were made based on the analysis of HSQC, 1H-1H COSY, HMBC (Fig. 2), and NOE correlations (Fig. 3).
Fig. 3. Key NOE correlations of pyrrospirones C (1) and D (2).
T. Song et al. / Tetrahedron 74 (2018) 884e891
Fig. 4. X-ray crystal structure of pyrrospirone D (2).
Fig. 5. DdS-R values for the MTPA esters (1c, 1d, 2c, and 2d).
Compounds 3 and 4 were originally isolated as a mixture, which was not able to be separated by HPLC using different HPLC columns. In order to separate 3 and 4, the mixture was acetylated and then separated by HPLC to give 3a and 4a, which were in return hydrolyzed using 3N LiOH to produce 3 and 4. The HRESIMS and 13C NMR data showed that 3 and 4 also have the same molecular formula of C32H41NO5, a CH2 unit less than 1 and 2. The 13C NMR data (Table 1) of 3 and 4 bore a close resemblance to those of 1 and 2, respectively, except for the different chemical shifts for C-19 to C-21 due to the absence of a methoxy at C-19 in 3 and 4, implying that 3 and 4 were the analogues of 1 and 2 without the methoxy at C-19. Just like 1 and 2, the structural difference between 3 and 4 is also due to the different configurations at C-17, which was indicated by the different 13C NMR chemical shifts for C-8 to C-12, C-16, C-20, C32 and the different 1H NMR chemical shifts and the coupling patterns for H-16 and H-17 (Tables 1 and 2). Compounds 3 and 4 were thus elucidated as new alkaloids, named as pyrrospirones E (3) and F (4). The UV, 1H and 13C spectra of 5 suggested that 5 is also an analogue of pyrrospirone alkaloids. Its HRESIMS spectrum showed [MþH]þ ion at m/z 518.2896 and [MþNa]þ ion at m/z 540.2722, corresponding to a molecular formula C32H39NO5, two protons less than that of 3. Detailed comparison of the NMR data of 5 with those of 3 suggested that the structural difference between 5 and 3 was that the oxymethine (dC 71.7; dH 5.26) at C-17 in 3 was replaced by a carbonyl group (dC 208.9) in 5. The significant divergence in 13C NMR chemical shifts (Table 1) for C-10, C-12, C-16, C-17, and C-32 between 5 and 3 were observed because of the different substitutes at C-17 in 5 and 3. Compound 5 was thus identified as a new compound, named as pyrrospirone G. The 13C and 1H NMR data of 5 were listed in Tables 1 and 3, respectively. Compound 6 is also a pyrrospirone alkaloid based on the UV and NMR spectroscopic analyses. Its molecular formula C32H41NO4 was deduced from its HRESIMS ions at m/z 504.3118 for [MþH]þ and 526.2927 for [MþNa]þ, 16 mass units less than that of 3. Further NMR comparison indicated that 6 differs from 3 only in the substitute at C-17. In the NMR spectra of 3, the signals for an oxymethine (dC 71.7; dH 5.26) was replaced by the signals for a methylene (dC 41.6; dH 1.45, 2.47) in 6. Therefore, 6 was assigned as
887
an analogue of pyrrospirone E (3) with a methylene at C-17. Compound 6 is a new alkaloid, named as pyrrospirone H, and its 13C and 1 H NMR data were showed in Tables 1 and 3. The molecular formula C32H39NO4 for 7, established by its HRESIMS ions at m/z 502.2952 of [MþH]þ and 524.2767 of [MþNa]þ, is two protons less than that of 6. Compared to 6, the NMR spectra of 7 showed the existence of an additional pair of double bond (dC 132.4, 140.7; dH 5.26, d, 9.2 Hz, 5.47, d, 9.2 Hz) without the two methylenes (dC 36.6, 41.6) at C-16 and C-17 in 6. Accordingly, the structure of 7 was elucidated as an analogue of pyrrospirone H (6) with a double bond at C-16 and C-17. Compound 7 is also a new pyrrospirone alkaloid, named as pyrrospirone I. The 13 C and 1H NMR data of 7 see Tables 1 and 3. Pyrrospirones A (1a) and B (1b), as unique alkaloids with a spiro ring system and a new type of pyrrocidines,30 were previously isolated from an endophytic fungus Neonectria ramulariae Wollenw KS-246. Although pyrrospirones CI (1e7) were related to pyrrospirones A (1a) and B (1b), their structures are different in the substituent at C-12 with a methyl for 1e7 and a hydrogen for 1a and 1b. It was reported that pyrrospirones A (1a) and B (1b) could biosynthetically originate from pyrrocidine B (26).31 Therefore, it is suggested herein that pyrrospirones CI (1e7) might derive from GKK1032A1 (27) or GKK1032A2 (28). The possible biosynthetic origin of pyrrospirones CI (1e7) has been proposed in Fig. 6. Pyrrospirones CI (1e7) could be the results from the unusual 6endo-tet electrophilic cyclization of the epoxide derivatives (29, 30) of GKK1032A1 (27) or GKK1032A2 (28) after oxidation of the vinyl group. To the best of our knowledge, this study is the first report of this type of pyrrospirones CI (1e7). Pyrrospirones CI (1e7) were assayed for their activity in inhibiting the proliferation of glioma U87MG, U251, SHG44, and C6 cells by sulforhodamine B (SRB) assay. Doxorubicin (DOX, a chemotherapeutic drug)32 was used as a positive control. The results (Table 4) indicated that pyrrospirone G (5) had good activity with IC50 values of 1.06e8.52 mM, as compared to the activity of the positive control DOX with IC50 values of 0.47e8.03 mM. Pyrrospirones CF (1e4), H (6), and I (7) showed moderate activity with IC50 values of 7.44e26.64 mM. Pyrrospirones CI (1e7) were also evaluated for their activity in suppressing the growth of MRSA, E. coli, and C. albicans by using micro broth dilution method. Gentamicin (an antibiotic against both Gram-positive and Gram-negative bacteria) and amphotericin B (an antifungal drug) were used as positive controls. It has been found (Table 4) that pyrrospirones CF (1e4), H (6), and I (7) showed activity against both MRSA and E. coli (MIC: 2.0e19.0 mg/ mL) with pyrrospirone F (4) as the most active compound with MIC values of 2.0e3.0 mg/mL. None of the seven tested alkaloids showed antifungal activity. 3. Conclusion Marine-derived fungi of the genus Penicillium are able to synthesize both previously known and novel compounds with diverse structures and bioactivities.33 In this study, a marine crabassociated fungus Penicillium sp. ZZ380 was found to produce new pyrrospirones CI (1e7) as well as 18 known compounds with different structural classes. The structures of the new pyrrospirones were determined based on their NMR and HRESIMS data as well as the Mosher and X-ray diffraction methods. To the best of our knowledge, this study is the first report of this type of pyrrospirone alkaloids with both a spiro junction and a methyl at C-12. Bioactive assay demonstrated that pyrrospirone G (5) showed potent activity against the proliferation of four different glioma cells with IC50 values of 1.06e8.52 mM and that pyrrospirones C (1), F (4), and I (7) had antimicrobial activity in suppressing the growth of both MRSA
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T. Song et al. / Tetrahedron 74 (2018) 884e891
Table 3 1 H NMR data of pyrrospirones GI (5e7, 500 MHz, in pyridine-d5, J ¼ Hz). H
4a
1a; 1b 2 3a; 3b 4 5 7 8 9 11 13 16a 16b 17 20
1.84, 1.70, 1.67, 1.93, 1.02, 1.91, 3.02, 4.57, 5.26, 4.04, 1.75, 3.12, 4.51, 2.11, 3.81, 3.24, 3.49, 7.39, 7.17, 7.15, 7.44, 0.89, 1.16, 1.19, 1.78, 1.64, 8.05, 9.93, 1.96,
21 23 24 26 27 28 29 30 31 32 OH-19 NH OAc-17
5 m; 0.67, t (12.3) m m; 0.53, q (12.0) m dd (11.3, 8.1) d (13.0) m dd (8.1, 5.2) br s d (9.6) dd (12.9, 4.7); t (12.9, 12.9) dd (12.9, 4.7) d (14.3); d (14.3) d (13.1); d (13.1) dd (8.2, 2.2) dd (8.2, 2.5) dd (8.5, 2.5) dd (8.5, 2.2) d (6.3) d (6.3) s s s br s s s
1.83, 1.75, 1.72, 1.99, 1.16, 2.11, 3.53, 4.92, 5.09, 4.00, 2.59, 2.71, e 2.04, 3.48, 3.23, 3.48, 7.32, 7.17, 7.11, 7.40, 0.90, 1.24, 1.31, 1.77, 1.89, 8.19, 10.3, e
6 m; 0.71, t (11.9) m m; 0.54, q (12.1) m dd (11.5, 8.1) d (13.2) m dd (8.1, 6.4) br s d (6.3) d (16.3); d (16.3) d (14.8); d (14.8) d (13.4); d (13.4) dd (8.1, 2.2) dd (8.1, 2.5) dd (8.6, 2.5) dd (8.6, 2.2) d (6.3) d (6.3) s s s br s s
7
1.88, 1.74, 1.70, 1.98, 1.05, 2.01, 3.25, 4.70, 4.98, 3.81, 1.16,
m; 0.71, t (12.0) m m; 0.53, q (12.2) m dd (11.4, 7.9) d (13.9) m dd (7.9, 5.6) br s d (7.6) m; 2.03, m
1.85, 1.72, 1.69, 1.97, 1.12, 2.05, 3.31, 4.79, 5.10, 4.13, 5.26,
m; 0.70, t (12.3) m m; 0.54, q (12.0) m dd (11.3, 8.1) d (13.4) m dd (8.1, 5.9) br s d (7.7) d (9.2)
1.45, 2.01, 3.62, 3.23, 3.50, 7.32, 7.13, 7.09, 7.46, 0.90, 1.19, 1.28, 1.83, 1.46, 8.01, 9.72, e
m; 2.47, m d (14.1); d (14.1) d (13.3); d (13.1) dd (8.0, 2.2) dd (8.0, 2.5) dd (8.6, 2.5) dd (8.6, 2.2) d (6.3) d (6.3) s s s br s s
5.47, 2.22, 3.63, 3.27, 3.52, 7.34, 7.16, 7.14, 7.45, 0.90, 1.22, 1.27, 1.81, 1.65, 8.13, 9.92, e
d (9.2) d (14.3); d (14.3) d (13.2); d (13.2) dd (8.0, 2.0) dd (8.0, 2.3) dd (8.3, 2.3) dd (8.3, 2.0) d (6.3) d (6.3) s s s br s s
Fig. 6. Possible biosynthetic origin of pyrrospirones CI (1e7).
Table 4 Activities of pyrrospirones CI (1e7) against the proliferation of glioma cells and the growth of MRSA and E. coli. Compounds
1 2 3 4 5 6 7 DOX Gentamicin
Glioma cells (IC50: mM)
Bacteria (MIC: mg/mL)
U87MG
U251
SHG44
C6
MRSA
E. coli
12.15 ± 0.61 9.95 ± 0.50 16.24 ± 0.68 12.44 ± 0.81 1.06 ± 0.05 12.89 ± 0.64 13.67 ± 0.53 1.20 ± 0.06 e
22.12 ± 1.11 23.39 ± 1.27 26.64 ± 2.14 22.82 ± 1.15 1.28 ± 0.06 23.92 ± 1.20 13.46 ± 0.76 8.03 ± 1.20 e
10.03 ± 0.10 13.74 ± 0.55 15.76 ± 1.20 8.93 ± 0.76 2.14 ± 0.11 13.02 ± 0.79 7.44 ± 0.97 0.67 ± 0.10 e
13.87 ± 1.26 14.56 ± 0.95 21.03 ± 3.15 14.87 ± 1.66 8.52 ± 1.01 23.24 ± 2.24 19.18 ± 2.11 0.47 ± 0.10 e
4.0 12.0 10.0 2.0 >50 19.0 4.0 e 0.5
5.0 3.0 11.0 3.0 >50 4.0 2.0 e 1.0
T. Song et al. / Tetrahedron 74 (2018) 884e891
and E. coli with MIC values of 2.0e5.0 mg/mL. 4. Experimental section 4.1. General experimental procedures UV spectra were recorded on a METASH UV-8000 (Shanghai METASH Instruments Co. Ltd., China). Optical rotation and ECD spectra were measured on a JASCO DIP-370 digital polarimeter and a JASCO J715 spectropolarimeter (JASCO, Japan), respectively. HRESIMS data were acquired on an Agilent 6230 TOF LC/MS spectrometer. NMR spectra were acquired on a Bruker 500 spectrometer using standard pulse programs and acquisition parameters. Chemical shifts were expressed in d (ppm) and referred to the NMR solvent used. Octadecyl-functionalized silica gel (ODS, Cosmosil 75C18-Prep, Nacalai Tesque Inc., Japan) was used for column chromatography. HPLC separation was performed on Agilent 1260 HPLC system (HPLC A), or CXTH 3000 prepared HPLC system (HPLC B, Beijing Chuangxintongheng Science & Technology Co. Ltd., China), or SHIMADZU 20A prepared HPLC system (HPLC C). All solvents used for this study were purchased from the Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). Human glioma U87MG (JDS2568), U251 (XB-0439), SHG44 (RX-J150) cells and rat glioma C6 (XB-003) cells were obtained from the Cell Bank of the Chinese Academy of Sciences. Methicillin-resistant Staphylococcus aureus (MRSA) ATCC 43300, Escherichia coli ATCC 25922, and Candida albicans were gifts from Drs. Zhongjun Ma, Pinmei Wang, and Bin Wu, respectively. Doxorubicin (DOX, >98.0%) was ordered from Sigma-Aldrich, and gentamicin (99.6%) and amphotericin B (>95.0%) from Meilune Biotechnology Co. Ltd. (Dalian, China). 4.2. Isolation and taxonomic identity of strain ZZ380 Strain ZZ380 was isolated from a sample of wild crab (Pachygrapsus crassipes), which had been living on the seaside rocks of Putuo Mountain (Zhoushan, China). A voucher sample (PCPT201603) was authenticated by one of the authors (T. Song) and deposited in the Laboratory of Institute of Marine Biology, Ocean College, Zhoushan Campus, Zhejiang University, China. Briefly, a crab was dipped in 75% ethyl alcohol for 10 s and then mashed in sterile water. The mash was centrifugated (5000 r/min, 5 min) to produce a supernatant. The supernatant (1 mL) was diluted into 10%, 1%, and 0.1% (V/V) with sterile water. The 0.1% diluted suspension of 200 mL was covered on the surface of PDA (Potato Dextrose Agar) solid medium in a petri dish and then incubated at 28 C for seven days. The single colony was picked and then transferred to another PDA dish. After several days of growth at 28 , the single colony (strain ZZ380, Fig. S1) that grew well was transferred onto PDA slants, which were stored at 4 C for further use. The strain ZZ380 was identified using ITS rDNA sequence analysis by Legenomics (Hangzhou, China) and its DNA sequence using BLAST (nucleotide sequence comparison) was compared to the GenBank database. The ITS rDNA sequence of strain ZZ380 has been deposited in GenBank (accession number: MF503479). A voucher strain (Penicillium sp. ZZ380) was preserved at the Laboratory of Institute of Marine Biology, Ocean College, Zhoushan Campus, Zhejiang University, China.
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which contains 250 mL of BMPM medium (glucose 20 g, glycerol 20 g, soy flour 10 g, cotton seed embryo meal 10 g, (NH4)2SO4 1 g, CaCO3 10 g, tap water 1000 mL). All flasks were incubated at 28 C for 30 days in stationary state. A total of 40.0 L culture was made for this study. 4.4. Extraction and isolation of compounds 125 The 40.0 L culture was centrifuged to give fermentation broth and mycelia. The mycelia were extracted with MeOH three times to afford a mycelia extract after the removal of MeOH. The fermentation broth was partitioned with EtOAc three times to give an EtOAc extract after the removal of EtOAc. A mixture of the mycelia extract and the EtOAc extract was fractionated on a ODS column eluted successively with 80% MeOH, 90% MeOH, and 100% MeOH to give six fractions (Fr. 1 to Fr. 6) based on the results of TLC analysis. Each fraction was further separated by ODS column, following by HPLC purification, to give compounds 1e25. The isolation procedure and separation conditions were depicted in Fig. S4. 4.4.1. Pyrrospirone C (1) Colorless amorphous powder; molecular formula C33H43NO5; [a]25 D þ5.5 (c 0.50, MeOH); ECD (10 mg/L, MeOH) lmax (Dε) 211 (þ150.1), 233 (23.0), 288 (26.4) nm; UV (MeOH) lmax (log ε) 205 (4.49), 228 (4.01), 279 (3.39) nm; 13C NMR data (125 MHz, in pyridine-d5), see Table 1, 1H NMR data (500 MHz, in pyridine-d5), see Table 2; HRESIMS m/z [MþH]þ 534.3217 (calcd for C33H44NO5, 534.3219), [MþNa]þ 556.3030 (calcd for C33H43NNaO5, 556.3039), and [MþK]þ 572.2777 (calcd for C33H43KNO5, 572.2778). 4.4.2. Pyrrospirone D (2) Colorless amorphous powder; molecular formula C33H43NO5; [a]25 D þ13.6 (c 0.38, MeOH); ECD (10 mg/L, MeOH) lmax (Dε) 212 (þ36.2), 233 (þ0.86), 280 (4.4) nm; UV (MeOH) lmax (log ε) 207 (4.40), 227 (4.08), 277 (3.38) nm; 13C NMR data (125 MHz, in pyridine-d5), see Table 1, 1H NMR data (500 MHz, in pyridine-d5), see Table 2; HRESIMS m/z [MþH]þ 534.3216 (calcd for C33H44NO5, 534.3219), [MþNa]þ 556.3030 (calcd for C33H43NNaO5, 556.3039), and [MþK]þ 572.2781 (calcd for C33H43KNO5, 572.2778). 4.4.3. Pyrrospirone E (3) Colorless amorphous powder; molecular formula C32H41NO5; [a]25 D þ80.3 (c 0.15, MeOH); ECD (10 mg/L, MeOH) lmax (Dε) 212 (þ73.5), 235 (11.5), 288 (12.6) nm; UV (MeOH) lmax (log ε) 205 (4.19), 230 (3.68), 279 (3.05) nm; 13C NMR data (125 MHz, in pyridine-d5), see Table 1, 1H NMR data (500 MHz, in pyridine-d5), see Table 2; HRESIMS m/z [MþH]þ 520.3064 (calcd for C32H42NO5, 520.3063), [MþNa]þ 542.2879 (calcd for C32H41NNaO5, 542.2882), and [MþK]þ 558.2610 (calcd for C32H41KNO5, 558.2622).
4.3. Large culture of strain ZZ380
4.4.4. Pyrrospirone F (4) Colorless amorphous powder; molecular formula C32H41NO5; [a]25 D þ99.9 (c 0.20, MeOH), ECD (10 mg/L, MeOH) lmax (Dε) 213 (þ138.2), 237 (þ5.7), 281 (16.4) nm; UV (MeOH) lmax (log ε) 205 (4.19), 228 (3.77), 280 (2.97) nm; 13C NMR data (125 MHz, in pyridine-d5), see Table 1, 1H NMR data (500 MHz, in pyridine-d5), see Table 2; HRESIMS m/z [MþH]þ 520.3057 (calcd for C32H42NO5, 520.3063), [MþNa]þ 542.2875 (calcd for C32H41NNaO5, 542.2882), and [MþK]þ 558.2622 (calcd for C32H41KNO5, 558.2622).
Colonies of the strain ZZ380 from the PDA slants were inoculated into a 500 mL Erlenmeyer flask containing 250 mL of PDB (Potato Dextrose Broth) medium and then incubated at 28 C for three days on a rotary shaker (180 rpm) to produce seed broth. The seed broth (5 mL) was inoculated into a 500 mL Erlenmeyer flask,
4.4.5. Pyrrospirone G (5) Colorless amorphous powder; molecular formula C32H39NO5; [a]25 D þ24.6 (c 0.50, MeOH); ECD (10 mg/L, MeOH) lmax (Dε) 212 (þ164.5), 235 (94.7), 280 (þ3.6) nm; UV (MeOH) lmax (log ε) 205 (4.23), 227 (3.95), 277 (2.87) nm; 13C NMR data (125 MHz, in
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pyridine-d5), see Table 1, 1H NMR data (500 MHz, in pyridine-d5), see Table 3; HRESIMS m/z [MþH]þ 518.2896 (calcd for C32H40NO5, 518.2906), [MþNa]þ 540.2722 (calcd for C32H39NNaO5, 540.2726), and [MþK]þ 556.2455 (calcd for C32H39KNO5, 556.2465). 4.4.6. Pyrrospirone H (6) Colorless amorphous powder; molecular formula C32H41NO4; [a]25 D þ18.5 (c 0.50, MeOH); ECD (10 mg/L, MeOH) lmax (Dε) 211 (þ190.4), 235 (24.7), 286 (26.6) nm; UV (MeOH) lmax (log ε) 207 (4.36), 227 (3.86), 276 (2.90) nm; 13C NMR data (125 MHz, in pyridine-d5), see Table 1, 1H NMR data (500 MHz, in pyridine-d5), see Table 3; HRESIMS m/z [MþH]þ 504.3118 (calcd for C32H42NO4, 504.3114), [MþNa]þ 526.2927 (calcd for C32H41NNaO4, 526.2933), and [MþK]þ 542.2672 (calcd for C32H41KNO4, 542.2673). 4.4.7. Pyrrospirone I (7) Colorless amorphous powder; molecular formula C32H39NO4; [a]25 D þ118.2 (c 0.10, MeOH); ECD (10 mg/L, MeOH) lmax (Dε) 211 (þ87.9), 232(32.7), 283 (9.7) nm; UV (MeOH) lmax (log ε) 206 (4.22), 278 (3.22) nm; 13C NMR data (125 MHz, in pyridine-d5), see Table 1, 1H NMR data (500 MHz, in pyridine-d5), see Table 3; HRESIMS m/z [MþH]þ 502.2952 (calcd for C32H40NO4, 502.2957) and [MþNa]þ 524.2767 (calcd for C32H39NNaO4, 524.2777). 4.5. Acetylation of 3 and 4
HPLC (column: Zorbax SB-C18, 250 4.6 mm, 5 mm; mobile phase: MeOH/H2O, 92/8; flow rate: 1.3 mL/min). Two compounds of SMTPA ester of pyrrospirone C (1c, 5.6 mg, tR 16.1 min) and R-MTPA ester of pyrrospirone C (1d, 5.8 mg, tR 15.8 min) were obtained for NMR analysis. Through the same process, MTPA esterification of pyrrospirone D (2, 5.0 mg) gave S-MTPA ester of pyrrospirone D (2c, 2.7 mg, tR 17.0 min) and R-MTPA ester of pyrrospirone D (2d, 2.4 mg, tR 16.7 min). 4.8. X-ray crystallographic analysis of pyrrospirone D (2) X-ray diffraction analysis was carried out on an Xcalibur Atlas Gemini Ultra diffractometer (Agilent Technologies) with Cu Ka radiation (l ¼ 1.541 84 Å) at 293 K. The structures were solved by direct methods (SHELXS-2008) and refined with full-matrix leastsquares on F2 (SHELXL-2015). All non-hydrogen atoms were refined anisotropically, and all hydrogen atoms were placed in idealized positions and refined as riding atoms with the relative isotropic parameters. Crystallographic data for 2 have been deposited at the Cambridge Crystallographic Data Centre (CCDC Number: 1580841). Copies of the data can be obtained, free of charge, through application to the Director, CCDC, 12 Union Road, Cambridge CB2 1EZ, UK (fax: þ 44 (0)1223-336033, or e-mail: http://[email protected]. uk/[email protected]).
The mixture (25 mg) of 3 and 4 dissolved in Ac2O/pyridine (1: 2, 3 mL) was allowed to stand at room temperature for 24 h. After addition of 1 mL of water, the resulting mixture was dried in vacuo to give a residue. This residue was applied to an ODS column eluted successively with water and MeOH. The MeOH fraction was further separated by Agilent 1260 HPLC (column: Agilent Zorbax SB-C18, 250 9.2 mm, 5 mm; mobile phase: ACN/H2O, 87/13; flow rate: 2.0 mL/min) to give acetylation products (3a and 4a) of 3 and 4.
4.8.1. Crystallographic data of pyrrospirone D (2) Colorless needle crystals, C33H43NO5, Mr 533.70, monoclinic, space group P 1 21 1, a ¼ 6.74932 (14) Å, b ¼ 13.8982(2) Å, c ¼ 16.8690(3) Å, a ¼ 90.00 , b ¼ 100.4959(18) , g ¼ 90.00 , V ¼ 1555.90 (5) Å3, Z ¼ 2, D ¼ 1.208 g/cm3, crystal size 0.49 0.16 0.13 mm3, F(000) ¼ 612.0, T ¼ 293 K, The final R1 value is 0.0459 (wR2 ¼ 0.1179) for 5090 reflections [I 2s (I)]. CCDC Number: 1580841.
4.5.1. Compound 3a Colorless amorphous powder; molecular formula C34H43NO6; 13 C NMR data (125 MHz, in pyridine-d5), see Table 1, 1H NMR data (500 MHz, in pyridine-d5), see Table 2; HRESIMS m/z [MþH]þ 562.3166 (calcd for C34H44NO6, 562.3169) and [MþNa]þ 584.2984 (calcd for C34H43NNaO6, 584.2988).
4.9. Culture of glioma cells
4.5.2. Compound 4a Colorless amorphous powder; molecular formula C34H43NO6; 13 C NMR data (125 MHz, pyridine-d5), see Table 1, 1H NMR data (500 MHz, pyridine-d5), see Table 3; HRESIMS m/z [MþH]þ 562.3162 (calcd for C34H44NO6, 562.3169) and [MþNa]þ 584.2980 (calcd for C34H43NNaO6, 584.2988). 4.6. Alkaline hydrolysis of 3a and 4a Each (5 mg) of 3a and 4a dissolved in a mixture of MeOH/H2O (3:1, 2 mL) was hydrolyzed by using 1 mL 3 N LiOH in MeOH/H2O (3:1) for 12 h at room temperature. The reaction mixture was neutralized with 3N HCl to pH 6e8 and then concentrated under reduced pressure to give deacetylation products 3 and 4. 4.7. MTPA esterification of pyrrospirones C (1) and D (2) Pyrrospirone C (1, 11 mg) in 2 mL anhydrous pyridine was divided into 2 equal portions. Mosher regent R-MTPA-Cl or SMTPA-Cl (each 50 mL) was added to each portion. Each mixture was stirred at room temperature for 2 h and the reaction was terminated by an addition of 1 mL MeOH. Each resulting mixture was dried in vacuo to give a residue, which was purified by Agilent 1260
Human glioma U87MG cells were cultured in MEM (Minimum Essential Medium, Gibco) with 10% FBS (Fetal Bovine Serum, PAA Laboratories Inc.), human glioma U251 and rat glioma C6 cells in DMEM (Dulbecco's Modified Eagle Medium, Gibco), and human glioma SHG44 in RPMI-1640 Medium (Roswell Park Memorial Institution 1640 Medium, Gibco). All cells were incubated at 37 C in a humidified incubator with 5% CO2. Cells after the third generation were used for the experiment. 4.10. Sulforhodamine B (SRB) assay The SRB assay8,13 was used to evaluate the activities of compounds 1e7 against the proliferation of glioma U87MG, U251, SHG44, and C6 cells. Doxorubicin (DOX) was used as a positive control. 4.11. Antimicrobial assay Micro broth dilution method11 was applied to determine the antimicrobial activities of compounds 1e7 against the growth of MRSA, E. coli, and C. albicans. Gentamicin (an antibiotic against both Gram-positive and Gram-negative bacteria) and amphotericin B (an antifungal drug) were used as positive controls. Conflicts of interest The authors declare no conflict of interest.
T. Song et al. / Tetrahedron 74 (2018) 884e891
Acknowledgments This study was supported by the National Natural Science Foundation of China (81273428, 81773587, and 81773769). We appreciate Mrs. Jianyang Pan (Pharmaceutical Informatics Institute of Zhejiang University) for performing the NMR spectrometry and Dr. Jiyong Liu (Department of Chemistry of Zhejiang University) for performing the X-ray diffraction analysis. Appendix A. Supplementary data Supplementary data related to this article can be found at https://doi.org/10.1016/j.tet.2018.01.015. References 1. 2. 3. 4. 5. 6. 7. 8. 9.
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