English

• 1.   Introduction

  Marine sponges of the genus Agelas (class Demospongiae, order Agelasida, family Agelasidae) are important source of bioactive natural products with major representatives such as pyrrole alkaloids [1-4], diterpene alkaloids [5-7], glycolipids [8-10] and so on. The natural pyrrole alkaloids are a unique family structurally featured by a bromopyrrole-2-carboxamide core and diverse linear side chains [2, 3], fused rings [4], and dimmer structure patterns [1]. Since 1971 [11], more than 130 pyrrole alkaloids have been isolated from Agelas sponges. Many of these pyrrole alkaloids showed promising biological activities represented by the matrix metalloproteinase inhibitor ageladine A [12], antihistaminic taurodispacamide A [13], and antimicrobial nagelamide J [14]. In our continuing search for novel secondary metabolites with potential bioactivity from Agelas sponges collected off Xisha Islands (Paracel Islands) [15], an investigation on another species of A. nakamurai afforded three new non-brominated pyrrole alkaloids, nakamurines A-C (1-3) (Fig. 1). We herein describe the isolation, structure elucidation and biological activities of compounds 1-3.

  图 1

  

  图 1  Structures of compounds 1–3.

  Figure 1.  Structures of compounds 1–3.

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  2.   Results and discussion

  Nakamurine A (1) was obtained as a white amorphous solid having a molecular formula of C₇H₁₀O₂N₂ deduced from the positive HR-ESIMS (m/z 177.0632 [M + Na]⁺, calcd. 177.0634) and ¹³C NMR data (Table 1). The UV absorption band at λ_max 267 nm was attributed to a substituted pyrrole chromophore [16, 17], and IR absorption bands at 3286, 1643, 1558 cm^-1 indicated the occurrence of NH and amide carbonyl groups [18, 19]. A pyrrole-2-carboxamide moiety was readily verified by the amino group at δ_H 11.53 (br s, 1H, H-1), and the aromatic proton resonances at δ_H 6.89 (br s, 1H, H-2), 6.86 (br s, 1H, H-4), and 6.09 (m, 1H, H-3) in the ¹H NMR spectrum, which was consistent with the carbon resonances at δ_C 161.1 (C-6), 125.7 (C-5), 122.1 (C-2), 110.8 (C-4), and 108.7 (C-3) [20]. The speculation for the pyrrole-2-carboxamide moiety was further confirmed by consecutive ¹H-¹H COSY correlations of H-1/ H-2/H-3/H-4 and HMBC correlations from H-2 and H-3 to C-5, and from H-7 (δ_H 8.75, br t, 1H, J = 6.15 Hz) to C-6 (Fig. 2). Additionally, a methoxylated methylene group showing notable deshielded resonances at δ_H 4.62 (d, 2H, J = 6.15 Hz, H₂-8) and δ_C 70.5 (C-8) was connected to N-7 by ¹H-¹H COSY correlation of H-7/H₂-8 and HMBC correlations of H₂-8 with C-6 and C-OMe (δ_C 54.9), and of H₃-OMe [δ_H 3.21(s, 3H)] with C-8 (Fig. 2). Thus, nakamurine A (1) was determined as a debromo-analogue of 5-bromopyrrole-2-(N-methoxymethyl)carboxamide which was isolated from the Papua New Guinean sponge A. nakamurai [21].

  表 1

  

  表 1  ¹H (500 MHz) and ¹³C (125 MHz) NMR data for compounds 1–3 in DMSO-d₆.

  Table 1.  ¹H (500 MHz) and ¹³C (125 MHz) NMR data for compounds 1–3 in DMSO-d₆.

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  图 2

  

  图 2  Key ¹H-¹H COSY and HMBC correlations for compounds 1–3.

  Figure 2.  Key ¹H-¹H COSY and HMBC correlations for compounds 1–3.

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  Nakamurine B (2), a colorless oil, was determined to have the molecular formula of C₁₀H₁₂O₃N₂ on the basis of positive HR-ESIMS (m/z 209.0918[M + H]⁺, calcd. 209.0921) and ¹³C NMR data(Table 1). 1D NMR and HSQC data of 2 revealed the presence of a pyrrole-2-carboxamidemoiety.Besides, thesignalsofonemethoxy[δ_H 3.64(s, 3H)/δ_C 51.5], one methylene [δ_H 4.01(t, 2H, J = 4.30 Hz, H₂-8)/δ_C 39.4 (C-8)], two methines [δ_H 5.87(d, 1H, J = 15.75 Hz, H-10)/δ_C 119.9 (C-10), δ_H 6.91(dt, 1H, J = 15.75, 4.65 Hz, H-9)/δ_C 146.8 (C-9)], and one carboxyl group [δ_C 166.1 (C-11)] were observed, which strongly suggested that compound 2 could be an analogue of acanthamide A isolated from the sponge Acanthostylotella sp. [22]. The spin-spin coupling system of H-7/H₂-8/H-9/H-10 deduced from ¹H-¹H COSY spectrum and HMBC correlations from H-9 and H₃-OMe to C-11 (Fig. 2) further indicated that the methyl crotonate moiety was connected to N-7. In addition, a large coupling constant of J_H-9/H-10 = 15.75 Hz indicated the 9E-geometry. Accordingly, the structure of nakamurine B (2) was concluded as shown in Fig. 1.

  Nakamurine C (3) was isolated as a colorless oil. The molecular formula of 3 was determined to be C₇H₈O₃N₂ by positive HR-ESIMS (m/z 191.0426 [M + Na]⁺, calcd. 191.0427). The NMR spectra of 3 (Table 1) obviously showed the presence of a 2-carboxamide pyrrole core. Careful comparison of the NMR data of 3 with those of 1 disclosed an extra quaternary carbon at δ_C 152.0 (C-8) in 3 instead of the methylene group in 1, indicating the presence of an N-formyl-carboxamide moiety in 3 [20]. The methoxy group [δ_H 3.69 (s, 3H)/δ_C 51.9] was assigned at C-8 by a HMBC correlation of H₃-OMe with C-8 (Fig. 2). Therefore, the structure of nakamurine C (3) was established as shown in Fig. 1.

  Compounds 1-3 were evaluated for cytotoxic activities against four human tumor cell lines (K562, HL-60, A549, and HCT-116), antiviral activity against H1N1 influenza A virus, and antimicrobial activities against Staphylococcus aureus, Escherichia coli and Candida albicans. Compounds 1-3 showed no activity in the cytotoxicities tests (the inhibition ratios < 30% at a concentration of 50 μmol/L) and in the antiviral activity assays (the inhibition ratios < 3% at a concentration of 50 μg/mL). While in antimicrobial assay, compound 2 exhibited weak inhibitory activity against Candida albicans with MIC of 60 μg/mL (positive drug fungicidin MIC = 3.75 μg/mL).

  3.   Conclusion

  Since 1971, only 10 non-brominated pyrrole-2-carboxamide metabolites have been isolated from the marine sponges of the genus Agelas, including the Great Barrier Reef sponge A. oroides [20], Solomon Islands sponges A. mauritiana [23] and A. cf. mauritiana [24, 25], and Caribbean sponges A. conifera [26] and A. clathrodes [27]. Our present study on the sponge A. nakamurai collected from the Xisha Islands of the South China Sea yielded three new non-brominated pyrrole-2-carboxamide alkaloids, nakamurines A-C (1-3). Their structures were established on the basis of comprehensive spectroscopic analyses. It was noticed that the sponge Agelas genus derived linear pyrrole-2-carboxamide metabolites containing an ester group at the end of the side chain were only found in the South China Sea [4]. In the bioassay, compound 2 showed weak antimicrobial activity against Candida albicans with MIC of 60 μg/mL, whereas no activity was found in the cytotoxicities tests and in the antiviral activity assays for nakamurines A-C (1-3).

  4.   Experimental

  4.1   General experimental procedures

  UV spectra were measured on a Beckman DU640 spectrophotometer (Beckman Coulter Inc., Brea, CA, USA). IR spectra were taken on a Nicolet NEXUS 470 spectrophotometer (International Equipment Trading Ltd., Vernon Hills, IL, USA) in KBr discs. NMR experiments were performed on a JEOL JNMECP 500 spectrometer (JEOL Ltd., Tokoyo, Japan). The 2.50 ppm and 39.50 ppm resonances of DMSO-d₆ were used as internal references for ¹H NMR and ¹³C NMR spectra, respectively. HR-ESIMS spectra were acquired using a Micromass Q-TOF Ultima Global GAA076 LC mass spectrometer (Thermo Fisher Scientific Inc., Waltham, MA, USA). HPLC separation was carried out on an Agilent 1100 series instrument with DAD detector (Agilent Technologies, Palo Alto, CA, USA), equipped with a semi-preparative ODS column (YMC-Pack ODS-A, 5 μm, 250 mm × 10 mm). MPLC utilized a self-made ODS column (310 mm × 26 mm, 15 mL/min). Silica gel (200-300 mesh, 300-400 mesh, Qingdao Haiyang Chemical Co. Ltd., Qingdao, China) and ODS silica gel (50 μm, Merck, Darmstadt, Germany) were used for column chromatography (CC). Precoated silica gel plates (GF254, Qingdao Haiyang Chemical Co. Ltd., Qingdao, China) were used for thin layer chromatography (TLC) analyses, and spots were visualized by heating silica gel plates sprayed with 10% H₂SO₄ in EtOH.

  4.2   Animal material

  The sponge A. nakamurai featuring bright orange color was collected from the Xisha Islands of the South China Sea in October 2012, at a depth of about 10 meters. The specimen was identified by Nicole J. de Voogd, a coauthor in National Museum of Natural History, Leiden, The Netherlands. A voucher specimen (NO. XS-2012-09) was deposited in the school of Medicine and Pharmacy, Ocean University of China, China.

  4.3   Extraction and isolation

  The frozen organism (5.0 kg, wet weight) was homogenized and extracted with MeOH four times (each time, 5 L, 3 days) at room temperature. The combined solutions were concentrated in vacuum and desalted by anhydrous MeOH three times (0.6 L, 0.5 L, 0.4 L) to yield a residue (110.0 g). The crude extract was subjected to silica gel vacuum liquid chromatography (VLC), eluting with a gradient of petroleum ether-acetone (from 50:1 to 1:1, v/v) and subsequently CH₂Cl₂-MeOH (from 20:1 to 1:1, v/v) to obtain seven fractions(A1-A7). Fr. A4 and Fr. A5 were found to seemingly contain pyrrole alkaloids by TLC and HPLC analysis. Fr. A4 (3.4 g) was subjected to silica gel CC using petroleum ether-acetone (from 40:1 to 1:1, v/v) as eluent to give five subfractions (A4.1-A4.5). Then Fr. A4.4 (1.3 g) was separated by ODS CC with stepwise elution of MeOH-H₂O (30:70, 40:60, 45:55, 50:50, 60:40, 70:30 and 100:0, v/v) as the mobile phase to yield six subfractions (A4.4.1-A4.4.6). Fr. A4.4.3 (77.0 mg) was further purified by semi-preparative HPLC (ODS, 5 μm, 250 mm × 10 mm; MeOH-H₂O, 15:85, v/v; 1.5 mL/min; UV detection at 280 nm) to afford compound 1 (2.5 mg, t_R = 30 min). Fr. A5 (9.5 g) was subjected to silica gel CC (petroleum ether-acetone, from 30:1 to 1:1, v/v) to give seven subfractions (A5.1-A5.7). Fr. A5.5 (1.8 g) was then separated by ODS CC with stepwise elution of MeOH-H₂O (25:75, 35:65, 45:55, 55:45, 65:35, 75:25, 85:15 and 100:0, v/v) to get four subfractions (A5.5.1-A5.5.4). Fr. A5.5.2 (110.0 mg) was finely purified by semi-preparative HPLC (ODS, 5 μm, 250 × 10 mm; MeOH-H₂O, 20:80, v/v; 1.5 mL/min; UV detection at 280 nm) to afford compound 2 (15 mg, t_R = 55 min). While Fr. A5.6 (1.1 g) was fractionated by MPLC (ODS, 310 mm × 26 mm; MeOH-H₂O, 15:85, v/v; 15 mL/min) and was further purified by semi-preparative HPLC (ODS, 5 μm, 250 mm × 10 mm; MeOH-H₂O-TFA, 8:92:0.1, v/v; 2.0 mL/min; UV detection at 210 nm) to afford compound 3 (4.5 mg, t_R = 50 min).

  Nakamurine A (1): White amorphous solid; UV (MeOH) λ_max (log ε) 206 (2.12), 267 (2.60) nm; IR (KBr) v_max 3286, 2958, 1643, 1558, 1521, 1326, 1064, 746 cm^-1; ¹H NMR (DMSO-d₆, 500 MHz) and ¹³C NMR (DMSO-d₆, 125 MHz) data, see Table 1; HRESIMS m/z 177.0632 [M + Na]⁺ (calcd. for C₇H₁₀O₂ N₂Na, 177.0634).

  Nakamurine B (2): Colorless oil; UV (MeOH) λ_max (log ε) 207 (2.79), 266 (2.84) nm; IR (KBr) v_max 3377, 2937, 1735, 1682, 1656, 1540, 1456, 1201, 1039, 970, 761 cm^-1; ¹H NMR (DMSO-d₆, 500 MHz) and ¹³C NMR (DMSO-d₆, 125 MHz) data, see Table 1; HR-ESIMS m/z 209.0918 [M + H]⁺ (calcd. for C₁₀H₁₃ O₃N₂, 209.0921).

  Nakamurine C (3): Colorless oil; UV (MeOH) λ_max (log ε) 205 (2.24), 283 (2.75) nm; IR (KBr) v_max 3252, 2947, 1733, 1690, 1650, 1538, 1471, 1235, 740 cm^-1; ¹H NMR (DMSO-d₆, 500 MHz) and ¹³C NMR (DMSO-d₆, 125 MHz) data, see Table 1; HR-ESIMS m/z 191.0426 [M + Na]⁺ (calcd. for C₇H₈O₃N₂Na, 191.0427).

  4.4   Biological assays

  In vitro cytotoxicities against four tumor cell lines (human leukemia, K562; human myeloid leukemia, HL-60; human lung carcinoma, A549; and human colon carcinoma, HCT-116) were evaluated by MTT [28] and SRB [29] methods with adriamycin (doxorubicin, ADM) as positive control. The antiviral activity against H1N1 influenza A virus was evaluated by the CPE inhibition assay [30] using MDCK cells. Ribavirin was used as positive control. Antimicrobial activity was determined by the filter paper disc diffusion method and conventional broth dilution assay [31]. Three microbial strains including Staphylococcus aureus, Escherichia coli and Candida albicans were used. The control drugs penicillin G for Staphylococcus aureus, streptomycin sulfate for Escherichia coli and fungicidin for Candida albicans were included in each assay.

  Acknowledgments

  This work was supported by the National Natural Science Foundation of China (Nos. 41376142, 41476107, 41522605), NSFCShandong Joint Fund for Marine Science Research Centers (Nos. U1406402), AoShan Talents Program Supported by Qingdao National Laboratory for Marine Science and Technology (No. 2015ASTP) and Science and Technology Program of Applied Basic Research Projects of Qingdao Municipal Government (No. 14-2-4-32-jch). Special thanks are given to L. Liu (Marine Biomedical Research Institute, Qingdao, China), Dr. W. Wang (School of Medicine and Pharmacy, Ocean University of China, Qingdao, China) for the cytotoxicity and antivirus tests.

• 1. [1]

     Cychon C., Lichte E., Köck M.. The marine sponge Agelas citrina as a source of the new pyrrole-imidazole alkaloids citrinamines A-D and Nmethylagelongine[J]. Beilstein J. Org. Chem., 2015, 11:  2029-2037. doi: 10.3762/bjoc.11.220

  2. [2]

     Kusama T., Tanaka N., Kashiwada Y., Kobayashi J.. Agelamadin F and tauroacidin E, bromopyrrole alkaloids from an Okinawan marine sponge Agelas sp[J]. Tetrahedron Lett., 2015, 56:  4502-4504. doi: 10.1016/j.tetlet.2015.05.114

  3. [3]

     Tanaka N., Kusama T., Takahashi-Nakaguchi A.. Nagelamides U-W, bromopyrrole alkaloids from a marine sponge Agelas sp[J]. Tetrahedron Lett., 213, 54:  3794-3796.

  4. [4]

     Zhu Y., Wang Y., Gu B.B.. Antifungal bromopyrrole alkaloids from the South China Sea sponge Agelas sp[J]. Tetrahedron, 2016, 72:  2964-2971. doi: 10.1016/j.tet.2016.04.020

  5. [5]

     Abdjul D.B., Yamazaki H., Kanno S.. Structures and biological evaluations of agelasines isolated from the Okinawan marine sponge Agelas nakamurai[J]. J. Nat. Prod., 2015, 78:  1428-1433. doi: 10.1021/acs.jnatprod.5b00375

  6. [6]

     Kubota T., Iwai T., Takahashi-Nakaguchi A.. Agelasines O-U, new diterpene alkaloids with a 9-N-methyladenine unit from a marine sponge Agelas sp[J]. Tetrahedron, 2012, 68:  9738-9744. doi: 10.1016/j.tet.2012.09.040

  7. [7]

     Appenzeller J., Mihci G., Martin M.T.. Agelasines J K, and L from the Solomon Islands marine sponge Agelas cf. mauritiana[J]. J. Nat. Prod., 2008, 71:  1451-1454. doi: 10.1021/np800212g

  8. [8]

     Costantino V., Fattorusso E., Imperatore C.. Clathrosides and isoclathrosides, unique glycolipids from the Caribbean sponge Agelas clathrodes[J]. J. Nat. Prod., 2006, 69:  73-78. doi: 10.1021/np050331v

  9. [9]

     Pettit G.R., Xu J.P., Gingrich D.E.. Isolation and structure of agelagalastatin from the Papua New Guinea marine sponge Agelas sp[J]. Chem. Commun., 1999, 10:  915-916.

  10. [10]

      Costantino V., Fattorusso E., Mangoni A.. Immunomodulating glycosyl ceramides from the marine sponge Agelas dispar[J]. Tetrahedron, 1996, 52:  1573-1578. doi: 10.1016/0040-4020(95)00986-8

  11. [11]

      Forenlza S., Minale L., Riccio R.. New bromo-pyrrole derivatives from the sponge Agelas oroides[J]. Chem. Commun., 1971, 18:  1129-1130.

  12. [12]

      Fujita M., Nakao Y., Matsunaga S.. Ageladine A:an antiangiogenic matrixmetalloproteinase inhibitor from the marine sponge Agelas nakamurai[J]. J. Am. Chem. Soc., 2003, 125:  15700-15701. doi: 10.1021/ja038025w

  13. [13]

      Fattorusso E., Taglialatela-Scafati O.. Two novel pyrrole-imidazole alkaloids from the mediterranean sponge Agelas oroides[J]. Tetrahedron Lett., 2000, 41:  9917-9922. doi: 10.1016/S0040-4039(00)01764-0

  14. [14]

      Araki A., Tsuda M., Kubota T.. Nagelamide J:a novel dimeric bromopyrrole alkaloid from a sponge Agelas species[J]. Org. Lett., 2007, 9:  2369-2371. doi: 10.1021/ol0707867

  15. [15]

      Li T., Wang B., de Voogd N.J.. Two new diterpene alkaloids from the South China Sea sponge Agelas aff. nemoechinata[J]. Chin. Chem. Lett., 2016, 27:  1048-1051. doi: 10.1016/j.cclet.2016.05.017

  16. [16]

      Iwai T., Kubota T., Fromont J.. Nagelamide I and 2, 2-Didebromonagelamide B, new dimeric bromopyrrole-imidazole alkaloids from a marine sponge Agelas sp[J]. Chem. Pharm. Bull., 2014, 62:  213-216. doi: 10.1248/cpb.c13-00821

  17. [17]

      Tsuda M., Uemoto H., Kobayashi J.. Slagenins A~C, novel bromopyrrole alkaloids from marine sponge Agelas nakamurai[J]. Tetrahedron Lett., 1999, 40:  5709-5712. doi: 10.1016/S0040-4039(99)01075-8

  18. [18]

      Tilvi S., Moriou C., Martin M.T.. Agelastatin E agelastatin F, and benzosceptrin C from the marine sponge Agelas dendromorpha[J]. J. Nat. Prod., 2010, 73:  720-723. doi: 10.1021/np900539j

  19. [19]

      Uemoto H., Tsuda M., Kobayashi J.. MukanadinsA-C, newbromopyrrolealkaloids from marine sponge Agelas nakamurai[J]. J. Nat. Prod., 1999, 62:  1581-1583. doi: 10.1021/np9902542

  20. [20]

      König G.M., Wright A.D.. Two new naturally occurring pyrrole derivatives from the tropical marine sponge Agelas oroides[J]. Nat. Prod. Lett., 1994, 5:  141-146. doi: 10.1080/10575639408044048

  21. [21]

      Iwagawa T., Kaneko M., Okamura H.. New alkaloids from the Papua New Guinean sponge Agelas nakamurai[J]. J. Nat. Prod., 1998, 61:  1310-1312. doi: 10.1021/np980173q

  22. [22]

      Ebada S.S., Edrada-Ebel R., de Voogd N.J.. Dibromopyrrole alkaloids from the marine sponge Acanthostylotella sp[J]. Nat. Prod. Commun., 2009, 4:  47-52.

  23. [23]

      Vergne C., Appenzeller J., Ratinaud C.. Debromodispacamides B and D:isolation from the marine sponge Agelas mauritiana and stereoselective synthesis using a biomimetic proline route[J]. Org. Lett., 2008, 10:  493-496. doi: 10.1021/ol702866m

  24. [24]

      Appenzeller J., Tilvi S., Martin M.T.. Benzosceptrins A and B with a unique benzocyclobutane skeleton and nagelamide S and T from pacific sponges[J]. Org. Lett., 2009, 11:  4874-4877. doi: 10.1021/ol901946h

  25. [25]

      Schroif-Grégoirea C., Appenzellera J., Debitus C.. Debromokeramadine from the marine sponge Agelas cf. mauritiana:isolation and short regioselective and flexible synthesis[J]. Tetrahedron, 2015, 71:  3609-3613. doi: 10.1016/j.tet.2014.12.081

  26. [26]

      Shen X., Perry T.L., Dunbar C.D.. Debromosceptrin, an alkaloid from the Caribbean sponge Agelas conifera[J]. J. Nat. Prod., 1998, 61:  1302-1303. doi: 10.1021/np980129a

  27. [27]

      Morales J.J., Rodriguez A.D.. The structure of clathrodin, a novel alkaloid isolated from the Caribbean Sea sponge Agelas clathrodes[J]. J. Nat. Prod., 1991, 54:  629-631. doi: 10.1021/np50074a051

  28. [28]

      Alley M.C., Scudiero D.A., Monks A.. Feasibility of drug screening with panels of human tumor cell lines using a microculture tetrazolium assay[J]. Cancer. Res., 1988, 48:  589-601.

  29. [29]

      Skehan P., Storeng R., Scudiero D.. Newcolorimetric cytotoxicity assay for anticancer-drug screening[J]. J. Natl. Cancer Inst., 1990, 82:  1107-1112. doi: 10.1093/jnci/82.13.1107

  30. [30]

      Wang W., Zhang P., Yu G.L.. Preparation and anti-influenza A virus activity of κ-carrageenan oligosaccharide and its sulphated derivatives[J]. Food Chem., 2012, 133:  880-888. doi: 10.1016/j.foodchem.2012.01.108

  31. [31]

      Pierce C.G., Uppuluri P., Tristan A.R.. A simple and reproducible 96-well plate-based method for the formation of fungal biofilms and its application to antifungal susceptibility testing[J]. Nat. Protoc., 2008, 3:  1494-1500. doi: 10.1038/nprot.2008.141

• 

  Figure 1  Structures of compounds 1–3.

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  Figure 2  Key ¹H-¹H COSY and HMBC correlations for compounds 1–3.

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  Table 1.  ¹H (500 MHz) and ¹³C (125 MHz) NMR data for compounds 1–3 in DMSO-d₆.

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•