Note pubs.acs.org/jnp
Antipodal Crambescin A2 Homologues from the Marine Sponge Pseudaxinella reticulata. Antifungal Structure−Activity Relationships Matthew T. Jamison† and Tadeusz F. Molinski*,†,§ †
Department of Chemistry and Biochemistry and §Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, 9500 Gilman Drive MC-0358, La Jolla, California 92093-0358, United States S Supporting Information *
ABSTRACT: Investigation of antifungal natural products from the marine sponge Pseudaxinella reticulata from the Bahamas led to the discovery of new crambescin homologues (1, 2) and enantiomers (3, 4) of known natural products. The cyclic-guanidine structures were solved through analysis of 2D NMR, MS-MS, and CD data. The absolute configurations of 1−4 were established as 13Ropposite of known homologues reported from Crambe crambe obtained from the Mediterranean Seaby comparison of their CD spectra with predicted Cotton effects obtained from DFT calculations. Antifungal activities of 1−4 against the pathogenic strains Candida albicans and Cryptococcus sp. were observed to correlate potency (MIC50 and MIC90) with the length of the alkyl side chain. ince the first isolation of the crambescins by Berlinck and Braekmann1 from Crambe crambe, a remarkable diversity of guanidine-containing natural products has been uncovered from marine sponges.2 Many guanidine alkaloids exhibit potent biological activity, including suppression of HIV-1−T-cell fusion,3 inhibition of laccase responsible for catecholaminedependent melanization in Cryptococcus neoformans,4 and increased NO production in macrophages.5 During investigations of marine sponges, it was observed that a MeOH extract of Pseudaxinella reticulata, collected in the Bahamas, exhibited broad-spectrum antifungal activity. Bioassay-guided purification of the active compounds led to the isolation of two new crambescin A2 homologues, (+)-1 and (+)-2, and dextrorotatory enantiomers of two known compounds, crambescin A2 4206 (3) and Sch 5759487 (4) (Figure 1). Herein are reported the complete structural characterization, including assignment of absolute stereostructures, and the antifungal activities of the new natural products and related compounds. The MeOH extract of the marine sponge P. reticulata was progressively partitioned against hexane, CH2Cl2, and n-BuOH. The n-BuOH partition was subjected to gel filtration (Sephadex LH-20) and elution with MeOH to produce a series of antifungal fractions. LC-MS analysis of the latter fractions revealed molecular masses with m/z values in the ranges corresponding to the guanidine-containing natural products batzelladines, ptilomycalins, and crambescins. Analysis of the minor components revealed a fraction with m/z values that did not match known guanidine-containing compounds. Purification by repeated reversed-phase HPLC of the latter fraction gave compounds 1−4. Crambescin A2 392 (1)8 has the molecular formula C20H36N6O2 as established by HRESITOFMS (m/z 393.2970 [M + H]+). The 1H NMR spectrum of 1 (acetone-d6) showed
S
© XXXX American Chemical Society and American Society of Pharmacognosy
Figure 1. Crambescin A2 homologues (1−5, free bases).
three downfield exchangeable signals (δH 11.27, s, 9.11, s, and 8.13, bs) consistent with the presence of a substituted guanadino group. A combination of COSY and HSQC data established three contiguous diastereotopic pairs of methylene 1 H NMR signals, H-9′ab (δH 3.34 (1H, ddd, J = 2.8, 8.0, 18.2 Hz), 3.05 (1H, ddd, J = 9.7, 9.7, 18.2 Hz; δC 31.3), H-10′ab (δH 2.3 m, 2.16 m; δC 22.3), and H-11′ab (δH 3.93 (1H, dd, J = 2.3, Special Issue: Special Issue in Honor of William Fenical Received: December 26, 2014
A
DOI: 10.1021/np501052a J. Nat. Prod. XXXX, XXX, XXX−XXX
Journal of Natural Products
Note
9.1 Hz), 3.81 (1 H, dd, J = 9.1, 15.3 Hz); δC 48.1) (Table 1). A shared HMBC cross-peak from H-9 and H-11 to C-8 (δC Table 1. 1H and 13C NMR Data for 1, TFA Salt (acetone-d6) locant
δ 13Ca
1 2
159.3 41.2
3 4 5 6 7 8 9
26.0c 26.0c 64.2 165.6 102.7 152.4 31.3 31.3
10 11 12 13 14 15−19 20 a
22.3 22.3 48.1 48.1 153.4 50.3 37.0 29.8c 14.0
δ 1H (mult, J, integ.)b 3.30 (2H, 18.2) 1.73 (2H, 1.79 (2H, 4.21 (2H,
gHMBC (1H→13C)
COSY
ddd, J = 2.8, 8.0,
1, 3
3
m) m) t, J = 5.9)
2, 3, 5 4, 6
5 4
7, 8, 10
10
6, 8, 10
10
3.34 (1H, ddd, J = 2.8, 8.0, 18.2) 3.05 (1 H, ddd, J = 9.7, 9.7, 18.2) 2.30 (1H, m) 2.16 (1H, m) 3.93 (1H, dd, J = 2.3, 9.1) 3.81 (1H, dd, J = 9.1, 15.3) 4.38 (1H, t, J = 5.6) 1.55 (2H, m) 1.29c(m) 0.89 (m)
8 11 8, 9, 10 10
9, 11 9, 11 10 10
6−8, 12, 14, 15
14 13
18, 19
19
Figure 2. MS-MS fragmentation of compounds 1−4.
The specific rotations of 1, 3, and 4 in MeOH were dextrorotatory ([α]D +10, +18, and +21, respectively, Table 2). Table 2. Reported Specific Rotations ([α]D, MeOH) of Crambescin Alkaloid Salts
a
cmpd
CF3COOH salt
HCl salt
3 4 5
+18 +21 +7.1
+20 +28
ref a a
11b
b
This work. The protonation state for 5 was not explicitly defined, although, from the description of the experimental conditions, it is likely to be a TFA salt.
Although the optical rotations of 36 and 47 were unreported, previous work by the Thomas group established the 13S absolute configuration of crambescins A2, B1, and C1 from Mediterranean C. crambe by CD and comparisons of the observed Cotton effects (CEs) with those predicted from DFT calculations.11 All three crambescin A2 homologues from C. crambe were also dextrorotatory ([α]D +7.1, +12.1, +35.9).11 Surprisingly, the CD spectra of 1−4 (Figure 3) showed positive CEs of equal magnitude arising from asymmetric perturbation of the vinylogous amide chromophore [for example, 1, λ 248 nm (Δε +1.92), 289 (+0.83)], but opposite in sign of that of Thomas’ norcrambescin A2 [5, λ 248 nm (Δε −2.22), 291 (−0.99)].11 The latter comparative CD analysis unambiguously established that 1−4, from P. reticulata, are antipodal to 5 from C. crambe;12 however, a paradox remained: all compounds 1−5 from both sponges were reported as dextrorotatory. The crambescins in our work were isolated uniformly as guanidinium trifluoroacetate salts (CF3COO− counterion). We and others13 have observed that the [α]D values of some chiral ammonium salts are strongly influenced by the nature of the counterion. The paradox may be explained if specific optical rotations were measured under different protonation states, for example, if the crambescins from C. crambe were measured as different salts or even as free bases.11 We exchanged the counterions of the salt forms of 3 and 4 from CF3COO− to Cl− and remeasured the optical activities (Table 2). Although modest increases in magnitudes of [α]Ds were observed (e.g., 3· TFA, [α]D +18; 3·HCl, [α]D +20), the sign of rotation remained unchanged. It can only be speculated that the compositions of salts of 3−511 were less well-defined and possibly comprised mixed protonation states or perhaps tautomers. Regardless, the CD measurements of all compounds 1−5 are more reliable determinants of absolute configuration, as they were made on dilute solution (6.0 ± 0.5 mm). Antifungal Microbroth Dilution Assay. A clear 96-well plate was prepared with a serial dilution of 10 μL of drug in DMSO and 190 μL of Difco Antibiotic Medium #3 (BD).14 A 2-fold dilution series was prepared from 100 μL of the stock solution across 12 wells for each compound tested. Overnight liquid cultures of C. albicans ATCC 14503, C. neoformans var. gattii, C. glabrata, and C. krusei were diluted to an OD of 0.01−0.02, and an aliquot (100 μL) of the diluted culture was added to each well and made up with medium to a final volume of 200 μL. Measurements of OD (λ = 600 nm) of each well were conducted after a further incubation (35 °C, 48 h). Nonlinear regression15 was applied to fit the OD data to a sigmoidal curve and extract MIC50 and MIC90 values. Preparation of Crambescin A2 Hydrochloride Salts. The TFA salts of 3 (2.2 mg) and 4 (1.3 mg) were separately dissolved in 0.1 M HCl (200 μL) and dried under reduced pressure. The residues were redissolved in H2O (200 μL) and dried under reduced pressure to give 3·HCl: colorless glass; [α]D +28 (c 1.0, MeOH); 4·HCl: colorless glass; [α]D +20 (c 1.0, MeOH). (+)-Crambescin A2 392 (1): colorless glass, TFA salt; [α]D +10 (c 0.10, MeOH); UV (MeOH) λmax 209 nm (ε log10 3.49), 288 (3.74); FTIR (ATR, ZnSe plate) ν 3356, 3188, 2920, 2850, 1710, 1662, 1633, 1590, 1379, 1350, 1131, 1092, 1030, 1006 cm−1; CD (MeOH, c 2.28 × 10−4 M) λ 248 nm (Δε +1.92), 289 (+0.83); 1H and 13C NMR, see Table 1 (acetone-d6); HRESITOFMS m/z 393.2970 [M + H]+ (calcd for C20H37N6O2, 392.2973).
EXPERIMENTAL SECTION
General Experimental Procedures. Optical rotations were measured on a JASCO P-2000 at the D-double emission line of Na. UV−vis spectra were measured on a JASCO V-630 spectrometer. FTIR spectra were collected on thin film samples using a JASCO FTIR-4100 fitted with an ATR accessory (ZnSe plate). CD spectra were measured on a JASCO J-810 spectropolarimeter in quartz cells (1 or 5 mm path length). Inverse-detected 2D NMR spectra were measured on a JEOL ECA (500 MHz) spectrometer, equipped with a 5 mm 1H{13C} 5 mm probe, or a Bruker Avance III (600 MHz) NMR spectrometer with a 1.7 mm 1H{13C} microcryoprobe. 13C NMR spectra were collected on a Varian NMR spectrometer (125 MHz) equipped with a 5 mm Xsens 13C{1H} cryoprobe. NMR spectra were referenced to residual solvent signals (acetone-d6) (δH 2.05, δC 29.8). High-resolution ESITOF analyses were carried out on an Agilent 1200 HPLC coupled to an Agilent 6350 TOFMS. Low-resolution MS measurements were made on a Thermoelectron Surveyor UHPLC coupled to an MSQ single-quadrupole detector. HPLC was performed on an Agilent 1200 HPLC. Optical densities of yeast cultures (OD, λ 600 nm) in microplate wells were measured using a Molecular Devices Spectramax 384 Plus. Animal Material. The sponge Pseudaxinella reticulata (07-08-058) was collected in 2007 at Sweetings Cay, Bahamas, at a depth of 17 m. A voucher sample of the sponge is archived at UC San Diego. Extraction and Isolation. The frozen lyophilized sponge, 07-08058 (51 g wet; 9.29 g dry wt) was extracted with MeOH (2 × 300 mL, 12 h), the combined MeOH extracts were concentrated, and the water content was adjusted to approximately 1:9 H2O−MeOH prior to repeated extraction with hexane (300 mL × 2). Concentration of the hexane-soluble layer gave fraction A (0.506 g). The aqueous-MeOH D
DOI: 10.1021/np501052a J. Nat. Prod. XXXX, XXX, XXX−XXX
Journal of Natural Products
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(+)-Crambescin A2 406 (2): colorless glass, TFA salt; UV (MeOH) λmax 209 (ε log10 3.51), 288 (3.80); FTIR (ATR, ZnSe plate) ν 3369, 1681, 1442, 1204, 1138, 845, 803, 727 cm−1; CD (MeOH, c 2.24 × 10−4 M) λ 247 (Δε +2.22), 289 (+0.90); 1H and 13C NMR, see Table 1 (acetone-d6); HRESITOFMS m/z 407.3131 [M + H]+ (calcd for C21H39N6O2, 407.3129). (+)-Crambescin A2 420 (3): colorless glass, TFA salt; [α]D +21 (c 1.0, MeOH); CD (MeOH, c 2.97 × 10−4 M) λ 248 (Δε +2.34) 289 (+1.02); 1H NMR spectra were consistent with published data;6 HRESITOFMS m/z 421.3291 [M + H]+ (calcd for C22H42N6O2, 421.3286). (+)-Sch 575948 (4): colorless glass, TFA salt; [α]D +18 (c 1.0, MeOH); CD (MeOH, c 3.57 × 10−4 M) λ 248 (Δε +1.52), 289 (+0.63); 1H NMR spectra were consistent with published data;7 LRESIMS m/z 364.4 [M + H]+. Molecular Mechanics Calculations. The molecular structure of a truncated analogue (i) of compound 1 was energy minimized at the molecular mechanics level (MMFF, iSpartan, Wavefunction, Irvine, CA, USA), and the lowest energy conformers were determined by Monte Carlo searching (see Figure 4).
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Romminger, S.; Morais, R. P.; Bandeira, K.; Mizuno, C. M. Nat. Prod. Rep. 2010, 27, 1871−1907. (c) Berlinck, R. G. S. Nat. Prod. Rep. 1999, 16, 339−365. (3) Chang, L.; Whittaker, N. F.; Bewley, C. A. J. Nat. Prod. 2003, 66, 1490−1494. (4) Dalisay, D. S.; Saludes, J. P.; Molinski, T. F. Bioorg. Med. Chem. 2011, 19, 6654−6657. (5) Makarieva, T. N.; Ogurtsova, E. K.; Denisenko, V. A.; Dmitrenok, P. S.; Tabakmakher, K. M.; Guzii, A. G.; Pislyagin, E. A.; Es’kov, A. A.; Kozhemyako, V. B.; Aminin, D. L.; Wang, Y.-M.; Stonik, V. A. Org. Lett. 2014, 16, 4292−4295. (6) Mai, S. H.; Nagulapalli, V. K.; Patil, A. D.; Truneh, A.; Westley, J. W. Marine compounds as HIV inhibitors. U.S. patent WO9301193 (A1), January 21, 1993. (7) Yang, S.-W.; Chan, T.-M.; Pomponi, S. A.; Chen, G.; Wright, A. E.; Patel, M.; Gullo, V.; Pramanik, B.; Chu, M. J. Antibiot. 2014, 56, 970−972. (8) The original name “crambine” is here changed to “crambescin”, as proposed by Rinehart (see ref 10), to resolve ambiguity with the trivial name of a previously described peptide: Van Etten, C. H.; Nielsen, H. C.; Peters, J. E. Phytochemistry 1965, 4, 467−473. Following the convention of Rinehart, the names of crambescin-like molecules are given a suffix corresponding to the unitary molecular mass and “A2”, which refers to the bicyclic alkaloid structure with the shorter, four-carbon, N-alkyl-guanidino side chain. (9) Snider, B.; Shi, Z. J. Org. Chem. 1993, 58, 3828−3839. (10) Jares-Erijman, E. A.; Ingrum, A. A.; Sun, F.; Rinehart, K. L. J. Nat. Prod. 1993, 56, 2186−2188. (11) Bondu, S.; Genta-Jouve, G.; Leiros, M.; Vale, C.; Guigonis, J.M.; Botana, L. M.; Thomas, O. P. RSC Adv. 2012, 2, 2828−2835. (12) We cannot exclude the possibility that compounds 1−5 are partially racemic. (13) (a) O’Malley, D. P. Total Synthesis of Dimeric Pyrrole-Imidazole Alkaloids, Ph.D. Thesis, Scripps Research Institute, 2008. (b) Ma, Z.; Wang, X.; Wang, X.; Rodriguez, R. A.; Moore, C. E.; Gao, S.; Tan, X.; Ma, Y.; Rheingold, A. L.; Baran, P. S.; Chen, C. Science 2014, 346, 219−224. (14) CLSI. Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts. Approved Standard 3rd ed.; CLSI document M27-A3; Clinical and Laboratory Standards Institute: Wayne, PA, 2008. (15) Brown, A. M. Comput. Methods Programs Biomed. 2000, 65, 191−200.
ASSOCIATED CONTENT
* Supporting Information S
1
H and 13C NMR and 2D NMR spectra of 1 and 2. MS-MS fragmentation data for 1−4. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*Tel: +1 (858) 534-7115. Fax: +1 (858) 822-0386. E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank E. Rogers and C. Skepper for assistance with collection of Pseudaxinella reticulata, and S. Zea (Universidad Nacional de Colombia, InveMar) for taxonomic identification. We would like to thank the government of the Bahamas for permission to collect samples through an unnumbered permit for operations in their territorial waters, and J. R. Pawlik (University of North Carolina, Wilmington) and the captain and crew of the R/V Seward Johnson II for expedition logistics. We are grateful to Y. Su for measurements of HRMS, B. Duggan and A. Mrse (UCSD) for assistance with NMR measurements, and two anonymous reviewers for helpful suggestions. The 500 MHz NMR spectrometer and the HPLC TOF mass spectrometer were purchased with funds provided by the NSF (Chemical Research Instrument Fund, CHE0741968) and the NIH (Shared Instrument Grant, S10RR025636), respectively. This work was supported by a grant from the NIH (AI1007786).
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DEDICATION Dedicated to Dr. William Fenical of Scripps Institution of Oceanography, University of California−San Diego, for his pioneering work on bioactive natural products.
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REFERENCES
(1) Berlinck, R. G. S.; Braekman, J. C.; Daloze, D.; Hallenga, K.; Ottinger, R. Tetrahedron Lett. 1990, 31, 6531−6534. (2) The literature on guanidine alkaloids has been extensively reviewed. For example, see the recent reviews: (a) Berlinck, R. G. S.; Trindade-Silva, A. E.; Santos, M. F. C. Nat. Prod. Rep. 2012, 29, 1382− 1406. (b) Berlinck, R. G. S.; Burtoloso, A. C. B.; Trindade-Silva, A. E.; E
DOI: 10.1021/np501052a J. Nat. Prod. XXXX, XXX, XXX−XXX
