Collacyclumines A–D from the endophytic fungus Colletotrichum salsolae SCSIO 41021 isolated from the mangrove Kandelia candel

Phytochemistry 171 (2020) 112237

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Collacyclumines A–D from the endophytic fungus Colletotrichum salsolae SCSIO 41021 isolated from the mangrove Kandelia candel

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Xiuping Lina, Wen Aic, Meng Lib, Xuefeng Zhoua, Shengrong Liaoa, Junfeng Wanga, Juan Liua, Bin Yanga,∗, Yonghong Liua,∗∗ a CAS Key Laboratory of Tropical Marine Bio-resources and Ecology, Guangdong Key Laboratory of Marine Materia Medica, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou, 510301, China b Beijing Key Lab of Plant Resource Research and Development, Beijing Technology and Business University, Beijing, 100048, China c College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, 310000, China

A R T I C LE I N FO

A B S T R A C T

Keywords: Mangrove plant Endophytic fungus Colletotrichum salsolae Collacyclumine

Four undescribed alkaloids, namely collacyclumines A–D, along with a known analogue, agrocybenine, were isolated from the endophytic fungus Colletotrichum salsolae SCSIO 41021 derived from the mangrove plant Kandelia candel (L.) Druce. Collacyclumine A represents the first case of dimeric pyrrolidine alkaloid in nature. The structures of these compounds were elucidated by a combination of NMR spectra, HRESIMS data, and X-ray diffraction experiment. A proposed biosynthetic pathway of these isolated compounds were also discussed. None of compounds showed cytotoxic effects against ten cell lines.

1. Introduction Pyrrolidine/piperidine alkaloids, which are widely distributed in nature, including various plants, marine creatures, insects, and microbes, are a class of structurally interesting and biologically important natural products that trigger, or at least inspire, investigations relating to drug discovery, organic synthesis, and chemical ecology (Jiang and Gerwick, 1991; Michael, 2008; O'Hagan, 2000; Pinder, 1989; Plunkett, 1994). Compared with other naturally occurring alkaloids, microbederived pyrrolidine/piperidine alkaloids are rare, but their biological activity such as antitumor, and anti-inflammatory, is well studied, including a number of excellent studies reported in recent years (Gu et al., 2018; Ibrahim et al., 2015; Phainuphong et al., 2018). Mangrove plants, distributed in tropical and subtropical intertidal forest wetlands, harbor a variety of endophytic fungi, which are widely recognized as a prolific source of structurally unique and biologically active natural products that could be used for the development of new medicinal agents (Chen et al., 2018b; May Zin et al., 2017; Yang et al., 2018; Zhou et al., 2018). In our previous research, forty-five endophytic fungal strains were isolated from a mangrove plant Kandelia candel (L.) Druce (Rhizophoraceae) sample, collected from Daya Bay, China, in March 2012. In our continuing search for structurally interesting natural products from mangrove endophytic fungi, selected on the basis of

interesting HPLC-UV profile, a series of bioactive derivatives with antiprotein tyrosine phosphatase-1B (anti-PTP1B), anti-silent information regulator T1 (anti-SIRT1) activities and cyclooxygenase 2 (COX-2) inhibitor have been isolated (Ai et al., 2014; Ju et al., 2015). As part of a program to search for structurally unique and biologically interesting natural products, we investigated the endophyte Colletotrichum salolae SCSIO 41021 (Glomerellaceae) that was derived from the inner part of a K. candel stem and identified four undescribed alkaloids, collacyclumines A–D (1–4), along with a known compound, agrocybenine (5) (Fig. 1). Herein, we report the isolation, structure determination, bioactivity evaluation, as well as plausible biosynthetic pathway of the undescribed compounds. 2. Results and discussion 2.1. Characterization and identification of isolated strain SCSIO 41021 The strain SCSIO 41021 was isolated from the inner part of stem of mangrove plant K. candeland stored on MA (Kjer et al., 2010) slants at 4 °C and deposited at CAS Key Laborary of Tropical Marine Bio-resources and Ecology. The morphological analysis of strain SCSIO 41021 showed vegetative hyphae was estate and branched. Conidiophores formed directly on the hyphae. One or two conidia yield on one

∗

Corresponding author. CAS Key Laboratory of Tropical Marine Bio-resources and Ecology, Guangdong Key Laboratory of Marine Materia Medica, South China Sea Institute of Oceanology, Chinese Academy of Sciences, 164 West Xingang Road, 510301, Guangzhou, China. ∗∗ Corresponding author. E-mail addresses: [email protected] (B. Yang), [email protected] (Y. Liu). https://doi.org/10.1016/j.phytochem.2019.112237 Received 27 December 2018; Received in revised form 20 December 2019; Accepted 20 December 2019 0031-9422/ © 2019 Published by Elsevier Ltd.

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GS, ITS and TUB2) phylogenetic tree (Fig. 3), which showed strain SCSIO 41021 resided inside of C. salsolae with strong statistical support (ML, 100%). On the basis of its multi-locus phylogenetic and morphological analyses, strain SCSIO 41021 was identified as C. salsolae and designated as C. salsolae SCSIO 41021. 2.2. Structure elucidation Compound 1 was isolated as yellow crystals, and the HRESIMS provided molecular formula of C15H20N2O2 from an ion peak at m/z 261.1602 [M + H]+ (calculated for C15H21N2O2, m/z 261.1598), requiring seven degrees of unsaturation. Inspection of the 1H NMR spectrum of 1 (Table 1) showed six methyls at δH 2.52 (s, H-12), 1.94 (s, H-15), 1.49 (s, H-13 and H-14) and 1.32 (s, H-16 and H-17), and a olefinic singlet at δH 7.54 (s, H-4). The combined analysis of its 13C (Table 1), DEPT-135, HSQC, and HMBC spectra allowed the designation of 13 carbon resonances for 15 carbons as six methyls, one vinylic methine, and eight quaternary carbons, including four vinylic quaternary carbons (δC 105.6, 162.9, 163.2, and 168.7) and two ketone carbonyls (193.3, and 208.3). Since the above data accounted for five degrees of unsaturation, the presence of two rings was required to

Fig. 1. The structures of the compounds 1–5.

conidiophore. Conidia were cylindrical, straight, round at one end and acute at the other, sometimes round at both ends (Fig. 2 C‒E). Sexual stage didn't been observed. Aspersoria were single and climate with locate edges (Fig. 2 F‒G). By running RAxML V.7.0.3, the maximum likelihood method was used to constructed a multi-locus (CAL, GAPDH,

Fig. 2. Colony appearance and micromorphology of strain SCSIO 41021. A‒B. colony appearance after 7 days at 25 °C (MB medium); C‒E. Conidia; F‒G. appressoria. Bars: 10 μm (C–G). 2

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Fig. 3. Phylogenetic assignment of C. salsolae SCSIO 41021 inferred from the maximum likelihood analysis of CAL, GAPDH, GS, ITS and TUB2 nucleotide sequences by running RAxML v.7.0.3. The RAxML bootstrap support values (> 50%) are given at the nodes. Scale bar indicates 0.04 expected changes per site. Accession numbers of nucleotide sequences are provided in PHYTO Table S1.

satisfy the molecular formula of 1. In the HMBC spectrum, the correlations from H-4 to C-2, C-3, C-5, and C-11, H3-12 to C-3, and C-11, and H3-13 to C-2, C-3 and C-14, led to the assignment of the ring A, and the ring B was also assigned based on the HMBC correlations from the H3-

15 to C-6, C-7, and C-8, and H3-16 to C-8, C-9 and C-17. Fortunately, single crystals suitable for an X-ray diffraction experiment were finally obtained by slow evaporation and slow diffusion of methanol. The connection of the rings A and B via C-5/C-6 linkage was further 3

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Koshino et al., 1996), except for the presence of a keto-carbonyl group and the concomitant absence of a methylene in 3. In the HMBC spectrum of 3, correlations from H3-13 to C-7, and C-8 revealed the presence of a keto-carbonyl group at C-7. Therefore, the structure of collacyclumine C (3) was defined as depicted. Compound 4 was isolated as a colorless oil. Its molecular formula, C13H15NO3, was determined by the HRESI-MS of a molecule at m/z 232.0982 [M ‒ H]‒ (calcd for 232.0979), corresponding to seven degrees of unsaturation. The 1H NMR spectrum contained signals for two olefinic protons at δH 6.72 (s, H-7) and 7.35 (s, H-5), and four methyls at δH 1.77 (s, H-11 and H-12), 1.91 (s, H-13), and 2.83 (s, H-14). The 13 C NMR spectra showed signals for one ketone moiety, one carbonyl, two sp2C methines, four methyls and four of five quaternary carbons. The HMBC cross-peaks from H3-11 to C-2, C-3, and C-12; H3-12 to C-2, C-3, and C-11; H3-13 to C-3, C-4, and C-5; and H-5 to C-3 and C-6 enabled the establishment of a 2,2,4- trimethyazacyclohex-4-en-3-one ring A. The another six-membered ring B fused with ring A at C-6−N-1 was confirmed by the HMBC correlations from H-7 to C-5, C-6, C-8, and C-9; H3-14 to C-8, C-9, and C-10, the downfield chemical shifts of C-2 (δ 67.8) and C-10 (δ 167.6), and the indices of hydrogen deficiency. Therefore, the structure of 4 was established, and was given the trivial name collacyclumine D. By comparing the 1H, 13C NMR and MS data with the literature values, the known compound (5) was identified as agrocybenine (Chen et al., 2018a). From a biosynthetic aspect, compounds 1, and 3–5 could be generated from compound 2, via different reaction cascades as illustrated in the hypothetical biosynthetic pathway (Fig. 5). It is interesting to note that all of these isolates contain one or more basic units of a N-(2methyl-4-oxopentan-2-yl) formamide, which allowed the classification of these compounds as pyrrolidine/piperidine alkaloids. Compound 2B might firstly undergo a condensation reaction at C-2/C-6 to form a pyrrolidine ring, and then the other amide part of this molecule could further undergo condensation with pyruvic acid, reduction of the carbonyl group, and followed with an intromolecular condensation, respectively, to form the second pyrrolidine ring, which was then deaceylated to provide the final compound 1 (Mackay et al., 1980). Compound 2B might also undergo a condensation reaction at C-2/C-6′ to form a piperidine ring 2C, then followed with another condensation to afford the 6/5 framework of compounds 5, which was ultimately oxidized to form compound 3. Compound 4 might be generated from the same precursor 2C (Padwa et al., 1999, 2000; Pyridones Tobinaga et al., 2018). The cytotoxicities of 1–5 were evaluated against a panel of ten human tumor cell lines (K562, A549, Huh-7, H1975, MCF-7, U937, BGC823, HL60, HeLa, MOLT-4). The results indicated that none of compounds exhibited cytotoxicity.

Table 1 NMR spectroscopic data (500 MHz, MeOH) of 1–4. Pos.

1 δC

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

80.5 168.7 131.6 162.9 163.2 105.6 208.3 63.3 193.3 26.8 22.1 22.1 5.8 22.6 22.6

2 δH, mult. (J in Hz)

7.54 s

δC

δH, mult. (J in Hz)

31.6 209.6 51.7 53.5

2.14 s

161.0 27.0 27.0

2.52 1.49 1.49 1.94 1.32 1.32

s s s s s s

3

3.00 s

1.44 s 1.44 s

δC

4 δH, mult. (J in Hz)

67.0 202.2 99.9 149.5 166.4 202.2 60.7 28.4 28.4 7.59 29.7 29.7

1.34 1.34 1.73 1.44 1.44

s s s s s

δC

67.8 199.2 123.8 135.6 126.9 114.2 143.4 117.1 170.7 26.9 26.9 15.3 14.5

δH, mult. (J in Hz)

7.35 s 6.72 s

1.77 1.77 1.91 2.83

s s s s

Fig. 4. X-ray structure of 1.

determined by the single-crystal X-ray diffraction analysis (Fig. 4). Finally, the final structure of collacyclumin A (1) was unambiguously confirmed as shown in Fig. 1. Compound 2 was isolated as a colorless oil. Its HR-ESI-MS spectrum showed the [M + Na]+ molecular ion peak at m/z 307.1632 (calcd 307.1628) corresponding to the molecular formula of C14H24N2O4, with four degrees of unsaturation. The 1H NMR data of 2 (Table 1), together with the molecular formula and the DEPT-135 spectra, suggested that 2 had two symmetrical structural units. The 1H NMR data showed singals for one methylene group at δH 3.00 (s, H-3), three methyl groups at δH 2.14 (s, H-1), 1.44 (s, H-7 and H-8). The 13C NMR (Table 1) and DEPT spectra of 2 showed it has 7 carbons in one unit, including one methylene carbon (δC 51.7, C-3), three methyl carbons, and three quaternary carbons (including one ketone carbon at δC 209.6, C-2 and one carbonyl carbon at δC 161.0, C-6). In the HMBC spectrum of 2, the correlations of H3-1/C-2, and C-3; H-3/C-2, and C-4; H3-7/C-3, C-4 and C-8; and H3-8/C-3, C-4 and C-7 suggested that there was a N-(2-methyl4-oxopentan-2-yl)formamide fragment. In consideration of the 1H and 13 C NMR data, combined with the molecular formula information, compound 2 was proposed as a symmetrical molecule. Therefore, collacyclumin B (2) was identified as a dimeric N-(2-methyl-4-oxopentan2-yl) formamide as shown in Fig. 1. Compound 3 was isolated as a colorless oil and had a molecular formula of C12H16N2O2 as determined by HRESIMS at m/z 221.1288 [M + H]+ (calcd 221.1285), with six degrees of unsaturation. In a detailed comparison of 1D and 2D NMR data (Table 1), compound 3 showed the similar planar structure as that of agrocybenine (5). (Chen et al., 2018a;

3. Conclusions In conclusion, four undescribed alkaloids with diverse skeletons, along with a known analogue, agrocybenine, were isolated from the endophytic fungus C. salsolae SCSIO 41021 derived from the mangrove plant K. candel (L.) Druce. To the best of our knowledge, collacyclumine A (1) is the first example of a dimeric pyrrolidine alkaloid. Meanwhile, collacyclumine B (2) is the first case of dimeric N-(2-methyl-4-oxopentan-2-yl)formamide. Collacyclumine C (3) and agrocybenine (5) are piperidine alkaloids, which have been isolated as plant constituents. Collacyclumine D (4) is a relatively rare quinolizidine alkaloid. Until now, only few quinolizine analogs have been isolated (Aoyagi et al., 1998; Tokuyama et al., 1987). Our study, therefore, suggests that marine-derived fungus C. salsolae is one of the promising sources to provide the chemical diversity and complexity of alkaloids.

4

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Fig. 5. Plausible biogenetic pathways of 1−5.

4. Experimental section

4.4. Phylogenetic and morphological analyses

4.1. General experimental procedures

Based on concatenated multi-locus dataset (CAL, GAPDH, GS, ITS, TUB2) and used maximum Likelihood (ML) method, phylogenetic analyses of Colletotrichum gloeosporioides species complex were carried out in this study. Applied RAxML v.7.0.3 (Stamatakis, 2006) with 1000 replicates under the GTR-GAMMA model, ML analyses was performed. Used the cover technique described previously (Zhou et al., 1998), the morphological features of the conidia, appressoria and mycelia of strain SCSIO 41021 in the 7-d cultures grown on MA medium were observed with an OLYMPUS BX53 light microscope.

HR-ESIMS were determined with a Bruker maXis Q-TOF in positive/ negative ion mode. The NMR spectra were recorded on a Bruker AC 500 NMR spectrometer with TMS as an internal standard. All chemical shifts were assigned with δ-values. Column chromatography (CC) was performed on silica gel (200–300 mesh, 300–400 mesh), and Sephadex LH20 (Amersham Biosciences, Sweden), respectively. TLC were carried out on silica gel GF254 (10–40 μm) plates (Qingdao Marine Chemical Factory, China). All solvents used were of analytical grade (Tianjin Fuyu Chemical and Industry Factory). Semipreparative HPLC (Agilent Technologies, 1260 infinity series) was performed using an ODS column (YMC-pack ODS-A, 10 × 250 mm, 5 μm, 1.5 mL/min).

4.5. Fermentation, isolation and purification Strain stored on PDA slants at 4 °C was cultured on PDA agar plates and incubated for 7 days. Seed medium (potato 200 g, dextrose 20 g, NaCl 2.5 g, distilled water 1000 mL) in 50-mL Erlenmeyer flasks was inoculated with strain and incubated at 25 °C for 48 h on a rotating shaker (180 rpm). Production medium of 200-mL PDB in 1000 mL flasks was inoculated with 10 mL seed solution. Flasks were incubated at 25 °C under static stations and daylight. After 15 days, cultures from 100 flasks were harvested for the isolation of substances. The total 20L of PDB culture was crushed and extracted with acetone three times. The acetone extract was evaporated under reduced pressure to afford an aqueous solution. The aqueous solution was extracted three times with EtOAc to give another EtOAc solution (63 g)The EtOAc portion was subsequently separated by silica gel column chromatography using CHCl3–MeOH gradient elution to give ten fractions (Frs. S1–S10). Fr. S3 was purified by Sephadex LH-20 using CHCl3/MeOH = 1:1 to yield 5 portions, and portion 3 was further purified with semi-preparative HPLC, eluting with MeOH/ H2O = 57:43 at a flow rate of 2 mL/min, to afford 1 (4.2 mg, tR = 35.4) and 4 (2.6 mg, tR = 46.0). Fr. S4 was purified by Sephadex LH-20 using CHCl3/MeOH = 1:1 to yield 3 portions, and portion 2 was further purified with semi-preparative HPLC, eluting with MeOH/ H2O = 60:40 at a flow rate of 2 mL/min, to afford 5 (3.2 mg, tR = 21.7). Fr. S5 was purified by Sephadex LH-20 using CHCl3/ MeOH = 1:1 to yield 7 portions, and portion 6 was further purified with semi-preparative HPLC, eluting with MeOH/H2O = 55:45 at a

4.2. Fungal material The fungal strain Colletotrichum salsolae SCSIO 41021 (Glomerellaceae) was obtained from the inside of the stem of the mangrove plant Kandelia candel (L.) Druce (Rhizophoraceae) collected in Daya Bay, China (N22°44′19.82″, E114°31′58.18″) in March 2012. It was isolated on MA medium (malt extract 15 g, sea salt 10 g, agar 15 g, distilled water 1000 mL, pH 7.4–7.8), stored on MA slants at 4 °C and deposited at CAS Key Laborary of Tropical Marine Bio-resources and Ecology (Guangzhou City).

4.3. DNA extraction, PCR amplification and sequencing Genomic DNA was isolated from cells with a modified CTAB protocol as described by Guo et al. (2000). Five loci, including the glutamine synthetase (GS), calmodulin (CAL) and beta-tubulin (TUB2) genes, an intron of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene, and the 5.8S nuclear ribosomal gene with the two flanking transcribed spacers (ITS), were amplified and sequenced. The primers used in this study are shown in Table S2. The obtained DNA fragments were sequenced using forward and reverse primers. 5

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flow rate of 2 mL/min, to afford 2 (4.1 mg, tR = 29.8) and 3 (2.9 mg, tR = 45.2). Collacyclumine A (1): yellow crystals; 1H and 13C NMR data, see Table 1; HRESIMS: m/z: 261.11602 [M + H]+ (calcd for C15H21N2O2 261.1598), m/z: 283.1422 [M + Na]+ (calcd for C15H20N2NaO2 283.1417). Collacyclumine B (2): colorless oil; 1H and 13C NMR data, see Table 1; HRESIMS: m/z: 285.1812 [M + H]+ (calcd for C14H25N2O4 285.1809), m/z: 307.1632 [M + Na]+ (calcd for C14H24N2NaO4 307.1628). Collacyclumine C (3): colorless oil; 1H and 13C NMR data, see Table 1; HRESIMS: m/z: 221.1288 [M + H]+ (calcd for C12H17N2O2 221.1285), m/z: 243.1140 [M + Na]+ (calcd for C12H16N2NaO2 243.1104). Collacyclumine D (4): colorless oil; 1H and 13C NMR data, see Table 1; HRESIMS: m/z: 232.0982 [M ‒ H] ‒ (calcd for C13H14NO3 232.0979).

Foundation of Guangdong Province (2018A0303130219), Project from Institution of South China Sea Ecology and Environmental Engineering, CAS (ISEE2018PY04). We are grateful to Dr. Z. Xiao, A. Sun, C. Li, and Y. Zhang in the analytical facility at SCSIO for recording spectroscopic data. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.phytochem.2019.112237. References Ai, W., Wei, X., Lin, X., Sheng, L., Wang, Z., Tu, Z., Yang, X., Zhou, X., Li, J., Liu, Y., 2014. Guignardins A-F, spirodioxynaphthalenes from the endophytic fungus Guignardia sp KcF8 as a new class of PTP1B and SIRT1 inhibitors. Tetrahedron 70, 5806–5814. Aoyagi, S., Hasegawa, Y., Hirashima, S., Kibayashi, C., 1998. Total synthesis of (+)-homopumiliotoxin 223G. Tetrahedron Lett. 39, 2149–2152. Chen, Q.-B., Gao, J., Zou, G.-A., Xin, X.-L., Aisa, H.A., 2018a. Piperidine alkaloids with diverse skeletons from Anacyclus pyrethrum. J. Nat. Prod. 81, 1474–1482. Chen, S., Ding, M., Liu, W., Huang, X., Liu, Z., Lu, Y., Liu, H., She, Z., 2018b. Anti-inflammatory meroterpenoids from the mangrove endophytic fungus Talaromyces amestolkiae YX1. Phytochemistry 146, 8–15. Gu, B.-B., Jiao, F.-R., Wu, W., Jiao, W.-h., Li, L., Sun, F., Wang, S.-P., Yang, F., Lin, H.-W., 2018. Preussins with inhibition of IL-6 expression from Aspergillus flocculosus 16D-1, a fungus isolated from the marine sponge Phakellia fusca. J. Nat. Prod. 81, 2275–2281. Guo, L.D., Hyde, K.D., Liew, E.C.Y., 2000. Identification of endophytic fungi from Livistona chinensis based on morphology and rDNA sequences. New Phytol. 147, 617–630. Ibrahim, S.R.M., Mohamed, G.A., Moharram, A.M., Youssef, D.T.A., 2015. Aegyptolidines A and B: new pyrrolidine alkaloids from the fungus Aspergillus aegyptiacus. Phytochem. Lett. 12, 90–93. Jiang, Z.D., Gerwick, W.H., 1991. Novel pyrroles from the Oregon red alga Gracilariopsis lemaneiformis. J. Nat. Prod. 54, 403–407. Ju, Z., Lin, X., Lu, X., Tu, Z., Wang, J., Kaliyaperumal, K., Liu, J., Tian, Y., Xu, S., Liu, Y., 2015. Botryoisocoumarin A, a new COX-2 inhibitor from the mangrove Kandelia candel endophytic fungus Botryosphaeria sp KcF6. J. Antibiot. 68, 653–656. Kjer, J., Debbab, A., Aly, A.H., Proksch, P., 2010. Methods for isolation of marine-derived endophytic fungi and their bioactive secondary products. Nat. Protoc. 5, 479–490. Koshino, H., Lee, I.K., Kim, J.P., Kim, W.G., Uzawa, J., Yoo, I.D., 1996. Agrocybenine, novel class alkaloid from the Korean mushroom Agrocybe cylindracea. Tetrahedron Lett. 37, 4549–4550. Mackay, D., Dao, L.H., Dust, J.M., 1980. The reaction of nitrosocarbonyl compounds with 2,5disubstituted furans-Synthesis of 1,4,2-dioxazine and 1,4,2-dioxazole derivatives. J. Chem. Soc. Perk. T. 1, 2408–2416. May Zin, W.W., Buttachon, S., Dethoup, T., Pereira, J.A., Gales, L., Inacio, A., Costa, P.M., Lee, M., Sekeroglu, N., Silva, A.M.S., Pinto, M.M.M., Kijjoa, A., 2017. Antibacterial and antibiofilm activities of the metabolites isolated from the culture of the mangrove-derived endophytic fungus Eurotium chevalieri KUFA 0006. Phytochemistry 141, 86–97. Michael, J.P., 2008. Indolizidine and quinolizidine alkaloids. Nat. Prod. Rep. 25, 139–165. Mosmann, T., 1983. Rapid colorimetric assay for cellular growth and survival –application to proliferation and cyto-toxicity assays. J. Immunol. Methods 65, 55–63. O'Hagan, D., 2000. Pyrrole, pyrrolidine, pyridine, piperidine and tropane alkaloids. Nat. Prod. Rep. 17, 435–446. Padwa, A., Heidelbaugh, T.M., Kuethe, J.T., 2000. Using the Pummerer cyclization-deprotonation-cycloaddition cascade of imidosulfoxides for alkaloid synthesis. J. Org. Chem. 65, 2368–2378. Padwa, A., Sheehan, S.M., Straub, C.S., 1999. An isomunchnone-based method for the synthesis of highly substituted 2(1H)-pyridones. J. Org. Chem. 64, 8648–8659. Phainuphong, P., Rukachaisirikul, V., Saithong, S., Phongpaichit, S., Sakayaroj, J., Srimaroeng, C., Ontawong, A., Duangjai, A., Muangnil, P., Muanprasat, C., 2018. Asperidines A-C, pyrrolidine and piperidine derivatives from the soil-derived fungus Aspergillus sclerotiorum PSU-RSPG178. Bioorg. Med. Chem. 26, 4502–4508. Pinder, A.R., 1989. Pyrrolidine, piperidine, and pyridine alkaloids. Nat. Prod. Rep. 6, 515–521. Plunkett, A.O., 1994. Pyrrole, pyrrolidine, pyridine, piperidine, and azepine alkaloids. Nat. Prod. Rep. 11, 581–590. Pyridones Tobinaga, H., Kameyama, T., Oohara, M., Kobayashi, N., Ohdan, M., Ishizuka, N., Kume, M., Tomari, M., Tanaka, Y., Takahashi, F., Kinoshita, H., Shimada, S., Shinohara, S., Kai, H., 2018. Pyrrolinone derivatives as a new class of P2X3 receptor antagonists. Part 1: initial structure-activity relationship studies of a hit from a high throughput screening. Bioorg. Med. Chem. Lett 28, 2338–2342. Stamatakis, A., 2006. RAxML-VI-HPC: maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models. Bioinformatics 22, 2688–2690. Tokuyama, T., Nishimori, N., Shimada, A., Edwards, M.W., Daly, J.W., 1987. New classes of amidine, indolizidine and quinolizidine alkaloids from a poison-frog, (dendrobatidae). Tetrahedron 43, 643–652. Yang, B., Dong, J., Lin, X., Tao, H., Zhou, X., Liu, Y., 2014. Xylaolide A, a new lactone from the fungus Xylariaceae sp DPZ-SY43. Nat. Prod. Res. 28, 967–970. Yang, B., Tao, H., Lin, X., Wang, J., Liao, S., Dong, J., Zhou, X., Liu, Y., 2018. Prenylated indole alkaloids and chromone derivatives from the fungus Penicillium sp. SCSIO041218. Tetrahedron 74, 77–82. Zhou, G., Qiao, L., Zhang, X., Sun, C., Che, Q., Zhang, G., Zhu, T., Gu, Q., Li, D., 2018. Fusaricates H-K and fusolanones A-B from a mangrove endophytic fungus Fusarium solani HDN15-410. Phytochemistry 158, 13–19. Zhou, Z.H., Liu, Z.H., Qian, Y.D., Kim, S.B., Goodfellow, M., 1998. Saccharopolyspora spinosporotrichia sp. nov., a novel actinomycete from soil. Int. J. Syst. Bacteriol. 48, 53–58.

4.5. X-ray crystallographic data of 1 Chemical formula: C15 H20 N2 O2 (Mw = 260.33), Triclinic, space group P-21 with a = 7.9886(7) Å, b = 10.3895(5) Å, c = 9.4840(8) Å, V = 716.25(11) Å3, Z = 2, Dcalc = 1.207 Mg/m3, final R indices [I > 2sigma(I)] R1 = 0.0334, wR2 = 0.0907, R indices (all data) R1 = 0.0346, wR2 = 0.0915. Collacyclumine A (1) was crystallized from methanol to give white crystals. A single crystal with dimensions was selected and measured on a CrysAlis PRO CCD area detector diffractometer with graphite-monochromated Cu Kα radiation (λ = 1.541 78 Å). Crystallographic data for structure 1 in this paper have been deposited with the Cambridge Crystallographic Data Centre as supplementary publication number CCDC 1887534. Copies of the data can be obtained, free of charge, on application to CCDC, 12 Union Road, Cambridge CB21EZ, UK [fax: +44 (0)-1223-336033 or e-mail: [email protected]. ac.uk]. 4.6. Cytotoxic bioassay Compounds 1–5 were tested in vitro cytotoxic activity against several human tumour cell lines, including H1975, U937, K562, BGC823, MOLT-4, MCF-7, A549, HeLa, HL7702 and Huh-7 cell lines by the MTT method reported (Mosmann, 1983; Yang et al., 2014). Cultured cells were inoculated into each well (96-well plate) with 200 mL of the medium. After pre-incubation (24 h), aliquots of test compounds in MeOH were added to the culture wells. After incubation for 2 days, the toxic effect of each compound was observed under a microscope. The IC50 values were measured using the MTT colorimetric method. MTT solution (15 mL, 5mgmL21 in phosphate-buffered saline) was added to each well and incubated for 3 h. The medium was removed by aspiration and the residual formazan was dissolved in 100 mL of DMSO. The absorbance was measured at 560 nm with a Tecan Sunrise microplate reader. Declaration of competing interest All authors declare that they have no conflict of interest. Acknowledgments This work was supported by grants from the Open Research Fund Program of Beijing Key Lab of Plant Resource Research and Development, Beijing Technology and Business University (PRRD2017-ZD3), the National Natural Science Foundation of China (21977102, 41406187, 81973235, 81860626, 21772210, 41776169), Special Funds for Promoting Economic Development (Marine Economic Development) of Guangdong Province (GDOE[2019]A28), Guangzhou Science and Technology Project (201804010462), the Natural Science 6