Note pubs.acs.org/jnp
Cyclic 3‑Alkyl Pyridinium Alkaloid Monomers from a New Zealand Haliclona sp. Marine Sponge Vidhiya Damodaran,† Jason L. Ryan,‡ and Robert A. Keyzers*,† †
Centre for Biodiscovery and School of Chemical & Physical Sciences, Victoria University of Wellington, PO Box 600, Kelburn, Wellington 6140, New Zealand ‡ Integrated Bioactive Technologies Group, Callaghan Innovation, Gracefield, PO Box 31310, Lower Hutt 5040, New Zealand S Supporting Information *
ABSTRACT: Bioassay and NMR approaches have been used to guide the isolation of one known and two new cyclic 3-alkyl pyridinium alkaloid (3-APA) monomers from the New Zealand marine sponge Haliclona sp. The new compounds, dehydrohaliclocyclins C (3) and F (4), are the first reported examples of cyclic 3-APA monomers with unsaturation in the alkyl chain. The known compound haliclocyclin C (2) was also isolated from a mixture with 4. The structures of compounds 2−4 were elucidated using NMR spectroscopy, mass spectrometry, and chemical degradation.
3-Alkyl pyridinium alkaloids (3-APAs) are common secondary metabolites from sponges of the order Haplosclerida,1 possessing a wide range of activities including antibacterial, antifungal, and cytotoxic properties.2−4 3-APAs are found as polymeric, linear, or cyclic compounds varying in length, degree of unsaturation, and branching of the alkyl chain.1 Linear 3APAs can vary in the terminal group and the number of pyridinium subunits from monomers (e.g. viscosalines5) to polymers (such as the halitoxins6), while cyclic 3-APAs vary in the number of pyridinium subunits from monomers to hexamers.1,4,7 Reported cyclic 3-APAs have been dominated by the dimeric cyclostellettamines. Cyclic 3-APA monomers were unknown until Köck and co-workers isolated haliclocyclin F (1), with a fully saturated C14 alkyl chain.4 This was followed later by the isolation of the C13 homologue haliclocyclin C (2),8 a known synthetic intermediate.9,10 There have been no reports of cyclic 3-APA monomers with unsaturation in the alkyl chain to date. Herein, 2 has been isolated along with two new congeners containing unsaturation in the alkyl chain from an extract of the New Zealand marine sponge Haliclona sp. In accordance with the naming system proposed by Köck,8 these compounds were named dehydrohaliclocyclin C (3) and dehydrohaliclocyclin F (4). The structures of 3 and 4 were elucidated using 1D and 2D NMR experiments, HRESIMS, and oxidative degradation in combination with MS/MS studies. Saccharomyces cerevisiae was used to identify antifungal leads due to its local availability and its potential for mode of action studies using yeast chemical genomics, available in-house.11,12 An extract library of Pacific marine invertebrates was screened for S. cerevisiae inhibitory activity, with one “hit” coming from © XXXX American Chemical Society and American Society of Pharmacognosy
the extract of a sponge identified as Haliclona sp. collected at D’Urville Island, New Zealand. Using a joint bioassay and spectroscopy-guided process, the MeOH extract of the Haliclona sp. was iteratively fractionated using both normaland reversed-phase stationary phases, culminating in three iterations of reversed HPLC to finally isolate dehydrohaliclocyclin C (3) (0.36 mg), dehydrohaliclocyclin F (4) (0.25 mg), and the known compound haliclocyclin C (2) (0.16 mg). Dehydrohaliclocyclin C (3) was isolated as a colorless oil. The NMR data of 3 closely resembled those of 2, suggesting that they are related compounds. Positive ion mode HRESIMS showed a singly charged molecular ion of m/z 258.2216, indicating a formula of C18H28N+ requiring 5.5 degrees of unsaturation. The molecular formula of 3 differed from that of 2 by having two less hydrogens, suggesting that 3 was an Received: August 13, 2013
A
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unsaturated analogue. The 1H, 13C, and multiplicity-edited HSQC NMR spectra were used to establish the typical 3-APA aromatic system. Briefly, the multiplicities of the aromatic protons in the 1H NMR spectrum (δH 9.60, broad doublet; 8.83, singlet; 8.17, doublet; 8.03, doublet of doublets) and presence of five aromatic carbon resonances indicated a 1,3disubstituted heteroaromatic ring with a nitrogen atom at the 1position. HMBC correlations from H-2 (8.83 ppm) to C-7 (62.7 ppm), C-19 (32.3 ppm), and C-3 (143.4 ppm), from the downfield proton resonance of H-6 (9.60 ppm) to C-7, and from H-4 (8.17 ppm) to C-19 completed the aromatic substructure. This was confirmed by the long-range couplings observed (COSY) for most of the aromatic protons (Figure 1) and was consistent with a 3-APA structure.
alkyl chain resonances in a 3-APA to be completely resolved using the COSY spectrum. Even at 600 MHz, the methylene resonances usually overlap to give a methylene envelope. In 3, the position of the double bond in the alkyl chain seems to have differentiated the proton resonances enough that each one was resolved (Supporting Information S4). The proton resonances from the alkene were also clearly distinguishable as two separate multiplets integrating for one proton each. Several approaches were used to determine the geometry of the double bond. First, the 13C chemical shifts of C-11 and C14 (δ C 27.1 and 25.2, respectively) suggested a Zgeometry.7,13,14 Next, a 2D NOESY experiment appeared to show correlations from one alkene proton to the other; however, the correlations were weak and so close to the diagonal that this was inconclusive (Supporting Information S9). A fully coupled HSQC experiment was used to extract a coupling of 10 Hz between the alkene resonances (Supporting Information S10), providing further evidence for a Zgeometry.15 Finally, the most conclusive evidence for the Zgeometry came from a homonuclear decoupling experiment irradiating both the allylic resonances of H2-11 and H2-14 concurrently, nullifying the coupling between the methylene and alkene protons. This experiment resolved the alkene protons from multiplets into doublets with a mutual coupling constant of 10.8 Hz (Supporting Information S11) consistent with a Z-geometry alkene and confirming that proposed from the 13C NMR, 2D NOESY, and coupled HSQC data. This information, together with the singly charged molecular ion identified in the HRESIMS, led to the proposal of 3 as the structure for dehydrohaliclocyclin C. Dehydrohaliclocyclin F (4) was isolated as a colorless oil. Positive ion mode HRESIMS analysis detected a singly charged molecular ion of m/z 272.2378, indicating a formula of C19H30N+ requiring 5.5 degrees of unsaturation and differing from dehydrohaliclocyclin C (3) by the addition of one methylene group. The NMR spectra of 4 closely resembled those of 3, especially in the aromatic region. The 1,3disubstituted pyridinium ring was established from the diagnostic 1H NMR resonances of H-2 and H-4 to H-6 combined with their HMBC correlations as detailed for 3. Unlike the clearly resolved 1H NMR spectrum of 3, the additional conformational flexibility imparted by the extra methylene group caused coalescence of the methylene signals into an envelope in the spectrum of 4, typical of the alkyl chains of most 3-APAs (Supporting Information S12), and prevented the use of COSY data to determine the double-bond position. A series of 1D TOCSY experiments were used to establish the connection between the pyridinium ring and the overlapping alkene resonances of H-13 and H-14. Consequently, irradiation of both H2-7 and H2-20 with increasing spin-lock mixing times (15−150 ms) resulted in TOCSY energy transfer to the alkene protons H-13 and H-14, with the relevant reciprocal transfer from H-13/14 to both H 2 -7 and H 2 -20 (Supporting Information S17−S19). Finally, the position and geometry of the alkene in 4 needed to be determined. As for 3, a Z-geometry alkene was indicated by the 13C chemical shifts of C-12 and C-15 (both δC 26.8).7,13,14 A homonuclear decoupling experiment was also performed, but unfortunately the results of this experiment were not as clear as for 3, producing two overlapping doublets of doublets from a highly second order multiplet (Supporting Information S20). Despite this, the measured coupling constant was between 10 and 11 Hz and could not be greater than 12
Figure 1. Key COSY and HMBC correlations establishing the structure of dehydrohaliclocyclin C (3). COSY correlations are shown in bold, and key HMBC correlations are shown by arrows.
It was noted, however, that 3 is unusual, as the H-2 singlet is usually the most downfield resonance in the 1H NMR spectrum, while the H-6 resonance of 3 has been shifted downfield to 9.60 ppm. The reason for this difference between the recorded spectra and the literature NMR data has not been determined. The alkyl chain of 3 was established from a contiguous chain of resolved COSY correlations from H2-7 to H2-19 via alkene protons H-12 (5.17 ppm) and H-13 (5.23 ppm), completing the final structure of 3 as shown (Figure 1) that was supported by HMBC correlations (Table 1). It is very unusual for the Table 1. NMR Spectroscopic Data of Dehydrohaliclocyclin C (3) (CDCl3, 1H 600 MHz; 13C 150 MHz) position
δC
type
1
2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
144.1 143.4 145.2 128.5 143.6 62.7 31.1 25.1 28.41 27.1 129.8 130.5 25.2 28.36 27.3 26.6 29.0 32.3
CH C CH CH CH CH2 CH2 CH2 CH2 CH2 CH CH CH2 CH2 CH2 CH2 CH2 CH2
189
8.83, s
3, 4, 7, 19
171 171
8.17, 8.03, 9.60, 5.01, 2.06, 1.04, 1.36, 1.96, 5.17, 5.23, 1.94, 1.42, 1.27, 1.17, 1.81, 2.94,
2, 5, 6, 19 4, 3 2, 4, 5, 7 2, 6, 8, 9 7, 9 7, 8, 10, 11 8, 9, 11, 12 9, 10, 12, 13 10, 11, 14 11, 14, 15 12, 13, 15, 16 13, 14, 16, 17 14, 15, 17, 18 15, 16, 18, 19 3, 16, 17, 19 2, 3, 4, 17, 18
JCH
150 124 121 124 125 154 154 124 124 118 122 127 130
δH (J in Hz)
d (7.8) dd (8.1, 6.0) brd (5.3) m m m m m m m m quin (6.5) m m m m
HMBCa
a
HMBC correlations, optimized for 6 Hz, are from proton(s) stated to the indicated carbon. B
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Scheme 1. Onium Reactions of the Possible Dialdehyde 5 to Form MS/MS Fragments 7 and 9 and a McLafferty-like Rearrangement of the Possible Dialdehyde 6 to Form the MS/MS Fragment 8 (shown with calculated masses)
Hz, supporting the proposal of a Z-geometry alkene. A fully coupled HSQC was also performed, which showed that the coupling between the resonances was 11.0 Hz (Supporting Information S21). With this evidence, a Z-geometry was proposed for the alkene of 4. To determine the position of the double bond, an oxidation was performed using ruthenium trichloride and sodium periodate in a CHCl3/MeCN/H2O solution.16 This successfully oxidatively cleaved the alkene of 4 to form a dial, as shown by the MS ion detected at m/z 304.2269, which could be either 5 or 6 depending on the parent alkene placement. Collisioninduced dissociation (CID) ESIMS/MS analysis of the dialdehyde was then used to identify key fragment peaks whose masses would be dictated by the alkyl chain lengths.3,17 A reproducible fragment mass of m/z 192.1382 was identified that could be attributed to two possible fragments, 7 or 8. Dial 5, with alkyl chains of seven carbons, could fragment to 7 via an onium reaction,18 or a methyl pyridinium with a chain length of six (8) could arise from a McLafferty-like rearrangement of dialdehyde 6 (Scheme 1).17 The strong reproducible fragment ion detected at m/z 106.0650 (Supporting Information S22) that is produced from fragmentation of a protonated pyridinium ring (9) is characteristic of an onium fragmentation (Scheme 1),17,18 indicating that the fragment ion at m/z 192.1382 was the product of an onium reaction from 5; therefore the alkene of 4 linked carbons 13 and 14. This proposal was consistent with the 1D TOCSY NMR data, which showed that TOCSY transfer from alkene protons H-13 and H-14 to H2-7 and H2-20 occurred at the same mixing time, indicating that the alkyl chains linking the alkene to the pyridinium were of approximately the same length. In the case of a Δ14,15 alkene, as required to generate dial 6, TOCSY transfer would proceed through five and seven carbon chains and would be expected to result in transfer to H2-7 and H2-20 at different mixing times. Haliclocyclin C (2) is known from both natural and synthetic sources.8−10 In the current study it was isolated as a colorless oil from a mixture with 4. All spectroscopic data (1H and 13C NMR, HRESIMS) were consistent with those reported in the literature.8−10 Isolation of the 3-APAs was initially guided by testing for inhibition of S. cerevisiae growth. They showed potent activity (IC50 1.5 ± 1 μg/mL) in initial assays; however, upon isolation of the pure 3-APA compounds, a paucity of material prevented testing activity against both S. cerevisiae and other micro-
organisms. For this reason, the inhibitory activity of the pure 3APA monomers was measured only against the more clinically relevant pathogens Enterococcus faecalis (Gram-positive bacteria) and Candida albicans (fungi) in disk-diffusion assays. Unfortunately, dehydrohaliclocyclin C (3), dehydrohaliclocyclin F (4), and haliclocyclin C (2) showed no activity against either microbe when tested at 20, 8, and 2 μg loadings. In conclusion, two new unsaturated 3-APA monomers have been isolated from a New Zealand marine Haliclona sponge. The structures of dehydrohaliclocyclins C (3) and F (4) were established using a comprehensive combination of NMR analysis and chemical oxidation coupled with tandem MS/MS experiments.
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EXPERIMENTAL SECTION
General Experimental Procedures. NMR spectra were obtained using a Varian DirectDrive spectrometer equipped with a triple resonance HCN cryogenic probe operating at 600 MHz for 1H and 150 MHz for 13C nuclei. The chemical shifts of 1H and 13C spectra were referenced to the residual solvent peaks (CDCl3: δC 77.0, δH 7.26; CD3OD: δC 49.0, δH 3.31).19 Quantitative 1H NMR with nitromethane as an internal quantification standard was used to measure the mass of the isolated compounds. HRESIMS results were obtained from an Agilent 6530 Q-TOF mass spectrometer equipped with an Agilent 1260 HPLC for solvent delivery (C18 Zorbax Extend, 2.1 × 50 mm, 1.8 μm particle size) utilizing a JetStream electrospray ionization source in positive and negative ion modes. Water and MeCN solvents were both made up as 5 mM solutions with ammonium formate. CID was performed using nitrogen as collision gas at various energies (10−100, arbitrary units). Normal-phase column chromatography was performed using Supelco Discovery DSC-DIOL functionalized silica: 3-(2,3-dihydroxypropoxy)propylsilica (Diol). Reversed-phase column chromatography was performed using Supelco Diaion HP20 and HP20ss poly(styrene-divinylbenzene) resins. Two separate HPLC systems were used, the first using a Rainin Dynamax SD-200 solvent delivery system with 25 mL pump heads and a Varian Prostar 335 photodiode array detector for UV/vis detection (monitoring at 210 and 270 nm). The second HPLC system was an Agilent Technologies 1260 Infinity HPLC equipped with a quaternary pump, a thermostated column compartment, and a diode array detector (DAD). Following the DAD, a Quicksplit flow splitter directed 5% of the flow to an Agilent 380 evaporative light scattering detector with 95% of the flow directed toward collection. For HPLC purification, a C18 analytical (Phenomenex Prodigy, 4.6 × 250 mm, 5 μm particle size) column was used. All solvents used were HPLC grade (Fisher Scientific) with the exception of hexanes and CH2Cl2, which were Optima grade. Water was distilled prior to use, and in the C
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case of HPLC the H2O was further filtered through a membrane with a pore size of 0.45 μm before use. Animal Material. The sponge was collected at Port Hardy, D’Urville Island, New Zealand, off a rock surface in April 2000 (40.46.2 S; 173.59.7 E) at a depth of 10−15 m. The sponge was identified as a species of Haliclona (order Haplosclerida, family Chalinidae). The sponge was massive and encrusting, approximately 4 cm thick. The surface was mottled tan in color with a similar colored interior. Small (
