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Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX
Peloruside E (22-Norpeloruside A), a Pelorusane Macrolide from the New Zealand Marine Sponge Mycale hentscheli, Retains Microtubule-Stabilizing Properties Sa Weon Hong,†,‡ A. Jonathan Singh,*,‡,§ Vimal Patel,‡,∥ Euan R. Russell,‡,∥,# Jessica J. Field,‡,∥ John H. Miller,‡,∥ 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
J. Nat. Prod. Downloaded from pubs.acs.org by KAOHSIUNG MEDICAL UNIV on 09/07/18. For personal use only.
‡
S Supporting Information *
ABSTRACT: A new peloruside congener, peloruside E (5), has been isolated in submilligram quantities from a specimen of the New Zealand marine sponge Mycale hentscheli. The structure of 5 differs from the parent compound peloruside A (1) by replacement of the C-10 gem-dimethyl moiety with a monomethyl substituent and represents the first structural deviation in the pelorusane scaffold. Peloruside E (5) is potently antiproliferative (HL-60, IC50 90 nM, cf. 1, 19 nM) and polymerizes purified tubulin, albeit at a rate lower than that of 1.
M
of the natural product and uncover new naturally occurring analogues to further explore the delicate structure−activity relationship. This has resulted in the identification of a new congener of 1, named peloruside E (5), which is discussed herein.
icrotubule-targeting agents, invariably, are small molecules that interfere with the ability of a cell to proliferate by disrupting the dynamic process of microtubule polymerization/depolymerization of their composite α,β-tubulin heterodimers.1 As such, compounds of this nature represent attractive targets for further development into cancer therapeutics. Marine organisms, in particular sponges, have been shown to be a source of microtubule-targeting agents, including discodermolide, laulimalide, and zampanolide.2 In 2000, the isolation of peloruside A (1), a polyoxygenated macrolide, was reported from the New Zealand poecilosclerid sponge Mycale (Carmia) hentscheli (Bergquist and Fromont, 1988).3 Peloruside A is highly cytostatic and cytotoxic in nanomolar concentrations and shows antimitotic behavior through the stabilization of microtubule polymerization in a similar manner to paclitaxel (Taxol).4 More recently, the isolation of pelorusides B−D (2−4) and preparation of semisynthetic derivatives revealed the sensitivity of a finely tuned pharmacophore related to the pyran moiety.5,6 X-ray crystallography studies confirmed the binding of 1 (along with laulimalide) to a nontaxoid site on β-tubulin and also revealed a possible interaction of the pyran core with a second dimer on the neighboring protofilament.7 The pharmacological potential and complex structural characteristics have made 1 an attractive synthetic target, with six total syntheses reported to date.8−13 Furthermore, studies into the development of analogues have been reported.14 In our continuing efforts to study the pharmacological potential of peloruside A, we have undertaken to procure more © XXXX American Chemical Society and American Society of Pharmacognosy
A specimen of M. hentscheli collected from Pelorus Sound, New Zealand, was extracted with MeOH and subjected to previously described isolation techniques6 to access fractions enriched with peloruside A (1). Final purification by reversedand normal-phase HPLC ultimately yielded 1 and a new congener named peloruside E (5). The molecular formula for 5 was established by HRESIMS as C26H46O11, which is the same as for that peloruside B (2). Due to the minute amount of isolated material (30 μg, 56 nmol), carbon resonances were assigned from the protondetected HSQC and HMBC experiments. Nonetheless, comparison of NMR data of 5 with the parent compound 1 allowed for rapid assembly of the planar structure. Salient Received: July 9, 2018
A
DOI: 10.1021/acs.jnatprod.8b00557 J. Nat. Prod. XXXX, XXX, XXX−XXX
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features within the 1H NMR experiment revealed diagnostic resonances associated with the pelorusane scaffold, namely, macrolactonization at CH-15 (δC 70.8; δH 5.69), primary and vinylic methyls (δH 0.85, 1.65) at the branched C-16 side chain, and oxymethylation (δH 3.30, 3.40, 3.47). COSY and 1D-TOCSY experiments were used to establish the C-2 to C-8, C-11 to C-15, and C-17 to C-20 segments of the structure, which were found to be identical in sequence to that of 1. Connectivity of these units and placement of the three oxymethyls at C-3, C-7, and C-13 were established by HMBC correlations (Figure 1). The structural changes in 5 were found
Figure 1. Selected 2D-NMR correlations used to establish the planar and configurational structure of peloruside E (5).
Figure 2. In vitro tubulin (porcine brain) polymerization induced by pelorusides A (1, PelA) and E (5, PelE) and paclitaxel (PTX, positive control). CTRL = vehicle only. Compounds were tested at 2.8 μM, n = 3 technical replicates.
to be at C-10, where a monomethyl unit [CH-10: δC 39.8; δH 1.91, qd (7.1, 1.9 Hz), CH3-21: δC 6.5; δH 1.05, d (7.2 Hz)] had replaced the original gem-dimethyl unit found in 1−4. This feature marks the first significant change to the pelorusane scaffold. Connectivity between C-8 and C-9 was only observed from a correlation from 9-OH (δH 5.77) to C-8 (δH 1.05) in the HMBC experiment. Together with the similar chemical shift and NOE correlation data observed in the pyran systems of 1 and 5, the connectivity from C-5 to C-9 was firmly established. Through-space correlations observed in a 2D-NOESY experiment (Figure 1, mixing time = 500 ms) between H-10 and H-11, and H-8 and H3-21, indicate the formal loss of CH322 and orients all groups syn-facially. Transannular correlations observed from H-2 to H-11 and 9-OH, and 9-OH with H-5 and H3-21, point to a solution conformation that is similar3 in nature to 1. The measurement of the optical rotation of 5 was attempted [α −0.0025°, [α]20D −250 (c 0.001)]; however, due to the limited amount of material available, we do not believe this to be an accurate measurement. De Brabander’s synthesis of ent-18 not only established the correct absolute configuration of the natural product, but also demonstrated the greater than 500-fold difference in biological activity between the two enantiomers against human tumor cell lines. Considering the similarities between 1 and 5 in constitutional and through-space correlation data, and in the demonstrated antiproliferative and antimitotic activity (see Biological Activity), we propose the absolute configuration of 5 to be conveyed as depicted. Biological Activity. Cell proliferation assays (MTT, 48 h incubation) showed peloruside E (5) was growth inhibitory toward human promyeloctic leukemia HL-60 cells with an IC50 of 90 ± 1 nM (SEM, n = 3 replicates), approximately 5 times less potent than peloruside A (1) in the same study (IC50 = 19 ± 1 nM, n = 3 replicates). Pelorusides A and E were also tested in an in vitro tubulin polymerization assay to compare their relative microtubule-stabilizing activity (Figure 2). At equimolar concentrations, polymerization induced by peloruside E was much slower than either 1 or paclitaxel (positive control).
Tubulin polymerization was also observed for vehicle-only wells, albeit at a much lower rate. Using flow cytometry analysis, treatment of HL-60 cells with 1 or 5 induced mitotic arrest at the G2/M checkpoint, although treatment with 5 required three times the concentration needed for 1 to exert a similar response (Table 2). Accumulation of cells in G2/M of the cell cycle is a characteristic effect of antimitotic agents. All naturally occurring pelorusides reported to date contain all 24 carbons on the macrolide structure; the only differences come in 3 and 4, where deoxygenation or alternate ring formation occurs, respectively. The C-10 monomethyl unit in 5 represents the first occurrence of a structural change at this position for a natural peloruside congener. Most of the reported synthetic procedures that deviate from natural peloruside A (1), including the NaBH4 reduction product, have all targeted the pyran ring as the point of difference.14 Collectively, these endeavors demonstrate a significant reduction, if not the total loss of cytotoxicity, when the pyran is perturbed.14 Additionally, Taylor and co-workers indicate that any continued interest in 1 as a therapeutic agent may rely on “conformationally simplified” analogues, for which a change at C-10 may contribute; whether it is favorable or not requires further investigation.15 Our findings reinforce the structurally stringent nature of the peloruside pharmacophore but also introduces the possibility of more synthetically accessible microtubule-stabilizing analogues if the gemdimethyl moiety of the pelorusides is deemed to be nonessential for activity.
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EXPERIMENTAL SECTION
General Experimental Procedures. Optical rotations were measured on a Rudolph Research Analytical Autopol IV polarimeter. NMR spectra were obtained using a Varian DirectDrive spectrometer equipped with a triple-resonance HCN cryogenically cooled (25 K) probe, operating at 600 MHz for the 1H nucleus. Inverse-detected heteronuclear experiments (HSQC, gHMBC) were used to acquire carbon chemical shifts. Chemical shifts δ (ppm) were referenced to
B
DOI: 10.1021/acs.jnatprod.8b00557 J. Nat. Prod. XXXX, XXX, XXX−XXX
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Table 1. NMR Data (600 MHz in CDCl3) for Peloruside E (5) position
δC, mult.a
1 2 3
173.9, C 70.6, CH 77.9, CH
3-OMe 4a
56.0, CH3 32.2,b CH2
4b 5 6a
63.4, CH 32.2,b CH2
6b
δH, mult. (J in Hz) 4.52, s 4.32, dd (10.8, 5.4) 3.30, s 1.83, m 2.16, m 4.30, tdd (10.9, 5.4, 2.3) 1.54, q (12.0) 1.87, ddd (11.2, 5.1, 2.0) 3.73, ddd (11.6, 5.0, 3.2) 3.40, s 3.91, d (3.0)
7
75.2, CH
7-OMe 8 9 9-OH
55.9, CH3 65.8, CH 101.1, C
10
39.8, CH
11
68.3, CH
1.91, qd (7.1, 1.9) 5.25, d (11.8)
12a
36.9, CH2
1.13, m
% accumulation COSY 3(w) 2(w), 4a, 4b 3,
2.29, ddd (16.5, 12.5, 4.8) 3.92, m
HMBC 1 3-OMe 3 3
4b(w), 5 3, 4a(w), 5 4a, 4b, 6a
3, 5
5, 6b, 7
4, 5, 7
6a, 7
7, 8
6a, 6b, 8
7
5.77, s
12b
11, 21
3, 5, 9-OH, 11 2, 3-OMe, 4b, 11, 23 3, 4b, 23 4b, 5 3, 3-OMe, 4a 2, 4a, 6b, 7(w), 9-OH(w) 6b 5, 6a, 7, 7OMe
7 7
6b, 7, 8 7, 7-OMe, 21
8, 9 9
2, 5(w), 11(w), 21 11, 12a, 21
11, 13
2, 3, 9-OH(w), 10, 12a 10, 11, 12b, 14a 12a, 13, 21
13
12b, 13-OMe, 14b 13
77.8, CH
13OMe 14a
58.8, CH3
3.47, s
36.2, CH2
14b, 15
15
12a, 14b, 23
13, 14a, 15
13
13, 14a, 15
14a, 14b, 17
23
14b, 18
23
19a, 23, 24a 15, 20(w), 24b
15
70.8, CH
16 17 18
136.5, C 131.1, CH 43.3, CH
5.01, d (10.5) 2.61,c m
19a
24.6, CH2
1.14, m
14b
12b, 14b
20
12.2, CH3
0.85, t (7.5)
15, 18, 23 17, 19b, 24a, 24b 19b(w), 20 18, 19a(w), 20 19a, 19b
21
6.5, CH3
1.05, d (7.2)
10
9, 10, 11
22 23
17.5, CH3
1.65, d (1.2)
17
67.0, CH2
3.36c, t (10.2)c 3.63, dd (10.6, 3.8)
15, 16, 17 17
19b
24a 24b
1.41, m
18, 24b 18, 24a
17, 19b, 20 17
compound
G1
S
G2/M
vehicle paclitaxel, 1000 nM peloruside A (1), 100 nM peloruside A (1), 500 nM peloruside E (5), 300 nM peloruside E (5), 1500 nM
62 28 27 30 38 29
20 19 23 23 22 23
19 53 50 47 39 49
delivery utilizing a JetStream electrospray ionization source in positive-ion mode. Reversed-phase column chromatography was achieved using Supelco Diaion HP20 poly(styrene-divinylbenzene) (PSDVB) chromatographic resin. Supelco Diol-functionalized silica gel (DIOL) was used for normal-phase flash chromatography. HPLC was performed using either an Agilent Technologies 1260 Infinity HPLC equipped with a diode array detector or an Agilent 380 evaporative light-scattering detector (ELSD). C18 and HILIC HPLC column (Phenomenex) sizes were either semipreparative (10 mm × 250 mm, 4 mL/min) or analytical (4.6 mm × 250 mm, 1 mL/min) scale, unless otherwise stated. Solvents used for reversed-phase column chromatography were of HPLC or analytical grade quality. All other solvents were purified by distillation before use and filtered. Solvent mixtures are reported as % vol/vol unless otherwise stated. Sponge Material. Mycale hentscheli was hand collected and identified by M. J. Page, NIWA, using scuba from Pelorus Sound, New Zealand, and held at −20 °C until extracted. Isolation of Peloruside E. A single specimen (1.2 kg frozen weight) of M. hentscheli was extracted twice with MeOH (2 × 2.5 L) for 24 h. The second and then first extracts were passed through PSDVB (HP-20, Supelco) with successive dilution of the eluents with H2O and further recycling through the column until a 3-fold dilution was achieved. The loaded column was washed with H2O and eluted with (i) 20% acetone/H2O (fraction A), (ii) 40% acetone/H2O (fraction B), (iii) 60% acetone/H2O (fraction C), and (iv) acetone (fraction D). Fraction B was diluted 3-fold with H2O, recycled onto a second PSDVB column, and then eluted with increasing concentrations of MeOH/H2O (60−100%, fractions E−I). The 70% MeOH/H2O fraction (fraction F, 198 mg) was further fractionated using DIOL (30 mL) with EtOAc/CH2Cl2 (0−100% fractions J−O) and MeOH (fraction P). The CH2Cl2 fraction (fraction J, 61.9 mg) was purified by C18 HPLC with ELSD detection (10 μm, 10 × 250 mm, MeOH/H2O, 35−100%) to afford 10 fractions (Q−Z), from which fraction U afforded 27.1 mg of peloruside A (1). Fraction S (1 mg) was subjected to normal-phase HPLC (HILIC, 5 μm, 4.6 × 250 mm, 2-propanol/n-hexane, 10−20%) to give 30 μg of peloruside E (5, tR = 15.7−16.5 min). Peloruside A (1): colorless film; all other data as previously described.3 Peloruside E (5): colorless film; NMR data, Table 1; HRESIMS m/ z 557.2938 [M + Na]+ (calcd for C26H46O11Na, 557.2932, Δ = +1.1 ppm). Cell Proliferation Assay. A 48 h MTT [3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide] cell proliferation assay was performed using the human promyelocytic leukemia (HL-60) cell line as previously described.17 Tubulin Polymerization Assay. A fluorescence-based, in vitro tubulin polymerization assay kit (Cytoskeleton, Inc.) was used to determine whether peloruside E enhanced microtubule stabilization. The assay was prepared as per manufacturer instructions, in the absence of microtubule-associated proteins, and optimized for the detection of polymerization enhancers by omitting glycerol from the polymerization buffer. Pelorusides A (1) and E (5) and paclitaxel (positive control) were tested at 2.8 μM final concentration (n = 3 replicates, single preparation). Each well of a 96-well plate contained 5 μL of vehicle, positive control (paclitaxel, 2.8 μM final concentration)
5(w),6b, 7-OMe, 8
10, 12a, 12b 11, 12b 11, 12a, 13
NOESY
13
2.01, dd (15.6, 10.8) 2.11, dd (15.6, 10.2) 5.69, d (10.9)
Table 2. Cell Cycle Arrest Effect of Pelorusides A (1) and E (5) against the HL-60 Cell Line
19a, 20
18(w), 19a, 19b 8, 9-OH, 10, 12b 3, 3-OMe, 14a, 17 17, 24b 18, 24a
a
Chemical shifts obtained from HSQC and HMBC experiments. Interchangeable. cObtained from a 1D-TOCSY experiment.
b
the residual solvent peak (CDCl3: δC 77.16, δH 7.26).16 Highresolution masses were obtained from an Agilent 6530 Q-TOF mass spectrometer equipped with an Agilent 1260 HPLC for solvent C
DOI: 10.1021/acs.jnatprod.8b00557 J. Nat. Prod. XXXX, XXX, XXX−XXX
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or drug, and 50 μL of tubulin buffer solution with porcine brain tubulin added immediately before data acquisition. Measurements were taken at 1 min intervals over 100 min, using a PerkinElmer EnSpire multimode plate reader, preheated to 37 °C, λex 360 nm/λem 450 nm. Cell Cycle Arrest. Cell cycle analysis was carried out by flow cytometry as previously described.18 After 18 h of treatment with drug (1, 5, or paclitaxel), HL-60 cells were resuspended in 0.05 mg/mL propidium iodide, 0.1% sodium citrate, 0.1% Triton-X100, and 100 μg/mL RNase and incubated in the dark for 30 min. The DNA content of 10 000 cells was assessed in a FACSCanto II flow cytometer with Diva software (Becton Dickson). Scan data were analyzed using FlowJo software (v 10.0.4, Tree Star).
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(10) Ghosh, A. K.; Xu, X.; Kim, J.-H.; Xu, C.-X. Org. Lett. 2008, 10, 1001−1004. (11) Evans, D. A.; Welch, D. S.; Speed, A. W. H.; Moniz, G. A.; Reichelt, A.; Ho, S. J. Am. Chem. Soc. 2009, 131, 3840−3841. (12) McGowan, M. A.; Stevenson, C. P.; Schiffler, M. A.; Jacobsen, E. N. Angew. Chem., Int. Ed. 2010, 49, 6147−6150. (13) Hoye, T. R.; Jeon, J.; Kopel, L. C.; Ryba, T. D.; Tennakoon, M. A.; Wang, Y. Angew. Chem., Int. Ed. 2010, 49, 6151−6155. (14) Brackovic, A.; Harvey, J. E. Chem. Commun. 2015, 51, 4750− 4765. (15) Larsen, E. M.; Wilson, M. R.; Taylor, R. E. Nat. Prod. Rep. 2015, 32, 1183−1206. (16) Gottlieb, H. E.; Kotlyar, V.; Nudelman, A. J. Org. Chem. 1997, 62, 7512−7515. (17) Hood, K. A.; West, L. M.; Northcote, P. T.; Berridge, M. V.; Miller, J. H. Apoptosis 2001, 6, 207−219. (18) 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
* Supporting Information S
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.8b00557. Isolation scheme, 1D- and 2D-NMR spectra, cell proliferation and cell cycle arrest assay data (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 Peter T. Northcote: 0000-0002-2086-9972 Present Address #
E. R. Russell: Department of Microbiology and Immunology, University of Otago, Dunedin, New Zealand. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors thank I. Vorster (Victoria University of Wellington) for NMR and MS assistance and M. Page (National Institute of Water and Atmospheric Research) for collection of sponge material and for the image of Mycale hentscheli used in the TOC graphic.
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REFERENCES
(1) Akhmanova, A.; Steinmetz, M. O. Nat. Rev. Mol. Cell Biol. 2015, 16, 711−726. (2) Miller, J. H.; Field, J. J.; Kanakkanthara, A.; Owen, J. G.; Singh, A. J.; Northcote, P. T. J. Nat. Prod. 2018, 81, 691−702. (3) West, L. M.; Northcote, P. T.; Battershill, C. N. J. Org. Chem. 2000, 65, 445−449. (4) Hood, K. A.; West, L. M.; Rouwé, B.; Northcote, P. T.; Berridge, M. V.; Wakefield, S. J.; Miller, J. H. Cancer Res. 2002, 62, 3356−3360. (5) Singh, A. J.; Xu, C.-X.; Xu, X.; West, L. M.; Wilmes, A.; Chan, A.; Hamel, E.; Miller, J. H.; Northcote, P. T.; Ghosh, A. K. J. Org. Chem. 2010, 75, 2−10. (6) Singh, A. J.; Razzak, M.; Teesdale-Spittle, P.; Gaitanos, T. N.; Wilmes, A.; Paterson, I.; Goodman, J. M.; Miller, J. H.; Northcote, P. T. Org. Biomol. Chem. 2011, 9, 4456−4466. (7) Prota, A. E.; Bargsten, K.; Northcote, P. T.; Marsh, M.; Altmann, K.-H.; Miller, J. H.; Díaz, J. F.; Steinmetz, M. O. Angew. Chem., Int. Ed. 2014, 53, 1621−1625. (8) Liao, X.; Wu, Y.; De Brabander, J. K. Angew. Chem., Int. Ed. 2003, 42, 1648−1652. (9) Jin, M.; Taylor, R. E. Org. Lett. 2005, 7, 1303−1305. D
DOI: 10.1021/acs.jnatprod.8b00557 J. Nat. Prod. XXXX, XXX, XXX−XXX
