Article pubs.acs.org/jnp
Peroxide Natural Products from Plakortis zyggompha and the Sponge Association Plakortis halichondrioides−Xestospongia deweerdtae: Antifungal Activity against Cryptococcus gattii Matthew T. Jamison,† Doralyn S. Dalisay,†,‡ 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: Cryptococcus gattii is a human pathogen and causative agent of a pernicious, sometimes fatal, disseminated fungal disease. Investigation of antifungal extracts of the marine sponge association Plakortis halichondrioides−Xestospongia deweerdtae and the sponge Plakortis zyggompha from the Bahamas led to the discovery and isolation of 6-epi-7,8-dihydroplakortide K (1), plakortide AA (2), and three new plakinic acids, N−P (4−6; unstable 1,2-dioxolanes bearing benzyl-substituted conjugated dienes), along with known plakinic acids L, K, and M.5 Chiroptical comparisons and DFT calculations of 13C NMR chemical shifts were used to assign the absolute stereostructure of 4. The stereospecific base-promoted rearrangement−saponification of 1 to 10 was briefly investigated and showed tight kinetic control and stereospecific formation of the new C-2 stereocenter with inversion at C-3. Plakinic acid M and plakortides 9 and 11 exhibited antifungal activity against C. gattii (MIC90 = 2.4 to 36 μM), but plakinic acids N−P were inactive under the same conditions.
T
from the Bahamas revealed antifungal plakinic acids I−M,5a,b compounds containing 1,2-dioxolane and 1,2-dioxanes embedded in long-chain, ω-phenylcarboxylic acids. Here, we report 6epi-7,8-dihydroplakortide K (1)6b and plakortide AA (2) from P. zyggompha, and from P. halichondroides−X. deweerdtae, plakinic acid M (3) and three unstable 1,2-dioxolanes, plakinic acids N−P (4−6), containing conjugated benzyl-substituted 1,3-dienes.13 Labile peroxides from Plakortis have been noted earlier, for example, compound 7, first described by Stierle and Faulkner from P. halichondrioides,14 although it had degraded before complete characterization. Recently, Andersen and coworkers reported methyl capucinoate A (8) and related peroxides from Dominican species of P. halichondrioides and Plakinastrella onkodes without comment on their stabilities.15 The antifungal structure−activity relationships of 1−3 and the plakortide acids 9, 11, and 10rearrangement product of 1 were explored by an in vitro assay against a panel of pathogenic fungi.
he human fungal pathogen Cryptococcus gattii has been responsible for an expanding number of outbreaks of human diseaseprimarily, pneumonia and disseminated mycoses of the central nervous systemin British Columbia, Canada, the Pacific Northwest, and, recently, Southern California.1,2 Cryptococcosis caused by this emergent pathogen is especially pernicious because the causative agent is acquired environmentally from both soil and certain tree species and can cause disease in healthy individuals as well as immunocompromised patients. Between 1999 and 2007, 218 regional cases of cryptococcal pneumonia caused by C. gattii were reported, including 19 deaths, a case-fatality rate of 8.7%.3 The environmental reservoirs of pathogenic molecular types VGIII MATα and MATa of C. gattii in Southern California were recently identified through DNA sequencing of samples and bioinformatic analysis.1b In our search for antifungal leads from marine sponges that inhibit Cryptococcus spp., we investigated extracts of Bahamian sponges that exhibited antifungal activity. Marine sponges within the genera Plakortis and Plakinastrella are consistent sources of cyclic peroxide-containing natural products (both 1,2-dioxolanes and 1,2-dioxanes) that exhibit antiparasitic,4 antifungal,5 antiplasmodial,6 antibacterial,7 and cytotoxic8 properties. Previous research on Plakortis zyggompha (de Laubenfels, 1934) led to several classes of nonperoxide polyketides including volatile ketones,9 isospiculoic acid,10 (+)-zyggomphic acid,11 and the peroxide plakortide Q.12 Our earlier analyses of antifungal extracts of the symbiotic sponge association Plakortis halichondrioides−Xestospongia deweerdtae © XXXX American Chemical Society and American Society of Pharmacognosy
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RESULTS AND DISCUSSION
A MeOH−CH2Cl2 extract from of Plakortis zyggompha, collected at Plana Cays Bahamas, was redissolved in MeOH− Special Issue: Special Issue in Honor of John Blunt and Murray Munro Received: October 23, 2015
A
DOI: 10.1021/acs.jnatprod.5b00951 J. Nat. Prod. XXXX, XXX, XXX−XXX
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of 1 (5% KOH, EtOH−H2O, 0 °C) was carried out and delivered two isomeric carboxylic acids in a 2:1 ratio. The major product 9 was the expected carboxylic acid, as shown by analysis of the MS and 1H and 13C NMR spectra. The NMR spectra of the minor product revealed a new stereocenter and hydroxy substituent at C-2 (δC 72.6, δH 4.16) that was integrated into a contiguous H-2−H-5 spin system (COSY). An HMBC cross-peak between H-3 (δH 3.75) and C-6 (δC 88.2) secured the tetrahydrofuran structure 10. Exposure of 2 to the same conditions gave the corresponding homologues: carboxylic acids 11 and 12. Faulkner and co-workers obtained a similar tetrahydrofuran upon treatment of plakortin with NaOMe in MeOH and proposed a base-catalyzed mechanism with a transient C-2−C3 epoxide. 17 In our hands, the reaction of 1 was diastereoselective: compound 10 was the only stereoisomer obtained. Faulkner and co-workers did not assign the C-2 configuration of their product, but we were sufficiently curious to investigate the stereoselectivity of the rearrangement of 1. The C-2 stereocenter in 10 was assigned as 2S by Kusumi’s phenylglycine methyl ester (PGME) method18 (Figures 1b, and
H2O (9:1), and the solution was progressively partitioned against hexanes, CH2Cl2, and n-BuOH. The hexanes-soluble fraction was separated by successive silica gel flash chromatography, guided by an antifungal bioassay, to yield a fraction enriched in plakortide methyl esters. Reversed-phase HPLC (C18) was used to obtain pure 6-epi-7,8-dihydroplakortide K (1) and plakortide AA (2). The HRESITOFMS m/z 595.4183 [M2 + Na]+ of 1 indicated a molecular formula of C16H30O4, and the 1H NMR spectrum contained a diasterotopic methylene (δH 3.02, 1H, dd, J = 15.6, 9.6 Hz and δH 2.36, 1H, dd, J = 15.6, 3.3 Hz) characteristic of H2-2 in 1,2-dioxolane natural products.5a The presence of three methyl triplets (δH 0.91, t, J = 7.4; δH 0.88, t, J = 7.4; δH 0.85, t, J = 7.4) indicated a diethyl-substituted 1,2-dioxane appended to an unbranched pentyl side chain. The 1H NMR chemical shift of the C-4 methine (δH 4.48, ddd, J = 3.3, 5.2, 9.0 Hz) had been shown earlier by Faulkner and co-workers14,17 to be characteristic of the H-4eq−H-5ax orientation found in plakortin. The relative configuration of 1 was supported by analysis of the NOESY spectrum. The specific rotation of 1 ([α]D −191) is similar in magnitude but of opposite sign of that of plakortin ([α]D +189), suggesting a 3S*,4S*,6R* relative configuration of 1. Hydrogenation of 1 followed by modified Mosher’s analysis16 of the resulting diol established the absolute configuration of 1 as 3S,4S,6R (Figure S1, Supporting Information). Compound 2 showed a protonated molecule in the mass spectrum 14 amu higher than 1, but an 1H NMR spectrum almost identical with that of 1; thus 2 is the higher homologue of 1 with an extended alkyl chain. While the fresh MeOH extract of P. zyggompha exhibited strong antifungal activity, after prolonged storage in MeOH and fractionation, none of the fractions or isolated compounds were active. We suspected adventitious Fischer esterification had occurred on storage in solvent and that the methyl ester products were inactive. Plakortides undergo facile esterification when stored in MeOH, as has been noted previously.10 In order to recover the active plakortide carboxylic acids, saponification
Figure 1. (a) Proposed mechanism of selective rearrangement− hydrolysis of 1 to 10. (b) Assignment of the configuration of 10 from Δδ values of R- and S-phenylglycineamide methyl esters18 (PGME, Δδ = δ(S-PGME) − δ(R-PGME); see Supporting Information).
S2). The diastereoselectivity of the rearrangement arises from tightly orchestrated tandem reactions. Base-promoted formation of i, the kinetic Z-enolate of 1,19 followed by intramolecular attack of the electrophilic peroxide upon the si face of the CC bond of i generates an incipient epoxide, ii, which undergoes “rebound” SN2 attack of the liberated alkoxide upon the epoxide ring to form 10 with inversion of configuration at C-3. We propose that the same relative configuration arises in the corresponding base-promoted rearrangement of plakortin.17 The unambiguous stereoassignments of 1 and 10 reveal two remarkable features of this “Faulkner rearrangement”. Electrophilic capture of peroxide by syn-facial attack of the si face of ZB
DOI: 10.1021/acs.jnatprod.5b00951 J. Nat. Prod. XXXX, XXX, XXX−XXX
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enolate i (Figure 1) is kinetically controlled; it occurs faster than equilibration to the thermodynamically more stable Eenolate or rotation around the C-2−C-3 bond. The next step is committed: rebound capture of the epoxide is slower, of necessity, to allow rotation of the C-3−C-4 bond of iia and alignment of the C-3−O bond (Figure 1, iib) for the correct trajectory of SN2 back-side attack by alkoxide and exo-trig20 ring opening of the epoxide with inversion of C-3 to give iii. Subsequent hydrolysis of iii delivers 10. Although formation of ii is familiarit is analogous to the rebound step in the mechanism of formation of α,β-epoxy ketones and esters by treatment of α,β-unsaturated carbonyl compounds with alkaline alkylhydroperoxidethe stereospecificity and coordination of both steps are notable. A second sponge with antifungal activity, P. halichondrioides− X. deweerdtae, was extracted with CH2Cl2 instead of MeOH in order to suppress the spontaneous esterification observed previously. Following the same solvent partition described for P. zyggompha, a hexane-soluble fraction was obtained that retained antifungal activity and was separated by successive silica gel flash chromatography to yield a fraction enriched in plakinic acids. The mixture was separated by reversed-phase HPLC (Phenyl-Hexyl and pentafluorophenyl (PFP) solid supports) to obtain known compound plakinic acid M (3)5a,21 and other plakinic acids5 as colorless oils. The CH2Cl2-soluble fraction was subjected to gel filtration (Sephadex LH-20) to give a fraction containing trace amounts of new unsaturated plakinic acids, 4−6. Rapid decomposition of the latter compounds was observed after HPLC purification (gradient H2O−0.1% CF3COOH−CH3CN). In order to minimize decomposition, the organic solvent was removed under reduced pressure, the remaining aqueous solution reextracted with EtOAc and concentrated, and the resulting compound characterized immediately.22 The 1H NMR spectrum of plakinic acid N (4, C24H34O4; HRESITOFMS m/z 385.2389 [M − H]−) showed two deshielded signals, H-10 (δH 5.64, 1H, s) and H-12 (δH 5.43, 1H, t, J = 7.3 Hz), indicating a substituted 1,3-diene. HMBC correlations from an exceptionally deshielded methylene H-13 (δH 3.44, 2H, d, J = 7.3 Hz)both benzylic and allylicto C12 (δC 127.6) and C-11 (δC 133.6) allowed placement of the substituted 1,3-diene at the homobenzylic position. Correlations from H-10 (δH 5.64, 1H, s) to C-11, C-9 (δC 134.1), C-23 (δC 17.7), and C-24 (δC 17.3) verified the locations of the methyl branches. The 9E,11E geometry of the conjugated diene was confirmed through NOESY correlations from H-10 to H-8 (δH 1.84, 1H, dd, J = 12.7, 7.6 Hz; 1.97, 1H, dd, J = 12.7, 6.4 Hz) and H-13 to H-24 (δH 1.82, 3H, s). Analysis of COSY correlations assembled the remainder of alkyl chain spin system, H-6 (δH 1.65, dd, J = 14.4, 3.7 Hz; 1.44, dd, J = 14.4, 8.3 Hz), H-7 (δH 1.85, m), and H-8 and H-22 (δH 0.90, d, J = 6.3 Hz). Plakinic acid N (4) lacked the characteristic methine (δH, 4.45) of 3; instead the presence of two methyl singlets, H20 (δH 1.47, 3H, s) and H-21 (δH 1.37, s), was compatible with a 1,2-dioxolane (peroxide) ring; the latter was confirmed by HMBC correlations from H-4 (δH 2.48, 2.21, 2H) to C-3 (δC 83.3), C-5 (δC 87.2), C-20 (δC 23.5), and C-21 (δC 24.3). The relative configuration of the Me groups located at C-3 and C-5 within the 1,2-dioxolane ring of 4 was shown to be syn by NOESY correlations from H-4b (δH 2.21) to both methyl signals, H-20 (δH 1.47, s) and H-21 (δH 1.37, s), while H4a (δH 2.48) showed only one correlation to H-20. Finally, a longrange correlation observed from the remaining methylene H-2
(δH 2.77, 2.70) to C-3 and C-1 (δC 175.2) allowed completion of the constitution of the carboxylic acid 4. Plakinic acid O (5, C25H34O4; m/z 397.2388 [M − H]−) has a mass that differs from 4 by 12 amu; this is consistent with a higher homologue of 4 containing an additional double-bond equivalent. Comparison of the 1H NMR data of 4 and 5 (Table Table 1. 1H NMR Dataa for 1 and 2 (CDCl3; δ 1H, mult (J Hz)) no. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 a
1
2
3.02, dd (15.6, 9.6) 2.36, dd (15.6, 3.3) 4.48, ddd (9.0 3.3, 5.2) 2.15, m 1.54, m 1.26,b m
3.02, dd (15.6, 9,6) 2.70, dd (15.6, 3.1) 4.48, ddd (9.1, 4.6, 3.1) 2.15, d (12.4) 1.54, m 1.26,b m
1.43, m 1.26,b m 1.26,b m 1.26,b m 1.30, m 0.88, t (7.4) 1.14, dddd (7.4, 7.4, 7.4, 14.2) 1.21, m 0.91, t (7.4)
1.41, m 1.26,b m 1.26,b m 1.26,b m 1.26,b m 1.30, m 0.88, t (7.4)
1.55, m 2.03, dddd (7.4, 7.4, 7.4, 14.4) 0.85, t (7.4) 3.70, s
1.14, dddd (7.4, 7.4, 7.4, 14.1) 1.21, m 0.91, t (7.4) 1.52, 2.03, 0.85, 3.71,
m dddd (7.4, 7.4, 7.4, 14.3) t (7.4) s
600 MHz. bOverlap.
2) revealed a similar conjugated 1,3-diene (H-11, δH 5.63, s; H13, δH 5.42, t, J = 7.4 Hz) and dimethyl-substituted 1,2dioxolane spin system (H-21, δH 1.46, s; H-22, δH 1.38, s). Two new deshielded vicinally coupled vinyl signals (H-2, δH 6.07, d, J = 15.8 Hz, and H-3, δH 7.04, d, J = 15.8 Hz) both showed HMBC correlations to C-1 (δC 170.0) and are consistent with an α,β-unsaturated carboxylic acid similar to plakortide E23 (7, δH 6.07 and 6.85, δC 166.9). The large coupling constant between H-2−H-3 (J = 15.6 Hz) defined the 2E configuration. Plakinic acid P (6, HRESITOFMS m/z 383.2232 [M − H]−) is a lower homologue of 5. Examination of the 1H NMR spectrum of 6 revealed only one vinyl methyl signal, H-24 (δH 1.69, 3H, s), and an additional vinyl proton signal, H-12 (δH 6.30, dd, J = 15.0, 10.9 Hz). A 10E,12E-conjugated diene was assigned based on the large vicinal coupling between H-13 (δH 5.71, ddd, J = 15.0, 7.0, 7.0 Hz) and H-12 (δH 6.30, dd, J = 15.0, 10.9 Hz) and an NOESY cross-peak between H-11 and the vinyl methyl group H-24 (δC 1.69, s). The relative configuration of the 1,2-dioxolane in 6 was confirmed through observation of NOESY cross-peaks from H-5b (δH 2.29) to both of the methyl groups, H-21 and H-22. A contiguous spin system in 6 arising from H-14 (δH 3.42, t, J = 7.1 Hz), H-13 (δH 5.71, ddd, J = 15.0, 7.0, 7.0 Hz), H-12, and H-11 verified the same carbon framework of 5, except for the absence of the C-12-Me signal. Assignment of the configurations of remote alkyl branch points in saturated acyclic polyketide chains is challenging. C
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Table 2. 1H NMRa Data for 3−6 (CDCl3; δ 1H, mult (J Hz)) no. 1 2
3 4a 4b 5a 5b 6
7
3 3.01, dd (15.8, 9.2) 2.50, m 4.45, ddd (8.8, 4.8, 4.5) 2.45, m
4
5
2.77, d (14.9)
6.07, d (15.8)
6.06, d (15.8)
7.04, d (15.8)
7.06, d (15.8)
2.43, d (12.3) 2.29, d (12.3)
2.43, d (12.3) 2.29, d (12.3)
1.57, dd (14.9, 4.1) 1.42, dd (14.9, 8.1)
1.57, dd (14.4, 4.1) 1.42, dd (14.4, 8.3)
1.97, dd (12.8, 6.5) 1.84, dd (12.8, 7.8)
1.97, dd (13.2, 6.7) 1.84, dd (13.2, 7.7)
5.63, s
5.77, d (10.9) 6.30, dd (15.0, 10.9) 5.71, ddd (15.0, 7.0, 7.0) 3.43, d (7.0)
2.48, d (12.4) 2.21, d (12.4)
1.65, dd (14.4, 3.7) 1.44, dd (14.4, 8.3) 1.85, m
1.31, m 8
1.65, m
9
1.31, m
1.84, dd (12.7, 7.6) 1.97, dd (12.7, 6.4)
1.14, m
a
10 11 12
1.29b 1.29b 1.29b
5.64, s
13
1.29b
3.44, d (7.3)
14 15 16 17 18 19 20 21 22 23 24 25 26 27
1.29b 1.29b 1.29b 1.29b 2.60, t (7.7) 7.24, 7.29, 7.22, 7.29, 7.24, 0.87, 1.39, 0.92,
5.43, t (7.3)
m t (8.2) m t (8.2) m d (6.8) s d (6.8)
6
2.70, d (14.9)
1.47, m 1.44, m
1.48, m
As might be expected, the largest differences in 13C chemical shift [Δδ = δ(S) − δ(R), Table S5] occur for C-5 (Δδ = +1.6 ppm), C-6 (+2.5 ppm), and C-8 (+5.0 ppm). Peroxides 5 and 6 have δC values that correspond closely to those of 4 from C-1 to C-10 (see Table 3, Δδ < 0.3 ppm); therefore, both compounds share the same configuration at the exocyclic stereocenter (8R). This configuration is consistent with the corresponding Me branch point, 8R in 13 (established independently by liposomal ECD5), and reasonable from biosynthetic considerations. The absolute configurations of 4−6 were addressed by chiroptical comparisons with known natural products and published values of [α]D of synthetic compounds. The ECD spectra of 5 and 6 exhibited a positive Cotton effect of moderate magnitude (λ 234 nm, Δε +2.9 and +2.6, respectively), but the ECD spectra of 3 or 4 were essentially baseline. The latter observations demonstrate the dominant influence of the 1,2-dioxolane ring within the first sphere of asymmetry (C-4) upon the π−π* electronic transition of the α,β-unsaturated carboxylic acid; however, this alone was insufficient to assign absolute configurations to 5 and 6. An examination of [α]D of chiral 1,2-dioxolane and 1,2-dioxanes of well-defined configuration allows deconvolution of the molecular contributions to sign and magnitude according to van’t Hoff’s rule of optical superposition.26 Compounds 4−6 were optically active, with [α]D values (Table 4) of the same positive sign and similar magnitudes, indicating they shared the same absolute configuration of the substituted 1,2-dioxolane ring. The replacement of a terminal acetic acid (e.g., 4) with acrylic acid (5 and 6) does not change the sign of [α]D. Sun and co-workers prepared all four diastereomers of 3-(4′,6′diethyl(dioxolan-3′-yl)acrylic acids 14a−c (Figure 2) and their enantiomers through total synthesis27 and used their specific rotations to unambiguously assign the stereostructure of natural plakortide E methyl ester (14).23 Dussalt and co-workers prepared two series of stereodefined “plakinates” (3′dioxolanyl-substituted acetic methyl esters 15a−c,28 Figure 2) and compared their specific rotations to assign the absolute configurations in plakinic acid A (15)29 and 15d, an unnamed analogue.30 The specific rotations of stereoisomers 13a−c are dominated by the C-4 and C-6 configurations (or C-3 and C-5 in 15a−d), but the signs are insensitive to stereocenters remote from the dioxolane ring. In Dussalt’s compounds 15a−d, the sign of rotation is invariant between carboxylic acids and their methyl esters or by replacement of the n-C16H33 chain at C-5 with a more complex ω-phenyl-unsaturated substituent (15d). By employing a diradical ring opening of cyclopropanes and subsequent asymmetric capture by molecular oxygen, Tian and co-workers synthesized stereodefined trans-3,5-dimethyl- and diethyl-1,2-dioxolanes (including plakinic acid F and epiplakinic acid F) and reported chiroptical trends for [α]D that parallel the foregoing observations.31 It is reasonable, therefore, to conclude from the specific rotations of 4−6 ([α]D = +92, +84, and +82, respectively)being comparable to 14 and 14c ([α]D = +87.0 and +75.0, respectively)that the absolute configurations are uniform: (3S,5S)-4 and the (4S,6R) configuration for 5 and 6 (note change in CIP priorities). Using these observations, we can now also assign the configurations of the known plakinic acids C and D methyl esters, first reported by Davidson from a Fijian Plakortis sp., as (3S,5S).8d The surprising finding shows an inversion of conf iguration at the two stereocenters within the dioxolane rings of 4−6 with
5.42, t (7.4) 3.44, d (7.4)
7.20, 7.29, 7.19, 7.29, 7.20, 1.47, 1.37, 0.90, 1.73, 1.82,
d (7.5) t (7.6) t (7.2) t (7.6) d (7.5) s s d (6.3) s s
7.20, 7.29, 7.18, 7.29, 7.20, 1.46, 1.38, 0.89, 1.71, 1.81,
d (7.6) t (7.6) t (7.3) t (7.6) d (7.6) s s d (6.4) s s
7.20, 7.29, 7.19, 7.29, 7.20, 1.47, 1.38, 0.89, 1.69,
d (7.1) t (7.8) t (6.5) t (7.8) d (7.1) s s d (6.4) s
600 MHz. bOverlap.
Earlier, we had developed liposomally ordered electronic circular dichroism (L-ECD)5 for independent assignment of the absolute configuration of remote methyl-branched stereocenters in plakinic acids K and L (e.g., C-8 of plakinic acid L, 13).5a Unfortunately, the lability of 4−6 precluded further chemical manipulations. Instead, we were able to successfully assign the configuration of C-7 using density functional theory (DFT) predictions of 13C NMR chemical shifts by gaugeindependent atomic orbital (GIAO) calculations.24 MMFF and DFT minimized conformations of both 7S and 7R epimers of 4 were calculated (Gaussian 09; see Supporting Information), and 13C chemical shifts were calculated and then averaged by Boltzmann weightings. Goodman DP4 probabilities were evaluated for the two possibilities.25 The 7R configuration of 4 was predicted with high confidence (96.4%) over its epimer. D
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Table 3. 13C NMR Data for 1−6 (CDCl3) no. 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 26 27 a
1a 172.2, 31.3, 78.6, 34.3, 33.0, 82.4, 36.3, 22.2, 22.5, 32.4, 14.0, 25.2, 11.1, 24.6, 7.5, 51.9,
C CH2 CH CH CH2 C CH2 CH2 CH2 CH2 CH3 CH2 CH3 CH2 CH3 CH3
2a 172.2, 31.3, 78.6, 34.4, 33.0, 82.4, 36.4, 29.8, 22.5, 22.6, 31.7, 14.0, 25.2, 11.1, 24.7, 7.5, 51.8,
C CH2 CH CH CH2 C CH2 CH2 CH2 CH2 CH2 CH3 CH2 CH3 CH2 CH3 CH3
3a 176.8, 31.7, 79.5, 27.9, 37.2, 81.7, 48.5, 28.4, 38.8, 27.1, 29.7, 29.7, 29.8, 29.9, 30.0, 29.5, 31.7, 36.0, 142.3, 128.4, 128.2, 125.5, 128.2, 128.4, 17.2, 21.6, 21.9,
4b
C CH2 CH CH CH2 C CH2 CH CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 C CH CH CH CH CH CH3 CH3 CH3
175.2, 43.8, 83.3, 57.6, 87.2, 45.4, 27.7, 49.6, 134.1, 130.7, 133.6, 127.6, 34.5, 141.4, 128.3, 128.4, 125.8, 128.4, 128.3, 23.5, 24.3, 20.9, 17.7, 17.3,
C C CH2 C CH2 CH CH2 C C C C CH2 C CH CH CH CH CH CH3 CH3 CH3 CH3 CH3
5b 170.0, 117.9, 152.9, 84.1, 58.2, 87.1, , 44.9, 27.6, 49.4, 134.0, 130.6, 133.6, 127.6, 34.4, 141.3, 128.3, 128.4, 125.7, 128.4, 128.3, 23.6, 24.5, 20.8, 17.6, 17.2,
6b C C C C CH2 C CH2 CH CH2 C C C C CH2 C CH CH CH CH CH CH3 CH3 CH3 CH3 CH3
171.0, 117.8, 152.9, 84.0, 58.1, 87.1, 44.8, 27.6, 48.9, 135.4, 126.4, 127.6, 130.7, 39.3, 140.6, 128.5, 128.4, 126.0, 128.4, 128.5, 23.7, 24.6, 21.0, 16.4,
C C C C CH2 C CH2 CH CH2 C C C C CH2 C CH CH CH CH CH CH3 CH3 CH3 CH3
125 MHz. bIndirect detection (HSQC, HMBC).
respect to the co-occurring plakinic acids J (16) and L (13).5a We measured the specific rotations of known plakinic acids I− K (17, 16, 18) isolated from this sample of P. halichondriodes− X. deweerdtae (collected in 2011, Table 4) and found the signs and magnitudes were essentially identical to the samples first characterized from a different specimen of the same sponge5a collected in the same vicinity in 2008. While antipodal endoperoxide configurations have been reported in compounds obtained from Plakortis spp. collected from different geographical locations, our remarkable result is best explained if the biosynthesis of 4−6, with complex unsaturated side chains, diverges significantly from the stereospecific oxidation that generates the cyclic peroxide moiety in n-alkyl-substituted and ω-phenylalkyl-n-plakinic acids (e.g., 13 and 16) that are more commonly found in Plakortis spp. Interestingly, the one invariant stereocenter in plakortides and plakinic acids appears to be the first Me branch (C-7 or C-8), which is consistently R. From a biosynthetic perspective, it is noteworthy that 5 and 6 retain a “complete” polyketide chain, whereas most 1,2dioxolanes in the series (e.g., 4) appear to have undergone oxidative loss of one carbon from the carboxyl terminus with respect to 1,2-dioxanes (e.g., 3, 7,14 and 815). The loss of C1 observed in 4 and most other 1,2-dioxolanes, but not in 5 and 6 or 1,2-dioxanes, suggests that assembly of five-membered-ring peroxides may not be an obligatory consequence of oxidative cleavage. Regardless, it appears that the biosynthesis of all members of the series can be rationalized as originating from a phenylacetic acid starter unit, probably derived from phenylalanine, followed by conventional iterative ketide extensions (C2, acetate), namely, condensation of the growing chain with malonyl
CoA and loss of CO2, and occasional replacements by methylmalonyl CoA (C3, propionate). While methyl branches in polyketides may originate from alternate polyketide processing (methylation by SAM), the occurrence of ethylbranched members (e.g., 1, 2) is more compatible with a variation of the former mechanism, specifically, ketide extension by ethylmalonyl CoA (C4, butyrate). The antifungal properties of compounds 1−6 and 9−11 against a panel of pathogenic fungi, Candida albicans, Cryptococcus gattii, Cryptococcus grubii, and Candida krusei (Table 5), were investigated. As expected, the carboxylic acids 9 and 11 were active against all four strains of Candida and Cryptococcus (MIC90 = 2.7−36 μM) tested, while the corresponding methyl esters 1 and 2 were inactive (MIC90 > 100 μM). Plakinic acid M (3) was the most active of all peroxides tested (MIC90 = 2.7 μM, against fluconazole-resistant C. krusei), significantly more so than the ω-phenyl higher homologues 17 and 18. The rearranged product 10 was not active against C. albicans or C. gattii, demonstrating that both the endoperoxide and carboxylic acid functionalities are necessary for antifungal activity. A recent report showed that plakortide P (19) and dihydrofuran 20 cause mitotic arrest at G2/M and G0/G1 transitions, respectively, in the cell cycle of cultured human cancer cells, while the corresponding 1,2dioxolane was inactive. 8a One interpretation of these observations is that 1,2-dioxanes and 1,2-dioxolanes exhibit independent modes of action for cytotoxic and antifungal activities. When assayed against fungi, plakinic acids 4−6 were inactive, possibly due to decomposition during prolonged incubation. E
DOI: 10.1021/acs.jnatprod.5b00951 J. Nat. Prod. XXXX, XXX, XXX−XXX
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Table 4. Specific Rotations of Plakortis Peroxides and Synthetic Analogues (See Figure 2) cmpd 1 3 4 5 6 13 14 14a 14b 14c 15a 15b 15c 15d 15 16 17 18
[α]D
c (g/100 mL), solvent
−191 −137 −112 +92 +84 +82 −26.2 +75.1a +87.0b −86.0 −74.8 +75.0 −a +25b −23f −14f −14 −58a −43 −38 −113 −101c −113 −85
1.0, CHCl3c 1.31, CHCl3e 0.86, CHCl3d 0.54, CHCl3d 0.12, CHCl3d 0.43, CHCl3d 1.9, CHCl3e 2.23, CHCl3 0.85, CHCl3 0.26, CHCl3 0.39, CHCl3 0.15, CHCl3 1.0, CH2Cl2 1.0, CH2Cl2 1.0, CH2Cl2 −, − 1.15, − 0.044, CHCl3e 1.0, CHCl3d 0.044, CHCl3e 1.0, CHCl3d 3.39, CHCl3e 1.0, CHCl3d
comment, reference b
5a c c c c
plakinic acid L5a 23 27 27 27 unnamed30 28 28 28 g,28
plakinic acid A29 plakinic acid J5a c
plakinic acid I5a plakinic acid K5a c
a
Natural. bSynthetic. cThis work. dRedistilled HPLC grade, stabilized with pentenes (50 ppm). eSpec. grade, stabilized with “amylenes”. f Normalized from measured values to 100% ee. gPlakinic acid A synthetic stereoisomer of undefined side-chain configuration.
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CONCLUSION Two new plakortides (1 and 2) and four new plakinic acids (4−6) were isolated from the Bahamian marine sponges P. zyggompha and P. halichondrioides−X. deweerdtae, respectively. Compound 10 arises from base-promoted rearrangement of 1, a reaction that has a biomimetic analogy to Faulkner’s rearrangement that links plakortin to ring-contracted tetrahydrofuran natural products from Plakortis spp. Compounds 5 and 6 contained a previously unobserved combination of an α,β-unsaturated carboxylic acid and a benzyl-1,3-butadiene terminus. Compounds 3, 9, and 11 exhibited modest activity against C. gattii and showed that the presence of carboxylic acid and peroxide functional groups is obligatory for antifungal activity.
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Figure 2. Plakortide E methyl ester (14),23 synthetic stereoisomers (14a−c27), plakinic acid A (15),29 and synthetic compounds 15a−d.28
Table 5. Antifungal Activity (MIC90, μM)a of Peroxides 1−6 and 9−11 C. alb.b 1 2 3 4 5 6 9 10 11
EXPERIMENTAL SECTION
>100 >100 13 ± 2 >100 >100 >100 27 ± 1 >100 22 ± 2
C. gattiic >100 >100 2.4 ± 0.3 >100 >100 >100 36 ± 3 >100 9.6 ± 0.4
C. grubiic
C. kruseie
g
g
g
g
3.4 ± 0.2
2.7 ± 0.5
26 ± 3
10 ± 1
g
6.0 ± 0.5
g
4.3 ± 0.7
a
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. ECD spectra were measured on a JASCO J-810 spectropolarimeter in quartz cells (1 or 5 mm path length) at 23 °C. FTIR spectra were collected on thin film samples using a JASCO FTIR-4100 fitted with an ATR accessory (ZnSe plate). Inverse-detected 2D NMR spectra were measured on a JEOL ECA (500 MHz) spectrometer, equipped with a 5 mm 1H{13C} room-temperature probe, or a Bruker Avance II (600 MHz) NMR spectrometer with a 1.7 mm 1H{13C/15N} 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 (CDCl3, δH 7.26, δC 77.00 ppm). High-resolution ESITOF analyses were carried out on an Agilent 1200 HPLC coupled to an Agilent 6350
Antifungal activity was measured by the microbroth dilution assay following the protocol published by the Clinical and Laboratory Standards Institute (CLSI)35 using antibiotic medium 3 (Difco). b Candida albicans ATCC 14503. cCryptococcus gattii. cCryptococcus grubii. eCandida krusei. gNot tested. TOFMS at the Small Molecule MS Facility (UCSD). Low-resolution MS measurements were made using a Thermoelectron Surveyor UHPLC coupled to an MSD single-quadrupole detector. Semipreparative HPLC was performed on an Agilent 1100 HPLC or a Jasco system comprising dual pumps (PU-2086) and a mixed UV−vis detector (UV-2075) in tandem with an ELSD detector (Softa-A model 300). Candida krusei was obtained from a patient isolate from the Clinical Services, Microbiology, University of California Davis Health F
DOI: 10.1021/acs.jnatprod.5b00951 J. Nat. Prod. XXXX, XXX, XXX−XXX
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plakinic acid L (13, 4.6 mg, tR = 12.8 min).5b Fraction 4 (32 mg) was purified by reversed-phase HPLC (Phenomenex Luna Phenyl-Hexyl column, 250 × 10 mm, 2.5 mL/min; mobile phase H2O (0.1% TFA)− CH3CN, gradient 90−95% CH3CN over 20 min) to yield plakinic acid I5b (17, 3.6 mg, tR = 10.1 min) and plakinic acid K5a (18, 3.4 mg, tR = 10.5 min). Fraction 5 (89 mg) was purified by reversed-phase HPLC (Phenomenex Luna Phenyl-Hexyl column, 250 × 10 mm, 2.5 mL/ min; mobile phase H2O (0.1% TFA)−CH3CN, gradient 90−95% CH3CN over 20 min) to give a peak comprising a mixture of compounds (3.8 mg, tR = 11.5 min). Final reversed-phase HPLC purification (Phenomenex Kinetix PFP column, 250 × 4.6 mm, 1.3 mL/min; mobile phase H2O (0.1% TFA)−CH3CN, isocratic 35:65 CH3CN over 20 min, tR = 12.5 min) yielded pure plakinic acid M5a (3, 1.22 mg). Fraction B was separated by size-exclusion chromatography (Sephadex LH-20, MeOH) into 10 fractions, which were pooled according to TLC characteristics (visualized with p-anisaldehyde). Bioassay-guided separation located the most potent antifungal activity in fraction 17 (163 mg); the latter was separated by reversed-phase HPLC (Phenomenex Kinetix C18 column, 21.2 × 150 mm, H2O (0.1% TFA)−CH3CN, isocratic 35:65 over 40 min) to yield crude 4−6. Final reversed-phase HPLC purifications (Phenomenex Kinetix PFP column, 250 × 4.6 mm, 1.3 mL/min; mobile phase H2O (0.1% TFA)−CH3CN) provided 6 (520 μg, isocratic 50:50 over 30 min, tR = 22 min), 4 (680 μg, isocratic 50:50 over 30 min, tR = 23.5 min), and 5 (150 μg, isocratic 45:55 over 20 min, tR = 14.4 min). Plakinic acid M (3): colorless oil; [α]D −112 (c 0.86, CHCl3) [lit.5a −137 (c 1.31, CHCl3)]; FTIR (ATR, ZnSe plate) ν 2935, 2860, 1717, 1456, 1384, 1299, 1200, 1155 cm−1; 1H NMR (CDCl3), Table 2; 13C NMR (CDCl3), Table 3; HRESITOFMS m/z 431.3172 [M − H]− (calcd for C27H43O4, 431.3167). Plakinic acid N (4): colorless oil; [α]D +92 (c 0.54, CHCl3); UV (MeOH) λmax 235 (ε log10 3.45), 288 (3.80); Δε (MeOH) baseline, only; FTIR (ATR, ZnSe plate) ν 3369, 1681, 1442, 1204, 1138, 845, 803, 727 cm−1; 1H NMR (CDCl3), Table 2; 13C NMR (CDCl3), Table 3; HRESITOFMS m/z 385.2389 [M − H]− (calcd for C24H33O4, 385.2384). Plakinic acid O (5): colorless oil; [α]D +84 (c 0.12, CHCl3); UV (MeOH) λmax 236 nm (ε log10 3.80); ECD (c 6.10 × 10−4 M) λmax 234 nm (Δε +2.9); FTIR (ATR, ZnSe plate) ν 2935, 2860, 1717, 1456, 1384, 1299, 1200, 1155 cm−1; 1H NMR (CDCl3), Table 2; 13C NMR (CDCl3), Table 3; HRESITOFMS m/z 397.2388 [M − H]− (calcd for C25H33O4, 397.2384). Plakinic acid P (6): colorless oil; [α]D +82 (c 0.43, CHCl3); UV (MeOH) λmax 235 nm (ε log10 3.87); ECD (c 6.77 × 10−4 M, MeOH) λ 234 nm (Δε +2.6); FTIR (ATR, ZnSe plate) ν 2935, 2860, 1717, 1456, 1384, 1299, 1200, 1155 cm−1; 1H NMR (CDCl3), Table 2; 13C NMR (CDCl3), Table 3; HRESITOFMS m/z 383.2232 [M − H]− (calcd for C24H31O4, 383.2228). Preparation of Carboxylic Acids 9−12. Ester 1 (15.6 mg) was dissolved in EtOH−H2O (8:1, 500 μL) and cooled in an ice bath for 15 min before adding a 10% KOH solution (8:1 EtOH−H2O, 500 μL) and stirring for 1 h at 23 °C. The mixture was cooled in an ice−water bath for 15 min, then acidified with 10% aqueous citric acid. The solution was diluted with H2O (5 mL) and extracted with EtOAc (2×, 5 mL). The combined organic layers were dried and separated by flash chromatography (silica gel, elution with 10% EtOAc in hexanes) to obtain 9 (4.6 mg, 30% yield). Further elution (20% EtOAc in hexanes) gave 10 (2.0 mg, 13% yield). Compounds 11 and 12 were prepared from 2 using the same procedure. 2-((3S,4S,6R)-4,6-Diethyl-6-pentyl-1,2-dioxan-3-yl)acetic acid (9): colorless oil; [α]D −207 (c 0.48, MeOH); FTIR (ATR, ZnSe plate) ν 2960, 2935, 2873, 1711, 1462, 1384, 1299, 1247, 1005, 933 cm−1; 1H NMR (CDCl3) 4.46 (1H, ddd, J = 3.9, 5.1, 9.2), 3.06 (1H, dd, J = 9.7, 15.8), 2.41 (1H, dd, J = 3.4, 15.8), 2.18 (1H, m), 2.05 (1H, dddd, J = 7.4, 7.4, 7.4, 14.1), 1.56 (2H, m), 1.43 (1H, m), 1.32−1.20 (8H, m), 1.17 (1H, m), 0.93 (3H, t, J = 7.4), 0.90 (3H, t, J = 7.4), 0.88 (3H, t, J = 7.4); 13C NMR (CDCl3) 176.4 (C-1), 82.6 (C-6), 78.4 (C-3), 36.3 (C-7), 34.4 (C-4), 33.0 (C-5), 32.4 (C-10), 31.2 (C-2), 25.1 (C-12), 24.7 (C-14), 22.5 (C-9), 22.2 (C-8), 14.0 (C-11), 11.1 (C-13), 7.5 (C-
Care System. Cryptococcus grubii and Cryptococcus gattii were provided as gifts (courtesy of A. Gelli, University of California, Davis). Optical densities of yeast cultures (OD, λ = 590 nm) in microplate wells were measured using a UV−vis plate reader (SpectraMax Plus 384, Molecular Devices). Animal Material. The sponge Plakortis zyggompha (de Laubenfels, 1934) (11-20-032) was collected in 2011 from Plana Cays (22°36.320′ N, 73°33.360′ W), Bahamas, at depth of −34 m. The surface was smooth, of a yellow-green color. The tissue was a pliable, soft cushion texture, common to most Plakortis spp. Microscopic examination revealed the presence of diod spicules (
