Michal Issac

Maurice Aknin

Anne Gauvin-Bialecki

Nicole De Voogd

Alisson Ledoux

Michel Frédérich

Yoel Kashman

Shmuel Carmeli

Michel Frederich

Cyclotheonellazoles A-C, Potent Protease Inhibitors from the Marine Sponge Theonella aff. swinhoei

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

M arine sponges of the genus Theonella have been shown to be a rich source of unique bioactive secondary metabolites with diverse structures derived from different biosynthetic pathways such as polyketide macrolides, alkaloids, steroids, and peptides. Among these metabolites are the cytotoxic macrolides swinholides, [START_REF] Carmely | [END_REF] which were isolated also from a marine cyanobacterium, 2 the cyclic peptides cyclotheonamides, 3 oriamide, 4 keramamides, 5 and perthamides, 6 the linear peptides polytheonamides, 7 koshikamides, 8 and yaku'amides, 9 and the cyclic peptolides theopapuamide, 10 papuamides, 11 and callipeltin A. 12 Most of these metabolites are composed of both proteinogenic and nonproteinogenic amino acids. The latter nonproteinogenic amino acids are primarily derived from amino-acid precursors incorporating an acetate or propionate extender unit. The cyclic peptides cyclotheonamides, 3 oriamide, 4 keramamides, 5 discobahamins, 13 and calyamides 14 contain a high proportion of nonproteinogenic amino acids such as vinylogous amino acids, vinylogous thiazole or oxazole amino acids, α-keto-β-amino acids, and oxidized amino acids and are cyclized through diamino acids such as lysine, ornithine, or 2,3-diaminopropionic acid. These metabolites are biosynthesized by hybrid PKS-NRPS enzymes characteristic of bacteria, thus suggesting that symbiotic bacteria, in the sponges, synthesize these metabolites. 15 In the course of our ongoing research aimed at the isolation of potential biomedicines from marine invertebrates, we examined a sample of the sponge Theonella aff. swinhoei collected in Dos de la Baleine, Madagascar. The extract of the sponge material that exhibited antiplasmodial activity (IC 50 = 0.30 μg/mL) was separated on a Sephadex LH-20 column, and the semipure compounds were separated by reversed-phase HPLC on a C 18 preparative column to afford swinholide A, which turned out to be one of the antimalarial constituents (IC 50 = 4.75 μg/mL), and three new cyclic peptides, cyclotheonaellazoles A-C (1-exhibited promising in vitro antiplasmodial activity. The antiplasmodial activity was ascribed in part 3). These cyclic peptides were found to contain only two (or three) proteinogenic amino acids, while the rest were nonproteinogenic acids, among them, the unique 4-propenoyl-2-tyrosylthiazole (Ptt), 3-amino-4-methyl-2-oxohexanoic acid (Amoha), and diaminopropionic acid (Dpr). These metabolites are related in structure to oriamide (4), which was isolated a decade ago from a sample of the sponge Theonella sp. collected in Sodwana Bay, South Africa, 4 but are shown here for the first time to be potent serine protease inhibitors.

■ RESULTS AND DISCUSSION

Cyclotheonaellazole A (1) was isolated as an amorphous, colorless solid that presented a positive HRESIMS sodium adduct ion at m/z 1042.3029, corresponding to the molecular formula C 44 H 54 N 9 Na 2 O 14 S 2 and 22 degrees of unsaturation. The 1 H NMR spectrum (in DMSO-d 6 , Table 1) presented 10 exchangeable protons (based on the HSQC experiment), four two-proton doublets of two para-substituted phenols, a thiazole proton, two doublet protons of an isolated and conjugated trans double bond, six methine protons and eight methylene protons between 5.5 and 2.5 ppm, a methine proton, six methylene protons, and two doublet-and two triplet-methyl signals in the shielded region of the spectrum. The 13 C NMR spectrum (Table 1) exhibited a conjugated carbonyl (198.1 ppm), 12 sp 2 nonprotonated carbons between 175 and 149 ppm, two nonprotonated, three methine, and four doubled methine sp 2 carbons between 134 and 114 ppm, six methine and one methylene carbon between 61 and 47 ppm, and one methine, six methylene, and four methyl carbons between 43 and 11 ppm. Analysis of the 2D NMR data from the COSY, TOCSY, and HSQC spectra of cyclotheonellazole A (Table 1) allowed the assignment of the side chains of the proteinogenic (glycine and alanine) and nonproteinogenic amino acids (diaminopropionic acid, 2-aminopentanoic acid, and cysteic acid) and the partial assignment of the aromatic residue p-hydroxybenzoic acid (Figure 1, bold lines). Furthermore, this analysis established the structure of a modified isoleucine unit with a deshielded amino-methine proton (δ H 5.04) and three isolated spin-systems of the Ptt subunit (Figure 1). HMBC correlations of the α and/or β protons of the acid subunits with the amide carboxyl carbons assigned the latter to the side chains of phydroxybenzoic acid, glycine, alanine, Dpr, and cysteic acid (Cya) (Figure 1). The amide carbonyl of Apa was assigned through the NOE of Amoha-NH with Apa-H-2 and its HMBC correlation with the sp 2 carbon resonating at 174.9 ppm. The carbonyl and carboxyl of the modified isoleucine moiety (Amoha) were established through the HMBC correlation of Amoha-H-3 with a conjugated carbonyl carbon (δ C 198.1) and combination of an NOE of Amoha-H-3 with Ptt-NH and an HMBC correlation of the latter with the carboxyl carbon resonating at δ C 163.9, which resembled the chemical shifts of similar 2-oxo-amino acids. 4,5 The three short proton-spinsystems were assembled to the complete Ptt unit through the HMBC correlations of Ptt-H-2 with the conjugated carbonyl (δ C 164.9), of Ptt-H-3 and the thiazole proton, Ptt-H-5, with the nonprotonated thiazole sp 2 carbon (δ C 149. carbon (δ C 156.3). These eight subunits were assembled to the full planar structure of 1 based on the HMBC correlations of the p-hydroxybenzyl amide carbonyl with the NH of Gly, the amide carbonyl of Gly with the NH of Ala, the amide carbonyl of Ala with the α-NH of Dpr, the amide carbonyl of Dpr with the NH of Apa, the amide carbonyl of Apa with the NH of Amoha, the amide carbonyl of Amoha with the NH of Ptt, the amide carbonyl of Ptt with the NH of Cya, and the amide carbonyl of Cya with the β-NH of Dpr and the NOEs of Apa-H-2 with Amoha-NH and Amoha-H-3 with Ptt-NH (Figure 1). These correlations were further supported by additional NOE correlations (Table 1). The absolute configurations of the stereogenic centers of the amino acids in 1 were determined by a variant of the advanced Marfey's method. 16 Treatment of 1 with H 2 O 2 /NaOH 17 followed by acid hydrolysis afforded a mixture of the amino acids derived from 1 where the Amoha was converted to Ile. The sample was split into two portions; one reacted with L- FDAA and the other with D-FDAA. Analysis of the derivatized mixture of amino acids by LC-MS revealed that the Ala, Dpr, Apa, Amoha, Cya, and Ptt are of the L-form. An attempt to treat the cyclic peptide with ozone prior to the hydrolysis failed and did not produce any recognized amino acid. The absolute Sconfiguration of C-4 of Amoha was determined by comparison of the 13 C chemical shifts of the methyl signals in Amoha (C-5, 16.3 ppm, and C-7, 11.9 ppm) with those of L-Ile (C-5, 16.2 ppm, and C-6, 11.6 ppm) in micropeptin DR1056, 18 L-allo-Ile (C-5, 14.1 ppm, and C-7, 12.2 ppm) in micropeptin KT1042, 19 and D-allo-Ile (C-5, 13.8 ppm, and C-6, 12.0 ppm) in aeruginosin GE686. 20 On the basis of these arguments structure 1 is proposed for cyclotheonellazole A.

The structure elucidation of cyclotheonellazoles B (2) and C (3) was accomplished in a similar manner. Both shared the same basic structure with 1, in which leucine (in 2) and homoalanine (in 3) replaced the 2-aminopentanoic acid of cyclotheonellazole A (1). Cyclotheonellazole B (2) presented an HRESIMS protonated molecule at m/z 1034.3363 corresponding to the molecular formula C 45 H 57 N 9 NaO 14 S 2 and 22 degrees of unsaturation. Its NMR data (DMSO-d 6 , Table 2) were similar to those of 1, presenting, in the 1 H NMR spectrum, two doublet methyl signals (δ H 0.88 and 0.92) and an isopropyl-methine proton (δ H 1.79) instead of the triplet methyl signal and the diastereotopic methylene signals (δ H 1.53, 1.31) of Apa in 1. In the 13 C NMR spectrum of 2 (Table 2), two new methyl carbons (δ C 23.6 and 21.0) and a methine carbon (δ C 24.4) appear instead of the methyl and methylene signals (δ C 13.9 and 19.8, respectively) of Apa in 1. Interpretation of the 2D NMR data collected for 2 (Table 2) revealed that the two doublet methyl groups (δ H 0.88 and 0.92, δ C 23.6 and 21.0, respectively) were coupled to a methine (δ H 1.79, δ C 24.4), which in turn was coupled to a methylene (δ H 2.38, 1.26, δ C 38.2). The latter methylene was coupled to an αmethine of an amino acid (δ H 4.40, δ C 52.7), which was coupled through a COSY correlation to an amide proton (δ H 8.58). HMBC correlation of the α-methine (δ H 4.40) with an amide carbonyl carbon (δ C 175.2) established this amino acid as Leu. This assignment revealed that the Apa, in 1, was substituted by a Leu, in 2, while the rest of the molecule remained identical. The configurations of the stereogenic centers of the amino acids of 2 were established by a modified Marfey's method, 16,17,21 in which the retention times of the L-FDAA-derivatized mixture of amino acids from 2 were compared with those from 1 (correlated on the basis of their retention times and molecular MS ions) and by comparison with the retention times of L-FDAA-derivatized D,L-leucine. This procedure established all amino acids as of the L-form. On the basis of these arguments the structure of cyclotheonellazole B was established as 2.

Cyclotheonellazole C (3) presented a positive HRESIMS sodium adduct ion at m/z 1028.2869 corresponding to the molecular formula C 43 H 52 N 9 Na 2 O 14 S 2 and 22 degrees of unsaturation. These findings suggested that 3 contains one methylene less than 1. The NMR data of 3 (DMSO-d 6 , Table 2) were almost identical with those of 1. Small differences in the chemical shifts of the proton and carbon signals of the straight-chain nonproteogenic amino acid (Apa in 1) and the absence of one methylene (in 3 relatives to 1) were the only differences observed, when the NMR spectra of 1 and 3 were compared, identifying the amino acid as homoalanine. The structure of the homoalanine and the full structure of 3 were established unambiguously by the 2D NMR experiments summarized in Table 2. The configurations of the amino acids of 3 were established by a similar procedure to the one used for 2 and by comparison with L-FDAA-derivatized D,L- homoalanine. 16,17,21 This analysis established that all of the amino acids of cyclotheonellazole C are of the L-form, thus assigning its structure as 3.

The biological activities of these compounds were tested against the serine protease enzymes chymotrypsin and elastase and the malaria parasite Plasmodium falciparum. Cyclotheonellazoles A (1), B (2), and C (3) were inactive against P. falciparum (IC 50 's > 20 μg/mL). Cyclotheonellazoles A (1), B (2), and C (3) inhibited chymotrypsin with IC 50 values of 0.62, 2.8, and 2.3 nM, respectively, and elastase with IC 50 values of 0.034, 0.10, and 0.099 nM, respectively. On the basis of the X-ray structure of the trypsin-cyclotheonamide A complex that supports the formation of a covalent tetrahedral hemiketal between the α-ketoamide of cyclotheonamide A and Ser195 of trypsin, 22 one can assume that the cyclotheonellazole complexes with elastase (and similarly with chymotrypsin) exhibit a tetrahedral transition state involving the keto group of Amoha and Ser195 of elastase, while the side chain of Amoha fits in the enzyme S1 pocket. 23 The negatively charged oxygen is probably stabilized through a hydrogen bond with the His57 residue in the enzyme, and the cleavage cannot occur because carbon is a poor leaving group (Figure 2). The IC 50 values obtained for the inhibition of chymotrypsin and elastase by the cyclotheonellazoles illuminated some structure-activity relationships. Cyclotheonellazole A, which contains a 2-aminopentanoic acid residue, was found to be the most potent inhibitor. This is probably due to a better compatibility with the enzyme S2 subsite. Cyclotheonellazoles B and C contain the amino acids leucine and homoalanine, and it seems that the length and the branching of the aliphatic chain influence the activity.

In summary, we have isolated three new cyclic peptides, along with the known dimeric polyketide swinholide A, from a T. aff. swinhoei sponge collected in Madagascar. Cyclotheonellazoles A-C (1-3) are composed of a high proportion of nonproteinogenic amino acids and are related in structure to oriamide (4), which was isolated a decade ago from a sample of the sponge Theonella sp. collected in Sodwana Bay, South Africa. 4 These cyclic peptides are shown here for the first time to be potent elastase and chymotrypsin inhibitors. The newly isolated cyclotheonellazoles A-C further validate Piel's group's findings concerning the huge diversity of hybrid PKS-NRPS enzymes, characteristic of bacteria, found in T. swinhoei. 15 These compounds, which are presumably biosynthesized by such hybrid enzymes, append to the constantly growing list of "nonribosomally" biosynthesized cyclic peptides isolated from T. swinhoei. The context of the biosynthesis of these highly active protease inhibitors within the sponge is yet unknown, but they provide us with additional scaffolds that selectively inhibit homologous serine proteases of pharmaceutical significance.

■ EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotation values were obtained on a Jasco P-1010 polarimeter at the sodium D line (589 nm). UV spectra were recorded on an Agilent 8453 spectrophotometer. IR spectra were recorded on a Bruker Tensor 27 FT-IR instrument (Billerica, MA, USA). NMR spectra were recorded on Bruker Avance and Avance III spectrometers at 500.13 MHz for 1 H and 125.76 MHz for 13 C. DEPT, COSY-45, gTOCSY, gROESY, gHSQC, gHMQC, and gHMBC spectra were recorded using standard Bruker pulse sequences. NMR chemical shifts were referenced to tetramethylsilane, δ H and δ C = 0 ppm. High-resolution mass spectra were recorded on a Waters MaldiSynapt instrument (ESI), and LCMS spectra were recorded on a Waters Xevo TQD instrument (ESI). HPLC separations were performed on a Merck Hitachi HPLC system (L-6200 Intelligent pump and L-4200 UV-vis detector), a JASCO P4-2080 Plus HPLC system with a multiwavelength detector, and an Agilent 1100 Series HPLC system.

Biological Material. The sponge Theonella aff. swinhoei (BA-019) was collected in Dos de la Baleine (22°14.857 S, 43°10.892 E), Madagascar, at a depth of 28 m on December 2011. A voucher specimen (RMNH POR 7396) was deposited in the Naturalis Biodiversity Center, The Netherlands.

Extraction and Isolation. The sponge material (139 g dry weight after extraction) was chopped into small pieces and extracted by mixture of 1:1 CH 2 Cl 2 /MeOH (2 × 1.5 L, each 24 h) to afforded 1.2 g of extract. The extract was separated by size exclusion chromatography on a Sephadex LH-20 column eluted with a mixture of 1:1 MeOH/ CHCl 3 to afford 12 fractions.

1 Figure 1 .

 11 Figure 1. Key COSY, HMBC, and NOE correlations of 1.

Figure 2 .

 2 Figure 2. Schematic illustration of elastase inhibition mechanism.

  Fractions 9-12 (142 mg) were combined and separated on a Sephadex LH-20 column with MeOH as the eluent to afford 12 fractions. Fraction 10 (33.0 mg) was separated by HPLC on a preparative RP-8 column (YMC-Pack C-8 250 × 20 mm, 5 μm, DAD) with a mixture of 45:55 CH 3 CN/H 2 O to afford the three pure natural products, cyclotheonellazole A (1) (1.6 mg, retention time (t R ) 22.3 min, 0.0952% yield), cyclotheonellazole B (2) (1.4 mg, t R 25.5 min, 0.0833% yield), and cyclotheonellazole C (3) (1.2 mg, t R 19.8 min, 0.0714% yield). Cyclotheonellazole A (1): amorphous, colorless material; [α] 24 D

Table 1 .

 1 NMR Data (DMSO-d 6 ) of Cyclotheonaellazole A (1) a

	position	δ C , type b	δ H (J in Hz)	HMBC correlations c	NOE correlations d
	Hbza 1	166.3, C		Hbza-3, Gly-2a,2b,NH
	2	124.9, C		Hbza-4,4′
	3, 3′	129.4, CH	7.73, d (9.0)	Hbza-4,4′, Gly-NH	Hbza-4,4′, Gly-2a,2b,NH,
	4, 4′	115.0, CH	6.78, d (9.0)	Hbza-3,3′	Hbza-3,3′
	5	160.4, C		Hbza-3,3′,4,4′
	5-OH		9.96 brs
	Gly 1	168.9, C		Gly-2a,2b, Ala-2,NH
	2	42.7, CH 2	3.79, dd (16.5, 5.5)		Hbza-3,3′, Gly-NH, Ala-2,NH
			3.89, dd (16.5, 5.5)		Hbza-3,3′, Gly-NH, Ala-2,NH
	NH		8.46, t (5.5)		Hbza-3,3′, Gly-2a,2b, Ala-NH
	Ala 1	171.5, C		Ala-2,3, Dpr-α-NH
	2	47.9, CH	4.33, m	Ala-3,NH	Ala-3,NH, Gly-2a,2b, Dpr-α-NH
	3	18.6, CH 3	1.16, d (7.0)	Ala-2,NH	Ala-2,NH, Dpr-α-NH
	NH		8.00, d (7.5)		Ala-2,3, Gly-2a,2b,NH
	Dpr 1	171.3, C		Dpr-2,3b, Apa-NH
	2	51.1, CH	4.03, m	Dpr-α-NH	Dpr-3a,3b,α-NH, Cya-2, Apa-NH
	3	40.9, CH 2	2.49, m	Dpr-α-NH	Dpr-2,3b,α-NH,β-NH
			3.75, m		Dpr-2,3a,β-NH
	α-NH		7.93, d (7.5)		Dpr-2,3a,3b, Ala-2,3
	β-NH		7.85, brs		Dpr-3a, Cya-NH, Apa-NH
	Apa 1	174.9, C		Amoha-3,NH
	2	54.5, CH	4.32, m		Apa-3a,3b,4a,4b,NH, Amoha-NH
	3	32.3, CH 2	1.63, m	Apa-4b,5,NH	Apa-2,3b,4a,5,NH
			2.29, m		Apa-2,3a,4a,4b,5,NH
	4	19.8, CH 2	1.31, m	Apa-5	Apa-2,3a,3b,5
			1.53, m		Apa-2,3b,5,NH
	5	13.9, CH 3	0.92, t (7.0)	Apa-4a,4b	Apa-3b,4a,4b,NH, Amoha-4
	NH		8.61, d (7.0)		Apa-2,3b,4b,5, Dpr-2,β-NH
	Amoha 1	163.9, C		Ptt-NH
	2	198.1, C		Amoha-3
	3	60.2, CH	5.04, dd (9.5, 3.5)	Amoha-5,NH	Amoha-4,5,NH, Ptt-2,NH
	4	37.1, CH	2.39, m	Amoha-3,5,6b,7	Amoha-2,3,5,NH, Apa-5
	5	16.3, CH 3	0.77, d (7.0)	Amoha-3,6a,6b	Amoha-3,4,6a,6b
	6	23.4, CH 2	1.08, m	Amoha-5,7	Amoha-5,7,NH
			1.14, m		Amoha-5,7
	7	11.9, CH 3	0.72, t (7.5)	Amoha-6a,6b	Amoha-6a,6b
	NH		8.31, d (9.5)		Amoha-3,4, Apa-2
	Ptt 1	164.9, C		Ptt-2,3, Cya-NH
	2

Table 2 .

 2 NMR Data (DMSO-d 6 ) of Cyclotheonaellazoles B (2) and C (3) a

			cyclotheonaellazole B (2)		cyclotheonaellazole C (3)
	position	δ C , type b	δ H (J in Hz)	δ C , type b	δ H (J in Hz)
	Hbza 1	166.3, C		166.4, C
	2	124.9, C		124.9, C
	3,3′	129.4, CH	7.72, d (8.5)	129.4, CH	7.73, d (8.5)
	4,4′	115.0, CH	6.78, d (8.5)	114.9, CH	6.78, d (8.5)
	5	160.4, C		160.4, C
	5-OH		9.97 brs		9.95 brs
	Gly 1	168.9, C		168.9, C
	2	42.7, CH 2	3.78, dd (16.0, 6.0)	42.7, CH 2	3.78, m
			3.88, dd (16.0, 6.0)		3.89, m
	NH		8.45, t (6.0)		8.46, t (5.5)
	Ala 1	172.1, C		171.5, C
	2	48.5, CH	4.33, dq (7.5, 7.0)	47.7, CH	4.33, dq (7.5, 7.0)
	3	19.2, CH 3	1.15, d (7.0)	18.5, CH 3	1.16, d (7.0)
	NH		8.01, d (7.5)		8.00, d (7.5)
	Dpr 1	171.2, C		171.4, C
	2	51.2, CH	4.01, m	51.1, CH	4.03, m
	3	41.0, CH 2	2.45, m	40.6, CH 2	2.46, m
			3.76, m		3.77, m
	α-NH		7.90, d (7.5)		7.94, d (7.5)
	β-NH		7.84, brs		7.83, brs
	Leu/hAla 1	175.2, C		174.8, C
	2	52.7, CH	4.40, m	55.9, CH	4.27, m
	3	38.2, CH 2	1.26, m	23.3, CH 2	1.78, m
			2.38, m		2.19, m
	4	24.4, CH	1.79, m	11.7, CH 3	0.99, t (7.0)
	5	11.7, CH 3	0.92, d (6.5)
	6	11.7, CH 3	0.88, d (6.5)
	NH		8.58, d (7.0)		8.61, d (7.5)
	Amoha 1	164.1, C		163.9, C
	2	198.9, C		198.1, C
	3	60.4, CH	5.05, dd (9.5, 3.5)	60.2, CH	5.04, m
	4	38.1, CH	2.39, m	37.1, CH	2.39, m
	5	16.2, CH 3	0.76, d (6.5)	16.3, CH 3	0.77, d (6.5)
	6	23.5, CH 2	1.07, m	23.4, CH 2	1.08, m
			1.15, m		1.14, m
	7	11.9, CH 3	0.71, t (7.5)	11.9, CH 3	0.72, t (7.5)
	NH		8.36, d (9.5)		8.32, d (9.5)
	Ptt 1	165.0, C		165.0, C
	2	123.6, CH	6.76, d (15.0)	123.5, CH	6.76, m
	3	132.6, CH	7.38, d (15.0)	132.7, CH	7.39, d (15.0)
	4	149.7, C		149.7, C
	5	123.2, CH	7.84, brs	123.3, CH	7.85, brs
	6	170.8, C		170.7, C
	7	54.6, CH	5.20, q (7.5)	54.5, CH	5.21, m
	8	39.1, CH 2	3.01, m	39.2, CH 2	3.02, dd (14.0, 6.0)
			3.08, m		3.09, m
	9	127.6, C		127.6, C
	10,10′	130.6, CH	7.16, d (8.0)	130.6, CH	7.16, d (8.5)
	11,11′	115.3, CH	6.67, d (8.0)	115.3, CH	6.68, d (8.5)
	12	156.3, C		156.3, C
	12-OH		9.27, s		9.26, s
	NH		9.39, d (8.0)		9.40, d (8.0)
	Cya 1	170.9, C		170.7, C
	2	49.5, CH	4.73, m	49.5, CH	4.74, m
	3	50.7, CH 2	2.68, dd (13.5, 5.5)	50.7, CH 2	2.70, dd (14.0, 6.0)
			3.25, m		3.25, m
	NH				8.15, d (9.5)

a 500 MHz for 1 H, 100 MHz for

13

C.

b

Multiplicity and assignment from HSQC experiment.

■ ACKNOWLEDGMENTS

The authors express their gratitude to M. E. Remanevy for assistance in sponge collection; Y. Abudi for technical assistance; and N. Tal, the Mass Spectrometry Facility of the School of Chemistry, Tel Aviv University, for the measurements of the mass spectra. The present research was supported in part by a Tel Aviv University internal grant (S.C. and Y.K.). M.I. acknowledges the Ph.D. Fellowship from the School of Chemistry of Tel Aviv University. M.A., A.G.-B., and M.F. gratefully acknowledge financial support from the European Commission and the Regional Council of Reunion Island: BIOMOL-TCN program (Activiteś Theŕapeutiques, Cosmetologiques et Nutraceutiques de Molećules Issues de la BiodiversitéTerrestre, Marine et Microbienne de la Zone Sud-Ouest de l'Oceán Indien), ERDF (European Regional Development Fund).

D

-11 (c 0.2, MeOH); UV (MeOH) λ max (log ε) 207 (4.15) nm; IR (ATR Diamond) ν max 3321, 2917, 2849, 1658, 1546, 1515, 1207, 1044, 699 cm -1 ; NMR data, Tables 2 andS2; HRESIMS m/z 1028.2869 [M + Na] + , (calcd for C 43 H 52 N 9 Na 2 O 14 S 2 , 1028.2871); retention times of DAA derivatives: L-Ala-L-DAA, 11.10 min; L-Dpr-L-DAA, 20.59 min; L-homoAla-L-DAA, 12.28 min, D-homoAla-L-DAA, 12.30 min; L-Ile-L-DAA, 17.88 min; L-Ptt-L-DAA, 16.28 min.

Methods for Determination of the Absolute Configurations. To determine the absolute configuration of the amino acids, advanced Marfey's analysis was performed. A sample of 0.25 mg of compounds 1-3 was treated with H 2 O 2 /NaOH (2 h) followed by acid hydrolysis, enabling recovery of Ile from the Amoha moiety and the rest of the free amino acids. The hydrolysis of 1-3 was carried out at 104 °C for 60 min with 500 μL of 6 M HCl. The solution of 1 was divided into two portions, which were dried under vacuum to afford the dry mixture of amino acids, and the residue was dissolved in 100 μL of H 2 O. One portion was derivatized with L-FDAA and the other with D-FDAA. The hydrolysates of 2 and 3 were evaporated to dryness, and the residues were dissolved in 100 μL of H 2 O. Similar solutions were prepared from the reference amino acids D/L-Leu and D/Lhomoalanine. To the latter solution were added 20 μL of 1 M sodium bicarbonate, per each amino acid in the peptide, followed by L- or D-FDAA (30.5 × 10 -6 mol/L in acetone) at a molar ratio of 1:1.2 for each amino acid. These solutions were stirred at 40 °C for 60 min. The reactions were quenched by the addition of 20 μL of 2 N HCl for each amino acid. The solutions were evaporated and dissolved in 250 μL of CH 3 CN. The two samples of L-and D-FDAA derivatives were analyzed by ESI LCMS. The analysis was performed on an Acquity-UPLC (Waters Inc., USA) coupled with a UV detector (Acquity-TUV detector, Waters Inc.) and mass spectrometer (Xevo TQD, Waters Inc.) The stationary phase consisted of a C18 (1.7 μm, 2.1 × 100 mm) column (Waters Inc.), and the mobile phase compositions were A: 95:5 H 2 O/CH 3 CN + 0.1% formic acid (FA) and B: CH 3 CN + 0.1% FA. The elution gradient was as follows: starting conditions 1 min of 100% A, linear gradient to 40% B over 25 min, and hold for 4 min. Samples of 10 μL were injected, and the flow rate was 0.5 mL/min. The UV detector was set to 340 nm, while the mass spectrometer was operated in both negative and positive ion modes, scanning between 200 and 650 mass units. The interpretation of the data was conducted after the run on both positive and negative ion modes by MassLynx software (v4.1, Waters Laboratory Informatics).

In Vitro Antiplasmodial Assays. The P. falciparum strains utilized and details of the assay protocols have been previously reported. 24 The selectivity index was determined as the ratio of cytotoxicity over antiplasmodial activity (expressed in IC 50 values).

Protease Inhibition Assays. The samples for biological assays were dissolved in DMSO at a concentration of 1 mg/mL, and the IC 50 values were determined by series dilutions that produced a sigmoidshaped response pattern of the enzyme inhibition versus the concentration of the inhibitor, according to the four-parameter logistic model. 25 The assays were made in 96-well plates for the following enzymes.

Chymotrypsin. The assay was performed in a Tris buffer (0.6 g TRIS HCl/100 mL H 2 O, pH 7.5). The enzyme and the substrate Suc-Gly-Gly-phenylalanine-p-nitroanilide were dissolved in the buffer at a concentration of 1 mg/mL. To each well were added 100 μL of buffer, 10 μL of enzyme, and 10 μL of sample. Then, the plate was placed in the spectrophotometer for 5 min of incubation at 37 °C. Thereafter, to each well was added 100 μL of substrate solution, and the plate was replaced in the spectrophotometer for the kinetic measurement of the absorbance intensity over 30 min at a wavelength of 405 nm.

Elastase. The assay was performed in a Tris buffer (2.422 g TRIS HCl/100 mL H 2 O, pH 8.0). The enzyme and the substrate, N-Suc-Ala-Ala-Ala-p-nitroanilide, were dissolved in the buffer at a concentration of 75 and 0.9 mg/mL, respectively. A 150 μL amount of buffer solution, 10 μL of enzyme, and 10 μL of sample were added to each well. Then, the plate was placed in the spectrophotometer for 20 min of incubation at 30 °C. Thereafter, 30 μL of substrate solution was added to each well, and the plate was replaced in the spectrophotometer for the kinetic measurement of the absorbance intensity over 20 min at a wavelength of 405 nm.

■ ASSOCIATED CONTENT

* S Supporting Information

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.7b00028.

Spectroscopic data including 1D and 2D NMR spectra and HRMS data of compounds 1-3 and complete 2D NMR data of compounds 2 and 3 (PDF)

■ AUTHOR INFORMATION

Corresponding Author *Tel (S. Carmeli): +972-3-6408550. Fax: +972-3-6409293. Email: carmeli@pot.tau.ac.il.

ORCID

Alisson Ledoux: 0000-0002-4052-6336 Shmuel Carmeli: 0000-0002-9641-9943

Notes

The authors declare no competing financial interest.


Michal Issac

Maurice Aknin

Anne Gauvin-Bialecki

Christopher D Pond

Louis R Barrows

Yoel Kashman

Shmuel Carmeli

Mollecarbamates, Molleureas, and Molledihydroisoquinolone, o-Carboxyphenethylamide Metabolites of the Ascidian Didemnum molle Collected in Madagascar

) is a cyclic form of o-carboxyphenethylamide. We propose that these unique natural products are most probably produced by an unprecedented biosynthetic pathway that contains a yet unknown chorismate mutase variant. The structures of the compounds were elucidated by interpretation of the data from 1D and 2D NMR, HRESIMS, and MS/MS analyses of the positive ESIMS experiments. Compounds 1-8 were tested against pathogenic bacteria and in a cytoprotective HIV cell based assay but did not show any significant effects in these assays.

A scidians (tunicates), like sponges, have been shown to be a rich source of unique bioactive secondary metabolites with diverse structures derived from different biosynthetic pathways. 1 The ascidian Didemnum molle is an example of a marine organism that shows high variability of secondary metabolites between collection sites. [2][3][4][5][6][7][8][9] D. molle is well recognized for the production of heterocycle-containing cyclic hexapeptides, i.e., comoramides, 2 didmolamides, 3 mollamides B-F, [4][5][6] keenamide, 4 and hexamollamide, 7 and heptapeptides, i.e., mollamide, 8 cyclodidemnamide, 9 and mayotamides. 2 These metabolites have been shown to be biosynthesized in D. molle by the symbiotic cyanobacterium Prochloron didemni. 5 Other metabolites that have been isolated from this ascidian include the sulfated mannopolysaccharide kakelokelose 10 and more recently the urea derivatives N,N′-diphenethylurea (10) 11 and molleurea A (11). 6 N,N′-Diphenethylurea was initially described from Streptomyces sp. AM-2498 and reported to have an antidepressant effect 12 and, more recently, enhancement of adipocyte differentiation in 3T3-L1 cells. 11 Molleurea A presented a mild anti-HIV activity in both an HIV integrase inhibition assay and a cytoprotective cell-based assay. 6 In the course of our ongoing research aimed at the isolation of potential biomedicines from marine invertebrates, we isolated nine new natural products, mollecarbamates A-D (1-4), molleureas B-E (5-8), and molledihydroisoquinolone (9), alongside the known N,N′-diphenylethylurea (10) 11 and molleurea A (11) from the extracts of two samples of D. molle. 6 These metabolites are, as far as we know, the only compounds in the literature that contain ortho-carboxyphenethylamide derivatives in their skeleton. The structure elucidation and the proposed biogenesis of the new compounds are discussed below.

■ RESULTS AND DISCUSSION

The extracts of the wet ascidian material (MAY13-117) were separated using a Kupchan solvent partition procedure 13 and Sephadex LH-20 column chromatography. The semipure compounds were separated by reversed-phase HPLC on a C 18 preparative column to afford the eight new natural products mollecarbamates A-D (1-4) and molleureas B-E (5-8) alongside N,N′-diphenylethyl urea (10) and molleurea A (11). From a second sample of D. molle (MAD11-BA065), molldihydroisoquinolone (9) was isolated by a similar isolation scheme.

Mollecarbamate A (1) was isolated as an amorphous, colorless solid that yielded a positive HRESIMS sodium adduct ion at m/z 349.1524, corresponding to the molecular formula C 19 H 22 N 2 O 3 Na and 10 degrees of unsaturation. The 1 H NMR spectrum (in C 6 D 6 , Tables 1 andS1 in the Supporting Information) of 1 revealed two exchangeable protons (based on the HSQC experiment), four pairs of methylene protons, several aromatic protons, and a methoxy group. The 13 C NMR spectrum (Table 1) of 1 indicated the presence of mono-and disubstituted aromatic ring systems, one carboxylic carbon (170.4 ppm), and a shielded carbonyl (157.9 ppm). Analysis of the 2D NMR data from the COSY, TOCSY, and HSQC spectra of 1 (Table S1) allowed the assignment of four substructures (Figure 1, bold lines), and HMBC correlations (Figure 1, curved arrows) established two phenethylamine fragments. HMBC correlations, from the protons of the methoxy group resonating at 3.38 ppm and the protons of the methylene resonating at 3.50 ppm to the shielded carbonyl resonating at 157.9 ppm, established the structure of a carbamate moiety connected to a short aliphatic chain, as shown in Figure 1. The two fragments were assembled to the full planar structure based on an HMBC correlation between the amide carbonyl resonating at 170.4 ppm and the methylene protons at 3.31 ppm. On the basis of this analysis, the structure of mollecarbamate A was established as 1. The structure of 1 was verified by mass analysis of the product ions obtained from a positive mode ESIMS/MS experiment (Figure 2a), presenting characteristic ions that helped with the structure elucidation of the more complex members (3 and 4) of this group of metabolites.

The structure elucidation of mollecarbamates B (2), C (3), and D (4) was accomplished in a similar manner. They differ from 1 in the number of carboxyphenethyl amide units ranging from two to four, respectively. With the growing number of the Chart 1 carboxyphenethyl amide units, the structure elucidation became much more complicated and the support of the MS/MS fragmentation data was crucial.

Mollecarbamate B (2) presented an HRESIMS sodium adduct ion at m/z 496.2216 corresponding to the molecular formula C 28 H 31 N 3 NaO 4 and 15 degrees of unsaturation. Its proton NMR data (C 6 D 6 , Tables 1 andS2) were similar to those of 1, revealing additional signals of two pairs of methylene protons (δ H 3.69 and 2.65), an exchangeable proton (δ H 8.04), and aromatic protons (δ H 7.30-6.60). The 13 carboxyphenethyl amide unit. This subunit was connected through HMBC correlations to a phenethylamide moiety and to a carboxyphenethylcarbamate moiety similar to those in 1.

On the basis of these results the structure of mollecarbamate B was established as 2. The MS/MS data (Figure S14) supported the proposed structure. Mollecarbamate C (3) presented an HRESIMS sodium adduct ion at m/z 643.2897, corresponding to the molecular formula C 37 H 40 N 4 NaO 5 and 20 degrees of unsaturation. The 1 H and 13 C NMR spectra of 3 suggested the presence of four ethylamide groups (Table 1) and a carbamate unit (methoxy, δ H 3.39, δ C 51.6; carbonyl, δ C 158.1; and amide, δ H 7.21), three carbonyl carbons (δ C 171.4, 171.0, and 170.8), and highly crowded aromatic regions. Analysis of the 2D NMR data (COSY, TOCSY, NOESY, HSQC, HMBC, Table S3) revealed that 3 is composed of phenethylamide and three carboxyphenethylamides in a row, terminated with a carbamate unit. The proposed structure was supported by the MS/MS fragment ions at m/z 589, 399, 252, and 105 (Figure S21). Thus, structure 3 was assigned to mollecarbamate C.

Mollecarbamate D ( 4) presented an HRESIMS sodium adduct ion at m/z 790.3579 corresponding to the molecular formula C 46 H 49 N 5 NaO 6 and 25 degrees of unsaturation. The 1 H and 13 C NMR spectra of 4 (Table 1) were highly complicated for interpretation because all signals were concentrated in very narrow regions, especially in the aromatic area; however four carbonyl carbons, four ethylamide units, and a carbamate moiety were evident from these spectral data. The structure elucidation was determined in a similar manner to 1-3 and supported by MS/MS fragment ions at m/z 736, 546, 399, 252, and 105 (Figure S28), suggesting structure 4 for mollecarbamate D.

Molleurea B ( 5) was isolated as an amorphous, colorless solid that presented an HRESIMS sodium adduct ion at m/z 585.2844, corresponding to the molecular formula C 35 H 38 N 4 NaO 3 and19 degrees of unsaturation. The 1 H NMR spectrum (in C 6 D 6 , Tables 2 andS5) revealed eight groups of methylene protons (3.70-2.47 ppm), aromatic protons (7.33-6.77 ppm), and four exchangeable protons (8.23, 5.82, 5.13, and 4.50 ppm). Together with the 13 C NMR data (Table 2) the presence of two mono-and two disubstituted aromatic ring systems (139.7-126.7 ppm) was suggested. Furthermore, the 13 C NMR spectrum revealed two carboxylic carbons (171.7 and 171.0 ppm) and a shielded carbonyl (159.1 ppm). Analysis of the 2D NMR data from the COSY, TOCSY, and HSQC spectra of molleurea B (Table S5) allowed the assignment of the proton sequence (Figure 3, bold lines) and together with the HMBC correlations (Figure 3 MS fragmentation ions of 5 (Figure 2b) at m/z 442, 416, 252, 122, and 105 suggested a phenethylamide linked to two carboxyphenethylamide units in a row and through a urea bridge to a second phenethylamide unit. On the basis of these arguments, the structure of molleurea B was established as 5.

Molleurea C ( 6) was isolated as an amorphous, colorless solid that yielded an HRESIMS sodium adduct ion at m/z 585.2837 corresponding to the molecular formula C 35 H 38 N 4 NaO 3 and 19 degrees of unsaturation. Although 6 yielded an identical molecular formula to that of molleurea B (5), its 1 H and 13 C NMR spectra were much simpler than those of 5, containing half of the signals compared to those of 5, thus suggesting that 6 had C 2 symmetry. Its 1 H NMR spectrum (Table S6) revealed four signals of aromatic protons indicative S6) contained an odd number of signals, one of which, at 158.6 ppm, was half the height of the other nonprotonated signals, suggesting its position as the symmetry axis. The structure elucidation of 6 was accomplished in a similar manner to that of 5, in which two subunits of monoand disubstituted aromatic rings were connected based on an HMBC correlation between the methylene protons at 3.45 ppm and the amide carbonyl resonating at 170.0 ppm, and these two identical phenethylamide-carboxyphenethylamide subunits were further connected by an HMBC correlation from the methylene proton signal at 3.41 ppm to the urea carbonyl carbon, at 158.6 ppm. The MS/MS fragmentation ions of 6 (Figure S42) at m/z 295, 252, 122, and 105 supported the symmetric structure suggested for molleurea C. On the basis of the arguments described above, structure 6 was assigned to molleurea C.

The 1 H and 13 C NMR spectra of compounds 7 and 8 were highly complicated for interpretation, as all signals were concentrated in very narrow regions of the spectra (especially in the aromatic region) and did not allow unambiguous assignment of all signals. The NMR data of 7 and 8 are presented in Tables 2 andS7 and S8; thus their structure elucidation was secured by interpretation of their fragmentation pattern in the positive ESIMS/MS.

Molleurea D ( 7) was isolated as an amorphous, colorless solid that yielded an HRESIMS sodium adduct ion at m/z 732.3531, corresponding to the molecular formula C 44 H 47 N 5 NaO 4 and 24 degrees of unsaturation. The 147 mass unit difference between the molecular ion of 7 and the molecular ion of molleurea C (5) suggested the inclusion of an additional carboxyphenethylamide subunit to the structure of 7, relative to 5. This was supported by the appearance of five broad amide signals and 10 methylene signals in the 1 H NMR spectrum (Table 2) of 7. The terminal phenethylamide was linked to three carboxyphenethylamide units in a row and through a urea bridge to the second phenethylamide unit through NOESY correlations (Table S7). The structure of 7 was established unequivocally by interpretation of the MS/MS fragmentation ions (Figure S49) at m/z 589, 563, 544, 399, 269, 252, 122, and 105, thus assigning structure 7 to molleurea D.

Molleurea E ( 8) presented an HRESIMS sodium adduct ion at m/z 732.3529, corresponding to the molecular formula C 44 H 47 N 5 NaO 4 and 24 degrees of unsaturation. Although the molecular ion and formula of molleurea E (8) were identical with those of 7, and the number of methylene proton and carbon signals in their corresponding NMR spectra was identical, the distribution of the latter NMR signals in their spectra was different (Table 2). The 13 C NMR spectrum of 8 revealed signals of three amide and one urea carbon (170. 9, 170.6, 170.4, and 159.4 ppm, respectively) as for 7, suggesting that 8 differed from molleurea D (7) in the position of the urea bridge. The terminal phenethylamide was linked to two carboxyphenethylamide units in a row and through a urea bridge to the third carboxyphenethylamide that was connected through its carboxy group to the second phenethylamide unit. The connections of the subunits were deduced from HMBC correlations between the amide carbonyls and the adjacent methylenes and through NOESY correlations between the amide methylenes 3 and 30 from both sides of the urea bridge (3.30 and 3.47 ppm, respectively, Table S8). The structure of 8 was established unequivocally by interpretation of the MS/MS fragmentation ions (Figure S56) at m/z 442, 416, 269, 252, 122, and 105, thus assigning structure 8 to molleurea E.

Molledihydroisoquinolone (9), isolated from a different sample of D. molle, presented a positive high-resolution ASAPMS protonated molecule at m/z 148.0774, corresponding to the molecular formula C 9 H 10 NO and six degrees of unsaturation. The 1 H NMR spectrum of 9 revealed four aromatic protons (7.82-7.29 ppm), one exchangeable proton (7.90 ppm), and two pairs of methylene protons (3.35 and 2.89 ppm). The 13 C NMR spectrum revealed the presence of a disubstituted aromatic ring (139.5-127.6 ppm), a conjugated amide carbonyl (164.7 ppm), and two methylene carbons. COSY correlations established two fragmentsethyl amine and an ortho-disubstituted aromatic moietywhile HMBC correlations permitted assembly of the two fragments to the dihydroisoquinolone structure of 9. Dihydroisoquinolone ( 9) is the simplest cyclic derivative of o-carboxyphenethylamine. We examined the extract and fractions obtained from the extract for the presence of compounds 1-8 and related compounds, by LCMS and NMR, but none of these compounds were found.

Molleurea A, a tris-phenyl urea, isolated from the tunicate D. molle collected from Papua New Guinea, was found to inhibit the HIV virus in a cytoprotective cell based assay with an IC 50 value of 60 μM. 6 On the basis of this finding, compounds 1-8 were tested in the cytoprotective HIV assay at concentrations of 5.0 and 0.5 μg/mL and exhibited very weak activity (48-17% of protection at 5.0 μg/mL, Table S10). Compounds 1-9 were also tested for growth inhibition of the bacteria Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli at a single dose of 50 μg/mL but were all found inactive in these assays.

A literature search surprisingly revealed that apart from molleurea A 6 no natural product contains the unique ocarboxyphenethylamine subunit in its structure. Furthermore, no biosynthetic route to such a compound is known. The closest known metabolite, m-carboxyphenylalanine, is biosynthesized, in certain plants including Nicotina silvestris, 14 by a unique shikimate pathway, 15 via the catalysis of isochorismate mutase. We propose here a biogenesis scheme for the molleurea and mollecarbamate precursor o-carboxyphenethylamine. The proposed pathway is based on the shikimate pathway and includes a new type of chorismate mutase that converts the putative precursor chorismate isomer 12 into ocarboxyphenethylamine (Scheme 1). The proposed route, in which shikimic acid is converted to 4-enoylpyruvylshikimic acid-5-phosphate (instead of the 3-enoylpyruvylshikimic acid-5phosphate) and further undergoes a 1,2-phosphate elimination, produces intermediate 12, the 4-enoylpyruvate derivative of chorismic acid. A mutase can transform 12, through a Claisentype rearrangement, into isomeric prephenate 13. Intermediate 13 is proposed to be converted by an aminotransferase to the putative 14, which undergoes 1,4-water elimination by a putative dehydrase to give L-o-carboxyphenylalanine (15), which upon decarboxylation produces o-carboxyphenethylamine. Phenethylamine might be produced directly from phenylalanine or through the proposed pathway by decarboxylation of o-carboxyphenethylamine, as shown in Scheme 1.

Mollecarbamates 1-4 contain methyl carbamates that might be considered to be isolation artifacts. According to the chemistry literature, methyl carbamates can be synthesized from the corresponding primary urea derivatives. A recent publication describes the alcoholysis of urea and alkylurea in excellent yields by alkali-treated zeolite material but at elevated temperature (140-500 °C). 16 A recently accepted Chinese patent claims that urea and metal oxides can be used under mild reaction conditions for the production of alkylcarbamates, in good yields. 17 The procedure we utilized for the isolation of mollecarbamates 1-4 used methanol mixtures with dichloromethane and water for extraction and elution of the compounds from the columns but did not expose the compounds to temperatures above 50 °C. HPLC grade silica should not contain metal oxides. Thus, it is not reasonable that 1-4 were produced from the molleureas or the corresponding urea derivatives throughout the isolation procedure. Furthermore, our experience with the urea-bridge-containing anabaenopeptins suggests that substituted urea moieties do not hydrolyze under the usual acidic conditions (6 N HCl, 104 °C, 24 h) but rather by treatment with anhydrous hydrazine. 18 Mollecarbamates 1-4 and molleureaes 5-8 were isolated from a sample of the ascidian D. molle collected in Mayotte (Indian Ocean) alongside N,N′-diphenylethylurea (10) and molleurea A (11), which had been previously isolated from samples of the same ascidian collected in the Okinawa Prefecture 11 and in Papua New Guinea (Pacific Ocean), 6 respectively. The occurrence of the same metabolites in samples of D. molle collected in such remote locations suggests that they are indeed produced either by D. molle or by one of its common symbiotic microorganisms, Prochloron didemni, 5 or another yet unrecognized bacterium. The fact that N,N′-diphenethylurea was initially described from Streptomyces sp. AM-2498 12 may suggest that this group of metabolites is produced by a heterotrophic bacterium associated with D. molle. Dihydroisoquinolone ( 9), similar to N,N′-diphenylethylurea (10) 11 and molleurea A (11), 6 was isolated as the sole o-carboxyphenethylamine derivative from a sample of D. molle collected in December 2011 in Baie des Assassins, Madagascar. Although its structure differs from the 10 compounds isolated from the sample collected in May 2013 in Mayotte (an island northwest of Madagascar), we propose that all 11 of these metabolites originate from the same o-carboxyphenethylamine building block. The difference in the content of these two samples of D. molle might be a consequence of a difference in the genetics of the animals 19 or their symbiont 5 or of seasonality. 20 This issue may be verified in the future by collection of the ascidians from both locations in the same season and comparison of their genomes and metabolomes. The biogenesis proposed above for the biosynthesis of o-carboxyphenethylamine should be proved in the future by genome mining of D. molle and its symbionts, isolation of the enzymes involved in the biosynthesis, and mechanistic studies of the processes, a task that is beyond the expertise of our research groups.

■ EXPERIMENTAL SECTION

General Experimental Procedures. UV spectra were recorded on an Agilent 8453 spectrophotometer. IR spectra were recorded on a Bruker Tensor 27 FT-IR instrument. NMR spectra were recorded on Bruker Avance and Avance III spectrometers at 500.13 MHz for 1 H and 125.76 MHz for 13 C (referenced to TMS δ = 0). DEPT, COSY, gTOCSY, gROESY, gHSQC, gHMQC, and gHMBC spectra were recorded using standard Bruker pulse sequences. High-resolution mass spectra were recorded on a Waters MaldiSynapt instrument (ESI), and LCMS spectra were recorded on a Waters Xevo TQD instrument (ESI). HPLC separations were performed on a Merck Hitachi HPLC A sample of the tunicate D. molle (MAD11-BA065, 123 g, dry weight after extraction) was extracted with a mixture of 1:1 MeOH/ CH 2 Cl 2 and then with EtOAc (3 × 500 mL, each) to afford after evaporation the MeOH/CH 2 Cl 2 extract (1.39 g) and the EtOAc extract (0.74 g). A portion of the MeOH/CH 2 Cl 2 extract (300 mg) was separated on a Flash NP-Si column, eluted with 10% solvent polarity increments from petroleum ether to EtOAc, to afford 13 fractions. Fractions 10-11 (13.

Figure 1 .

 1 Figure 1. Representative COSY and HMBC correlations that support the structure elucidation of 1.

Figure 2 .

 2 Figure 2. MS/MS fragmentation patterns (a) of mollecarbamate A (1) and (b) of molleurea B (5).

Scheme 1 .

 1 Scheme 1. Biosynthetic Pathways to Phenylalanine and m-Carboxyphenylalanine and the Biogenesis of o-Carboxyphenethylamine

  0 mg) were combined and further separated by HPLC on a preparative RP-8 column (YMC-Pack C-8 250 × 20 mm, 5 μm, DAD) eluting with CH 3 CN/0.1% TFA in H 2 O (3:2) to afford molledihydroisoquinolone (9) (1.6 mg, t R 16.4 min, 0.53% yield). Mollecarbamate A (1): amorphous, colorless material; UV (MeOH) λ max (log ε) 202 (4.46) nm; IR (ATR Diamond) ν max

Table 2 .

 2 NMR Data(C 6 D 6 ) of Molleureas B-E(5-8) a C , type b δ H , mult. (J in Hz) δ C , type b δ H , mult. (J in Hz) δ C , type b δ H , mult. (J in Hz) δ C , type b δ H , mult. (J in Hz) of a 1,2-disubstituted benzene at 7.02, 6.99, 6.93, and 6.86 ppm and three signals of aromatic protons at 7.14, 7.07, and 7.06 ppm (integration 2:2:1) of a monosubstituted benzene ring, two amide protons at 5.52 and 5.10 ppm, and signals for four methylenes at 3.45, 3.41, 2.71, and 2.67 ppm. The 13 C NMR spectrum (Table

		5	6	7	8
	position	δ

; 2D NMR data, Table S9; HRASAPMS m/z 148.0774 [M + H] + (calcd for C 9 H 10 NO, 148.0762).

Cytoprotective HIV Cell-Based Assay. 21 Briefly, 1A2 cells (a subclone of CEM-SS TART cells that are more prone to apoptosis upon HIV infection) were cultured in RPMI/20% fetal bovine serum and plated in 96-well plates. The assay utilized controls that included cells without HIV, cells infected with HIV, and cells infected with HIV and treated with AZT at a final concentration of 50 μg/mL or test compounds. Uninfected control cells and HIV-exposed cells were allowed to incubate for 96 h at 37 °C in 5% CO 2 . Cell viability was then assessed with a standard MTT assay. 22 On each assay plate, each condition was performed in triplicate, and p-values were determined between groups to gauge the performance of the assay. For a valid assay, HIV growth had to be less than 50% of controls; AZT rescue needed to be at least 50% above HIV killing, and a test compound was considered active if it performed at 70% of the AZT value.

Bacterial Growth Inhibition Assay. 23 The bacterial strains utilized in this study, P. aeruginosa ATCC 47085, E. coli ATCC 25922, and S. aureus ATCC 29213, were purchased from the American Type Culture Collection (ATCC). P. aeruginosa was grown in cationadjusted Mueller-Hinton broth (Sigma), while E. coli and S. aureus were grown in LB (Lennox). Starter cultures were incubated for 24 h (37 °C, 5% CO 2 , aerobic conditions) and then diluted in fresh medium to obtain an optical density of 0.004 (OD600, Evolution 60, Thermo Scientific). All strains were tested using a single concentration of 50 μg/mL in 96-well flat-bottom plates (Corning). After 24 h of incubation, MTT (25 μL of a 1 mg/mL solution in H 2 O) was added to each well followed by additional incubation at 37 °C for 1 h. Results were obtained from two independent experiments, and each experiment was performed in triplicate.

■ ASSOCIATED CONTENT

* S Supporting Information

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.7b00123.

Spectroscopic data including 1D and 2D NMR spectra, complete tabulated 2D NMR data, HRMS data, and MS/ MS spectra of compounds 1-9 (PDF)

■ AUTHOR INFORMATION

Corresponding Author *Tel (S. Carmeli): +972-3-6408550. Fax: +972-3-6409293. Email: carmeli@post.tau.ac.il.

ORCID

Anne Gauvin-Bialecki: 0000-0001-5188-0704 Shmuel Carmeli: 0000-0002-9641-9943

Notes

The authors declare no competing financial interest.

■ ACKNOWLEDGMENTS

The present research was supported in part by a Tel Aviv University internal grant (S.C. and Y.K.).