**4. Conclusions**

In conclusion, one novel aminopyrimidone alkaloid compound, ernstine A (**1**), seven new aminoimidazole alkaloid compounds, phorbatopsins D-E (**2**, **3**), calcaridine C (**4**), naamines H–I (**5**, **7**), naamidines J–K (**6**, **8**) were isolated from a CH2Cl2-MeOH extract from *Ernsta naturalis* along with the known thymidine (**9**). To improve the investigation of this unstudied calcareous marine sponge, a metabolomic study by molecular networking was conducted. This strategy, based on the interpretation of MS/MS spectra of other compounds grouped in the same clusters than the isolated ones due to their structural feature similarities, allowed us to propose structural hypotheses for 16 compounds, 5 known and 11 potentially new.

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/md20100637/s1, Figure S1: HRESIMS spectrum for ernstine A (1), Figure S2: 1H NMR (600 MHz, CD3OD) spectrum for ernstine A (1), Figure S3: 1H-1H COSY NMR (600 MHz) spectrum for ernstine A (1), Figure S4: 1H-13C HSQC NMR (600 MHz) spectrum for ernstine A (1), Figure S5: 1H-13C HMBC NMR (600 MHz) spectrum for ernstine A (1), Figure S6: 1H-1H NOESY NMR (600 MHz) spectrum for ernstine A (1), Figure S7: HRESIMS spectrum for phorbatopsin D (2), Figure S8: 1H NMR (600 MHz, CD3OD) spectrum for phorbatopsin D (2), Figure S9: 13C NMR (125 MHz, CD3OD) spectrum for phorbatopsin D (2), Figure S10: 1H-13C HSQC NMR (600 MHz) spectrum for phorbatopsin D (2), Figure S11: 1H-13C HMBC NMR (600 MHz) spectrum for phorbatopsin D (2), Figure S12: HRESIMS spectrum for phorbatopsin E (3), Figure S13: 1H NMR (600 MHz, CD3OD) spectrum for phorbatopsin E (3), Figure S14: 1H-13C HSQC NMR (600 MHz) spectrum for phorbatopsin E (3), Figure S15: 1H-13C HMBC NMR (600 MHz) spectrum for phorbatopsin E (3), Figure S16: 1H-1H NOESY NMR (600 MHz) spectrum for phorbatopsin E (3), Figure S17: HRESIMS spectrum for calcaridine C (4), Figure S18: 1H NMR (600 MHz, CD3OD) spectrum for calcaridine C (4), Figure S19: 13C NMR (125 MHz, CD3OD) spectrum for calcaridine C (4), Figure S20: 1H-1H COSY NMR (600 MHz) spectrum for calcaridine C (4), Figure S21: 1H-13 C HSQC NMR (600 MHz) spectrum for calcaridine C (4), Figure S22: 1H-13C HMBC NMR (600 MHz) spectrum for calcaridine C (4), Figure S23: 1H-1H NOESY NMR (600 MHz) spectrum for calcaridine C (4), Figure S24: HRESIMS spectrum for naamine H (5), Figure S25: 1H NMR (600 MHz, CD3OD) spectrum for naamine H (5), Figure S26: 1H-13C HSQC NMR (600 MHz) spectrum for naamine H (5), Figure S27: 1H-13C HMBC NMR (600 MHz) spectrum for naamine H (5), Figure S28: 1H- 1H NOESY NMR (600 MHz) spectrum for naamine H (5), Figure S29: HRESIMS spectrum for naamidine J (6), Figure S30: 1H NMR (600 MHz, CD3OD) spectrum for naamidine J (6), Figure S31: 13C NMR (125 MHz, CD3OD) spectrum for naamidine J (6), Figure S32: 1H-1H COSY NMR (600 MHz) spectrum for naamidine J (6), Figure S33: 1H-13C HSQC NMR (600 MHz) spectrum for naamidine J (6), Figure S34: 1H-13C HMBC NMR (600 MHz) spectrum for naamidine J (6), Figure S35: 1H-1H NOESY NMR (600 MHz) spectrum for naamidine J (6), Figure S36: HRESIMS spectrum for naamine I (7), Figure S37: HRESIMS spectrum for naamidine K (8), Figure S38: 1H NMR (600 MHz, CD3OD) spectrum for mixture of naamine I (7) and naamidine K (8), Figure S39: 1H-13C HSQC NMR (600 MHz) spectrum for mixture of naamine I (7) and naamidine K (8), Figure S40: 1H-13 C HMBC NMR (600 MHz) spectrum for mixture of naamine I (7) and naamidine K (8), Figure S41: 1H NMR (600 MHz, CD3OD) spectrum for thymidine (9), Figure S42: 13C NMR (125 MHz, CD3OD) spectrum for thymidine (9), Figure S43: 1H-1H COSY NMR (600 MHz) spectrum for thymidine (9), Figure S44: 1H-13C HSQC NMR (600 MHz) spectrum for thymidine (9), Figure S45: 1H-13C HMBC NMR (600 MHz) spectrum for thymidine (9), Figure S46: MS/MS spectra of the isolated compounds were deposited in the GNPS spectral libraries under following identifier, Figure S47: MS/MS spectrum of ernstine A (1), Figure S48: MS/MS spectrum of Phorbatopsin D (2), Figure S49: MS/MS spectrum of Phorbatopsin E(3), Figure S50: MS/MS spectrum of Naamine H (5), Figure S51: MS/MS spectrum of Naamine I (7), Figure S52: MS/MS spectrum of Naamidine J (6), Figure S53: MS/MS spectrum of Naamidine K (8), Figure S54: MS/MS spectrum of Calcaridine C (4).

**Author Contributions:** A.G.-B., G.L.G. and J.O. designed the project, supervised the whole experiment, and prepared the manuscript. G.H., L.F., F.T., P.C. and P.-E.C. performed the chemical experiments (extraction, isolation, and structural identification of the compounds). P.-E.C. and L.F. wrote the first draft of the manuscript. A.G.-B. organized the sponge collection, and the sponge was identified by N.J.d.V. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the TASCMAR project, which is funded by the European Union under gran<sup>t</sup> agreemen<sup>t</sup> number 634674.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The MS/MS and NMR data presented in this study are openly available in Zenodo at https://doi.org/10.5281/zenodo.7152302. The obtained molecular network can be accessed at: https://gnps.ucsd.edu/ProteoSAFe/status.jsp?task=527472b15d4247dfad0534aa80f7ebfa.

**Acknowledgments:** The authors express their gratitude to the SALSA platform at ICOA for the access to Bruker Data Analysis 4.4 and MetaboScape 4.0 software and for the technical help for the retreatment of UPLC/HRMS/MS analysis.

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