Unprecedented Polyketides from a Marine Sponge-Associated

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Unprecedented Polyketides from a Marine Sponge-Associated Stachylidium sp. Celso Almeida,† Ekaterina Eguereva, Stefan Kehraus, and Gabriele M. König* Institute for Pharmaceutical Biology, University of Bonn, Nussallee 6, 53115 Bonn, Germany S Supporting Information *

ABSTRACT: From the marine sponge-derived fungus Stachylidium sp. six novel phthalide-related compounds, cyclomarinone (1), maristachones A−E (2−5), and marilactone (6), were isolated. The structure of compound 1 comprises a hydroxycyclopentenone ring instead of the furanone ring characteristic for phthalides and represents a new carbon arrangement within polyketides. In the epimeric compounds 5a and 5b the phthalide (=isobenzofuranone) nucleus is modified to an isobenzofuran ring with ketal and acetal functionalities. Biosynthetically the structural skeletons of cyclomarinone (1) and maristachones A (2), C (4), D (5a), and E (5b) are most unusual due to the presence of an additional carbon atom when compared to the basic polyketide skeleton. This special biosynthetic feature also holds true for the likewise isolated polyketide marilactone (6).

I

n fungi, polyketide metabolism gives rise to a myriad of diverse structures. Some of these compounds are of immense medical importance, such as mycophenolic acid1 and myriocin as a lead structure for the immunosuppressant fingolimod.2 The herein reported study focused on a marine-derived fungus and evaluated its metabolic and pharmacological potential. Recently we reported the isolation of the novel human leucocyte elastase (HLE)-inhibiting phthalimidines marilines A−C3 as well as the unusual antiplasmodial phthalide derivatives marilones A−C4 from the sponge-derived fungus Stachylidium sp. This very same fungus also produces the tyrosine-derived stachylines A−D,5 if cultured under different conditions. Impressed by the high potential of this fungus to produce new natural products, we varied culture conditions and here report the phthaliderelated polyketides cyclomarinone (1), maristachones A−E (2−5), and marilactone (6). The majority of polyketides in Stachylidium sp. have a nearly identically substituted aromatic ring, suggesting that they are biosynthetically closely related.

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RESULTS AND DISCUSSION The molecular formula of 1 was deduced by accurate mass measurement (HRESIMS) to be C11H12O4, requiring six sites of unsaturation. The 13C NMR and DEPT135 spectra revealed 11 carbon resonances: two methyls, one methylene, one sp2 methine, and one sp3 methine, as well as six quaternary carbons. The 1H NMR spectrum of 1 showed a singlet resonance for the aromatic methine (CH-6) at δ 6.87, indicating together with UV and 13C NMR data the presence of a pentasubstituted benzene ring. The methyl group CH3-11 (δC 10.0) was linked to C-4 of the aromatic ring due to heteronuclear long-range correlations of the methyl protons with C-3, C-4, and C-5. The methoxy group protons OCH3-10 (δH 4.01) had an HMBC correlation to C-3 of the aromatic ring, thus clearly delineating its position. The sp3 methine CH-1 displayed a 13C NMR © XXXX American Chemical Society and American Society of Pharmacognosy

resonance signal typical for a carbon bound to oxygen (δC 67.1) and showed 1H−1H coupling with H2-9 (J = 6.5 and 1.6 Hz). H-1 had HMBC correlations with C-2, C-3, and C-7 of the aromatic ring, suggesting that it is connected to C-2 of the aromatic moiety. All of the protons, H-1, H-6, and H2-9 had HMBC couplings to a carbonyl carbon (C-8, δC 206.4), which is thus placed between C-7 of the aromatic ring and CH2-9, establishing a second ring in 1. For compound 1 the name cyclomarinone is suggested. Special Issue: Special Issue in Honor of Lester A. Mitscher Received: September 27, 2012

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sp3 methylene CH2-14, completing the substituent at C-13 of the aromatic ring. On the basis of HMBC and COSY correlations we found that the substituent at C-8 of the aromatic moiety was an aliphatic chain, giving rise to two 1H−1H spin systems, interrupted by a ketone functionality (C-5, δC 214.8). Thus, the COSY spectrum revealed a spin system ranging from H3-1 to H2-4, including the oxygenated methine proton H-3 (δH 3.85) and from H-6 to H3-16 and H2-7. H2-4, H-6, H2-7, and H3-16 displayed HMBC correlations to the ketone carbon C-5 and thus connected the partial structures that were deduced from 1H coupling data. Heteronuclear long-range correlations also established the connection of CH2-7 to C-8 of the aromatic ring. For compound 4 the name maristachone C is suggested. The molecular formulas of 5a and 5b were deduced by HREIMS and found to be identical, C23H34O5. NMR data showed that compounds 5a and 5b have the same planar structure, but differ in configuration. For clarity in the description of the structure elucidation, only the spectroscopic data of 5a will be described initially. The 13C NMR and DEPT135 spectra allowed us to conclude that the molecule consisted of eight methyl, three sp2 methines, one sp3 methine, and three methylene groups, whereas eight resonances were assigned to quaternary carbons. Comparison with spectroscopic data for 1−4 indicated that 5a was a closely related structure, especially concerning the aromatic moiety. In detail, the NMR spectra revealed signals for three methoxy groups, OCH3-10, OCH3-12, and OCH3-13 (δC at 59.8, 49.7, and 54.2, respectively). Heteronuclear long-range correlations from H3-12 and H3-13 to C-8 and C-1, respectively, allowed location of the methoxy moieties at the respective carbons, whereas OCH3-10 was bound to C-3 as in compounds 1−4. The carbons C-1 (δC at 105.4) and C-8 (δC at 111.4) displayed strongly downfield shifted resonances for sp3 carbons, indicating that both were connected to two oxygen atoms. Furthermore, HMBC couplings from H3-9 with C-7 and C-8 connected this methyl group to C-8. The methine proton H-1 had HMBC correlations to C-3, C-7, and C-8, which together with mass spectrometric and 13C NMR data allowed us to deduce a furan moiety with ketal and acetal functionalities as the second ring in 5a. Differences between 5a and 5b were established to be related to the relative configuration between C-1 and C-8. This was evident from NOE interactions, which were significantly different concerning the two molecules. Namely, in 5a the protons of the methyl group CH3-9 had an NOE correlation with OCH3-13, while OCH3-12 had an NOE correlation with H-1. In 5b the NOE correlations were the opposite; that is, CH3-9 correlates with H-1 and OCH3-13 correlates with OCH3-12 (see Supporting Information, Figure S16). For compounds 5a and 5b the names maristachones D and E are suggested, respectively. These compounds may arise by a chemical reaction of MeOH from the isolation process with 6-acetyl-2,4-dihydroxy3-methylbenzaldehyde (compound 7, Supporting Information, Figure S24), a possible reactive intermediate in the biosynthetic process. Spectroscopic data for compound 6 revealed that it belongs to a different structural class than 1−5, as no benzene ring was discernible. The molecular formula of 6 was found by HREIMS to be C8H10O3. The 13C NMR spectrum indicated two methyls, an sp2 methine, and one sp3 methylene group. Four further resonances were due to quaternary carbons, which made up half of the carbons encountered in the molecule. The methyl

The molecular formula of 2 was deduced by HRESIMS to be C12H18O4, requiring four sites of unsaturation. Spectroscopic data clearly suggested that compound 2 shares the pentasubstituted aromatic ring with compound 1. 1H NMR coupling from the methine H-8 to H3-9 (J = 6.3 Hz) was discerned in the 1H NMR spectrum, with C-8 having a carbon shift (δC 65.9) characteristic for oxygen substitution, thus giving evidence for a hydroxyethyl (C-8/C-9) function. The methoxy function OCH3-12 was connected to the methylene CH2-1 as deduced from heteronuclear long-range correlations of the methoxy protons to C-1. Compound 2 is thus a monocyclic compound, for which we propose the trivial name maristachone A. The molecular formula of 3 was deduced by HRESIMS to be C11H16O4, requiring as in the case of 2 four sites of unsaturation. The structure of 3 was found to be almost identical to that of 2 (Tables 1 and 2). The only difference between these Table 1. 1H NMR Spectroscopic Data for Compounds 1−3, 5a, and 5b 1 δH (J in Hz)

2

3

δHa (J in Hz)

δH (J in Hz)

a

pos. 1

5.54, dd (1.6, 6.5)

6 8 9

6.87, s

10 11 12 13 1′ 2′ 4′ 5′ 6′ 8′ 9′ 10′ a

a: 3.08, dd (6.5, 19.1) b: 2.50, dd (1.6, 19.1) 4.01, s 2.23, s

a

5a

5b

δH (J in Hz)

δ Ha (J in Hz)

a

a: 4.39, d (9.9) b: 4.43, d (9.9) 6.91, s 5.06, q (6.3) 1.33, d (6.3)

4.53, s

6.32, s

6.02, s

6.77, s 4.64, s

6.60, s

6.59, s

1.62, s

1.55, s

3.67, s 2.09, s 3.32, s

3.74, s 2.15, s 3.42, s

3.90, s 2.07, s 2.85, s 3.38, s 4.64, d (6.6) 5.48, t (6.6) 2.08, m 2.11, m 5.11, m 1.64, br s 1.75, br s 1.59, br s

3.89, s 2.06, s 2.94, s 3.50, s 4.63, d (6.6) 5.48, t (6.6) 2.07, m 2.11, m 5.10, m 1.64, br s 1.75, br s 1.58, br s

Acetone-d6 (2, 5a, 5b), MeOH-d4 (1 and 3), 300 MHz.

molecules was discerned to be the substitution of C-8. In the case of compound 2 this carbon was substituted with the methyl group CH3-9, whereas in compound 3 C-8 was an oxygenated methylene group. Consequently, the 1H NMR spectrum of 3 exhibited a singlet resonance for a methylene group at δH 4.64. We propose the trivial name maristachone B for compound 3. Compound 4 has a molecular formula of C18H28O4 as deduced by HRESIMS and, thus, five sites of unsaturation. The 13 C NMR and DEPT135 spectra allowed the assignment of 18 resonances to five methyl, four sp3 methylene, one sp2 methine, and two sp3 methine groups, whereas six resonance signals accounted for quaternary carbons. Compound 4 shares the nearly identical pentasubstituted aromatic ring as found for 2 and 3 (carbons/hydrogens have different numbering for 4), as proven by HMBC correlations from H-12 to C-8, C-10, C-11, C-13, and C-14. The methyl group CH3-15 is attached to the B

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Table 2. 13C NMR Spectroscopic Data for Compounds 1−3, 5a, and 5b

a

1

2

3

5a

5b

pos.

δC, mult.a

δC, mult.a

δC, mult.a

δC, mult.a

δC, mult.a

1 2 3 4 5 6 7 8 9 10 11 12 13 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 10′

67.1, 139.4, 158.4, 128.1, 159.8, 103.3, 137.2, 206.4, 48.4, 61.1, 10.0,

66.1, 119.6, 159.3, 116.1, 157.1, 108.5, 147.2, 65.9, 25.4, 61.8, 9.1, 57.8,

66.9, 120.3, 160.0, 117.5, 157.9, 111.6, 141.4, 62.6,

105.4, 121.4, 154.8, 119.0, 160.4, 100.2, 141.7, 111.4, 28.1, 59.8, 9.3, 49.7, 54.2, 66.1, 120.8, 141.1, 40.1, 27.0, 124.6, 132.0, 25.8, 16.6, 17.7,

104.8, 121.5, 154.9, 119.2, 160.4, 100.2, 141.3, 110.8, 27.8, 59.8, 9.3, 50.5, 55.5, 66.2, 120.9, 141.1, 40.1, 27.0, 124.7, 132.0, 25.8, 16.7, 17.7,

CH C C C C CH C C CH2 CH3 CH3

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

CH2 C C C C CH C CH2

62.2, CH3 9.2, CH3 58.1, CH3

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

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

Acetone-d6 (2, 5a, 5b), MeOH-d4 (1 and 3), 75.5 MHz. Implied multiplicities determined by DEPT.

a NF-κB protein complex assay, but showed no significant biological activities (see Supporting Information). The herein investigated sponge-derived Stachylidium sp. (strain 220) is closely related to S. bicolor and was originally isolated from the tropical sponge Callyspongia sp. Strain 220 produces a multitude of closely related polyketide-derived secondary metabolites, including the phthalides marilones A−C,4 the nitrogen-containing phthalimidines marilines A−C,3 and the herein reported cyclomarinone (1), maristachones A−H (2−5), and marilactone (6). All of these compounds are biosynthetically related, strikingly evident in most cases by the almost identically substituted aromatic ring. Whereas the biosynthesis of simple polyketides such as compound 3, which is composed of four acetate units and in essence is a derivative of the common fungal metabolite methylorsellinic acid,9 is easily descernable, the origin of the methyl group CH3-9 in 2, 5a, and 5b (or its equivalent CH3-15 in 4 or CH3-7 in 6) is not so clear. As was earlier reported for the phthalimidines marilines A−C3 and marilones A−C4 the biosynthesis of compounds 1, 2, and 4−6 requires either a propionate starter unit (B in Figure S24) or a C-methylation (e.g., via an SAM-dependent methyltransferase) of the starter unit acetic acid (A in Figure S24). This also applies to the methylene group CH2-9 in compound 1, which appears to be equivalent to the respective carbon. The structurally unique molecule 1 may have arisen from a reactive intermediate like 6-acetyl-2,4-dihydroxy-3-methylbenzaldehyde (compound 7, Figure S24), from which it may be formed by an aldol reaction. In this case CH3-9 would get incorporated into a hydroxycyclopentenone ring to give a new carbon arrangement within polyketides. The hydroxycyclopentenone ring present in cyclomarinone (1) is very rare in nature and was described before in claulansine E10 and with an additional methyl substituent in the pterosins.11 However, while

protons H3-7 coupled with H2-6 (J = 7.5 Hz), as evident from 1 H NMR data, and both had HMBC correlations with C-5, the latter being a quaternary carbon connected to oxygen (δC 164.9). The methyl group CH3-8 had HMBC correlations with C-1, C-2, and C-3 and was located at C-2 (δC 98.2). C-3 was characterized by a downfield shifted carbon resonance, which indicated it was connected to oxygen (δC 165.6). The HMBC correlations of H-4 and H3-8 to C-3 placed this hydroxylated quaternary carbon between C-2 and C-4. The quaternary carbon C-1 (δC 166.0) was identified as being part of a carboxylic functionality, also evidenced by an IR absorption at 1645 cm−1. Consideration of the molecular formula required compound 6 to be monocyclic. Thus, C-1 was connected to C-5 through an oxygen atom, forming a six-membered lactone ring. We propose the trivial name marilactone for compound 6, which is already described as a synthetic compound, for which, however, only 1H NMR spectroscopic data are reported.7,8 Compounds 1 and 2 both possess a single stereogenic center at C-1 and C-8, respectively. However, measurement of the optical rotation for these compounds yielded values close to zero. Thus, we assume the presence of racemic mixtures for these chiral compounds, as already reported for the marilines and marilones isolated from the same fungus.3,4 Racemic mixtures are most probable for compounds 5a and 5b, as their specific rotation values are also close to zero. All attempts to separate these racemic mixtures with chiral-phase HPLC methods were not successful. The configurations of the stereogenic centers C-3 and C-6 of compound 4 could not be determined due to the low yields of the respective compound. Compounds 1−6 were evaluated for antibacterial, antifungal, antialgal, psychoactive, and antiviral activities, for inhibition of protein kinases and proteases, and for activity in an antidiabetic activity assay panel, in a 3-T3-L1 murine adipocyte assay, and in C

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petroleum ether−acetone (13:1) (fraction 3 of 4), which was finally purified with 40% MeOH−60% H2O using RP-HPLC (fraction 2 of 2, 5.8 mg, tR 12 min). Cyclomarinone, 3,6-dihydroxy-4-methoxy-5-methyl-2,3dihydro-1H-inden-1-one (1): white, amorphous solid (2.5 mg; 250 μg/L); [α]23D ±0 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 220 (4.17), 266 nm (3.73); IR (ATR) νmax 3268 (br), 2930, 1697, 1605 cm−1; 1H and 13C NMR (Tables 1 and 2); LRESIMS m/z 206.9 [M − H]−; HRESIMS m/z 207.0650 [M − H]− (calcd for C11H11O4, 207.0652). Maristachone A, 5-(1-hydroxyethyl)-3-methoxy-4-(methoxymethyl)-2-methylphenol (2): white, amorphous solid (4.2 mg; 420 μg/L); [α]23D ±0 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 212 (4.04), 226 (3.82), 271 nm (3.27); IR (ATR) νmax 3338 (br), 2929, 1610, 1458, 1414 cm−1; 1H NMR and 13C NMR (Tables 1 and 2); LRESIMS m/z 224.9 [M − H]−; HRESIMS m/z 249.1101 [M + Na]+ (calcd for C12H18O4Na, 249.1097). Maristachone B, 5-(hydroxymethyl)-3-methoxy-4-(methoxymethyl)-2-methylphenol (3): white, amorphous solid (4.0 mg; 400 μg/L); UV (MeOH) λmax (log ε) 210 (4.25), 229 (3.94), 276 nm (3.18); IR (ATR) νmax 3347 (br), 2929, 1602, 1457, 1418 cm−1; 1 H NMR and 13C NMR (Tables 1 and 2); LRESIMS m/z 211.1 [M − H]−; HRESIMS m/z 235.0948 [M + Na]+ (calcd for C11H16O4Na, 235.0941). Maristachone C (4): white, amorphous solid (0.7 mg; 70 μg/L); [α]23D −33 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 206 (4.14), 275 nm (3.12); IR (ATR) νmax 3361 (br), 2929, 1702, 1609, 1457, 1407 cm−1; 1H NMR (acetone-d6, 300 MHz) δ 6.51 (1H, s, H-12); 3.85 (1H, m, H-3); 3.65 (3H, s, H3-17); 2.87 (1H, m, H-6); 2.81 (1H, m, H-7a); 2.53 (1H, m, H-7b); 2.52 (2H, m, H2-14); 2.51 (1H, m, H-4b); 2.41 (1H, dd, J = 4.4, 16.5 Hz, H-4a); 2.08 (3H, s, H3-18); 1.35 (2H, m, H2-2); 1.10 (3H, s, H3-15); 0.94 (3H, d, J = 6.7 Hz, H3-16); 0.85 (1H, d, J = 7.3 Hz, H3-1); 13C NMR (acetone-d6, 75 MHz) δ 214.8 (C, C-5); 159.2 (C, C-9); 155.6 (C, C-11); 141.9 (C, C-13); 122.1 (C, C-8); 115.6 (C, C-10); 111.5 (CH, C-12); 69.4 (CH, C-3); 60.4 (CH3, C-17); 49.5 (CH2, C-4); 48.1 (CH, C-6); 30.6 (CH2, C-2); 29.8 (CH2, C-7); 26.3 (CH2, C-14); 15.9 (CH3, C-16); 15.8 (CH3, C-15); 10.1 (CH3, C-1); 9.4 (CH3; C-18); LRESIMS m/z 307.6 [M − H]−, 309.5 [M + H]+; HRESIMS m/z 331.1886 [M + Na]+ (calcd for C18H28O4Na, 331.1880). Maristachone D (5a): white, amorphous solid (1.6 mg; 160 μg/L); [α]23D ±0 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 214 (4.09), 272 nm (2.98); IR (ATR) νmax 2930, 1610, 1463 cm−1; 1H NMR and 13C NMR (Tables 1 and 2); LREIMS m/z 330.2 [M]+; HREIMS m/z 390.2417 [M]+ (calcd for C23H34O5, 390.2406). Maristachone E (5b): white, amorphous solid (1.7 mg; 170 μg/L); [α]23D ±0 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 214 (4.10), 272 nm (2.98); IR (ATR) νmax 2925, 1610, 1464 cm−1; 1H NMR and 13 C NMR (Tables 1 and 2); HREIMS m/z 390.2412 [M]+ (calcd for C23H34O5, 390.2406). Marilactone, 6-ethyl-4-hydroxy-3-methyl-2H-pyran-2-one (6): amorphous solid (5.8 mg; 580 ug/L); UV (MeOH) λmax (log ε) 207 (4.05), 285 nm (3.69); IR (ATR) νmax 3330 (br), 2929, 2674, 1645, 1578 cm−1; 1H NMR (acetone-d6, 300 MHz) δ 6.02 (1H, s, H-4); 2.42 (2H, q, J = 7.5 Hz, H2-6); 1.13 (3H, t, J = 7.5 Hz, H3-7); 1.81 (3H, s, H3-8); 13C NMR (acetone-d6, 75 MHz) δ 166.0 (C, C-1); 165.6 (C, C-3); 164.9 (C, C-5); 99.1 (CH, C-4); 98.2 (C, C-2); 27.1 (CH2, C-6); 11.3 (CH3, C-7); 8.5 (CH3, C-8); LRESIMS m/z 153.1 [M − H]−; HREIMS m/z 154.0628 [M]+ (calcd for C8H10O3,154.0630).

claulansine E is a carbazole alkaloid, the pterosins are sesquiterpenes (see Supporting Information Figure S25).

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured on a Jasco DIP 140 polarimeter. UV and IR spectra were obtained employing a Perkin-Elmer Spectrum BX instrument. All NMR spectra were recorded in MeOH-d4 or acetone-d6 employing a Bruker Avance 300 DPX spectrometer. Spectra were referenced to residual solvent signals with resonances at δH/C 3.35/49.0 for MeOHd4 and δH/C 2.04/29.8 for acetone-d6. HREIMS were recorded on a Finnigan MAT 95 spectrometer. HRESIMS were recorded on a Bruker Daltonik micrOTOF-Q time-of-flight mass spectrometer with ESI source. HPLC was carried out using a system composed of a Waters 515 pump together with a Knauer K-2300 differential refractometer. HPLC columns were from Knauer (250 × 8 mm, Eurospher100 Si and 250 × 8 mm, Eurospher-100, C18, 5 μm; flow 2 mL/min) and Macherey-Nagel (Nucleodur C18 EC Isis 250 × 4.6 mm, 5 μm, flow: 1 mL/min). Merck silica gel 60 (0.040−0.063 mm, 70−230 mesh) was used for vacuum liquid chromatography (VLC). Columns were wetpacked under vacuum using petroleum ether. Before applying the sample solution, the columns were equilibrated with the first designated eluent. Standard columns for extract fractionation had dimensions of 13 × 4 cm. Fungal Material. The marine-derived fungus Stachylidium sp. was isolated from the sponge Callyspongia sp. cf. C. f lammea (collected at Bear Island, Sydney, Australia) and identified by P. Massart and C. Decock, BCCM/MUCL, Catholic University of Louvain, Belgium (see Supporting Information S26). A specimen is deposited at the Institute for Pharmaceutical Biology, University of Bonn, isolation number “293K04”, culture collection number “220”. Cultivation, Extraction, and Isolation. Compounds 1−6 were isolated from a 60-day culture (40 Fernbach flasks, each containing 250 mL (= 10 L; 25 °C) of Stachylidium sp. on an agar-biomalt medium (10 g/L biomalt, 15 g/L agar) supplemented with sea salt [(g/L): KBr (0.1), NaCl (23.48), MgCl2 × 6H2O (10.61), CaCl2 × 2H2O (1.47), KCl (0.66), SrCl2 × 6H2O (0.04), Na2SO4 (3.92), NaHCO3 (0.19), H3BO3 (0.03)]. An extraction with 5 L of EtOAc yielded 4.8 g of extract, which was subjected to a VLC fractionation in an open column using silica as the solid phase and a gradient solvent system with petroleum ether− acetone of 10:1, 5:1, 2:1, 1:1, 100% acetone, and 100% MeOH, resulting in six VLC fractions. Compound 1 was isolated from VLC fraction 3, followed by NPHPLC fractionation using petroleum ether−acetone (5:1) (fraction 9 of 10). This subfraction was further purified using 50% MeOH−50% H2O (RP-HPLC, Isis column; fraction 1 of 4). Finally we achieved purification using 30% MeOH−70% H2O (RP-HPLC, Isis column, fraction 6 of 6, 2.5 mg, tR 14 min). Compound 2 was isolated from VLC fraction 3, followed by NPHPLC fractionation using petroleum ether−acetone (5:1) (fraction 4 of 10). This subfraction was further purified using 50% MeOH−50% H2O (RP-HPLC, Isis column; fraction 2 of 4, 4.2 mg, tR 10 min). Compound 3 was isolated from VLC fraction 3, followed by NPHPLC fractionation using petroleum ether−acetone (5:1) (fraction 7 of 10). This subfraction was further purified using 50% MeOH−50% H2O (RP-HPLC, Isis column; fraction 2 of 4, 4 mg, tR 3 min). Compound 4 was isolated from VLC fraction 2, followed by NPHPLC fractionation using petroleum ether−acetone (11:1) (fraction 3 of 7). This sample was further fractionated with 90% MeOH−10% H2O (RP-HPLC, Isis column, fraction 2 of 4, 0.7 mg, tR 15 min). Compounds 5a and 5b were isolated from VLC fraction 2, followed by NP-HPLC fractionation using petroleum ether−acetone (11:1) and further RP-HPLC fractionation, which was performed with 85% MeOH−15% H2O (compound 5a, subfraction 1 of 2; 1.6 mg, tR 9 min, and compound 5b, subfraction 2 of 2; 1.7 mg, tR 11 min). Compound 6 was isolated from VLC fraction 3, followed by NP-HPLC fractionation using petroleum ether−acetone (5:1) (fraction 3 of 10). This subfraction was further separated using

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ASSOCIATED CONTENT

S Supporting Information *

Spectroscopic data and other relevant information are included for the new compounds. This material is available free of charge via the Internet at http://pubs.acs.org. D

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AUTHOR INFORMATION

Corresponding Author

*Tel: +49228733747. Fax: +49228733250. E-mail: g.koenig@ uni-bonn.de. Present Address †

Fundación Medina, Avenida del Conocimiento 3, Parque Tecnológico de Ciencias de la Salud, E-18100 Armilla, Granada, Spain.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the efforts of Dr. K. Dimas (Biomedical Research Foundation of Academy of Athens, Athens, Greece) for the cytotoxicity assays. We kindly thank Dr. M. Diederich (Fondation Recherche sur le Cancer et les Maladies du Sang, Laboratoire de Biologie Moléculaire et Cellulaire du Cancer (LBMCC), Luxembourg) for performing the NF-κB activity assays, and Dr. S. Paulsen (University of Tromsø, MabCent, Tromsø, Norway) for performing the antidiabetic activity assays. We also kindly thank the financial support from FCT (Science and Technology Foundation, Portugal). The Ki determinations and antagonist functional data were generously provided by the National Institute of Mental Health's Psychoactive Drug Screening Program, Contract # HHSN271-2008-00025-C (NIMH PDSP). The NIMH PDSP is directed by Bryan L. Roth MD, PhD at the University of North Carolina at Chapel Hill and Project Officer Jamie Driscol at NIMH, Bethesda MD, USA.

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DEDICATION Dedicated to Dr. Lester A. Mitscher, of the University of Kansas, for his pioneering work on the discovery of bioactive natural products and their derivatives.

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REFERENCES

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