An Aminopyrimidone and Aminoimidazoles Alkaloids from the Rodrigues Calcareous Marine Sponge Ernsta naturalis

A chemical study of the CH2Cl2−MeOH (1:1) extract from the sponge Ernsta naturalis collected in Rodrigues (Mauritius) based on a molecular networking dereplication strategy highlighted 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), along with the known thymidine (9). Their structures were established by spectroscopic analysis (1D and 2D NMR spectra and HRESIMS data). To improve the investigation of this unstudied calcareous marine sponge, a metabolomic study by molecular networking was conducted. The isolated molecules are distributed in two clusters of interest. Naamine and naamidine derivatives are grouped together with ernstine in the first cluster of twenty-three molecules. Phorbatopsin derivatives and calcaridine C are grouped together in a cluster of twenty-one molecules. Interpretation of the MS/MS spectra of other compounds of these clusters with structural features close to the isolated ones allowed us to propose a structural hypothesis for 16 compounds, 5 known and 11 potentially new.


Introduction
Calcispongiae (Calcarea Bowerbank, 1864), commonly called calcareous sponges are much less studied chemically compared to another class of Porifera, the Demospongiae, due both to their relatively low number of representatives within the Porifera phylum and to their low biomass [1]. The result is an underexplored source of natural products while these sponges are prolific sources of bioactive alkaloids, especially 2-aminoimidazole alkaloids. The biological activities reported for this kind of alkaloids include antifungal [2], antimicrobial [3], cancer cell toxicity [4], or Mammalian and Protozoan DYRK and CLK kinases inhibitors [5]. Nowadays, more than sixty 2-aminoimidazole alkaloids have been isolated from Calcarea sponges, almost all belonging to the genus Leucetta. A few studies described the chemical composition of other calcareous sponges' genera such as Clathrina [6], Leucosolenia [7], Leucascandra [8,9], or Pericharax [10,11]. The genus Ernsta (Klautau, Azevedo and Cóndor-Luján, 2021) belongs to the order Clathrinida, and comprises 20 species, and despite a ubiquitous distribution, there is no report of chemical investigations of sponges belonging to this genus so far. The genus Ernstia was erected by Klautau et al. in 2013 after a thorough molecular evaluation and some species formerly belonging to the genus
Ernstine A (1) was obtained as a yellow solid. The molecular formula, C 19 H 19 N 3 O 3 , was established from HRESIMS molecular ion peak at m/z 338.1497 [M+H] + . Analysis of the 1D and 2D 1 H, and 13 C NMR data for 1 (CD 3 OD, Table 1) revealed resonances and correlations consistent with those of two para-substituted phenol, but not linked with an aminoimidazolone moiety, such as calcarine A, but linked with an aminopyrimidone moiety ( Figure 2). The 1 H NMR spectrum of 1 recorded in CD 3 OD showed the presence of two AA'BB' spin systems at δH 6.94 and 6.74 (each 2H, d, J = 8.8 Hz) and at δH 7.08 and 6.88 (each 2H, d, J = 8.8 Hz), one singlet at δH 3.78 (3H, s), one singlet at δH 3.73 (3H, s), and one singlet at δH 3.56 (2H, s). Analysis and comparison of HSQC and HMBC correlations pointed out one methylene C-7 (δH 3.56; δC 39.6), two oxymethyl carbons C-12, C-17 (δH 3.73, 3.78; δC 55.4, 55.4), eight aromatic methines C-9, C-9 , C-10, C-10 , C-14, C-14 , C-15, C-15 , (δH 2 × 6.94, 2 × 6.74, 2 × 7.08, 2 × 6.88; δC 2 × 129.9, 2 × 114.1, 2 × 132.2, 2 × 114.1) of four chemically equivalent spin-pairs indicating a symmetry in the aromatic moieties, four quaternary aromatic carbons C-8, C-11, C-13, C-16 (δC 130.4, 158.4, 126.5, 159.1), two quaternary sp 2 carbons due to the double bond C-5, C-6 (δC 115.9, 154.8), a guanidine-like carbon C-2 (δC 158.4) and one amide carbonyl group C-4 (δC 163.0). The COSY correlations between H-9 and H-10 in addition of the HMBC correlations between H-9 and C-7, C-9 and C-11 and between H-10 and C-8 and C-10 indicated a symmetry and the presence of a para-phenolic group linked to the methylene C-7 in C-8. The HMBC correlations between H-12 and C-11 confirmed the substitution of the aromatic moiety in C-11 by the methoxy group C-12. In the same way, the COSY correlations between H-14 and H-15 in addition to the HMBC correlations between H-14 and C-14 and C-16, between H-15 and C-13, and C-15 and between H-17 and C-16 also revealed the presence of symmetry and a second para-phenolic moiety substituted by the methoxy group C-17 in C-16. The connection of the different moieties is confirmed by the NOE correlation cross-peaks ( Figure 2). The HMBC correlation between H-14 and C-5 allowed linking the second nonprotonated carbon of this moiety C-13 to the quaternary sp 2 carbon C-5. HMBC correlations between H-7 and C-2, C-4, C-5, and C-6, in addition to the molecular formula, C 19 H 19 N 3 O 3 indicating 12 degrees of insaturations, revealed the presence of the aminopyrimidone moiety. This is the first report of an aminopyridine alkaloid from a calcareous sponge. Ernstine A (1) was obtained as a yellow solid. The molecular formula, C19H19N3O3, was established from HRESIMS molecular ion peak at m/z 338.1497 [M+H] + . Analysis of the 1D and 2D 1 H, and 13 C NMR data for 1 (CD3OD, Table 1) revealed resonances and correlations consistent with those of two para-substituted phenol, but not linked with an aminoimidazolone moiety, such as calcarine A, but linked with an aminopyrimidone moiety ( Figure 2). The 1 H NMR spectrum of 1 recorded in CD3OD showed the presence of two AA'BB' spin systems at δH 6.94 and 6.74 (each 2H, d, J = 8.8 Hz) and at δH 7.08 and 6.88 (each 2H, d, J = 8.8 Hz), one singlet at δH 3.78 (3H, s), one singlet at δH 3.73 (3H, s), and one singlet at δH 3.56 (2H, s). Analysis and comparison of HSQC and HMBC correlations pointed out one methylene C-7 (δH 3.56; δC 39.6), two oxymethyl carbons C-12, C-17 (δH 3.73, 3.78; δC 55.4, 55.4), eight aromatic methines C-9, C-9′, C-10, C-10′, C-14, C-14′, C-15, C-15′, (δH 2×6.94, 2×6.74, 2×7.08, 2×6.88; δC 2×129.9, 2×114.1, 2×132.2, 2×114.1) of four chemically equivalent spin-pairs indicating a symmetry in the aromatic moieties, four quaternary aromatic carbons C-8, C-11, C-13, C-16 (δC 130.4, 158.4, 126.5, 159.1), two quaternary sp 2 carbons due to the double bond C-5, C-6 (δC 115.9, 154.8), a guanidine-like carbon C-2 (δC 158.4) and one amide carbonyl group C-4 (δC 163.0). The COSY correlations between H-9 and H-10 in addition of the HMBC correlations between H-9 and C-7, C-9′and C-11 and between H-10 and C-8 and C-10′ indicated a symmetry and the presence of a para-phenolic group linked to the methylene C-7 in C-8. The HMBC correlations between H-12 and C-11 confirmed the substitution of the aromatic moiety in C-11 by the methoxy group C-12. In the same way, the COSY correlations between H-14 and H-15 in addition to the HMBC correlations between H-14 and C-14′ and C-16, between H-15 and   Phorbatopsin D (2) was obtained as a yellow solid. The molecular formula, C11H13N3O3, was established from HRESIMS molecular ion peak at m/z 236.1029 [M+H] + . Analysis of the 1D and 2D 1 H, and 13 C NMR data for 2 (CD3OD, Table 2) revealed resonances and correlations ( Figure 2) consistent with those of a para-substituted phenol linked with an aminoimidazolone group, such as phorbatopsin B and C [16]. The 1 H NMR spectrum of 2 recorded in CD3OD showed the presence of an AA'XX' spin system at δH Phorbatopsin D (2) was obtained as a yellow solid. The molecular formula, C 11 H 13 N 3 O 3 , was established from HRESIMS molecular ion peak at m/z 236.1029 [M+H] + . Analysis of the 1D and 2D 1 H, and 13 C NMR data for 2 (CD 3 OD, Table 2) revealed resonances and correlations ( Figure 2) consistent with those of a para-substituted phenol linked with an aminoimidazolone group, such as phorbatopsin B and C [16]. The 1 H NMR spectrum of 2 recorded in CD 3 OD showed the presence of an AA'XX' spin system at δH 7.01 and 6.64 (each 2H, d, J = 8.4 Hz), one singlet at δH 3.17 (3H, s), and one AB spin system at δH 3.00 and 2.95 (each 1H, d, J = 13.7 Hz). Analysis of the HSQC and HMBC correlations and the comparison with the latter compounds pointed out one methylene C-6 (δH 3.00 and 2.95; δC 41.9), one oxymethyl C-11 (δH 3.17; δC 51.6), four aromatic methines C-8, C-8 , C-9, C-9 (δH 2 × 7.01, 2 × 6.64; δC 2 × 132.4, 2 × 115.9) of two chemically equivalent spin-pairs indicating a symmetry in the aromatic moiety, two quaternary aromatic carbons C-7, C-10 (δC 125.7, 157.1), one quaternary carbon of hemiaminal C-5 (δC 95.2) and one amide carbonyl group C-4 (δC 188.3). Compound 2 was different from phorbatopsin C by the presence of the oxymethyl C-11 and the quaternary carbon of hemiaminal C-5 instead of one aminomethine. The COSY correlations between H-8 and H-9 in addition to the HMBC correlations between H-8 and C-6, C-8 and C-10, and between H-9 and C-7 and C-9 indicated a symmetry and the presence of a para-phenolic group linked to the methylene C-6 in C-7. The HMBC correlation between H-6 and C-4, C-5, C-7, and C-8 indicated the substitution of the methylene by the para-phenolic core and by the quaternary carbon of hemiaminal C-5. The HMBC correlation between H-11 and C-5 indicated the substitution of the quaternary carbon of hemiaminal C-5 by a methoxy group. The chemical shift of the amide carbonyl group C-4 (δC 188.0) of phorbatopsin D (2) was close to that of phorbatopsin C (δC 188.7) but 17.0 ppm higher than that of phorbatopsin B (δC 171.0) described by Nguyen et al. [16]; this difference could be explained by the annular tautomerism (as classified by Katritzky and Lagowski [17]) of the aminoimidazolone moiety. to the C-4 of the tautomer b [18,19]. Indeed, Krawczyk et al. [18], had demonstrated that for creatinines substituted at position 5 with an electron-withdrawing substituent, the amine tautomer a is preferred in a polar solvent. Compound 2 was named phorbatopsin D according to phorbatopsin B and C reported in 2012 [16]. Table 2. The 1D and 2D NMR spectroscopic data ( 1 H, 13 C 600/150 MHz, CD 3 OD) for phorbatopsin D (2) and E (3). 7.01 and 6.64 (each 2H, d, J = 8.4 Hz), one singlet at δH 3.17 (3H, s), and one AB spin system at δH 3.00 and 2.95 (each 1H, d, J = 13.7 Hz). Analysis of the HSQC and HMBC correlations and the comparison with the latter compounds pointed out one methylene C-6 (δH 3.00 and 2.95; δC 41.9), one oxymethyl C-11 (δH 3.17; δC 51.6), four aromatic methines C-8, C-8′, C-9, C-9′ (δH 2 × 7.01, 2 × 6.64; δC 2 × 132.4, 2 × 115.9) of two chemically equivalent spinpairs indicating a symmetry in the aromatic moiety, two quaternary aromatic carbons C-7, C-10 (δC 125.7, 157.1), one quaternary carbon of hemiaminal C-5 (δC 95.2) and one amide carbonyl group C-4 (δC 188.3). Compound 2 was different from phorbatopsin C by the presence of the oxymethyl C-11 and the quaternary carbon of hemiaminal C-5 instead of one aminomethine. The COSY correlations between H-8 and H-9 in addition to the HMBC correlations between H-8 and C-6, C-8′ and C-10, and between H-9 and C-7 and C-9′ indicated a symmetry and the presence of a para-phenolic group linked to the methylene C-6 in C-7. The HMBC correlation between H-6 and C-4, C-5, C-7, and C-8 indicated the substitution of the methylene by the para-phenolic core and by the quaternary carbon of hemiaminal C-5. The HMBC correlation between H-11 and C-5 indicated the substitution of the quaternary carbon of hemiaminal C-5 by a methoxy group. The chemical shift of the amide carbonyl group C-4 (δC 188.0) of phorbatopsin D (2) was close to that of phorbatopsin C (δC 188.7) but 17.0 ppm higher than that of phorbatopsin B (δC 171.0) described by Nguyen et al. [16]; this difference could be explained by the annular tautomerism (as classified by Katritzky and Lagowski [17]) of the aminoimidazolone moiety. The chemical shift of C-4 in phorbatopsin D and phorbatopsin C corresponded to the C-4 of the tautomer a (Figure 3), whereas the chemical shift of C-4 in phorbatopsin B corresponds to the C-4 of the tautomer b [18,19]. Indeed, Krawczyk et al. [18], had demonstrated that for creatinines substituted at position 5 with an electron-withdrawing substituent, the amine tautomer a is preferred in a polar solvent. Compound 2 was named phorbatopsin D according to phorbatopsin B and C reported in 2012 [16].    Table 2) revealed resonances and correlations consistent with those of a para-substituted phenol linked with an aminoimidazolone group, such as phorbatopsin D (2). Compound 3 was different from 2 by the presence of the oxymethyl C-12 (δH 3.73; δC 55.2) instead of an alcohol group. This is confirmed by the HMBC correlations between H-12 and C-10 and NOE correlations between H-12 and H-9/H-9 .
Calcaridine C (4) was obtained as a yellow solid. The molecular formula, C 18 H 19 N 3 O 4 , was established from HRESIMS molecular ion peak at m/z 342.1449 [M+H] + . Analysis of the 1D and 2D 1 H, and 13 C NMR data for 4 (CD 3 OD, Table 3) revealed resonances and correlations consistent with those of two para-substituted phenol linked with an aminoimidazolone moiety, such as calcarine A [3]. Compound 4 was different from calcaridine A by the presence of an alcohol group in C-15 instead of a methoxy group and the substitution of N-1 which was substituted by a proton instead of a methyl. Moreover, the chemical shift of the amide carbonyl group C-4 (δC 189.8) of calcaridine C (4) was 15.8 ppm higher than that of calcaridine A (δC 174.0) described by Edrada et al. [3], calcaridine C corresponded to the tautomer a (Figure 3) whereas calcaridine A corresponded to tautomer b. This difference in isomeric protonation states could be explained by differences in the isolation protocol, herein all the compounds had been isolated in acidic conditions (0.1% formic acid) while Edradra et al. had isolated calcaridine A without acid. Compound 4 was named calcaridine C according to calcaridine A reported in 2003 [3] and calcaridine B reported in 2018 [20].  Table 4) showed that it was closely related to naamine A to G [4,[21][22][23][24][25][26][27], namely resonances and correlations consistent with those of two para-substituted phenol linked with a 2-aminoimidazole group (Figure 2). Compound 5 was different from naamine A by the lack of a methyl group attached to the N-3 of the 2-aminoimidazole ring. Naamidine J (6) was obtained as a yellow solid. The molecular formula, C 22 H 21 N 5 O 4 , was established from HRESIMS molecular ion peak at m/z 420.1664 [M+H] + . Analysis of the 1D and 2D 1 H, and 13 C NMR data for 6 (CD 3 OD, Table 5) showed that it was closely related to naamine H (5) and to naamidines A to I [22,23,28,29]. Namely, as naamine H (5), resonances and correlations were consistent with those of two para-substituted phenol linked with a 2-aminoimidazole ring but herein this 2-aminoimidazole ring was also linked to a hydantoin ring. The substitution of the benzyl rings was the same as naamine H (5), namely, one alcohol function and one methoxy group and the substitution of the hydantoin ring was the same as naamidine A, by one methoxy group on nitrogen. Naamine I (7) and Naamidine K (8) were obtained as a yellow solid mixture. The molecular formula, C 17 H 17 N 3 O 2 , of naamine I was established from HRESIMS molecular ion peak at m/z 296.1389 [M+H] +, and the molecular formula, C 21 H 19 N 5 O 4 , of naamidine K was established from HRESIMS molecular ion peak at m/z 406.1502 [M+H] + . Analysis of the 1D and 2D 1 H, and 13 C NMR data of the mixture of 7 and 8 (CD 3 OD, Tables 4 and 5) showed that it was closely related to naamine H (5) and to naamidine J (6). They were only differing by the substitution of the benzyl rings by two hydroxyls instead of one hydroxyl and one methoxy group.

Dereplication of the Crude Extract
To obtain the first molecular fingerprint of the unstudied Rodrigues calcareous marine sponge Ernsta naturalis, the CH 2 Cl 2 -MeOH extract was profiled by HPLC-HRMS/MS. These data were subsequently processed by GNPS [30]. Beforehand, the mass spectra of the eight new molecules isolated and characterized by NMR were submitted to the library of the GNPS (Accession codes of the isolated compounds). These molecules could thus be reported directly in the molecular network of the extract. The molecular network ( Figure 4) contains 167 nodes including 111 clustered molecules. The isolated molecules are distributed in two clusters of interest. Naamine and naamidine derivatives grouped together with ernstine A (1) in the first cluster 1 of twenty-three molecules ( Figure 5). Phorbatopsin derivatives (2, 3) and calcaridine C (4) grouped together in cluster 2 of twenty-one molecules ( Figure 6).
Using the same approach, the cluster of phorbatopsin derivatives could be partially characterized. This cluster is divided into two parts. The first one contains two nodes identified as calcaridine C (4) with similar spectra but different retention times. Calcari- interpretation of compound spectra, so no additional annotation was added in this part of the cluster. Finally, phorbatopsin D and E (2, 3) were projected in the second part of the cluster where six other molecules could be proposed (Table 7). For these molecules, the losses of neutrals CO, CH 4 O, and C 2 H 2 N 2 O due to fragmentation in the 2-aminoimidazolin-4-one cycle and the characteristic ions 107.0492 m/z [C 7 H 7 O] + and 121.0646 m/z [C 8 H 9 O] + which correspond to the phenolic group without and with a methoxy are found. Two known molecules with the same 2-aminoimidazolin-4-one moiety were proposed to be phorbatopsins A and C, along with three new compounds. Only one molecule with a different moiety has been proposed, leucettamine C, with a loss of C 3 H 6 N 2 O corresponding to a 2-imino-3-methyl-imidazolidin-4-one moiety. The naamidine derivatives cluster was characterized by the presence of neutral loss (C7H8O and C6H6O) which correspond to the methoxylated or hydroxylated phenolic group. In addition to the loss of neutral, characteristic ions were observed, as 160.0758 m/z [C10H10NO] + which determines the presence of the phenolic group with the 2-aminoimid- to the phenolic group without and with a methoxy are found. Two known molecules with the same 2-aminoimidazolin-4-one moiety were proposed to be phorbatopsins A and C, along with three new compounds. Only one molecule with a different moiety has been proposed, leucettamine C, with a loss of C3H6N2O corresponding to a 2-imino-3-methylimidazolidin-4-one moiety.  The naamidine derivatives cluster was characterized by the presence of neutral loss (C 7 H 8 O and C 6 H 6 O) which correspond to the methoxylated or hydroxylated phenolic group. In addition to the loss of neutral, characteristic ions were observed, as 160.0758 m/z [C 10 H 10 NO] + which determines the presence of the phenolic group with the 2-aminoimidazole moiety. Moreover, the presence of the loss of neutral (C 3 H 3 NO 2 or C 3 H 4 N 2 O or C 4 H 6 N 2 O) corresponding to the fragmentation in the 3-methyl-imidazolidin-4-one ring helps to indicate whether the group corresponds to either 3-methylimidazolidine-2,4-dione (as naamidine), or 2-imino-3-methyl-imidazolidin-4-one or 2-methylimino-3methyl-imidazolidin-2-one. With these elements, it is possible to propagate the annotations of cluster 1 by characterizing other nodes. Thus, structural hypotheses of nine additional molecules have been proposed (Table 6), in addition to the spectral confirmation of the five compounds already isolated and characterized by NMR. With this methodology, two nodes seem to correspond to naamine D isomers [23] and one node to naamidine D [22], two molecules isolated from the calcareous sponge Leucetta, eight other nodes seem to correspond to new molecules and for the last seven ones, the hypothesis was too uncertain to propose a structural hypothesis.

Biosynthetic Pathway
Even if a clear definition of biosynthetic origin of the 2-aminoimidazole alkaloids from the calcareous sponges has not been established at the present time, different hypotheses of biological pathway have been proposed but no experimental confirmation has been reported [31]. Crews had proposed a biosynthesis pathway including an intermediate with one phenyl ring coming from guanidine and p-hydroxyphenylpyruvic acid [31,32]. The presence in the crude extract of Ernsta naturalis of a compound with an ion peak in HRESIMS at m/z 220.1078 [M+H] + corresponding to the molecular formula of the intermediate pmethoxyphorbatopsin C, in addition to the presence of both aminoimidazole alkaloids with one phenyl ring and aminoimidazole alkaloids with two phenyl rings are in agreement with this proposal.

General Experiment Procedures
Optical rotations were measured on a MCP 200 Anton Paar modular circular polarimeter at 25 • C (MeOH, c in g/100 mL). 1 H and 13 C NMR data were acquired with a Bruker Avance II+-600 MHz spectrometer equipped with a TCI Cryoprobe at 300 K with 2 mm o.d. Match NMR tubes. Chemical shifts were referenced using the corresponding solvent signals (δ H 3.31 and δ C 49.00 for CD 3 OD). The spectra were processed using 1D and 2D NMR MNova software (Version No. 14.1.1-24571, Mestrelab Research S. L., Santiago de Compostela, Spain). HRESIMS spectra were recorded using a Waters SYNAPT G2 HDMS mass spectrometer (Waters, Guyancourt, France).
The sponge was lyophilized with Cosmos −80 • C CRYOTEC. MPLC separations were carried out on a Buchi Sepacore flash system C-605/C-615/C-660 and glass column (230 × 15 mm i.d.) packed with Acros Organics C18-RP, 23%C, silica gel (40−63 µm). Precoated TLC sheets of silica gel 60, Alugram SIL G/UV254 were used, and spots were visualized on the basis of the UV absorbance at 254 nm and by heating silica gel plates sprayed with formaldehyde−sulfuric acid or Dragendorff reagents. HPLC analyses were carried out using a Phenomenex Gemini C 18 (150 × 4.6 mm i.d., 3 µm) column and were performed on a Thermo Scientific Dionex Ultimate 3000 system equipped with a photodiode array detector and a Corona detector with Chromeleon software. Semi-preparative HPLC was carried out using a Phenomenex Geminin C 18 (250 × 10 mm i.d., 5 µm) column and was performed on a Thermo Scientific Dionex Ultimate 3000 system equipped with a photodiode array detector. All solvents were analytical or HPLC grade and were used without further purification.

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 CH 2 Cl 2 -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.