Zampanolides BâE from the Marine Sponge Cacospongia

Article pubs.acs.org/jnp

Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX

Zampanolides B−E from the Marine Sponge Cacospongia mycofijiensis: Potent Cytotoxic Macrolides with MicrotubuleStabilizing Activity Taitusi Taufa,†,‡,# A. Jonathan Singh,‡,§ Chloe R. Harland,†,‡ Vimal Patel,‡,|| Ben Jones,‡,||,□ Tu′ikolongahau Halafihi,⊥ John H. Miller,‡,|| Robert A. Keyzers,*,†,‡ and Peter T. Northcote*,‡,§ †

School of Chemical and Physical Sciences, Victoria University of Wellington, Wellington 6012, New Zealand Centre for Biodiscovery, Victoria University of Wellington, Wellington 6012, New Zealand § Ferrier Research Institute, Victoria University of Wellington, Wellington 6012, New Zealand || School of Biological Sciences, Victoria University of Wellington, Wellington 6012, New Zealand ⊥ Ministry of Fisheries, Sopu, Tongatapu, Kingdom of Tonga

J. Nat. Prod. Downloaded from pubs.acs.org by RMIT UNIV on 10/29/18. For personal use only.

‡

S Supporting Information *

ABSTRACT: Four new compounds (2−5) structurally related to the microtubule-stabilizing agent (−)-zampanolide (1) have been isolated from the Tongan marine sponge Cacospongia mycof ijiensis. Three of these new structures, zampanolides B−D (2−4), exhibit nanomolar cytotoxicity toward the HL-60 cell line, are antimitotic, and induce in vitro tubulin polymerization at levels comparable to 1. Zampanolide E (5), saturated at C-8/C-9, was significantly less potent and does not stabilize purified tubulin, even at 10-fold higher concentrations. The structural differences across these compounds reveal a plasticity of the zampanolide pharmacophore. While unsaturation is required at Δ8, the configuration of this alkene and those of Δ4 and Δ4′ have little effect on tubulin polymerization. The first natural co-occurrence of 1 and (−)-dactylolide (6) from the same sponge extract is also noted.

I

5) from a Tongan collection of the sponge Cacospongia mycof ijiensis, from which the opportunity to investigate the structure−activity relationship of naturally occurring congeners is presented. We also note the first simultaneous occurrence of 1 and (−)-dactylolide (6) and provide further evidence for their biogenetic relationship.

n recent years, attention has been given to the marine natural product (−)-zampanolide (1), a highly unsaturated 20-membered macrolide, first reported from the marine sponge Fasciospongia rimosa and later from Cacospongia mycof ijiensis.1,2 Zampanolide binds to the luminal site of β-tubulin, adjacent to the paclitaxel-binding site, thus disrupting the function of the microtubule and arresting cells in mitosis.2,3 Irreversible covalent binding was postulated and later confirmed by mass spectrometry,3 which may be the mechanism by which 1 overcomes multidrug resistance developed from overexpression of the P-glycoprotein drug efflux pump. The recent report of a high-resolution crystal structure of 1 complexed with α,βtubulin confirmed the covalent binding via Michael addition of His-229 at C-9 of 1.4 Zampanolide’s distinctive effects on tubulin assembly and its activity profile compared with that of paclitaxel against several multidrug-resistant cancer cell lines5 suggest that this macrolide could be a valuable addition to the existing set of anticancer drugs that target microtubules. Total syntheses of (−)-1,6−9 its antipode (+)-1,10,11 and simplified analogues12 have been achieved, yet there are limited chemical variants with supporting biological data to strengthen the structure−activity profile of the zampanolide structural class. In this paper, we describe the NMR-guided isolation and structure elucidation of zampanolides B−E (2− © XXXX American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION

The marine sponge C. mycof ijiensis was collected from Cathedral Cave, ‘Eua in the Kingdom of Tonga. Methanolic extracts of the sponge were purified using a combination of reversed- (PSDVB) and normal-phase (silica gel) chromatography. Several known biologically active compounds, including dendrolasin, mycothiazole, latrunculin A, 6,7-epoxylatrunculin A, laulimalide, neolaulimalide, and isolaulimalide, were identified,13−18 which typifies the rich chemistry associated with this sponge. Zampanolide-enriched fractions were subjected to reversed-phase HPLC methods (C18, CH3CN/ H2O), which led to the isolation of 1, new compounds Received: July 31, 2018

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DOI: 10.1021/acs.jnatprod.8b00641 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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Chart 1

Table 1. 1H NMR (600 MHz, CDCl3) Data for Zampanolide (1) and Zampanolides B−E (2−5) δH, mult. (J in Hz) position 2 3 4 6a 6b 8 9 10a 10b 11 12a 12b 14a 14b 15 16 18a 18b 19 20 21 22 23 2′ 3′ 4′ 5′ 6′ OH NH

1 5.95, 7.65, 6.11, 3.03, 4.13, 5.93, 6.82, 2.24, 2.37, 3.28, 1.92, 2.14, 1.95, 2.08, 3.96, 5.19, 2.20, 2.41, 5.28, 5.44, 1.80, 4.73, 1.72, 5.44, 6.46, 7.41, 6.05, 1.86,

d (15.0) dd (15.0, 11.7) d (11.6) d (13.5) d (13.7) d (16.6) ddd (15.9, 9.6, 4.7) dd (15.0, 9.7) m br t (10.7) m d (13.4) m d (13.4) ddd (11.0, 8.1, 2.5) d (7.8) dd (13.5, 11.5) d (13.9) ddd (11.1, 5.6, 1.9) br t (6.7) s br s, 2H s d (11.0) t (11.3) dd (15.0, 11.8) dq (15.0, 6.9) d (6.8)

6.34, d (7.7)

2 5.86, d (15.4) 7.53, dd (15.2,11.7) 6.20, d (11.8) 3.33, d (14.2) 3.48, d (14.2) 5.98, d (16.2) 7.00, ddd (16.2, 8.7, 6.0) 2.29, m 2.35, m 3.32, m 1.97, t (12.3) 2.13, m 1.89, ma 2.14, m 3.89, ddd (10.7, 8.0, 2.6) 5.26, d (7.5) 2.23, dd (14.2, 11.3) 2.47, d (13.5) 5.15, ddd (11.6, 6.8, 1.6) 5.39, br t (7.3) 1.89, s 4.72 br s, 2H 1.60, s 5.43, d (11.5) 6.44, t (11.7) 7.41, dd (15.0, 11.5) 6.04, dq (14.5, 6.8) 1.86, d (6.8) 3.79, br s 6.57, d (7.4)

3 5.78, 7.39, 6.16, 3.34, 3.42, 6.26, 6.04, 2.46, 2.78, 3.33, 1.99, 2.15, 1.95, 2.09, 3.88, 5.21, 2.30, 2.52, 5.19, 5.37, 1.97, 4.71, 1.65, 5.42, 6.43, 7.41, 6.03, 1.86, 3.79, 6.61,

d (15.1) dd (15.2, 11.4) d (11.6) d (15.0) d (15.0) d (11.7) m m m m dd (12.6, 12.4) d (12.6) dd (14.7, 11.8) d (14.7) ddd (11.8, 8.2, 2.5) d (8.2) dd (14.3, 10.8) d (13.7) ddd (10.5, 6.5, 1.8) br t (7.1) s br s, 2H s d (11.1) t (11.3) dd (15.0, 11.5) m d (6.9) br s d (7.6)

4 5.94, 7.64, 6.11, 3.05, 4.11, 5.95, 6.82, 2.25, 2.37, 3.39, 1.93, 2.14, 1.95, 2.09, 3.96, 5.20, 2.19, 2.42, 5.47, 5.47, 1.81, 4.73, 1.72, 5.73, 7.21, 6.15, 6.15, 1.86, 3.72, 6.36,

d (15.0) dd (15.0, 11.7) d (11.7) dd (13.8, 5.2) dd (13.8, 5.2 d (15.8) ddd (15.8, 9.4, 4.5) m m m m d (13.2) m d (13.6) ddd (11.0, 8.1, 2.7) d (8.1) m m br d (8.3) br d (8.3) s br s, 2H s d (15.2) dd (15.2, 9.5) dd (11.5, 9.5) m d (5.3) br s m

5 5.90, 7.52, 6.16, 3.26, 3.43, 2.44, 1.72, 1.44, 1.44, 3.21, 1.93, 2.07, 1.94, 2.08, 3.92, 5.25, 2.29, 2.51, 5.24, 5.41, 1.87, 4.70, 1.68, 5.42, 6.44, 7.41, 6.05, 1.86, 3.69, 6.48,

d (15.3) dd (15.3, 11.7) d (11.7) d (14.2) d (14.2) m, 2H m m m m m d (13.2) m d (13.6) ddd (11.1, 8.4, 2.6) m dd (14.2, 11.2) d (14.2) m br d (11.3) s br s, 2H s d (11.3) t (11.3) dd (15.0, 11.3) dq (15.0, 7.0) appt. dd (7.0, 1.4)b d (3.8) d (8.0)

a

Obscured by H3-21 and H3-6′. bPartially obscured by H3-21.

zampanolides B−E (2−5), and the first natural occurrence of (−)-dactylolide (6). Zampanolide B (2) was obtained as a colorless, amorphous solid. Positive ion HRESIMS analysis for 2 provided a sodiated adduct at m/z 518.2514, suitable for a molecular formula of C29H37NO6 that requires 12 degrees of unsaturation and is isobaric with 1. The 13C NMR spectrum revealed all 29 carbons, and the multiplicity-edited HSQC experiment showed

23 protonated carbons, including three methyls (δC 18.8, 17.3, 16.2), six methylenes (δC 108.9, 53.6, 42.2, 41.1, 40.7, 40.3), and 14 methines (δC 146.2, 143.6, 141.3, 140.2, 132.7, 131.6, 128.3, 128.2, 120.2, 117.1, 77.7, 76.9, 76.0, 71.7). The six nonprotonated carbons were assigned to three carbonyl centers (δC 198.0, 167.7, 167.9) and three nonprotonated alkene carbons (δC 130.7, 143.7, 143.7), while the two remaining proton resonances (δH 6.57, 3.79) were designated B



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zampanolide macrolide core, namely, the C-1−C-5 pentadienoyl, C-7−C-9 α,β-unsaturated enone, C-11−C-15 tetrahydropyran, C-22 exomethylene, and C-1′−C-6′ hexadienoyl moieties (Figure 1). The configurations of Δ2, Δ8, Δ2′, and Δ4′ were determined to be 2E, 8E, 2′Z, and 4′E by J-analysis (3JH‑2,H‑3 = 15.4 Hz, 3JH‑8,H‑9 = 16.2 Hz, 3JH‑2′,H‑3′ = 11.5 Hz, and 3JH‑4′,H‑5′ = 15.0 Hz) and were supported by NOESY correlation data. Through-space correlations also identified Δ4 and Δ16 as E, with the former being opposite that of 1 and the only point of difference between the two structures. The relative configuration for 2 was determined using NOE correlation data, J-analysis, and chemical shift comparison with 1 (Figure 1). A 1,3-diaxial relationship was established between H-11 and H-15 in the tetrahydropyran ring. Furthermore, NOE correlations observed from H3-23 to both H-15 and H19 place the latter, like in 1, as oriented on the same molecular face. The stereochemical relationship between H-19 and H-20 was deduced based on near-identical chemical shifts and vicinal coupling constants to those of 1. From these arguments, the macrolide core of 2, alkene isomerization notwithstanding, is biogenetically identical to 1, and thus its absolute configuration is proposed to be 11S, 15S, 19S, 20S. HRESIMS analysis of zampanolide C (3) exhibited a [M + Na]+ ion at m/z 518.2513, consistent with the molecular formula C29H37NO6, again identical to 1 and 2. A cursory examination of the NMR spectroscopic data (Tables 1, 2, and S3) revealed the presence of the same N-acyl hemiaminal side chain, again suggesting any modification was restricted to the macrolide core. Relative to 1, the deshielded shift of H-8 (δH 6.26), shielded shift of H-9 (δH 6.04), and decreased vicinal coupling constant (3JH‑8,H‑9 = 11.7 Hz, cf. 16.2 Hz in 1) were all consistent with a change in alkene configuration and established 3 as the 8Z isomer of 1. NOE correlation data supported the same relative configuration as 1 and 2. Zampanolide D (4), with a molecular formula of C 29 H 37 NO 6 , is isobaric with 1−3. The spectroscopic similarities between 1 and 4 (Tables 1, 2, and S4) suggested no alkene isomerization about the macrolide portion and differed only in the chemical shifts corresponding to the hexadienoyl side chain. Analysis of the 1H NMR spectrum revealed the coupling constants of H-2′−H-5′ (3JH‑2′,H‑3′ = 15.2 Hz; 3JH‑4′,H‑5′ = 11.5 Hz) were different from the corresponding resonances in 1, warranting a change in alkene configuration to

as exchangeable. With 10 of the 12 degrees of unsaturation accounted for, 2 requires a bicyclic structure. Given the high degree of similarity of NMR data with 1 (Tables 1, 2, and S2), the planar structure of 2 was established Table 2. 13C NMR (150 MHz, CDCl3) Data for Zampanolide (1) and Zampanolides B−E (2−5) δC, mult. position

1

2

3

4

5

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 1′ 2′ 3′ 4′ 5′ 6′

167.0, C 120.3, CH 140.4, CH 125.4, CH 144.0, C 45.3, CH2 198.1, C 131.5, CH 146.5, CH 40.3, CH2 76.6, CH 41.0, CH2 143.7, C 40.7, CH2 76.0, CH 130.1, CH 132.1, C 42.0, CH2 71.5, CH 75.6, CH 23.7, CH3 109.3, CH2 16.8, CH3 167.7, C 117.0, CH 143.8, CH 128.3, CH 140.3, CH 18.8, CH3


167.8, C 119.7, CH 140.9, CH 126.5, CH 143.7, C 47.5, CH2 198.7, C 128.5, CH 144.6, CH 35.9, CH2 77.3,a CH 40.2, CH2 144.5, C 40.6, CH2 75.4, CH 130.3, CH 132.7, C 42.2, CH2 71.7, CH 76.3, CH 26.2, CH3 108.9, CH2 16.3, CH3 167.8, C 117.2, CH 143.6, CH 128.3, CH 140.1, CH 18.8, CH3


167.3, C 120.4, CH 140.4, CH 126.7, CH 142.8, C 47.2, CH2 208.7, C 42.4, CH2 22.3, CH2 35.7, CH2 79.1, CH 41.1, CH2 144.5, C 40.8, CH2 75.8, CH 130.4, CH 131.8, C 41.6, CH2 71.2, CH 76.1, CH 24.8, CH3 108.7, CH2 16.3, CH3 167.8, C 117.0, CH 143.7, CH 128.3, CH 140.2, CH 18.8, CH3

a

Partially obscured by solvent peak.

through direct comparison of spectroscopic data. COSY and HMBC data identified structural fragments associated with the

Figure 1. Key 2D NMR correlations used to establish the planar structure and relative configuration of zampanolide B (2). C



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2′E, 4′Z, which is diametrically opposite to that of 1−3. Again, the relative configuration of 4 was established to be the same as 1−3. Zampanolide E (8,9-dihydrozampanolide, 5) was isolated as a colorless, amorphous solid. HRESIMS analysis of 5 presented a sodiated adduct ion suitable for a molecular formula of C29H39NO6 that differs from 1−4 by an increase of H2. Detailed analysis of the 1D and 2D NMR data for 5 (Tables 1, 2, and S5) revealed the absence of the resonances attributed to Δ8 in 1, to be replaced by two methylenes, CH2-8 and CH2-9, along with the corresponding deshielded shift of adjacent carbonyl C-7 (δC 208.7, cf. δC 198.1 in 1). The configurations of the remaining double bonds in 5 were determined to be identical to those of 1 through J-analysis and NOE correlation data. The remainder of the planar and configurational structure of 5 remained as in parent compound 1. The opposing optical activity of naturally occurring zampanolide [(−)-1] and dactylolide [(+)-6] has perplexed many organic chemists since the latter was first reported in 2001.19 Smith and co-workers synthesized the unnatural antipode ent-zampanolide [(+)-1]10,11 and yielded (+)-6 through thermolytic cleavage of the N-acyl side chain in benzene. Conversely, Hoye20 and Jennings21,22 independently prepared (−)-1 from synthetic (−)-6. Collectively, their results clearly indicate the stereochemical relationship between the two compounds; the sign of rotation is conserved with chemical interconversion. In 2009, Uenishi and co-workers hypothesized the existence of antipode (−)-6 as the natural precursor to (−)-1,6 and while their experimental findings did not reveal the presence of (−)-6 in the extracts of their source (Fasciospongia rimosa), they could not conclusively disprove its existence. We report here the first isolation of (−)-1 and (−)-6 from the same sponge extract; their co-occurrence is supportive of a common biogenetic relationship and confirms the macrolide core to be of the same absolute configuration. This also supports Chen and Kingston’s suggestion that natural 6 is actually levorotary.12 However, only a side by side comparison between Riccio’s sample of 6 from Dactylospongia sp. and (−)-6 from C. mycofijiensis can provide an unequivocal resolution to this argument. Furthermore, different solvents were used to measure the optical rotations for 1 (CH2Cl2)1 and 6 (MeOH)19 in their original reports. When presented with both 1 and 6 from the same source, we took the opportunity to complete the set of measurements in more than one solvent. In each case the resulting optical rotations measured in either solvent were levorotary (Table S1). Biological Activity. Cell proliferation assays (MTT, 72 h incubation) determined the new compounds to be potently cytotoxic toward the human promyelocytic leukemic (HL-60) cell line. Zampanolides B−D (2−4) inhibited cell growth at concentrations (IC50) in the range 3−5 nM (Table 3) and thus are equipotent with 1. The antiproliferative activity of (−)-6 correlates well with Jennings and Altmann’s synthetic versions across multiple cell lines.22,23 In cell cycle distribution assays (Table 4), 2−4 and (−)-6 display a similar antimitotic profile to 1 and paclitaxel (positive control) by causing cells to accumulate in mitosis at the G2/M checkpoint. Zampanolide E (5) did not elicit an antimitotic response. Zampanolides B−E (2−5) and (−)-6 were also tested in an in vitro tubulin polymerization assay (Figure 2) to compare their relative microtubule-stabilizing activity to 1. At equimolar concentrations (2.8 μM), polymerization of purified porcine tubulin induced by 2, 3, or 4 was comparable to 1, while being slower

Table 3. Cell Proliferation Activity of Zampanolides 1−5 and (−)-Dactylolide (6) against the HL-60 Cell Line mean IC50 ± SEM (nM)a

compound zampanolide (1) zampanolide B (2) zampanolide C (3) zampanolide D (4) zampanolide E (5) (−)-dactylolide (6)

3.2 3.3 3.8 5.3 300 240

± ± ± ± ± ±

0.5 1.4 0.4 1.1 8 25

a

n = 3 independent experiments, with two technical replicates per experiment.

Table 4. Cell Cycle Arrest Effect of Zampanolides 1−5 and (−)-Dactylolide (6) against the HL-60 Cell Linea % accumulation compound

G1

S

G2/M

untreated cells vehicle paclitaxel zampanolide (1) zampanolide B (2) zampanolide C (3) zampanolide D (4) zampanolide E (5) (−)-dactylolide (6)

50 51 18 18 16 16 19 44 17

28 27 24 28 30 29 26 25 29

22 22 58 54 54 54 55 30 54

All compounds were administered at 1 μM for 18 h at 37 °C, n = 2 replicates from a single experiment.

a

Figure 2. In vitro polymerization of tubulin (porcine brain) induced by 1−6 and paclitaxel (positive control). Compounds were tested at a final concentration of 2.8 μM (1−4 and paclitaxel, n = 3 replicates, single preparation) or 27 μM (5, n = 3 replicates, single preparation; 6, n = 1 sample from one preparation).

than paclitaxel (positive control). Zampanolide E (5) and (−)-6 were both tested at a 10-fold higher concentration of 27 μM but remained indistinguishable from vehicle-only treatment. D



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The absence of Δ8 in 5 reinforces the requirement of this alkene in 1 for optimal microtubule stabilization, as implied by its involvement as a Michael acceptor.4 Surprisingly, E/Z isomerization at Δ4 or Δ8 of the macrolide core (2 and 3, respectively) or Δ4′ of the hexadienoyl side chain (4) appears to have little to no impact on biological activity. Furthermore, the N-acyl side chain of 1 has been implicated in a secondary interaction in the tubulin-binding pocket through hydrogen bonding between Thr276 and 19-OH and C(O)-1′.4 Collectively, these findings provide valuable information relative to advancing the development of (−)-1 as a microtubule-targeting drug lead.

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CONCLUSIONS

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EXPERIMENTAL SECTION

acetone/H2O (fraction B), (iii) 80% acetone/H2O (fraction C), and (iv) acetone (fraction D). Fractions B and C were independently chromatographed over silica gel (EtOAc/CH2Cl2, 0−100%), from which zampanolide-containing fractions (30% EtOAc/CH2Cl2, fractions I and X, respectively) were selected for further purification. Adjoining fractions from both procedures (E−H, J−L from fraction B; T−W, Y, Z from fraction C) contained the known compounds dendrolasin, mycothiazole, latrunculin A, laulimalide, neolaulimalide, and isolaulimalide. Fraction I (133.5 mg) was purified further by silica gel chromatography (EtOAc/CH2Cl2, 0−100%), where the 10% EtOAc/CH2Cl2 fraction (fraction M, 2.1 mg) ultimately yielded (−)-dactylolide (6, 1.5 mg total) after C18 HPLC purification (50% CH3CN/H2O, tR = 42.2, 51.7 min). Fraction N (30% EtOAc/ CH2Cl2, 80.9 mg) was subjected to C18 HPLC (55% CH3CN/H2O) to afford 6,7-epoxylatrunculin A (14.1 mg), zampanolide (1, 10.8 mg, tR = 18.1 min), and four fractions (O−R) enriched with zampanolidelike compounds. These four fractions were independently purified by analytical C18 HPLC (CH3CN/H2O) to afford zampanolide B (2, 0.2 mg, 40%, tR = 88.2 min), zampanolide D (4, 0.6 mg, 50%, tR = 28.8 min), zampanolide C (3, 0.9 mg, 50%, tR = 36.3 min), and zampanolide E (5, 0.2 mg, 50%, tR = 36.4 min). Fraction X (57.5 mg) was treated in a similar manner to fraction I, from which fractions AA and AB (30−50% EtOAc/CH2Cl2, 15.2 mg total) were combined and purified by C18 HPLC (50−60% CH3CN/H2O) to yield 6,7epoxylatrunculin A (5.2 mg), 2 (0.3 mg), 4 (0.2 mg), 1 (5.0 mg), 3 (0.1 mg), and 5 (0.1 mg). Zampanolide (1): colorless, amorphous solid; [α]26D −113 (c 0.11, CH2Cl2), −155 (c 0.11, MeOH); UV (MeOH) λmax (log ε) 201 (3.40), 226 (3.32) 266 (3.27) nm; IR (film) νmax 2924, 2887, 2851, 1710, 1668, 1631, 1611, 1521, 1353, 1042 cm−1; NMR data, Tables 1 and 2, and as previously described;1 HRESIMS m/z 518.2516 [M + Na]+ (calcd for C29H37NO6Na, 518.2513). Zampanolide B (2): colorless, amorphous solid; [α]24D +27 (c 0.11, CH2Cl2); UV (MeOH) λmax (log ε) 227 (3.92), 263 (3.95) nm; IR (film) νmax 3239, 2908, 2864, 1670, 1685, 1647, 1579, 1422, 1016 cm−1; NMR data, Tables 1, 2, and S2; HRESIMS m/z 518.2514 [M + Na]+ (calcd for C29H37NO6Na, 518.2513). Zampanolide C (3): colorless, amorphous solid; [α]25D −267 (c 0.015, CH2Cl2); UV (MeOH) λmax (log ε) 201 (4.10), 264 (4.05) nm; IR (film) νmax 3316, 2929, 2854, 1657, 1635, 1521, 1432, 1152, 1051 cm−1; NMR data, Tables 1, 2, and S3; HRESIMS m/z 518.2513 [M + Na]+ (calcd for C29H37NO6Na, 518.2513). Zampanolide D (4): colorless, amorphous solid; [α]26D −114 (c 0.035, CH2Cl2); NMR data, Tables 1, 2, and S4; HRESIMS m/z 518.2509 [M + Na]+ (calcd for C29H37NO6Na, 518.2513). Zampanolide E (5): colorless, amorphous solid; [α]26D +240 (c 0.05, CH2Cl2); NMR data, Tables 1, 2, and S5; HRESIMS m/z 520.2674 [M + Na]+ (calcd for C29H37NO6Na, 520.2670). (−)-Dactylolide (6): colorless oil; [α]26D −80 (c 0.025, CH2Cl2), −40 (c 0.025, MeOH); UV (MeOH) λmax (log ε) 203 (4.02), 226 (3.87) 277 (3.74) nm; IR (film) νmax 3329, 3285, 2906, 2876, 2031, 1720, 1687, 1657, 1596 cm−1; NMR data for [(+)-6] as previously described;19 HRESIMS m/z 407.1834 [M + Na]+ (calcd for C23H28O5Na, 407.1829). Cell Proliferation Assay. An MTT assay was performed as previously described.25 Briefly, HL-60 cells were seeded at 1 × 104 cells/well in a 96-well plate and treated with a 2-fold serial dilution of 1−6 and paclitaxel (positive control). After incubation for 72 h at 37 °C, MTT (5 mg/mL in PBS) was added to each well, and the plate incubated for 2 h at 37 °C. The purple formazan crystals were solubilized overnight at 37 °C in 10% sodium dodecyl sulfate and 45% dimethylformamide, and the absorbance of the solutions in the wells was measured at 570 nm using an EnSpire 2300 multilabel reader (PerkinElmer). Cell Cycle Arrest. The proportion of cells in different parts of the cell cycle was determined by flow cytometry using a FACSCanto II with Diva software (Becton Dickinson Biosciences) as previously described.26 Briefly, HL-60 cells were seeded into 24-well plates at 1 × 105 cells/well and treated with 1−6 and paclitaxel (positive control)

Four new zampanolides were isolated from the Tongan marine sponge C. mycof ijiensis, three of which were determined to be potent microtubule-stabilizing agents akin to their archetypal congener (−)-1. Geometric variation within the macrolide core and side chain does not significantly impact on the bioactive pharmacophore, which opens the opportunity to further develop this family of potent cytotoxins into clinically valuable leads. Moreover, the co-isolation of both (−)-1 and (−)-6, the latter for the first time, resolves a longstanding conundrum regarding the biosynthetic origins and absolute configuration of the zampanolide macrolide core with respect to its close relative, dactylolide.

General Experimental Procedures. Optical rotations were measured using a Rudolph Autopol II polarimeter. UV/vis spectra were recorded on a Shimadzu UV-160 spectrophotometer. IR (film) spectra were recorded using a Bruker Platinum Alpha FTIR spectrometer. NMR spectra were obtained using a Varian DirectDrive spectrometer equipped with a triple-resonance HCN cryogenic probe, operating at 25 K at frequencies of 600 and 150 MHz for 1H and 13C nuclei, respectively. Chemical shifts were referenced to the residual solvent peak (CDCl3: δC 77.16, δH 7.26).24 High-resolution masses were obtained from an Agilent 6530 Q-TOF mass spectrometer equipped with an Agilent 1260 HPLC system for solvent delivery utilizing a JetStream electrospray ionization source in positive ion mode. Reversed-phase column chromatography was achieved using Supelco Diaion HP20 (PSDVB) chromatographic resin. HPLC was performed using either an Agilent Technologies 1260 Infinity HPLC equipped with a diode array detector or a Rainin Dynamax SD-200 solvent delivery system with 25 mL pump heads equipped with a Varian Prostar 335 photodiode array detector. Octadecyl-derivatized silica (C18, 5 μm, 100 Å) HPLC columns (Phenomenex) were either semipreparative (10 mm × 250 mm, 4 mL/min) or analytical (4.6 mm × 250 mm, 1 mL/min). Solvents used for reversed-phase column chromatography were of HPLC or analytical grade quality. All other solvents were purified by distillation and filtered before use. Solvent mixtures are reported as % vol/vol unless otherwise stated. Collection of Animal Material. The marine sponge Cacospongia mycof ijiensis was hand collected using scuba from ‘Eua, Kingdom of Tonga, in June 2016. Samples were stored at −18 °C until required for extraction. A voucher sample (PTN4_26A) is held at Victoria University of Wellington. Isolation of 1−6. An isolation chart (Figure S1) is provided in the Supporting Information. Briefly, methanolic extracts (2 × 2 L) of frozen C. mycof ijiensis (503 g) collected from ‘Eua were loaded onto PSDVB (HP20, 400 mL), followed by successive dilution and repetitive passing of the eluents with H2O until a final concentration of 25% MeOH/H2O was achieved. The loaded material on the stationary phase was washed with H2O, then eluted from the column with 1.2 L portions of (i) 40% acetone/H2O (fraction A), (ii) 60% E



Article

for 18 h at 37 °C. The cells were then incubated in the dark for 30 min with propidium iodide staining solution (0.05 mg/mL in 0.1% sodium citrate, 0.1% Triton-X100, 100 μg/mL RNase) before assessing the DNA content of the cells. DNA profile scan data were analyzed using FlowJo software (v 10.4.1, Tree Star). Tubulin Polymerization Assay. A fluorescence-based, in vitro porcine tubulin polymerization assay (Cytoskeleton, Inc.) was used to determine the microtubule stabilization properties of zampanolide (1), zampanolides B−E (2−5), and (−)-dactylolide (6). The assay was prepared as per the manufacturer’s instructions (Cytoskeleton Inc., v 4.0, cat. no. BK011P) in the absence of microtubule-associated proteins and optimized for the detection of polymerization enhancers by excluding glycerol from the polymerization buffer. Zampanolides 1−4 and paclitaxel (positive control) were tested at 2.8 μM final concentration, and zampanolide E (5) and dactylolide (6) tested at 27 μM. Wells were plated with 5 μL of vehicle, positive control (paclitaxel, 2.8 μM), or drug, followed with 50 μL of tubulin buffer solution added immediately before data acquisition. Measurements were taken at 1 min intervals over a 100 min period. A PerkinElmer EnSpire multimode plate reader was used in kinetic mode, preheated to 37 °C, λex 360 nm/λem 450 nm.

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S. J.; Singh, A. J.; Jiménez-Barbero, J.; Northcote, P.; Miller, J.; López, J. A.; Hamel, E.; Barasoain, I.; Altmann, K.-H.; Díaz, J. F. Chem. Biol. 2012, 19, 686−698. (4) Prota, A. E.; Bargsten, K.; Zurwerra, D.; Field, J. J.; Díaz, J. F.; Altmann, K.-H.; Steinmetz, M. O. Science 2013, 339, 587−590. (5) Field, J.; Northcote, P.; Paterson, I.; Altmann, K.-H.; Díaz, J.; Miller, J. Int. J. Mol. Sci. 2017, 18, 971. (6) Uenishi, J.; Iwamoto, T.; Tanaka, J. Org. Lett. 2009, 11, 3262− 3265. (7) Ghosh, A. K.; Cheng, X. Org. Lett. 2011, 13, 4108−4111. (8) Ghosh, A. K.; Cheng, X.; Bai, R.; Hamel, E. Eur. J. Org. Chem. 2012, 2012, 4130−4139. (9) Zurwerra, D.; Glaus, F.; Betschart, L.; Schuster, J.; Gertsch, J.; Ganci, W.; Altmann, K.-H. Chem. - Eur. J. 2012, 18, 16868−16883. (10) Smith, A. B., III; Safonov, I. G.; Corbett, R. M. J. Am. Chem. Soc. 2001, 123, 12426−12427. (11) Smith, A. B., III; Safonov, I. G.; Corbett, R. M. J. Am. Chem. Soc. 2002, 124, 11102−11113. (12) Chen, Q.-H.; Kingston, D. G. I. Nat. Prod. Rep. 2014, 31, 1202−1226. (13) Kakou, Y.; Crews, P.; Bakus, G. J. J. Nat. Prod. 1987, 50, 482− 484. (14) Crews, P.; Kakou, Y.; Quiñoà, E. J. Am. Chem. Soc. 1988, 110, 4365−4368. (15) Gulavita, N. K.; Gunasekera, S. P.; Pomponi, S. A. J. Nat. Prod. 1992, 55, 506−508. (16) Corley, D. G.; Herb, R.; Moore, R. E.; Scheuer, P. J.; Paul, V. J. J. Org. Chem. 1988, 53, 3644−3646. (17) Quiñoà, E.; Kakou, Y.; Crews, P. J. Org. Chem. 1988, 53, 3642− 3644. (18) Tanaka, J.; Higa, T.; Bernardinelli, G.; Jefford, C. W. Chem. Lett. 1996, 25, 255−256. (19) Cutignano, A.; Bruno, I.; Bifulco, G.; Casapullo, A.; Debitus, C. e. c.; Gomez-Paloma, L.; Riccio, R. Eur. J. Org. Chem. 2001, 2001, 775−778. (20) Hoye, T. R.; Hu, M. J. Am. Chem. Soc. 2003, 125, 9576−9577. (21) Ding, F.; Jennings, M. P. Org. Lett. 2005, 7, 2321−2324. (22) Ding, F.; Jennings, M. P. J. Org. Chem. 2008, 73, 5965−5976. (23) Zurwerra, D.; Gertsch, J.; Altmann, K.-H. Org. Lett. 2010, 12, 2302−2305. (24) Gottlieb, H. E.; Kotlyar, V.; Nudelman, A. J. Org. Chem. 1997, 62, 7512−7515. (25) Hood, K. A.; West, L. M.; Northcote, P. T.; Berridge, M. V.; Miller, J. H. Apoptosis 2001, 6, 207−219. (26) Field, J. J.; Kanakkanthara, A.; Brooke, D. G.; Sinha, S.; Pillai, S. D.; Denny, W. A.; Butt, A. J.; Miller, J. H. Invest. New Drugs 2016, 34, 277−289.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.8b00641. Isolation scheme for 1−6, comparison of specific rotation between 1 and 6, full tabulated NMR data for 2−5, NMR spectra for 1−5, biological activity data for 1−6 (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

A. Jonathan Singh: 0000-0003-1722-066X John H. Miller: 0000-0001-6383-1037 Robert A. Keyzers: 0000-0002-7658-7421 Peter T. Northcote: 0000-0002-2086-9972 Present Addresses #

T.T. School of Biological and Chemical Sciences, Faculty of Science, Technology and Environment, University of the South Pacific, Suva, Fiji. □ B.J. Ministry of Health, MedSafe, Wellington, New Zealand. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS T.T. acknowledges the New Zealand Commonwealth Scholarship for funding. I. Vorster (VUW) is thanked for NMR and MS assistance. Permission from the Tongan Ministry of Fisheries to collect samples is gratefully acknowledged, as is the assistance of K. and P. Stone (Dive Vava’u) and J. Laurie (Whale Swim, Fish & Dive Tours) for fieldwork.

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REFERENCES

(1) Tanaka, J.; Higa, T. Tetrahedron Lett. 1996, 37, 5535−5538. (2) Field, J. J.; Singh, A. J.; Kanakkanthara, A.; Halafihi, T.; Northcote, P. T.; Miller, J. H. J. Med. Chem. 2009, 52, 7328−7332. (3) Field, J.; Pera, B.; Calvo, E.; Canales, A.; Zuwerra, D.; Trigili, C.; Rodríguez-Salarichs, J.; Matesanz, R.; Kanakkanthara, A.; Wakefield, F


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