Antiprotozoal Linear Furanosesterterpenoids from ... - ACS Publications

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Antiprotozoal Linear Furanosesterterpenoids from the Marine Sponge Ircinia oros Giuseppina Chianese,† Johanna Silber,† Paolo Luciano,‡ Christian Merten,§ Dirk Erpenbeck,⊥ Bülent Topaloglu,∥ Marcel Kaiser,▽,△ and Deniz Tasdemir*,† †

GEOMAR Centre for Marine Biotechnology (GEOMAR-Biotech), Research Unit Marine Natural Products Chemistry, GEOMAR Helmholtz Centre for Ocean Research Kiel, Kiel 24106, Germany ‡ Department of Pharmacy, University of Naples Federico II, Naples 80131, Italy § Lehrstuhl für Organische Chemie 2, Ruhr-Universität Bochum, Bochum 44801, Germany ⊥ Department of Earth and Environmental Sciences and GeoBio-Center, Ludwig-Maximilians-Universität München, 80333 München, Germany ∥ Department of Marine Biology, Faculty of Fisheries, Istanbul University, Istanbul TR-34480, Turkey ▽ Department of Medical Parasitology and Infection Biology, Swiss Tropical and Public Health Institute, Basel CH-4002, Switzerland △ University of Basel, Basel CH-4003, Switzerland S Supporting Information *

ABSTRACT: Chemical investigation of the marine sponge Ircinia oros yielded four linear furanosesterterpenoids, including the known metabolites ircinin-1 (1) and ircinin-2 (2) and two new compounds, ircinialactam E (3) and ircinialactam F (4). Their chemical structures were elucidated by using a combination of [α]D, NMR, HRMS, and FT-IR spectroscopy. The absolute configuration of C-18 in compounds 1−3 was identified as R by electronic circular dichroism (ECD) spectroscopy coupled with time-dependent density functional theory calculations. Compounds 1−4 showed moderate leishmanicidal, trypanocidal, and antiplasmodial activities (IC50 values 28−130 μM). This is the second report of rare glycinyl lactam derivatives 3 and 4 from the genus Ircinia.

S

earlier reports.2,7 The absolute configuration of the stereogenic centers in linear Ircinia sesterterpene tetronic acids has relied solely on the specific rotation values.8 No further attempts have been made to confirm the absolute configurations. However, chiroptical and computational approaches such as those involving the comparison of the experimental and simulated electronic circular dichroism (ECD) spectrum generated by time-dependent density functional theory (TDDFT) represent a suitable strategy for stereochemical determinations with this structure class.9 In continuation of our ongoing investigation of marine sponges for identifying antiprotozoal secondary metabolites, we recently reported the in vitro antiparasitic activity of linear furanoterpenes obtained from the Southern Aegean Ircinia spinulosa against a small panel of protozoa (Trypanosoma brucei rhodesiense, T. cruzi, Leishmania donovani, and Plasmodium falciparum).10 Due to the emergence of drug resistance, there is an urgent need for viable alternatives to the existing therapeutic

ponges of the genus Ircinia (family Irciniidae, order Dictyoceratida, class Demospongiae, phylum Porifera) are prolific sources of linear furanosesterterpenes with a conjugated tetronic acid moiety.1 Sesterterpene tetronic acids have been reported to be of chemotaxonomic significance for the order Dictyoceratida, mainly for the sponge genera Ircinia, Psammocinia, and Sarcotragus.2 Many of these compounds contain furan and tetronic acid termini, while some others, including several types of norsesterterpenoids, have been postulated as degradation products of the tetronic acid group.1 Sesterterpene tetronic acids are of considerable interest due to their wide range of biological activities, including antimicrobial,3 anti-inflammatory,4 and cytotoxicity.5 They also have ecological significance based on their antifeedant and antifouling activities against diverse biosystems.6 Full chemical assignment of linear sesterterpene tetronic acids from Ircinia sponges has been ambiguous due to the presence of stereogenic carbons, which are difficult to assign by conventional NOESY spectroscopy, and multiple double bonds that are capable of geometric isomerism. NMR spectroscopy is routinely used for identification of their double-bond configuration; however there is inconsistency, particularly in © 2017 American Chemical Society and American Society of Pharmacognosy

Received: June 23, 2017 Published: August 25, 2017 2566

DOI: 10.1021/acs.jnatprod.7b00543 J. Nat. Prod. 2017, 80, 2566−2571

Journal of Natural Products

Note

Table 1. 1H NMR Data of Compounds 1−4 (600 MHz, CD3OD)

options against these parasitic diseases. In order to expand our library of antiprotozoal sponge terpenoids, we analyzed Ircinia oros collected from the Northern Aegean Sea. The fractionation and purification of the organic extract of the sponge afforded the known compounds ircinin-1 (1) and ircinin-2 (2) and two new, minor glycinyl lactam derivatives, ircinialactam E (3) and ircinialactam F (4). Full structural assignment of the compounds, including the known metabolites 1 and 2, relied on extensive 1D/2D NMR spectroscopy, FT-IR, HRMS, [α]D, and ECD/TDDFT computational calculations. Herein we describe the details of the isolation, structure elucidation, and the evaluation of the antiprotozoal activity of compounds 1−4. The sponge material was extracted successively with MeOH, MeOH/CHCl3 (1:1), and CHCl3. The combined organic extract (6.47 g) was partitioned by the modified Kupchan procedure.11 The CHCl3 and aqueous MeOH subextracts were subjected to repeated column and HPLC chromatography over C-18 bonded silica or chiral-phase columns to afford 1−4. The molecular formula of compound 1 was established as C25H30O5 by HRESIMS. The analysis of the 1D NMR spectra (Tables 1 and 2) in combination with the FT-IR and HSQC data suggested 1 to be ircinin-1, a linear sesterterpene with two furan rings and a methyl-tetronic acid terminal. However, the lack of the structure assignment of ircinin-1 based on 2D NMR experiments and the reports of both enantiomers in the literature7,8,12−14 prompted us to reinvestigate the structure of 1 in detail. A full planar structure of 1 was assembled on the basis of 2D COSY and HMBC spectra (Figure S1). The double-bond configuration at C-12 (E) was assigned based on 2D NOESY data (Figures S1 and S7), while the characteristic 13 C NMR shift for C-20 (δC 115.6)14 established a Z configuration at C-20. This left the configuration of the only stereogenic center, C-18, to be identified. In the literature, both positive and negative [α]D values have been reported for 1,7,8,13 suggesting the presence of both 18R-1 and 18S-1 enantiomers in Nature. Herein we measured a positive specific rotation value of [α]22D +33 (c 2.5, MeOH), which suggested an 18R configuration. The absolute configuration of 1 was confirmed by comparison of the experimental and simulated ECD spectrum generated by TDDFT on model 1 (Figure 1a). Through a DFT approach we have identified a series of conformers resulting from rotation around the C-18/C-20 bond in model 1 (Gaussian 03 software15). This systematic search afforded 15 rotamers for model 1, which were geometrically optimized at the DFT level using an mpw1pw91 functional and 6-31G(d) basis set. The relative energies of all conformations were

position 1 2 4 5

6 7 9 10 11

2

3

4

δH, mult (J in Hz)

δH, mult (J in Hz)

δH, mult (J in Hz)

7.40, 6.31, 7.31, 3.69,

7.40, 6.31, 7.32, 3.71,

br br br br

4.09, br s 6.87, br s

5.91, 7.09, 2.30, 1.57,

br s br s t (7.3) m

br br br br

s s s s

14 15 16

5.91, br s 7.09, br s 2.37, t (7.3) 2.20, dt (7.3, 7.1) 5.14, br t (7.1) 1.52, br s 1.97, m 1.37, ma

17

1.32, ma

18 19 20 25 1′

2.73, 1.05, 5.26, 1.73,

12

a

1 δH, mult (J in Hz)

m d (6.8) d (10.2) s

s s s s

2.00, t (7.8) 1.66, br s 5.11, t (7.0) 1.94, dd (15.7, 7.1) a 1.36, m b 1.42, m 2.71 (m) 1.04, d (6.8) 5.24, d (10.1) 1.70, s

3.54, br s

6.03, br s 7.12, br s 2.38, t (7.3) 2.22, dt (7.3, 7.1) 5.14, br t (7.1) 1.54, br s 1.97, m 1.37, ma a 1.38, ma b 1.28, ma 2.73, m 1.05, d (6.8) 5.26, d (10.2) 1.73, s 4.21, s

7.39, br s 6.27, br s 7.31, br s a 2.94, dt (14.9, 3.1) b 2.69, dd (14.9, 7.4) 4.42, ma 6.78, br s 2.23, ta 2.20, m 5.08, t (7.0) 1.55, br s 1.97, m 1.37, ma a 1.38, ma b 1.30, ma 2.73, m 1.05, d (6.8) 5.27, d (10.2) 1.73, s a 4.41, d (17.8) b 3.96, d (17.8)

Overlapped with other signals.

calculated, and the equilibrium room-temperature Boltzmann populations were obtained. As shown in Table S1, model 1 is characterized by seven of the 15 rotamers that have energies within 2 kcal/mol of the lowest energy conformation and are significantly populated (36.3%, 23.9%, 17.6%, 8.29%, 7.04% 1.38%, and 1.5%). The ECD spectrum of model 1 was simulated. The excitation energies as well as the oscillator and rotatory strengths of the electronic excitation were calculated for the lowest energy conformational families of each structure using the TDDFT methodology.16 The theoretical curve of model 1 closely resembled the experimental one (Figure 1b), strongly supporting the R absolute configuration at C-18 in compound 1. Combining the NMR and TDDFT data and the 2567



Note

Table 2. 13C NMR Data of Compounds 1−4 (150 MHz, CD3OD) position 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 1′ 2′

1

2

3

4

δC, type

δC, type

δC, type

δC, type

144.2, 112.3, 123.2, 141.0, 24.9, 155.6, 108.4, 127.1, 138.8, 26.2, 29.6, 125.5, 136.6, 16.1, 40.6, 26.9, 37.7, 32.0, 21.2, 115.6, 145.5, 166.0, 98.4, 174.1, 6.2,

CH CH C CH CH2 C CH C CH CH2 CH2 CH C CH3 CH2 CH2 CH2 CH CH3 CH C C C C CH3

144.1, 112.2, 123.2, 140.9, 24.8, 155.6, 108.1, 127.2, 138.7, 25.7, 29.4, 32.2, 136.5, 23.6, 126.2, 26.9, 38.7, 31.6, 21.1, 114.4, 146.5, 169.0, 95.5, 175.2, 6.2,

CH CH C CH CH2 C CH C CH CH2 CH2 CH2 C CH3 CH CH2 CH2 CH CH3 CH C C C C CH3

52.9, 140.5, 136.9, 173.6, 25.8, 153.1, 109.0, 127.1, 139.2, 26.2, 29.6, 125.3, 136.6, 16.1, 40.5, 26.9, 37.7, 31.9, 21.3, 115.3, 145.5, 165.9, 99.2, 174.0, 6.2, 44.9, 172.9,

CH2 CH C C CH2 C CH C CH CH2 CH2 CH C CH3 CH2 CH2 CH2 CH CH3 CH C C C C CH3 CH2 C

144.3, 112.5, 120.7, 141.8, 26.6, 62.7, 143.4, 139.3, 174.3, 26.5, 26.7, 124.8, 137.1, 15.6, 40.4, 26.7, 37.5, 31.9, 21.3, 115.4, 145.6, 166.3, 98.0, 174.1, 5.9, 43.2, 172.9,

Compound 2, also C25H30O5 by HRESIMS, was easily identified as an ircinin-2-type compound17 by comparison of its MS and NMR data with those of 1. The main structural differences with 1 were located in the position Δ13(15) instead of Δ12 and the opposite configuration (Z instead of E) of the trisubstituted double bond at the center of the molecule. Consistently, key HMBC correlations of the olefinic H3-14 (δH 1.66) with C-12, C-13, and C-15 defined the regiochemistry of the Δ13(15), while the 13C NMR chemical shift of CH3-14 (δC 23.6)14 and the NOE correlations between H3-14/H-15 and H2-12/H2-16 indicated the 13Z configuration. On the basis of the positive specific rotation value ([α]22D +11.9, MeOH) and comparison of the ECD spectrum of compound 2 with that of 1 (Figure S27), we confirm that 2 has the same absolute configuration at C-18 (R). Hence, compound 2 is (13Z,18R,20Z)-ircinin-2. The molecular formula of compound 3 was established by HRESIMS as C27H33NO7, requiring 12 double-bond equivalents. The 1H and 13C NMR data (Tables 1 and 2) revealed the presence of a conjugated tetronic acid moiety, a trisubstituted double bond, and a 1,3-disubstituted furan ring, accounting for eight unsaturation degrees. Compound 3 had identical NMR data to those of 1 across the C-7 to C-25 fragment. The main differences from 1 were the disappearance of the three terminal furan protons and the emergence of a broad olefinic singlet at δH 6.87 (H-2) and two pairs of highly deshielded methylene protons at δH 4.09 (H2-1, br s) and δH 4.21 (H2-1′, br s). The former (H2-1) displayed a COSY correlation with the olefinic singlet (H-2). The odd molecular weight confirmed the presence of a nitrogen atom within 3. The 13C NMR resonances and key HMBC correlations shown in Figure 2 confirmed the replacement of the terminal furanyl portion with a terminal glycinyl lactam moiety,2,14 which accounted for four degrees of unsaturation required by the molecular formula. The characteristic IR absorption at 1650 cm−1 further confirmed the presence of a lactam moiety. Diagnostic HMBC correlations of H-2 with C-1, C-3, C-4, and C-5 and of H-1′ with C-1, C-4, and C-2′ and finally the 3JCH coupling of H2-5 with the lactam carbonyl C-4 (δC 173.6) defined the regiochemistry of the glycinyl lactam as depicted in Figure 2. The 1H and 13C NMR resonances, supported by the 2D NOESY data, of 3 were almost identical to those of 1, corroborating the 12E and 20Z configuration. The absolute configuration of C-18 was assigned R by comparison of its ECD spectrum with that of compound 1 (Figure S27), given the structural identity of the chiral chromophore. A positive specific rotation value ([α]22D +31, MeOH) fully supported this assignment. Linear sesterterpene glycinyl lactams are very rare in Nature, and only a few of them with tetronic acid termini have been reported from two Irciniidae sponges.2,14 Compound 3 is the first example of a sesterterpene glycinyl lactam with an additional furan ring. Accounting for the lactam function and

CH CH C CH CH2 CH CH C C CH2 CH2 CH C CH3 CH2 CH2 CH2 CH CH3 CH C C C C CH3 CH2 C

Figure 1. (a) Simplified enantiomer (model 1) used for DFT calculation. (b) Experimental ECD spectrum of compound 1 (black); TDDFT-calculated curves of model 1 (Z-R) (red) and its enantiomer (Z-S) (blue).

positive [α]D value, compound 1 was unambiguously identified as (12E,18R,20Z)-ircinin-1.

Figure 2. COSY (in bold) and key H → C HMBC (arrows) correlations observed for 3 and 4. 2568



Note

following the previous series of ircinialactams,14 we named the new compound 3 (12E,18R,20Z)-ircinialactam E. (+)-HRESIMS analysis of compound 4 gave a protonated molecule [M + H]+ consistent with the same molecular formula (C27H33NO7) as 3. These data, together with the identical IR absorptions at 1650 cm−1, were indicative of another ircinialactam type of compound. A careful analysis of 1H and 13 C NMR spectra of compound 4 (Tables 1 and 2), along with its 2D COSY and HSQC NMR data, suggested 4 to be a regioisomer of 3, in which the terminal glycinyl lactam and the inner furanyl moieties were interchanged. As a result, a proton spin system including the diastereotopic methylene H2-5 (H-5a δH 2.94; H-5b δH 2.69), a methine (H-6, δH 4.42), and an olefinic proton (H-7, δH 6.78) appeared in the COSY spectrum. Diagnostic HMBC correlations (Figure 2) between H-6/C-9, H-6/C-1′, H-7/C-8, H-7/C-9, H-7/C-10, H2-10/C-9, H-1′/C6, H-1′/C-9, and H-1′/C-2′ confirmed the position of the 1,3substituted glycinyl lactam as shown. The latter function was connected to the terminal furan ring through additional HMBC cross-peaks of H2-5 with C-2, C-3, C-4, C-6, and C-7 (Figure 2). The almost identical 1H and 13C NMR chemical shifts with those of 1 and 3 across the C-12 to C-25 structural fragment indicate the same stereostructure as in 1. Given the positive specific rotation value ([α]D +21, MeOH) and analogy to 1, it is biosynthetically reasonable to assume that 4 has the same R configuration at C-18. Unlike 1−3, 4 has an additional stereogenic center at C-6. Due to minute amounts of compound 4, we were not able to investigate the absolute configuration at C-6 and C-18 by ECD. Thus, the new compound 4 was partially assigned as (12E,18R,20Z)ircinialactam F, leaving the C-6 configuration unassigned. Ircinialactam F (4) is the first sponge furanosesterterpene with a nonterminal glycinyl lactam moiety in Nature. Compounds 1−4 were tested in vitro for their antiprotozoal activity against P. falciparum, T. brucei rhodesiense, T. cruzi, and L. donovani. Results summarized in Table 3 show that 1 and 2

the glycinyl lactam counterparts (3 and 4). The latter type of compounds have been shown to modulate glycine receptor chloride channels.14 Ircinin-1 and ircinin-2 were originally reported as a mixture from I. oros in 1972.17 Since then, several studies have described these compounds from Ircinia and Sarcotragus sponges12−14 with both E/Z and 18R/18S configurations.7,8,12−14 Herein the absolute configuration of 1 (as well as 2 and 3) was confirmed as 18R based on TDDFT/ECD calculations. The comparison of experimental and calculated ECD spectra has successfully been applied to a linear marine homosesterterpene for assignment of a stereogenic carbon in the tetronic acid moiety.18 During the preparation of this paper, Afifi et al. reported both 18R and 18S enantiomers of 1 and 2 from the same sponge (Psammocinia sp.).19 They acquired the experimental ECD spectra of both enantiomers of 1, which are consistent with our assignment at C-18. The current study represents a successful application of DFT computational approach, confirming the absolute configuration of the only stereocenter (C-18) in ircinin-type marine linear sesterterpene tetronic acids. This study also enriches the linear terpenoid library of the genus Ircinia by adding two new and rare glycinyl lactam-type sesterterpenes (3, 4). Compounds 3 and 4 are the first examples of sesterterpene glycinyl lactams with an additional (inner or terminal) furan ring.

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Table 3. In Vitro Antiprotozoal Activity of Compounds 1−4a sample

T. b. rhodesiense

T. cruzi

L. donovani

P. falciparum

cytotoxicity

1 2 3 4 reference

97 65 130 130 0.015b

120 110 n.t.g n.t. 3.07c

31 28 120 95 0.51d

58 56 95 >100 0.009e

150 140 >200 >200 0.010f

EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotation measurements were conducted on a Jasco P-2000 polarimeter. UV spectra were obtained on a GE Healthcare NanoVue photometer. ECD spectra were recorded on a J-710 spectropolarimeter (Jasco) equipped with J-710 for Windows software (Jasco). IR spectra were measured on a PerkinElmer Spectrum Two FT-IR spectrometer. NMR spectra were recorded on a Bruker DRX 500 (500 and 125 MHz for 1H and 13C NMR, respectively) and a Bruker AV 600 spectrometer (600 and 150 MHz for 1H and 13C NMR, respectively) equipped with a Z-gradient triple resonance cryo probehead. The residual solvent signals of methanol-d4 were used as internal references (δH 3.31 and δC 49.0). High-resolution mass spectra of the samples were run on a Bruker micrOTOF II and on a Waters Xevo G2-XS QTof mass spectrometer in positive ESI mode. HPLC separations were performed on a MerckHitachi LaChrom Elite HPLC system consisting of an L-2450 diode array detector, an L-2200 autosampler coupled with a Hitachi L-7150 pump, and a VWR Hitachi Chromaster system consisting of a 5430 diode array detector, a 5310 column oven, a 5260 autosampler, and a 5110 pump. The eluents for HPLC separations were H2O (A) and MeCN (B). Routine HPLC separations were performed on a C18 Phenomenex Gemini-NX column (5 μm, 110A, Axia, 100 × 21.20 mm), whereas a Phenomenex Lux chiral-phase column (5 μm Amylose-2, 250 × 4.60 mm) was used in purification of ircinins 1 and 2. Animal Material and Taxonomical Identification. The sponge material was collected in June 2002 using scuba diving (−10 m) in Gökçeada (Northern Aegean Sea, Turkey). A voucher of the specimen is deposited in ethanol in the Bavarian State Collection of Paleontology and Geology, Munich, Germany, under the collection number SNSB-BSPG.GW30219. The specimen has been identified as Ircinia oros (Schmidt, 1864) by morphological methods plus corroboration by molecular taxonomy. Morphology clearly excludes all other Ircinia species reported from the Aegean Sea.20 The characteristic surface reticulation of I. retidermata Pulitzer-Finali & Pronzato, 1981 is absent. The perfectly round shape of the collagenous filament heads and high filament density are different from I. paucif ilamentosa Vacelet, 1961. The shape of the surface conules and oscula endings is different from I. variabilis (Schmidt, 1862). A dendritic growth form distinct for I. dendroides (Schmidt, 1862) is not present.

IC50 values are in μM. Cytotoxicity was evaluated against the L6 rat myoblast cells. bMelarsoprol as reference. cBenznidazole as reference. d Miltefosine as reference. eChloroquine as reference. fPodophyllotoxin as reference. gn.t.: not tested due to low amounts available. a

have identical antiprotozoal activity, with the highest potency against L. donovani (IC50 values 31 and 28 μM, respectively) and P. falciparum (IC50 values 58 and 56 μM). 2 had slightly better trypanocidal activity against T. brucei rhodesiense (IC50 values of 1 and 2 are 97 and 65 μM, respectively). The glycinyl lactam analogues (3, 4) appeared to be less active against all protozoan parasites, with IC50 values ranging from 95 to 130 μM. When tested for general cytotoxicity against the L6 rat myoblast cell line, all compounds displayed low or no toxic effects (Table 3). The analysis of the activity data reveals that the bifuran terminus as found in compounds 1 and 2 positively influences the in vitro antiprotozoal activity in comparison to 2569



Note

The other Mediterranean species20 also possess different growth forms: I. stipitata (digitate columns), I. digitata (digitate growth form), I. truncata (cup shaped), I. chevreuxi (vase shaped) [all (Topsent, 1894) from the Gulf of Sidra, Tunisia], and the Adriatic I. vestibulata (Szymanski, 1904) (Boletus growth form and different shape of conuli). I. favosa (Lieberkühn, 1859) is discussed as unrecognizable,21 while I. condensa (Topsent, 1894) and I. solida (Esper, 1794) are incertae sedis.20 Molecular taxonomy corroborated the morphological species determination using 344bp of the ITS-2 marker, which was as amplified and sequenced following Erpenbeck et al. 22 and subsequently compared with other Ircinia sequences as published in NCBI Genbank. The resulting fragment is 100% identical to I. oros JN65518323 and clusters with other I. oros sequences distant from other Ircinia species. Extraction and Isolation. The sponge material was homogenized and extracted successively with MeOH, MeOH/CHCl3 (1:1), and CHCl3. The extracts were combined and evaporated to dryness under vacuum, yielding 6.47 g of dry residue. The crude extract was subjected to a modified Kupchan partition scheme.11 For this, it was resuspended in a MeOH/H2O mixture (9:1, 200 mL) and partitioned against n-hexane (3 × 200 mL). The aqueous MeOH layer was adjusted to 70% MeOH (v/v) with H2O and partitioned against CHCl3 (3 × 200 mL). Evaporation of all extracts to dryness under vacuum gave three subextracts with yields of 0.39 g (n-hexane), 1.06 g (CHCl3), and 4.09 g (MeOH(aq)). The CHCl3 subextract was subjected to C18 flash column chromatography (FCC) eluting with a 10% gradient of MeOH in H2O. The fractions eluted with 100% MeOH were first separated over a Sephadex LH-20 column, affording a fraction (fr. 34-37) containing ircinins (100 mg). Repeated HPLC chromatography on C18 and Lux polysaccharide chiral-phase HPLC columns (gradient of H2O/MeCN from 20:80 to 3:97 in 8 min, flow 1.5 mL/min) of the fraction eluted at tR 12.5 min (16 mg) afforded pure 1 (6 mg) and 2 (1.5 mg). The aqueous MeOH subextract was also subjected to FCC over C18 material eluting with a 10% solvent gradient of MeOH in H2O. The FCC fraction eluted with MeOH/ H2O (6:4) was subjected to RP-HPLC chromatography (gradient of H2O/MeCN from 50:50 to 45:55 in 20 min), affording compound 4 (0.8 mg, tR 8.0 min). The FCC fraction eluted with a MeOH/H2O mixture (7:3) was subjected to RP-HPLC chromatography (gradient of H2O/MeCN from 50:50 to 45:55 in 20 min) to yield compound 3 (2.0 mg, tR 9.9 min). (12E,18R,20Z)-Ircinin-1 (1): yellow oil; [α]22D +33 (c 2.5, MeOH); UV (MeOH) λmax (log ε) 264 (4.04); ECD λext (Δε) (MeOH) 220 (+1.84), 240 (+3.71), 274 (+12.20) 301 (+4.68) nm; IR (film) νmax 3349, 2929, 1734, 1631, 1061 cm−1; 1H NMR (CD3OD, 600 MHz) and 13C NMR (CD3OD, 150 MHz), Tables 1 and 2; HRESIMS m/z 433.1996 [M + Na]+ (calcd for C25H30NaO5, 433.1985). (13Z,18R,20Z)-Ircinin-2 (2): yellow oil; [α]22D +11.9 (c 0.15, MeOH); UV (MeOH) λmax (log ε) 253 (3.03); ECD λext (Δε) (MeOH) 220 (−2.98), 240 (+1.65), 274 (+9.21) 301 (+4.75) nm; IR (film) νmax 3200, 2933, 1727, 1631, 1065 cm−1; 1H NMR (CD3OD, 600 MHz) and 13C NMR (CD3OD, 150 MHz), Tables 1 and 2; HRESIMS m/z 411.2190 [M + H]+ (calcd for C25H31O5, 411.2171). (12E,18R,20Z)-Ircinialactam E (3): yellow oil; [α]22D +31 (c 0.5, MeOH); UV (MeOH) λmax (log ε) 258 (4.10); ECD λext (Δε) (MeOH) 220 (+1.84), 240 (+3.71), 274 (+12.20) 301 (+4.68) nm; IR (film) νmax 3200, 2932, 1734, 1634, 1650, 1033 cm−1; 1H NMR (CD3OD, 600 MHz) and 13C NMR (CD3OD, 150 MHz), Tables 1 and 2; HRESIMS m/z 506.2151 [M + Na]+ (calcd for C27H33NNaO7, 506.2149). Ircinialactam F (4): yellow oil; [α]22D +21 (c 0.6, MeOH); UV (MeOH) λmax (log ε) 257 (4.08); IR (film) νmax 3200, 2975, 1748, 1635, 1650, 1033 cm−1; 1H NMR (CD3OD, 600 MHz) and 13C NMR (CD3OD, 150 MHz), Tables 1 and 2; HRESIMS m/z 484.2332 [M + H]+ (calcd for C27H34NO7, 484.2330). Computational Details. DFT calculations were performed using the Gaussian03 package (Multiprocessor). A systematic conformational search for model 1 around the C-18/C-20 bond was carried out at the mpw1pw91 level using the 6-31G(d) basis set (range: −12 to

+12; number of conformers = 15). All the conformers obtained were subsequently optimized at the mpw1pw91 level using the 6-31G(d,p) basis set. TDDFT calculations were run using the functional B3LYP and the basis set TZVP including at least 30 excited states in all cases and using IEF-PCM for MeOH. Bioactivity Assays. The antiparasitic activity of 1−4 (Table 3) against P. falciparum, T. cruzi, T. brucei rhodesiense, and L. donovani and the cytotoxicity against rat skeletal myoblast L6 cells were determined in vitro as described previously.24

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.7b00543. 1D and 2D NMR spectra of compounds 1−4, a photograph of the sponge, experimental ECD spectra of compounds 1−3, and conformational analysis table of model 1 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel (D. Tasdemir): +49-431-6004430. Fax: +49-431-6004441. E-mail: [email protected]. ORCID

Christian Merten: 0000-0002-4106-1905 Deniz Tasdemir: 0000-0002-7841-6271 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS C.M. thanks the Fonds der Chemischen Industrie (FCI) for a Liebig fellowship and furthermore acknowledges financial support by the Deutsche Forschungsgemeinschaft (DFG) through the Cluster of Excellence RESOLV (“Ruhr Explores SOLVation”, EXC 1069). The Turkish Ministry of Agriculture and Forestry is acknowledged for assistance in sample collection and transport.

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