*3.5. Evaluation for Antiproliferative Activity of* **1**–**5**

New bromopyrrole alkaloids **1**–**5** were evaluated for their antiproliferative activity against human cancer cell lines (HeLa, A549, and MCF7) according to the following procedure. The human cancer cell lines were cultured in Dulbecco's modified eagle medium (DMEM) supplemented with 5% fetal bovine serum (FBS). All cells were incubated at 37 ◦C in a humidified atmosphere with 5% CO2–95% air. Cells were seeded at 1 <sup>×</sup> 10<sup>4</sup> cells/well in a 96-well plate and preincubated for 24 h. Test samples were dissolved in small amounts of DMSO and diluted in the appropriate culture medium (final concentration of DMSO < 1%). After removal of the preincubated culture medium, 100 μL of medium containing various concentrations of test compound was added and further incubated for 48 h. A cell proliferation assay was performed with the Cell Counting Kit-8 (WST-8; Dojindo, Japan) according to the manufacturer's instruction. Briefly, the WST-8 reagent solution (10 μL) was added to each well of a 96-well microplate containing 100 μL of cells in the culture medium at various densities, and the plate was incubated for 2 h at 37 ◦C. Absorbance was measured at 450 nm using a microplate reader. Cisplatin was used as a positive control, whose IC50 values against HeLa, A549, and MCF7 cells were 11.7, 7.2, and 52.4 mM, respectively.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/1660-3397/18/9/455/s1. Figure S1: 1H NMR spectrum of agesasine A (**1**) in DMSO-*d*<sup>6</sup> (500 MHz), Figure S2: 13C NMR spectrum of agesasine A (**1**) in DMSO-*d*<sup>6</sup> (125 MHz), Figure S3: 1H-1H COSY spectrum of agesasine A (**1**) in DMSO-*d*<sup>6</sup> (500 MHz), Figure S4: HSQC spectrum of agesasine A (**1**) in DMSO-*d*<sup>6</sup> (500 MHz), Figure S5: HMBC spectrum of agesasine A (**1**) in DMSO-*d*<sup>6</sup> (500 MHz), Figure S6: ROESY spectrum of agesasine A (**1**) in DMSO-*d*<sup>6</sup> (500 MHz), Figure S7: HRESIMS spectrum (pos.) of agesasine A (**1**), Figure S8: Chiral HPLC chart of agesasine A (**1**), Figure S9: 1H NMR spectrum of agesasine B (**2**) in DMSO-*d*<sup>6</sup> (500 MHz), Figure S10: 13C NMR spectrum of agesasine B (**2**) in DMSO-*d*<sup>6</sup> (125 MHz), Figure S11: 1H-1H COSY spectrum of agesasine B (**2**) in DMSO-*d*<sup>6</sup> (500 MHz), Figure S12: HSQC spectrum of agesasine B (**2**) in DMSO-*d*<sup>6</sup> (500 MHz), Figure S13: HMBC spectrum of agesasine B (**2**) in DMSO-*d*<sup>6</sup> (500 MHz), Figure S14: HRESIMS spectrum (neg.) of agesasine B (**2**), Figure S15: 1H NMR spectrum of 9-hydroxydihydrodispacamide (**3**) in DMSO-*d*<sup>6</sup> (500 MHz), Figure S16: 13C NMR spectrum of 9-hydroxydihydrodispacamide (**3**) in DMSO-*d*<sup>6</sup> (125 MHz), Figure S17: 1H-1H COSY spectrum of 9-hydroxydihydrodispacamide (**3**) in DMSO-*d*<sup>6</sup> (500 MHz), Figure S18: HSQC spectrum of 9-hydroxydihydrodispacamide (**3**) in DMSO-*d*<sup>6</sup> (500 MHz), Figure S19: HMBC spectrum of 9-hydroxydihydrodispacamide (**3**) in DMSO-*d*<sup>6</sup> (500 MHz), Figure S20: HRESIMS spectrum (pos.) of 9-hydroxydihydrodispacamide (**3**), Figure S21: Chiral HPLC chart of 9-hydroxydihydrodispacamide (**3**), Figure S22: 1H NMR spectrum of 9-hydroxydihydrooroidin (**4**) in DMSO-*d*<sup>6</sup> (500 MHz), Figure S23: 13C NMR spectrum of 9-hydroxydihydrooroidin (**4**) in DMSO-*d*<sup>6</sup> (125 MHz), Figure S24: 1H-1H COSY spectrum of 9-hydroxydihydrooroidin (**4**) in DMSO-*d*<sup>6</sup> (500 MHz), Figure S25: HSQC spectrum of 9-hydroxydihydrooroidin (**4**) in DMSO-*d*<sup>6</sup> (500 MHz), Figure S26: HMBC spectrum of 9-hydroxydihydrooroidin (**4**) in DMSO-*d*<sup>6</sup> (500 MHz), Figure S27: HRESIMS spectrum (pos.) of 9-hydroxydihydrooroidin (**4**), Figure S28: ECD spectrum of 9-hydroxydihydrooroidin (**4**) in MeOH, Figure S29: 1H NMR spectrum of 9*E*-keramadine (**5**) in DMSO-*d*<sup>6</sup> (500 MHz), Figure S30: 13C NMR spectrum of 9*E*-keramadine (**5**) in DMSO-*d*<sup>6</sup> (125 MHz), Figure S31: 1H-1H COSY spectrum of 9*E*-keramadine (**5**) in DMSO-*d*<sup>6</sup> (500 MHz), Figure S32: HSQC spectrum of 9*E*-keramadine (**5**) in DMSO-*d*<sup>6</sup> (500 MHz), Figure S33: HMBC spectrum of 9*E*-keramadine (**5**) in DMSO-*d*<sup>6</sup> (500 MHz), Figure S34: ROESY spectrum of 9*E*-keramadine (**5**) in DMSO-*d*<sup>6</sup> (500 MHz), Figure S35: HRESIMS spectrum (pos.) of 9*E*-keramadine (**5**), Figure S36: 1H NMR spectrum of tauroacidin A in DMSO-*d*<sup>6</sup> (500 MHz), Figure S37: 1H NMR spectrum of taurodispacamide A in DMSO-*d*<sup>6</sup> (500 MHz), Figure S38: 1H NMR spectrum of oroidin in DMSO-*d*<sup>6</sup> (500 MHz), Figure S39: 1H NMR spectrum of keramadine in DMSO-*d*<sup>6</sup> (500 MHz), Figure S40: 1H NMR spectrum of 2-bromo-9,10-dihydrokeramadine in DMSO-*d*<sup>6</sup> (500 MHz), Figure S41: 1H NMR spectrum of nagelamide L in DMSO-*d*<sup>6</sup> (500 MHz), Figure S42: Structures of known bromopyrrole alkaloids, tauroacidin A, taurodispacamide A, oroidin, keramadine, 2-bromokeramadine, and nagelamide L, Figure S43: Antiproliferative activity of **1**–**5** against HeLa cells, Figure S44: Antiproliferative activity of **1**–**5** against A549 cells, Figure S45: Antiproliferative activity of **1**–**5** against MCF7 cells, Table S1: 1D and 2D NMR data for agesasine A (**1**) in DMSO-*d*6, Table S2: 1D and 2D NMR data for agesasine B (**2**) in DMSO-*d*6, Table S3: 1D and 2D NMR data for 9-hydroxydihydrodispacamide (**3**) in DMSO-*d*6, Table S4: 1D and 2D NMR data for 9-hydroxydihydrooroidin (**4**) in DMSO-*d*6, Table S5: 1D and 2D NMR data for 9*E*-keramadine (**5**) in DMSO-*d*6, Table S6: 1H NMR data for tauroacidin A and taurodispacamide A in DMSO-*d*6, Table S7: 1H NMR data for oroidin, keramadine, and 2-bromo-9,10-dihydrokeramadine in DMSO-*d*6, Table S8: 1H NMR data for nagelamide L in DMSO-*d*6.

**Author Contributions:** Conceptualization, S.L., N.T., D.T., S.-Y.K., M.K., K.I., J.K., and Y.K.; methodology, S.L. and N.T.; validation, S.L. and N.T.; formal analysis, S.L., N.T., and Y.K.; investigation, S.L., S.T., D.T., and S.-Y.K.; resources, J.K.; writing—original draft preparation, S.L.; writing—review and editing, N.T., M.K., K.I., and Y.K. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was partly supported by JSPS KAKENHI, grant number JP17K08337.

**Acknowledgments:** We thank Z. Nagahama for his help with the sponge collection.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


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