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

Abstract

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.

2.2. Structure of Magnificine A (1)

Magnificine A (1) (Figure 2) obtained as an optically active ([α]$\begin{array}{c}25\\ \mathrm{D}\end{array}$ = + 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. 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

). 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 (δ_H 2.03 and 1.33, ²J_7a,7b = 11.5 Hz), vicinal couplings from H-7a (³J_7a,8 = 4.2 Hz) and H-7b (³J_7b,8 = 11.5 Hz) to the oxygenated methine H-8 (δ_H 4.13, tt, J = 11.5, 4.2 Hz) were observed. Furthermore, H-8 exhibited additional vicinal (³J_HH) couplings to H-9a (δ_H 2.54, ddd, J = 11.5, 4.2, 1.8 Hz) and H-9b (δ_H 1.51, t, J = 11.5 Hz) completing the coupling system.

The ¹³C NMR resonances at δ_C 49.8 (CH₂, C-7), 65.1 (CH, C-8), and 47.9 (CH₂, C-9) are correlated to the protons at δ_H 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 (δ_C 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 δ_H/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 δ_C 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 δ_H/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 ³J_CH HMBC from H-3 (δ_H 5.72) to C-5 (δ_C 180.7) and C-10 (δ_C 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).

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).

The magnitude of the vicinal ³J_HH values between H-8 (tt, J = 11.5, 4.2 Hz) (Figure 6) and H-7b (δ_H = 1.33, ³J_8,7b = 11.5 Hz), and between H-8 and H-9b (δ_H = 1.51, ³J_8,9b = 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 ³J_HH values between H-8 and H-7a (δ_H = 2.03, ³J_8,7a = 4.2 Hz) and between H-8 and H-9a (δ_H = 2.54, ³J_8,9a = 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).

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.

2.3. Structure of Magnificine B (2)

Magnificine B (2) (Figure 2), an optically active ([α]$\begin{array}{c}25\\ \mathrm{D}\end{array}$ = −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. 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

) 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 (δ_H 4.33, quin., ³J = 3.5 Hz) instead of triplet of triplet (δ_H 4.13, tt, ³J = 11.5 and 4.2 Hz) in 1 suggested an opposite configuration of the OH moiety at C-8.

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.

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]$\begin{array}{c}25\\ \mathrm{D}\end{array}$ = −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. 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
5a 5b	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 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 δ_H/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 δ_H/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.

The racemic nature of 3 was confirmed from the absence of any optical activity ([α]$\begin{array}{c}25\\ \mathrm{D}\end{array}$ = 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.

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) (t_R = 13.0 min), 2 (1.2 mg) (t_R = 14.0 min) and 3 (2.7 mg) (t_R = 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) (t_R = 8.8 min) (17 mg) and 5 (3.5 mg) (t_R = 9.5 min).

3.4. Spectral Data of the Compounds

Magnificine A (1): Yellow oil; [α]$\begin{array}{c}25\\ \mathrm{D}\end{array}$ 70° (c 0.1, MeOH); UV (MeOH) λ_max (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]$\begin{array}{c}25\\ \mathrm{D}\end{array}$ −65° (c 0.1, MeOH); UV (MeOH) λ_max (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]$\begin{array}{c}25\\ \mathrm{D}\end{array}$ 0° (c 0.1, MeOH); UV (MeOH) λ_max (log ε): 205 (2.75), 274 (2.34) nm; IR (film): ν_max 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.