Note Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX
pubs.acs.org/jnp
Myrmenaphthol A, Isolated from a Hawaiian Sponge of the Genus Myrmekioderma Stephen M. Parrish,† Ram P. Neupane,† Mary Kay Harper,‡ John Head,† and Philip G. Williams*,† †
Department of Chemistry, University of Hawaii at Manoa, Honolulu, Hawaii 96822, United States Department of Medicinal Chemistry, University of Utah, Salt Lake City, Utah 84112, United States
‡
Downloaded via NOTTINGHAM TRENT UNIV on August 28, 2019 at 21:01:16 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.
S Supporting Information *
ABSTRACT: Four compounds (1−4) were isolated from a Hawaiian sponge of the genus Myrmekioderma. Myrmenaphthol A (1) incorporates two unusual elements into an oxidized steroidal core: a naphthyl AB-ring system and a hydroxy group at C-2. A comparison of the experimental and predicted electronic circular dichroism (ECD) spectra of 1 assigned an S configuration to the lone stereocenter (ΔESI = 0.75; similarity factor 0.8137). Known compounds, cinanthrenol A (2), 3,4-dihydroxypregna-5,17-diene-10,2-carbolactone (3), and 3,4-dihydroxypregna-5,20-diene-10,2-carbolactone (4), were also isolated. Despite literature reports of competitive inhibition at nanomolar levels for 2, neither 2 nor the structurally related 1 showed any activity against estrogen receptors at the concentrations tested.
A
13, -18, -17, and -16, and HMBC correlations from H3-20 to C-12, -14, and -17 allowed the assembly of fragment 2. Furthermore, HMBC correlations from H-1 to C-2, -3, -5, and -9 helped establish the phenol A ring, whereas correlations from H-11 to C-8, -9, and -10 connected fragments 1 and 2 to form the naphthyl nucleus. Correlations from H-7 to C-14 extended the ring system to generate fragment 3. At this point, the only remaining pieces in our chemical inventory were a nonprotonated carbon (δC 149.6, C-15), an oxygen, a hydrogen, and one degree of unsaturation. Therefore, the only existing option was to insert an enol moiety between carbons 14 and 16 while closing the ring to create structure 1. The relative configuration was determined through the analysis of rotating frame nuclear Overhauser effect spectroscopy (ROESY) correlations. A ROESY correlation between H3-20 and H-19 determined the configuration of the pregnane’s C-17 double bond to be E. Further ROESY correlations from H3-20 to H-11b suggest a cis orientation, and correlations from H3-20 to H-12a but not H-12b suggest that the methyl group adopts a conformation in which it is pseudoaxial. This is supported by the MM2 minimized energy conformer shown in Figure 2. The absolute configuration of 1 was determined through electronic circular dichroism (ECD) spectroscopy using Timedependent density functional theory (TDDFT) calculations, which have become a commonly used technique for determining the absolute configuration of various natural products.9 Conformational analysis of 1, using Monte Carlo multiple minimization (MCMM) and the OPLS-2005 force field in MacroModel with subsequent optimization in Gaussian 09 at the M06-2X/6-31+G level,10 identified four conformers within 5 kcal/mol of the lowest energy conformer, which
lthough there is large diversity of reported terpenoid and steroidal compounds isolated from marine invertebrates, few derivatives of the pregnane class of compounds have been reported,1−4 with the most recent being a set of pregnane-10,2carbolactones isolated by our group.5 Oxidations of the A ring into phenol pregnane derivatives are even rarer and lead to range of biological activities.6,7 Initial screening of a Hawaiian sponge belonging to the genus Myrmekioderma, collected off the southern coast of Lana‘i, displayed moderate activity ̅ against Herpes Simplex Virus-1 (HSV-1) and strong activity against Vesicular Stomatitis Indiana Virus (VSV) at 200 μg/ mL. When extracted on a larger scale, all fractions failed to reproduce this activity. Therefore, a chemical investigation of the sponge was conducted, resulting in the isolation of myrmenaphthol A (1) and known compounds 2−4.8,5 Myrmenaphthol A (1) was isolated as an amorphous yellow powder after multiple rounds of chromatography of the 75% MeOH fraction. High-resolution mass spectrometry (HRMS) indicated the presence of a protonated molecule with m/z 307.1135. This datum is consistent with the molecular formula C20H18O3, indicating 12 degrees of unsaturation. The 13C nuclear magnetic resonance (NMR) spectrum displayed 14 sp2 noncarbonyl carbons, and the heteronuclear multiple bond correlation (HMBC) spectrum displayed an additional sp2 carbon with a chemical shift indicative of an α,β-unsaturated keto group (δC‑16 190.9). Therefore, the molecule has seven double bonds, a keto group, and four rings to account for the degrees of unsaturation. The structure of 1 was assembled by analyses of correlation spectroscopy (COSY) and HMBC correlations (Figure 1). H6 showed a COSY correlation to H-7 as well as HMBC correlations to C-4, -5, -7, -8, and -10 to help establish part of fragment 1. This fragment was completed on the basis of a COSY correlation from H-4 to H-3. A COSY correlation between H-11 and H-12, HMBC correlations from H-19 to C© XXXX American Chemical Society and American Society of Pharmacognosy
Received: July 17, 2019
A
DOI: 10.1021/acs.jnatprod.9b00665 J. Nat. Prod. XXXX, XXX, XXX−XXX
Journal of Natural Products
Note
Figure 1. Key HMBC and COSY correlations of 1.
Figure 2. Relative configuration of 1. (A) Nuclear Overhauser effect (NOE) correlation from H3-20 to H-19 is evidence that the configuration of the C-17 olefin is E. (B) MM2 minimized energy conformer of the S enantiomer of 1, as seen looking down the carbonyl bond.
differed only in the direction of the two oxygen−hydrogen bonds. Figure 2B shows one of the minimized energy conformers of the 13S enantiomer looking down the carbonyl bond. The two conformers with the α-hydroxy proton in position to hydrogen bond to the carbonyl accounted for 99.7% of the conformers. The primary difference between these two conformers was the orientation of the O−H bond at C-2. (See Table S1.) A comparison with the experimental NMR data revealed no obvious contradictions in terms of expected coupling constants or chemical shifts validating the major conformers. TDDFT11 calculations were performed using Gaussian 0912 at the B3LYP13−16/6-31+G17,18 and the B3LYP/aug-cc-pVDZ19 levels to predict ECD spectra for the conformers of the 13S enantiomer. Finally, the Boltzmannweighted ECD spectrum of these conformers, calculated using SpecDis,20 was compared with the experimental data. These data (Figure 3) were in good agreement based on ΔESI = 0.75,20 with similarity factors of 0.8137 and 0.0647 for the 13S and 13R enantiomers, respectively. In an effort to obtain more of 1 from adjacent fractions, the inspection of the 50% MeOH fraction revealed multiple peaks with related chromophores at 280 nm. The collection of these peaks yielded 2 as a pure compound and both 3 and 4 as mixtures. Compound 2 shared many of the same proton resonances as 1. Perhaps the greatest similarity was that 2 displayed resonances consistent with the 2-naphthol spin system. HRMS provided a protonated molecule with m/z of 291.1386, in agreement with the molecular formula of C20H18O2 and 12 degrees of unsaturation. This coupled to two additional aromatic doublets facilitated our dereplication efforts to establish 2 as the known compound cinanthrenol A, previously isolated from a Cinachyrella sp. sponge.8
Figure 3. Predicted and observed ECD spectra of 1.
On the basis of HRMS and NMR data, 3 and 4 were determined to be 3,4-dihydroxypregna-5,20-diene-10,2-carbolactone (3) and 3,4-dihydroxypregna-5,17-diene-10,2-carbolactone (4). Compounds 3 and 4 had been previously isolated from Strongylophora sp. (Haplosclerida).5 Myrmenaphthol A (1) displays an uncommon naphthyl nucleus in a traditional steroid ring system, which is the first example of such a system isolated from a marine source. One of the few terrestrial examples of this moiety appears in equilenin,21 a potent estrogen receptor activator and a minor component of the FDA-approved drug Premarin. A major difference between 1 and nearly all similar natural products lies in the position of the oxidation of the A ring. Equilenin, like many known biological hormones such as testosterone and estradiol, is oxidized at C-3, whereas 1 has oxidation on the two-position. This rare oxidation of a steroid nucleus is only known to occur in cinanthrenol A, which was isolated simultaneously with 1. Previous studies have shown 2 to have affinity for estrogen receptors. Cinanthrenol A (2)8 was formerly isolated from a sponge dredged from the depths of the East China Sea, Japan. The phenanthracene compound displayed cytotoxicity against P-388 and He La cells with IC50 values of 4.5 and 0.4 μg/mL, respectively.8 Its strongest affinity was to estrogen receptor (ER1), which displayed competitive inhibition against estradiol with an IC50 of 10 nM8 in a radioactive receptor binding assay. The synthesis of ent-(+)-cinanthrenol A has recently been published, confirming the absolute configuration of cinanthrenol A and providing a pathway to synthesize structural B
DOI: 10.1021/acs.jnatprod.9b00665 J. Nat. Prod. XXXX, XXX, XXX−XXX
Journal of Natural Products
Note
Table 1. NMR Spectroscopic Data for 1 (1H 500 MHz,
13
C 125 MHz, CD3OD) 1
position
δC, type
1 2 3 4 5 6 7 8 9 10 11a 11b 12a 12b 13 14 15 16 17 18 19 20
106.8, CH 157.1, C 119.5, CH 131.1, CH 129.6, C 127.2, CH 124.6, CH 128.3, C 131.8, C 134.7, C 24.8, CH2 33.0, CH2 40.7, C 143.1, C 149.6, C 190.9, Ca 144.1, C 130.7, CH 14.8, CH3 22.3, CH3
δH (J in Hz)
COSY
HMBC (1H to 13C)
ROESY
7.33, d (2.2)
H-3
2, 3, 5, 9
H2-11
7.09, dd (8.8, 2.2) 7.70, d (8.8)
H-1, H-4 H-3
2, 5 1, 2, 5, 6, 9, 10
H-4 H-3
7.64, d (8.6) 8.07, d (8.6)
H-7 H-6
4, 5, 7, 8, 10 5, 8, 9, 10, 14
H-7 H-6
3.32, 3.22, 2.68, 1.82,
H-12b H2-12 H-11b, H-12b H2-11, H-12a
8, 9, 10, 12, 13 8, 9, 10, 12 9, 11, 13, 14 11, 13, 20
H-1, H-11 H-1, H-11, H-20 H-11b, H-12, H-19, H-20 H-12a
H-19 H-18
13, 16, 17, 19 13, 16, 17, 18 12, 13, 14
H-19 H-12a, H-18, H-20 H-11b, H-12a, H-19
m m dd (13.0, 6.1) td (12.5, 6.4)
6.63, q (7.5) 2.05, d (7.5) 1.31, s
a
Carbon chemical shifts determined from the HMBC experiment.
analogues, but no biological testing was reported.22 Because of 1’s similar framework to 2, testing of 1 and 2 against ER1 was conducted in parallel. Surprisingly, no significant inhibition was displayed for either compound, with only 30% inhibition at 100 μM against the ER-α binding domain. Indeed, multiple rounds of testing in ER biochemical assays failed to show any significant activity in our hands for either 1 or 2 as an agonist or an antagonist despite the nanomolar activity previously reported for 2.
■
skeleton is parchment-like and readily detachable, and it consists of a tangential layer of a smaller category of strongyles (∼265 × 9.8 μm). The choanosomal skeleton is largely confused with ascending tracts of larger strongyles (∼555 × 9.5 μm) among disorganized spicules including fine oxea/strongyles (∼325 × 2.5 μm) and abundant raphides in two sizes of trichodragmata (∼52.5 and 80 μm). All megascleres are smooth, and centrally curved or occasionally flexuous. The common Indo-Pacific species M. granulatum has larger megascleres and only one category of raphides. This specimen differs from another undescribed species of Myrmekioderma from Hawaii5 by producing mucus and having only smooth megascleres and a smaller size second category of raphides. Extraction and Isolation. Lyophilized sponge (100g) was extracted with 1:1 MeOH/CH2Cl2 three times overnight to yield 5.77 g of organic extract. The combined extracts were then subjected to a modified Kupchan partitioning using only MeOH, hexanes, and CH2Cl2. The CH2Cl2 partition (177.5 mg) was dry loaded on C8 silica gel and was subjected to a solid phase extraction procedure consisting of four steps of increasing MeOH/H2O content (0, 25, 50, 75, and 100%) and an isopropanol wash. Both the 50% MeOH and the 75% MeOH fractions contained the compounds of interest. The 50% fraction was subjected to reversed-phase HPLC on a Phenomenex column (Luna C18; 250 × 10 mm, 5 μ) using a flow rate of 2.8 mL/min and a concentration gradient of 40−80% (MeCN in H2O) over 20 min. This afforded pure compounds 1 (tR = 15.8 min, 0.6 mg, 0.01% yield), 2 (tR = 16.1 min, 1 mg, 0.02% yield), and 4 (tR = 9 min, 2.2 mg, 0.04% yield) along with 3 (tR = 8 min, 2.3 mg, 0.04% yield). Purity was assessed by NMR and determined to be 95, 88, 77, and 85% for 1, 2, 3, and 4, respectively. Myrmenaphthol A (1):(0.6 mg, 0.01% yield): yellow amorphous powder; [α]22D −79 (c 0.2, MeOH); ECD (0.05 mg/mL, MeOH), λmax (Δε) 380 (−89.5), 333 (22.6), 288 (44.8), 262 (34.8), 242 (−69.7), and 217 (133.5) nm; UV (MeOH) λmax (log ε) 362 (3.75), 240 (4.14), 203 (4.23) nm; IR (CaF2) νmax 3396, 1667, 1597, 1218 cm−1; NMR data, see Table 1; HRESI-TOFMS m/z 307.1335 [M + H]+ (calcd for C20H19O3, 307.1329). ER-α Assay. The assay was conducted through Thermo Fisher Scientific’s SelectScreen biochemical nuclear receptor profiling service employing the LanthaScreen TR-FRET Coregulator assay and using
EXPERIMENTAL SECTION
General Experimental Procedures. Optical rotations were measured on a Jasco DIP-370 digital polarimeter at the sodium line (589 nm). UV absorbances were measured on a Varian Cary 50 Bio UV−vis spectrophotometer. ECD spectra were recorded on a Jasco J815 CD spectrometer. IR spectroscopy was measured as a thin film on a CaF2 disk using a Shimadzu IRAffinity-1 Fourier-transform infrared (FTIR) spectrophotometer. 1H and 13C NMR and 2D NMR experiments on the natural products were carried out on a Varian Unity Inova at 500 MHz spectrometer. NMR spectra were referenced to the appropriate residual solvent signal (δH 3.30, δC 49.0 for CD3OD). The Heteronuclear single quantum coherence (HSQC) experiments were optimized for 1JC,H = 140 Hz and HMBC experiments were optimized for 3JC,H = 7 Hz. High-resolution mass spectrometric data were obtained using the electrospray ionization (ESI) source in positive mode on an Agilent 6545 QTOF apparatus. A binary gradient Shimadzu Prominence high-performance liquid chromatography (HPLC) system with an evaporative light scattering detector (ELSD) and a photodiode array (PDA) detector was used for all separations. Sponge Collection and Identification. An undescribed species of Myrmekioderma (class Demospongiae, order Axinellida, family Heteroxyiidae) (450 g) was collected in 40−60 fsw off the southern shore of Lana‘i at the second Cathedral (roughly 20°44′05.0″N, ̅ 156°55′25.7″W). The encrusting sponge has fingery projections and a smooth surface, produces clear mucus, and is cream colored in life and in spirit, and the interior has a pulpy consistency. The ectosomal C
DOI: 10.1021/acs.jnatprod.9b00665 J. Nat. Prod. XXXX, XXX, XXX−XXX
Journal of Natural Products
Note
17-β-estradiol as the stimulant. An in-house assessment of the ER inhibition was performed using Indigo Biosciences Human ER-α, reporter assay system with the same positive control. Computational Analysis. Conformers within 5 kcal/mol of the lowest energy conformer were searched using the MCMM method and the OPLS-2005 force field in MacroModel10 (Schrödinger). Each conformer within 5 kcal/mol of the lowest energy conformer was optimized in Gaussian 0912 at the M06-2X/6-31+G level, and the geometries of all conformers with similar energies were checked for redundancy. Density functional theory (DFT) was used to perform calculations, which were carried out in Gaussian 09. All ground-state geometries were optimized at the B3LYP/6-31+G level. The same DFT level was employed to evaluate the effects of solvation in MeOH using the SCRF/PCM method.23,24 TDDFT25,11 calculations at the B3LYP13−16/6-31+G17,18 and the B3LYP/aug-cc-pVDZ19 levels were conducted to calculate the electronic excitation energies and rotational strengths in MeOH. A Boltzmann-weighted ECD spectrum was calculated using SpecDis20 (σ = 0.24 eV at 5 nm shift; scaling factor 0.2369) for comparison with the experimentally determined data recorded in methanol at 0.05 mg/mL.
■
(7) Kittakoop, P.; Suttisri, R.; Chaichantipyuth, C.; Vethchagarun, S.; Suwanborirux, K. J. Nat. Prod. 1999, 62, 318−320. (8) Machida, K.; Abe, T.; Arai, D.; Okamoto, M.; Shimizu, I.; de Voogd, N. J.; Fusetani, N.; Nakao, Y. Org. Lett. 2014, 16, 1539−1541. (9) Nugroho, A. E.; Morita, H. J. Nat. Med. 2014, 68, 1−10. (10) MacroModel; Schrödinger, LLC: New York, 2019. (11) Runge, E.; Gross, E. K. U. Phys. Rev. Lett. 1984, 52, 997−1000. (12) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.; Mennucci, B.; Petersson, G. A.; Nakatsuji, H.; Caricato, M.; Li, X.; Hratchian, H. P.; Izmaylov, A. F.; Bloino, J.; Zheng, G.; Sonnenberg, J. L.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Vreven, T.; Montgomery, J. A., Jr.; Peralta, J. E.; Ogliaro, F.; Bearpark, M.; Heyd, J. J.; Brothers, E.; Kudin, K. N.; Staroverov, V. N.; Kobayashi, R.; Normand, J.; Raghavachari, K.; Rendell, A.; Burant, J. C.; Iyengar, S. S.; Tomasi, J.; Cossi, M.; Rega, N.; Millam, J. M.; Klene, M.; Knox, J. E.; Cross, J. B.; Bakken, V.; Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Martin, R. L.; Morokuma, K.; Zakrzewski, V. G.; Voth, G. A.; Salvador, P.; Dannenberg, J. J.; Dapprich, S.; Daniels, A. D.; Farkas; Foresman, J. B.; Ortiz, J. V.; Cioslowski, J.; Fox, D. J. Gaussian 09, revision C.01; Gaussian, Inc.: Wallingford, CT, 2009. (13) Becke, A. D. J. Chem. Phys. 1993, 98, 5648−5652. (14) Vosko, S. H.; Wilk, L.; Nusair, M. Can. J. Phys. 1980, 58, 1200− 1211. (15) Stephens, P. J.; Devlin, F. J.; Chabalowski, C. F.; Frisch, M. J. J. Phys. Chem. 1994, 98, 11623−11627. (16) Lee, C.; Yang, W.; Parr, R. G. Phys. Rev. B: Condens. Matter Mater. Phys. 1988, 37, 785−789. (17) Krishnan, R.; Binkley, J. S.; Seeger, R.; Pople, J. A. J. Chem. Phys. 1980, 72, 650−654. (18) McLean, A. D.; Chandler, G. S. J. Chem. Phys. 1980, 72, 5639− 5648. (19) Dunning, T. H., Jr. J. Chem. Phys. 1989, 90, 1007−1023. (20) Bruhn, T.; Schaumloffel, A.; Hemberger, Y.; Bringmann, G. Chirality 2013, 25, 243−249. (21) Bachmann, W. E.; Cole, W.; Wilds, A. L. J. Am. Chem. Soc. 1940, 62, 824−839. (22) Zhu, L.; Tong, R. J. Antibiot. 2016, 69, 280−286. (23) Cammi, R.; Tomasi, J. J. Comput. Chem. 1995, 16, 1449−1458. (24) Miertus̃, S.; Tomasi, J. Chem. Phys. 1982, 65, 239−245. (25) Gross, E. K. U.; Dobson, J. F.; Petersilka, M. Top. Curr. Chem. 1996, 181, 81−159.
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.9b00665. Copies of the 1H, 13C, and 2D NMR spectroscopic data for all new compounds, calculated conformers of 1, and a photo of the producing organism (PDF)
■
AUTHOR INFORMATION
Corresponding Author
*Tel. 808-956-5720. Fax: 808-956-5908. E-mail: philipwi@ hawaii.edu. ORCID
Philip G. Williams: 0000-0001-8987-0683 Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS We thank Dr. S. Cao for obtaining the ECD spectrum and W. Yoshida for help in obtaining NMR spectra. This work was funded by grants from the National Institute on Aging (5R01AG039468-03). Funds for the upgrades of the NMR instrumentation were provided by the CRIF program of the National Science Foundation (CH E9974921) and the Elsa Pardee Foundation. The purchase of the Agilent LC−MS was funded by grant 1532310 from the National Science Foundation. We gratefully acknowledge the advanced computing resources provided by the University of Hawaii Information Technology Service Cyberinfrastructure.
■
REFERENCES
(1) Corgiat, J. M.; Scheuer, P. J.; Rios Steiner, J. L.; Clardy, J. Tetrahedron 1993, 49, 1557−1562. (2) D’Auria, M. V.; Minale, L.; Riccio, R. Chem. Rev. 1993, 93, 1839−1895. (3) Kerr, R. G.; Baker, B. J. Nat. Prod. Rep. 1991, 8, 465−497. (4) Blackman, A. J.; Heaton, A.; Skelton, B. W.; White, A. H. Aust. J. Chem. 1985, 38, 565−573. (5) Dai, J.; Yoshida, W. Y.; Kelly, M.; Williams, P. J. Nat. Prod. 2016, 79, 1464−1467. (6) Tomono, Y.; Hirota, H.; Imahara, Y.; Fusetani, N. J. Nat. Prod. 1999, 62, 1538−1541. D
DOI: 10.1021/acs.jnatprod.9b00665 J. Nat. Prod. XXXX, XXX, XXX−XXX
