Magnificines A and B, Antimicrobial Marine Alkaloids Featuring a Tetrahydrooxazolo[3,2-a]azepine-2,5(3H,6H)-dione Backbone from the Red Sea Sponge Negombata magnifica.

Investigation of the Red Sea sponge Negombata magnifica gave two novel alkaloids, magnificines A and B (1 and 2) and a new β-ionone derivative, (±)-negombaionone (3), together with the known latrunculin B (4) and 16-epi-latrunculin B (5). The analysis of the NMR and HRESIMS spectra supported the planar structures and the relative configurations of the compounds. The absolute configurations of magnificines A and B were determined by the analysis of the predicted and experimental ECD spectra. Magnificines A and B possess a previously unreported tetrahydrooxazolo[3,2-a]azepine-2,5(3H,6H)-dione backbone and represent the first natural compounds in this class. (±)-Negombaionone is the first β-ionone of a sponge origin. Compounds 1-3 displayed selective activity against Escherichia coli in a disk diffusion assay with inhibition zones up to 22 mm at a concentration of 50 µg/disc and with MIC values down to 8.0 µM. Latrunculin B and 16-epi-latrunculin B inhibited the growth of HeLa cells with IC50 values down to 1.4 µM.

1. Introduction

Sponges belonging to the genus Negombata (formerly Latrunculia) [1] (pp. 698–699) are characterized by diverse secondary metabolites of different classes including macrolides (latrunculins) [2,3,4,5,6,7,8,9], pyrroloiminoquinone alkaloids (discorhabdins) [10,11,12,13,14,15,16,17], terpene peroxides [18,19], cyclic 2-oxecanone glycosides [20], diterpenes [21], ceramides [22,23], and peptides [24,25,26]. Reported latrunculins displayed anticancer, antiviral, antibiotic, antiangiogenic, antimigratory, and microfilament-disrupting activities [2,3,4,5,6,7,8,9]. Pyrroloiminoquinone alkaloids exhibited antimicrobial, immunomodulatory, caspase inhibition, antiviral, feeding deterrence, and antimalarial properties and present potent inhibition potential of mammalian topoisomerase II in vivo [10,11,12,13,14,15,16,17]. Additional pharmacological activities for other chemical entities identified from the genus Negombata include cytotoxicity [18,19,21], antifeeding [20], antiepileptic, and anti-inflammatory [22,23], potent inotropic effects and inhibition of the cardiac Na/Ca exchanger [24,25,26].

As a part of our growing interest to discover biologically active leads from marine resources [27,28,29], the organic extract of the sponge Negombata magnifica was examined. Two alkaloids, magnificines A and B (1 and 2), with a previously unreported tetrahydrooxazolo[3,2-a]azepine-2,5(3H,6H)-dione skeleton were purified. In addition, a new β-ionone derivative, (±)-negombaionone (3), with the previously reported latrunculin B (4) [2] and 16-epi-latrunculin B (5) [5] were obtained. Structural determinations of 1-5 were accomplished by HRESIMS and NMR spectral analyses.

2. Results and Discussion

2.1. Purification of 1-5

Fractionation of the methanolic extract of N. magnifica [30] (Figure 1) using partition (on silica gel), size exclusion (Sephadex LH 20), and purification of active fractions on HPLC afforded 1-5.

Figure 1

Underwater photograph of the Red Sea Negombata magnifica.

2.2. Structure of Magnificine A (1)

Magnificine A (1) (Figure 2) obtained as an optically active ([α] = + 70°) oil. The chemical structure of 1 was determined from interpretation of its MS and NMR spectra (Figures S1–S10). The HRESIMS data (m/z = 282.0961, C11H17NNaO6, [M + Na]+) supported molecular formula C11H17NO6, suggesting four degrees of unsaturation. Its 13C NMR spectrum and HSQC experiment exhibited 11 signals including four quaternary carbons, two oxygenated methines, two methylenes and three methyls (Figure 2 and Table 1). The combined 1H NMR spectrum and COSY experiment supported the existence of a single 1H-1H coupling system from H2-7 to H2-9 (CH2-7–CH-8–CH2-9) (Figure 3). Beside the geminal coupling between the protons at C-7 (δH 2.03 and 1.33, 2J7a,7b = 11.5 Hz), vicinal couplings from H-7a (3J7a,8 = 4.2 Hz) and H-7b (3J7b,8 = 11.5 Hz) to the oxygenated methine H-8 (δH 4.13, tt, J = 11.5, 4.2 Hz) were observed. Furthermore, H-8 exhibited additional vicinal (3JHH) couplings to H-9a (δH 2.54, ddd, J = 11.5, 4.2, 1.8 Hz) and H-9b (δH 1.51, t, J = 11.5 Hz) completing the coupling system.

Figure 2

Chemical structures of 1-5.

Table 1

NMR data of 1 (600 MHz for 1H and 150 for 13C, CDCl3).

No.	δC (mult.)	δH [mult., J (Hz)]	HMBC	NOESY
2	171.5, qC		H-3
3	113.3, CH	5.72 (s)		H3-11
5	180.7, qC		H-3, H2-7, H3-11, H3-12
6	35.0, qC		H3-11, H3-12, H2-7
7a	49.8, CH2	2.03 (ddd, 11.5, 4.2, 2.4)	H3-11, H3-12, H2-9
7b		1.33 (t, 11.5)
8	65.1, CH	4.13 (tt, 11.5, 4.2)	H2-7, H2-9	H-7b, H3-12, H3-13
9a	47.9, CH2	2.54 (ddd, 11.5, 4.2, 1.8)	H2-7, H3-13
9b		1.51 (t, 11.5)
10	86.4, qC		H3-13, H-3, H2-9
11	29.9, CH3	1.31 (s)	H3-12	H-3
12	25.1, CH3	1.27 (s)	H3-11	H-8, H3-13
13	25.6, CH3	1.59 (s)		H-8, H3-12

Figure 3

Subunits of 1 and 3, and COSY and HMBC of 1-3.

The 13C NMR resonances at δC 49.8 (CH2, C-7), 65.1 (CH, C-8), and 47.9 (CH2, C-9) are correlated to the protons at δH 2.03/1.33 (H-7a and H-7b), 4.13 (H-8), and 2.54/1.51 (H-9a and H-9b) in the HSQC experiment, supporting the assignment of these signals and the placement of OH group at C-8. The interruption of the spin-coupling system of H2-7–H-8–H2-9 on both sides suggests the quaternary nature of C-6 and C-10. The substituents at C-6 and C-10, and the existence of an amidic carbonyl (δC 180.7, C-5) were confirmed from the HMBC of H3-11/C-6, H3-12/C-6, H3-11/C-7, H3-12/C-7, H3-11/C-5, H3-12/C-5, H3-13/C-9, H3-13/C-10, H2-7/C-5, and H2-9/C-10 (Table 1 and Figure 3), completing the structure of the seven-membered ring (Fragment A). The remaining elements of C2H2O4 (Fragment B) displayed two signals in the 1H and 13C NMR spectra at δH/C 171.5 (qC, C-2) and 5.72/113.3 (CH, s, H-3/C-3) corresponding to a carbonyl of a lactone moiety and an oxygenated methine. The downfield chemical shift of C-10 at δC 86.4 (qC) supported its attachment to the heteroatoms (O and N) of the lactone and amide functionalities. The HMBC of H-3/C-10, H2-9/C-10 and H3-10/C-10 supported this assignment. The appearance of the NMR signals of H-3/C-3 at δH/C 5.72/113.3 supported the attachment C-3 to the N atom of the amidic group of the seven-membered ring as well as the presence of the remaining elements (OOH) at C-3, completing the molecular formula of 1. The attachment of the two fragments (five- and seven-membered rings) of 1 through N-4–C-10 was supported from 3JCH HMBC from H-3 (δH 5.72) to C-5 (δC 180.7) and C-10 (δC 86.4) (Figure 3), completing the planar structure of 1.

The planar structure of 1 as well as the substitution on both subunits of 1 were confirmed again from the MS ion peaks at m/z 249.09 (14.3%), 237.09 (13.3%), 219.08 (100%, base peak), 195.08 (13.1%) and 180.06 (2.3%) (Figure 4) in the ESIMS. The ion peak at m/z 249.09 results from the loss of OOH moiety [M − OOH + Na]+ from the parent ion peak at m/z 282.09 [M + Na]+. Consecutive loss of CO2H2 and H2O fragments from both sides of the compound results in an ion peak at m/z 219.08 (base peak) [M − CO2H2 − H2O + Na + H]+. Further loss of CH3 group from the base peak gives a minor ion peak at m/z 180.06 [M − CO2H2 − H2O − CH3]+. The loss of CO2H2 from the five-membered ring gives an ion peak at m/z 237.09 [M − CO2H2 + Na + H]+, which further lose H2O from the seven-membered ring resulting in an ion peak at m/z 195.08 [M − CO2H2 − H2O]+ (Figure 4).

Figure 4

Significant MS ion fragments of magnificine A (1).

The strong NOESY correlations between H-8 and H3-12, and between H-8 and H3-13 confirm the same relative configurations of such functionalities (Figure 5). Further, the NOESY between H-3 and H3-11 supported the same configuration as well as the opposite configuration to H-8, H3-12 and H3-13 (Table 1 and Figure 5).

Figure 5

Significant NOESY correlations of 1 and 2.

The magnitude of the vicinal 3JHH values between H-8 (tt, J = 11.5, 4.2 Hz) (Figure 6) and H-7b (δH = 1.33, 3J8,7b = 11.5 Hz), and between H-8 and H-9b (δH = 1.51, 3J8,9b = 11.5 Hz) suggests similar dihedral angles of 180° [31] between H-8 and both H-7b and H-9b (Figure 7). On the contrary, the values of the vicinal 3JHH values between H-8 and H-7a (δH = 2.03, 3J8,7a = 4.2 Hz) and between H-8 and H-9a (δH = 2.54, 3J8,9a = 4.2 Hz) suggest similar dihedral angles of 60° [31] between H-8 and H-7a, and between H-8 and H-9a (Figure 7).

Figure 6

Multiplicity of H-8 in 1 (blue) and 2 (red).

Figure 7

Anticipated dihedral angles between H-8 and adjacent methylenic protons (H-7a, H-7b, H-9a and H-9b) in 1 and 2.

The absolute configurations at the stereogenic carbons C-3, C-8 and C-10 of 1 were confirmed from comparison of the experimental and TDDFT-predicted ECD spectra (Figure 8). A good agreement between both ECD spectra was noticed. The sign of the unique Cotton Effect (CE) due to the n→π* transition of the lactone enabled the assignment of the configurations at the stereogenic centers as 3R,8S,10S. Accordingly, 1 was assigned as (3R,8S,9aS)-3-hydroperoxy-8-hydroxy-6,6,9a-trimethyltetrahydrooxazolo[3,2-a]azepine-2,5(3H,6H)-dione and named magnificine A.

Figure 8

Experimental and calculated ECD spectra of 1.

2.3. Structure of Magnificine B (2)

Magnificine B (2) (Figure 2), an optically active ([α] = −65°) compound, with molecular formula of C11H17NO6 (m/z = 282.0961, C11H17NNaO6, [M + Na]+). Interpretation of the MS and NMR spectra of 2 (Figures S11–S19) supported its structure determination. Inspection of the NMR spectra of 1 and 2 (Table 1 and Table 2) showed high similarity between the 1H and 13C chemical shifts, suggesting similar planar structure of both compounds. The appearance of oxymethine H-8 in 2 as a quintet (δH 4.33, quin., 3J = 3.5 Hz) instead of triplet of triplet (δH 4.13, tt, 3J = 11.5 and 4.2 Hz) in 1 suggested an opposite configuration of the OH moiety at C-8.

Table 2

NMR data of 2 (600 MHz for 1H and 150 MHz for 13C, CDCl3).

No.	δC (mult.)	δH [mult., J (Hz)]	HMBC	NOESY
2	171.9, qC		H-3
3	112.9, CH	5.70 (s)		H3-11
5	182.3, qC		H-3, H3-11, H3-12
6	35.9, qC		H-8, H3-11, H3-12, H2-7, H-3
7a	47.3, CH2	2.47 (td, 14.5, 3.5, 3.5)	H3-11, H3-12
7b		1.79 (dd, 14.5, 3.5)		H-8
8	66.8, CH	4.33 (quin, 3.5)		H-7b, H-9a, H-9b
9a	45.6, CH2	1.97 (td, 14.5, 3.5, 3.5)	H3-13	H-8
9b		1.53 (dd, 14.5, 3.5)		H-8
10	86.6, qC		H-3, H-8, H3-13
11	30.6, CH3	1.27 (s)	H3-12	H-3
12	26.4, CH3	1.47 (s)	H3-11	H3-13
13	27.0, CH3	1.78 (s)		H3-12

The NOESY cross-peaks between H-8 and H3-13, H-8 and H-7b, H-8 and H-9a, and between H3-12 and H3-13 supported the similar configuration of these moieties (Table 2 and Figure 5). Further, a NOESY between H3-11 and H-3 supported the same configuration (Figure 5). Additionally, the same 3J value of 3.5 Hz between H-8 (quin., J = 3.5 Hz) (Figure 6) and the four methylenic protons (H-7a, H-7b, H-9a and H-9b) proposed similar dihedral angles of 60° [31] between H-8 and these protons (H-7a, H-7b, H-9a and H-9b) (Figure 7).

The absolute configurations at C-3, C-8, and C-10 of 2 were determined by comparison between the predicted and the experimental ECD spectra (Figure 9). In comparison to compound 1, the sign of the unique CE was inverted in 2, suggesting opposite configuration at C-8 (Table 2 and Figure 5). Thus, the configurations at C-3, C-8 and C-10 was confirmed to be 3R,8R,10S. Thus, 2 was assigned as (3R,8R,9aS)-3-hydroperoxy-8-hydroxy-6,6,9a-trimethyltetrahydrooxazolo[3,2-a]azepine-2,5(3H,6H)-dione and named magnificine B.

Figure 9

Experimental and calculated ECD spectra of 2.

Magnificines A and B represent the first natural compounds with a tetrahydrooxazolo[3,2-a]azepine-2,5(3H,6H)-dione backbone. Their occurrence highlights exceptional biosynthetic and chemical biotransformation capabilities in marine sponges.

2.4. Structure of (±)-Negombaionone (3)

Compound 3 (Figure 2) was purified as an optically inactive ([α] = −65°) solid. The positive HRESIMS (m/z 245.1157, C13H18NaO3 [M + Na]+) supported the molecular formula of C13H18O3. The analyses of its NMR and MS spectra (Figures S20–S26) proved its chemical structure. Its 13C NMR spectrum revealed 13 resonances divided into four methyls, one methylene, two olefinic methines, and five quaternary carbons, as supported by the HSQC experiment (Table 3). The interpretation of 1H, 13C, COSY, HSQC and HMBC of 2 supported the assignment of two subunits in 3 as 2,3,4,6-terasubstituted cyclohex-2-en-1-one (subunit A) and buta-3-en-2-one (subunit B) linked together via C-3/C-8 (Figure 2). The 1H and 13C NMR resonances at δH/C 200.2 (qC, C-1), 128.8 (qC, C-2), 158.6 (qC, C-3), 36.7 (qC, C-4), 2.21 (1H, dd), 1.86(1H, t)/45.1 (CH2, H2-5/C-5), 4.37 (1H, dd)/69.3 (CH, C-6) (Table 3) supported the presence of subunit A. Vicinal couplings between H-6 and the geminal-coupled protons at C-5 (H-5a and H-5b) were observed. Further, HMBC of H-6/C-1, H2-5/C-1, H3-7/C-1, H3-7/C-2, H-8/C-2, H3-7/C-3, H-9/C-3, H3-12/C-3, H3-13/C-3, H2-5/C-4, H3-12/C-4, H3-13/C-4, H-6/C-5, H3-12/C-5, H3-13/C-5, H2-5/C-6, and H3-12/C-6 (Figure 3 and Table 3) confirmed the assignment of subunit A. Similarly, subunit B was assigned from the 1H/13C signals at δH/C 7.20 (1H, dd)/139.4 (CH, C-8), 6.22 (1H, d)/134.1 (CH, C-9), 197.3 (qC, C-10) and 2.36 (3H, s)/28.2 (CH3, C-11). The E configuration at C-8/C-9 was supported by a 3J value of 16.5 Hz between H-8 and H-9. The HMBC cross-peaks of H-8/C-9, H-8/C-10, H-9/C-10 and H3-11/C-10 (Figure 3 and Table 3) completed the assignment of subunit B. The connection of subunits A and B via C-3/C-8 was supported from HMBC of H-8/C-2 and H-9/C-3, completing the planar structure of 3.

Table 3

NMR data of 3 (600 MHz for 1H, 150 MHz for 13C, CDCl3).

No.	δC (mult.)	δH [mult., J (Hz)]	HMBC
1	200.2, qC		H-6, H2-5, H3-7
2	128.8, qC		H3-7, H-8
3	158.6, qC		H3-7, H-9, H3-12, H3-13
4	36.7, qC		H2-5, H3-12, H3-13
5a5b	45.1, CH2	2.21 (dd, 14.0, 6.0)1.86 (t, 14.0)	H-6, H3-12, H3-13
6	69.3, CH	4.37 (dd, 14.0, 6.0)	H2-5, H3-12
7	13.5, CH3	1.88 (d, 0.6)
8	139.4, CH	7.20 (dd, 16.5, 0.6)
9	134.1, CH	6.22 (d, 16.5)	H-8
10	197.3, qC		H-8, H-9, H3-11
11	28.2, CH3	2.36 (s)	H3-12
12	30.3, CH3	1.17 (s)	H3-13
13	25.7, CH3	1.35 (s)	H3-12, H2-5

The racemic nature of 3 was confirmed from the absence of any optical activity ([α] = 0°) as well as from the absence of any CE in the experimental ECD spectrum. Thus, 3 was confirmed to be a racemic mixture and was assigned as (±)-(E)-6-hydroxy-2,4,4-trimethyl-3-(3-oxobut-1-en-1-yl)cyclohex-2-en-1-one and named (±)-negombaionone.

Ionones represent a rare class of secondary metabolites in marine organisms. Only four candidates including 2-bromo-γ-ionone [32], (E)-3-oxo-β-ionone [33], dihydro-γ-ionone and ambra-aldehyde [34] are of marine origin (Figure 10). (±)-Negombaionone represents the first β-ionone of a sponge origin.

Figure 10

Representative examples of marine-derived ionones [32,33,34].

Compounds 4 and 5 were identified by an interpretation of their NMR (Figures S27–30) and MS data and by comparison with the data in the literature [30,31]. Accordingly, compounds 4 and 5 were characterized as latrunculin B [2] and 16-epi-latrunculin B [5], respectively.

Compounds 1-3 were investigated for their antimicrobial activities against three pathogens. Compounds 1-3 displayed selective activity against E. coli (ATCC 25922) at a concentration of 50 µg/disc in a disk diffusion assay with inhibition zones of 22, 20 and 20 mm, respectively. Further, 1-3 exhibited equal MIC values of 8, 8 and 8 µM, respectively, against E. coli in a microdilution assay. The compounds were inactive against S. aureus (ATCC 25923) and C. albicans (ATCC 14053). These results suggest selective activity of 1-3 against E. coli. These findings support the importance of marine sponges as a vigorous foundation of antimicrobial secondary metabolites and the potential of future development of 1-3 as antimicrobial leads.

In an MTT assay, latrunculin B (4) and 16-epi-latrunculin B (5) displayed growth inhibition of HeLa cells with IC50 values of 1.4 and 3.9 µM, respectively, suggesting the selectivity of 4 and 5 against HeLa cells.

3. Materials and Methods

3.1. General Experimental Procedures

The optical rotations and spectral data of 1-5 including UV, ECD, NMR and MS are acquired as previously reported [27,28,29].

3.2. Biological Materials

The brick-red sponge Negombata magnifica KellyBorges and Vacelet (order Poecilosclerida, suborder Mycalina, family Podospongiidae) [30] was collected as branched fingerlike strips by hands using SCUBA at depths of 20–25 from the Red Sea coast (N 021°39′17.5″, E 038°52′26.3″). A specimen with number KSA-119 was reserved at King Abdulaziz University.

3.3. Purification of Compounds 1-5

The freeze-dried material (85 g) was soaked in MeOH overnight (3 × 650 mL). Combined extracts were partitioned on a VLC silica column using hexane-EtOAc-MeOH gradients. The fraction eluted with 80% EtOAc in hexane was subjected twice to partition on Sephadex LH-20 using CH2Cl2-MeOH (1:1) to give four subfractions (Fr. A-D). The antibacterial fraction (Fr. B, 19 mg) (inhibition zone = 10 mm against E. coli, at 100 µg/disc) was purified on HPLC (C18, AR II Cosmosil 250 × 10 mm, 5 μm, Waters) using H2O-MeCN (60:40) at 3 mL/min to give compounds 1 (1.6 mg) (tR = 13.0 min), 2 (1.2 mg) (tR = 14.0 min) and 3 (2.7 mg) (tR = 15.0 min). Similarly, the cytotoxic fraction (Fr. C, 24 mg) (IC50 = 7 µg/mL against HeLa cells) was purified on HPLC (C18, Gemini® 5 μm, 250 × 0.64 mm, Phenomenex) using H2O-MeCN (40:60) at 1 mL/min to give 4 (17 mg) (tR = 8.8 min) (17 mg) and 5 (3.5 mg) (tR = 9.5 min).

3.4. Spectral Data of the Compounds

Magnificine A (1): Yellow oil; [α] 70° (c 0.1, MeOH); UV (MeOH) λmax (log ε): 201 (2.89), 274 (2.69) nm; ECD (MeOH) [Δε]212 nm +22.01; HRESIMS m/z 282.0961 (calcd for C11H17O6NNa, [M + Na]+, 282.0953).

Magnificine B (2): Yellow oil; [α] −65° (c 0.1, MeOH); UV (MeOH) λmax (log ε): 201 (2.80), 274 (2.66) nm; ECD (MeOH) [Δε]219 nm −6.00; HRESIMS m/z 282.0961 (calcd for C11H17O6NNa, [M + Na]+, 282.0953).

(±)-Negombaionone (3): Off-white solid; m.p.: 138 °C; [α] 0° (c 0.1, MeOH); UV (MeOH) λmax (log ε): 205 (2.75), 274 (2.34) nm; IR (film): νmax 3520, 1681, 1663, 1607, 1079, 984 cm−1; HRESIMS m/z 245.1157 (calcd for C13H18O3Na, [M + H]+, 245.1153).

3.5. Computational Details

The calculations of the DFT were carried out using Gaussian 16 [35] and as previously reported [28].

3.6. Disk Diffusion Assay

The evaluation of the antimicrobial activities of 1-3 against E. coli, S. aureus and C. albicans were carried out using a disk diffusion assay at 50 µg/disc as reported earlier [36,37,38].

3.7. MIC of the Compounds

The evaluation of the MIC values of compounds 1-3 against E. coli was performed using a macrodilution method as reported before [36,39].

3.8. MTT Assay

The evaluation of the growth inhibition activities of compounds 4 and 5 against HeLa cells (ATCC CCL-2) was carried out as previously reported using an MTT assay [27,40].

4. Conclusions

Sponges of the genus Negombata continue to provide profound chemical entities with previously unknown motifs. Two novel alkaloids, magnificines A (1) and B (2), together with a new β-ionone derivative, (±)-negombaionone (3), and the known latrunculin B (4) and 16-epi-latrunculin B (5), were purified from the antimicrobial and cytotoxic fractions of the sponge N. magnifica. The structural characterizations of 1-5 were supported by analyses of their NMR and MS data. Absolute configurations of 1 and 2 were established by comparison of the predicted and experimental ECD spectra. Magnificines A and B possess an unprecedented tetrahydrooxazolo[3,2-a]azepine-2,5(3H,6H)-dione backbone. Magnificines A and B and (±)-negombaionone displayed selective activity towards E. coli without any effect on S. aureus and C. albicans. On the other hand, latrunculin B and 16-epi-latrunculin B displayed significant growth inhibition activities towards HeLa cells. The current results suggest that 1-3 could be a foundation for the development of novel antibacterial leads.