Antiangiogenic Activity and Chemical Derivatization of the Neurotoxic

Note Cite This: J. Nat. Prod. 2018, 81, 1905−1909

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Antiangiogenic Activity and Chemical Derivatization of the Neurotoxic Acetogenin Annonacin Isolated from Asimina triloba Paige J. Monsen and Frederick A. Luzzio* Department of Chemistry, University of Louisville, 2320 South Brook Street, Louisville, Kentucky 40292, United States

J. Nat. Prod. 2018.81:1905-1909. Downloaded from pubs.acs.org by KAOHSIUNG MEDICAL UNIV on 08/24/18. For personal use only.

S Supporting Information *

ABSTRACT: Annonacin (1) was isolated from the North American pawpaw (Asimina triloba), as reported earlier from these laboratories. Natural 1 was submitted to the rat aortic ring bioassay for evaluation of antiangiogenic activity and was found to inhibit microvessel growth (IC50 value of 3 μM). 4,10,15,20-Tetraazido derivatives of 1 were prepared by permesylation followed by azide displacement or by iodination followed by azide displacement. The tetraazide derived from mesylation/azidation was antiangiogenic, while that derived from iodination/azidation exhibited no appreciable activity. The membrane permeability of natural 1 was evaluated using the parallel artificial membrane permeability assay and was found to be marginally permeable as compared to several clinically relevant compounds.

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associated acetogenin-containing extracts, derived from the commercially available Cell-Reg, an alternative medicine twig extract of pawpaw, were found to be active in the human umbilical vein endothelial cell (HUVEC) bioassay.7 It is reported herein that 1 is inhibitory when tested in the rat aortic ring (RAR) angiogenesis assay.8 Both the molecular targets and analogues relevant to acetogenins are of interest when considering their neurotoxic and antiangiogenic behavior, and, to this end, reported herein are new syntheses of the deoxytetraazido analogues (3 and 5) of annonacin that start from natural 1. The late-stage azidation of natural products introduces the possibility of reactive nitrogen-based analogue synthesis. Such analogues include those resulting from the attachment of biological probes as well as the creation of elaborate scaffolds based on “click” chemistry.9 Both the tetraazido derivatives reported herein were the result of replacing all four hydroxy groups in 1 whereby both 3 and 5 possess both the unnatural (reversed, 3) and natural stereochemistry (5) of the hydroxy groups in natural 1. Along with annonacin, the tetraazido derivatives 3 and 5 were evaluated for antiangiogenic activity in the RAR assay, and 3 was found to be active, while 5 was not appreciably active. Furthermore, preliminary experiments in which the tetraazide 3 was submitted to a test “click” reaction showed that 3 is a very efficient coreactant whereby all four azido groups were transformed to the corresponding tetra-triazole. Inasmuch as 1 is considered a confirmed environmental neurotoxin,10 one may speculate as to if or how efficiently the compound crosses the blood−brain

dible fruit of the plant family Annonaceae contain a group of biologically active compounds inclusive of the “annonaceous acetogenins”. These compounds are derivatives of longchain (C32 or C34) fatty acids that possess one or more disubstituted tetrahydrofuran rings, an alkyl chain of varying length, and a terminal butenolide.1 They are potent inhibitors of the mitochondrial complex I as well as inhibitors of cytoplasmic (anaerobic) production of adenosine triphosphate (ATP) and related nucleotides.2 Hence, the diverse biological activities of the annonaceous acetogenins have demonstrated their clear potential as experimental therapeutics or drug leads.3 Annonacin (1), a major representative of the annonaceous acetogenin group of compounds, has been isolated from Annona cherimolia, Annona densicoma (stem bark), Annona muricata (fruit, leaves), Annona squamosa (fruit), and Asimina triloba (L.) Dunal (bark/ fruit).4 Consequently, an interest in the neurotoxic properties of annonacin (1) grew from its isolation from fruits of annonaceous species endemic to Latin America, where their consumption by humans led to cases of atypical Parkinsonsim.5

Studies in our laboratories have confirmed that annonacin (1) is indeed preponderant in the fruits of the pawpaw, and efficient extraction techniques have been conducted to supply the pure natural product for both chemical derivatization and bioassays.6 Our interest in 1 as an angiogenesis inhibitor grew as we became aware of Nagle and Zhou’s study in which annonacin (1) and © 2018 American Chemical Society and American Society of Pharmacognosy

Received: April 9, 2018 Published: July 20, 2018 1905

DOI: 10.1021/acs.jnatprod.8b00284 J. Nat. Prod. 2018, 81, 1905−1909

Journal of Natural Products

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“inverted” 4,10,15,20-tetraiodide 4 (24%). Treatment of the tetraiodide 4 with sodium azide in warm dimethylformamide (DMF) over 20 h gave the reinverted tetraazide 5 (45%) after purification by flash column chromatography. Annonacin analogues 3 and 5 were then submitted to the RAR angiogenesis inhibition bioassay. Angiogenesis, or the sprouting of new vasculature, may be observed in an ex vivo assay consisting of rat aortic ring sections cultured on a matrix with growth factors and nutrients.15 The test compound or potential inhibitor is then added to the culture along with the appropriate controls in separate cultures, and the new microvessels are then quantified. The study of angiogenesis by culturing aortic rings is a well-developed assay and can be used to evaluate the morphological stages of the angiogenic process as well as the generation of extracellular matrix during vasculogenesis. The present studies of the antiangiogenic properties of 1 and tetraazides 3 and 5 entailed direct addition of the test compounds to the rat aortic ring cultures along with positive controls sorafenib (for 1)16b and TNP-470 (for 3 and 5).16a The RAR bioassays of 1 revealed distinct inhibition of angiogenesis (Figure 1, images 1A−C, graph 1D). Image 1A shows microvessel outgrowths on treatment of the aortic ring culture with only the dimethyl sulfoxide (DMSO) vehicle. Image 1B shows almost zero outgrowth on treatment of the aortic ring culture with 1 μM of the positive control sorafenib. On incubation of the ring culture with increasing concentrations of 1 (10 nM, 100 nM, 1 μM, 10 μM, graph 1D), microvessels were inhibited with an IC50 of 3 μM (image 1C). Evaluation of tetraazides 3 and 5 in the RAR assay involved treatment of the cultures with the DMSO vehicle control (images 2A, 3A) and incubation with the positive control TNP470 (images 2B, 3B) at 50 μM. Similar to image 1A, images 2A and 3A showed substantial microvessel growth, while the TNP470-treated cultures 2B and 3B showed substantial inhibition (graph 2D). Treatment of the ring cultures with synthetic tetraazide 3 (image 2C) showed inhibition (ca. 40%) at 50 μM, while image 3C showed almost no inhibition when the ring cultures were incubated with synthetic tetraazide 5 at 50 μM (image 3C, graph 3D). With respect to previous investigations in the neuroscience area, an interesting question remains as to how efficiently and through what mechanism a molecule with such a number of polar groups might cross the BBB.5c,17 Notwithstanding the permeability of the BBB, there are also the physiochemical consequences associated with permeation of the mitochondrial membranes as well, since the molecular target of these compounds is purported to be mitochondrial complex C.2c,18 Specifically, BBB and mitochondrial permeation is paramount to passing of such exemplary neurotoxic compounds such as rotenone or paraquat.19,20 Annonacin (1) was submitted to a standard PAMPA assay that utilized a lecithin/dodecane-treated polyvinylidene fluoride membrane/phosphate-buffered saline (PBS) system. The in vitro permeability (Pe) value of 1 using PBS at pH = 7.4 was 0.51 (×10−6 cm/s) when measuring against the controls, methotrexate and testosterone, which gave Pe values of 0.007 and 24.5 (×10−6 cm/s), respectively (Table 1). For comparison, the Pe values of dexamethasone (0.41 × 10−6), caffeine (1.8 × 10−6), and propranolol (12 × 10−6)21 are listed (Table 1) in order of increasing permeability (low ↔ high). Testosterone has been previously evaluated in a “BBBPAMPA” assay in which the membrane was treated with porcine brain lipid extracts, and the experimental Pe value was 17 as compared with the 24.5 and 18 found in the dodecane/lecithin

barrier (BBB) as well as the mitochondrial membranes.5c,11 Indeed, as a structural class, the acetogenins are linear combinations of polar (OH groups), moderately polar (butenolide), and nonpolar (lipid) functionalization, and it was deemed important to characterize the membrane permeation properties of annonacin (1) as compared with other drug-like molecules. Relevant to the present report, the issue of the permeability of vascular endothelial cells is of prime consideration when designing compounds that inhibit angiogenesis.12 Hence, natural annonacin (1) was submitted to the parallel artificial membrane permeability assay (PAMPA),13 and these results are reported herein. The synthesis of the tetraazido analogues 3 and 5 started with naturally occurring annonacin (1), which was isolated and purified as previously described (Scheme 1).6 The major Scheme 1. Conversion of Annonacin into Tetraazides 3 and 4a

Reagents/conditions: (a) CH3SO2Cl/Et3N/0 °C/3 h (44%). (b) NaN3/NMP/80 °C/3 h (52%). (c) PPh3/I2/imidazole/DCM/0 °C to rt/16 h (24%). (d) NaN3/DMF/45 °C (45%). a

departure from the previous isolation scheme was that freshly harvested pawpaw was collected and extracted as opposed to the commercially available frozen fruit pulp, of local origin but unknown shelf life, as previously reported. All four hydroxy groups of 1 were mesylated (CH3SO2Cl/triethylamine) in dichloromethane, giving the tetramesylate 2 (44%) as an oil after purification by flash column chromatography on silica gel. Treatment of the tetramesylate 2 with sodium azide in Nmethylpyrrolidone (NMP) at 80 °C afforded the 4,10,15,20tetraazide 3 (52%) after flash column chromatography. It should be noted that the mesylation−azidation results in inversion at all four stereocenters. The synthesis of tetraazide 5 then invoked a “double-inversion” type sequence that first entailed an iodinative-substitution of all hydroxy groups in 1.14 Thus, all four hydroxy groups of 1 were iodinated (I2/PPh3/imidazole) using the Garegg−Samuelsson method and provided the 1906



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Figure 1. Images of rat aortic ring assays: (1A) treatment with DMSO; (1B) treatment with 1 μM sorafenib; (1C) treatment with 1 μM annonacin (1); (2A) treatment with DMSO; (2B) treatment with 50 μM TNP-470; (2C) treatment with 50 μM tetraazidoannonacin (3); (3A) treatment with DMSO; (3B) treatment with 50 μM TNP-470; (3C) treatment with 50 μM tetraazidoannonacin (5). Inhibition of rat aortic ring microvessel growth: (1D) annonacin (1); (2D) tetraazidoannonacin (3); (3D) tetraazidoannonacin (5). New vessel sprouts are indicated with a blue arrow in the three control images.

PAMPA evaluation suggests that penetration is low compared to benchmark compounds that do permeate the BBB (as approximated by the PAMPA-BBB). The same issues may very well hold for the permeation of intracellular membranes en route to the mitochondrial machinery as well as those membranes in newly developing vasculature cells. The stepwise modification of the polar groups of 1 toward decreasing its polarity may increase its bioavailability through more efficient permeation and hence its bioactivity. Using this ready availability of natural annonacin (1), it should be quite possible to prepare and evaluate a great many analogues for further biological studies in cancer and in neuroscience.

Table 1. Permeability (Pe) in the PAMPA Assay for Standard Drugs and Annonacin (1) with Their Corresponding Assay Classification

a

Experimental values from PAMPA studies discussed in this report. Taken from ref 21. cTaken from ref 22.

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membrane.22 While the trends detailed in Table 1 indicate a very low permeability for 1 in vitro, active transport mechanisms may account for the permeability of compounds having multiple polar groups characteristic of many natural products and pharmacologically active compounds.23 In summary, the RAR bioassay confirms that naturally occurring 1 is an inhibitor of angiogenesis with an IC50 of 3.0 μM. Replacing all hydroxy groups of the natural compound with azido groups, all having either the same or reversed configuration, resulted in compounds that were less inhibitory than 1 in the RAR bioassay. While 1 was confirmed to cross the blood−brain barrier in vivo in previous studies,5a the present

EXPERIMENTAL SECTION

General Experimental Procedures. Unless otherwise specified, all solvents and reagents were ACS grade and were used as supplied. Optical rotations were determined on a JASCO P-2000 polarimeter. Infrared spectra (Fourier transform infrared spectroscopy, FTIR) were recorded with a PerkinElmer Spectrum 100 instrument. 1H and 13C NMR spectra were recorded on a Varian VNMRS 700 (13C NMR, 175 MHz), Varian VNMRS 400 (13C NMR, 100 MHz), or Oxford AS 500 MHz (13C NMR, 125 MHz) instrument using CDCl3 as solvent. Highresolution mass spectra (HRMS) were performed using electrospray ionization (ESI). Gravity column chromatography was carried out using silica gel 60 (E. Merck 7734, 70-230 mesh). Flash column 1907



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chromatography utilized silica gel 60 (E. Merck 9385-9, 230−400 mesh) with nitrogen gas pressurization. Thin-layer chromatograms were visualized during chromatographic and extraction runs by rapidly dipping the plates in anisaldehyde/ethanol/sulfuric acid stain and heating (hot plate). All air- and moisture-sensitive reactions were run in oven-dried glassware under an atmosphere of dry nitrogen. Due to the inherent neurotoxicity of acetogenins, extra safety precautions were taken. To minimize contact with natural annonacin, 1, and synthetic derivatives, 2, 3, 4, and 5, proper eye protection and safety gloves were worn at all times. All isolations, extractions, chemical reactions, and chromatographic purifications were performed in efficient fume hoods. Plant Material. The fruit of Asimina triloba was collected from trees growing wild at the Monroe farm, Indiana, in August 2016 and stored in the freezer. The species was identified by Robert Paratley, Department of Forestry, University of Kentucky. A voucher specimen (No. 57286) was deposited in the University of Kentucky Herbarium. Extraction and Isolation. Pawpaw fruit was peeled, and the seeds were removed, leaving only a soft, yellow pulp (∼1000 g). The pulp was placed in a commercial blender with methanol (1000 mL) and blended to a puree. The puree was stored overnight in a freezer. Half of the puree was vacuum filtered with a 17 cm glass Büchner funnel with a 500 mL methanol rinse, resulting in a bright orange filtrate. The second half of the puree was also vacuum filtered with a 17 cm Büchner funnel with a 500 mL methanol rinse. The filtrate collected from the second filtration was added to the previously filtered bright orange filtrate. The combined filtrates were concentrated to less than 1000 mL, then added to a 2000 mL separatory funnel. Liquid−liquid partitioning of the filtrate with ethyl acetate (3 × 200 mL) then chloroform (2 × 200 mL) yielded a crude organic extract that was concentrated to a yellow-orange syrup. Portions of the syrup (200−500 mg) were submitted to silica gel column chromatography using a 30 × 520 mm column with 150 mm of silica gel (chloroform−methanol, 97:3), which afforded 1 as a white, amorphous solid: [α]20D +17.3 (c 0.51, CHCl3); Rf 0.35 (chloroform− methanol, 9:1). The spectroscopic data were consistent with those described previously.6 (1R,6R,12R)-13-((S)-5-Methyl-2-oxo-2,5-dihydrofuran-3-yl)-1((2R,5R)-5-((R)-1((methylsulfonyl)oxy)tridecyl)tetrahydrofuran-2yl)tridecane-1,6,12-triyl Trimethanesulfonate (2). Annonacin (1) (30.0 mg, 0.05 mmol, 1.0 equiv) was dissolved in dichloromethane (1.5 mL), and the mixture was cooled to 0 °C under nitrogen. To the solution was added triethylamine (0.745 mmol, 15 equiv), and stirring was continued. Methanesulfonyl chloride (115 mg, 1.01 mmol, 20 equiv) was added to the reaction mixture dropwise by syringe under nitrogen. The reaction mixture was allowed to warm to room temperature and was stirred for 3 h. The reaction progress was monitored by TLC (chloroform−methanol, 9:1). Concentration of the reaction mixture and submission of the crude residue to flash column chromatography (toluene−-ethyl acetate, 2:1) provided pure 2 as a colorless oil (20 mg, 44%): Rf 0.17 (toluene−ethyl acetate, 2:1); FTIR νmax 2925, 2855, 1752, 1346, 1171, 1082, 909 cm−1; 1H NMR (CDCl3, 400 MHz) δ 7.28 (1H, s, H-33), 5.06 (1H, q, J = 6.8 Hz, H-34), 4.91 (1H, m, H-4), 4.70 (1H, m, H-10), 4.55 (2H, m, H-20, H-15), 4.11 (2H, m, H-19, H-16), 3.09 (3H, s, OSO2CH3), 3.07 (3H, s, OSO2CH3), 3.01 (3H, s, OSO2CH3), 3.01 (3H, s, OSO2CH3), 2.67 (2H, m, H-3), 2.08 (2H, m, H-17a, H-18a), 1.66−1.74 (12H, m, H-5, H-9, H-11, H-14, H-17b, H-18b, H-21), 1.44 (3H, d, J = 7.2 Hz, H-35), 1.25−1.50 (31H, m, H-6−H-8, H-12, H-13, H-22−H-31), 0.875 (3H, t, J = 6.8 Hz, H-32); 13C NMR (CDCl3, 700 MHz) δ 173.5 (C-1), 153.1 (C-33), 128.6 (C-2), 84.9 (C-16), 84.8 (C-19), 83.2 (C-10), 80.6 (C20), 79.8 (C-15), 78.0 (C-4), 77.9 (C-34), 38.9 (CH3, OSO2CH3), 38.7 (CH3, OSO2CH3), 34.2 (CH3, OSO2CH3), 31.5 (CH3, OSO2CH3), 30.4 (C-3), 29.6−22.6 (C-5−C-9, C-11−C-14, C-17, C18, C-21−C-31), 18.7 (C-35), 14.1 (C-32); HRESIMS (+) m/z 909.3844 [M + H]+ (calcd for C39H72O15S4, 909.3827). (S)-5-Methyl-3-((2S,8S,13S)-2,8,13-triazido-13-((2R,5R)-5-((S)-1azidotridecyl)tetrahydrofuran-2-yl)tridecyl)furan-2(5H)-one (3). Sodium azide (6.0 mg, 0.088 mmol, 8.0 equiv) was added to 2 (10.0 mg, 0.011 mmol, 1.0 equiv) under nitrogen. N-Methylpyrrolidone (350 μL) was added, and the reaction mixture was heated to 80 °C with stirring

for 3 h. The reaction progress was monitored by TLC (hexane−ethyl acetate, 2:1). Concentration of the reaction mixture and submission of the crude residue to flash column chromatography (hexane−ethyl acetate, 4:1) gave pure 3 as a colorless oil (4.0 mg, 52%): [α]20D −16.1 (c 0.51, CHCl3); Rf 0.48 (hexane−ethyl acetate, 3:1); FTIR νmax 2925, 2855, 1756, 1259, 1075 cm−1; 1H NMR (CDCl3, 400 MHz) δ 7.22 (1H, s, H-33), 5.06 (1H, q, J = 6.8 Hz, H-34), 4.07 (2H, m, H-16, H19), 3.57 (3H, m, H-4, H-20, H-15), 3.24 (1H, m, H-10), 2.53 (1H, dd, J = 13.2, 4.0 Hz, H-3a), 2.47 (1H, dd, J = 13.2, 9.6 Hz, H-3b), 1.99 (2H, m, H-17b, H-18b), 1.86 (2H, m, H-17a, H-18a), 1.44 (3H, d, J = 6.8 Hz, H-35), 1.26−1.50 (43H, m, H-5−H-9, H-11−H-14, H-21−H-31), 0.878 (3H, t, J = 6.8 Hz, H-32); 13C NMR (CDCl3, 700 MHz) δ 173.4 (C-1), 152.3 (C-33), 129.9 (C-2), 82.1 (C-16), 81.9 (C-19), 77.8 (C34), 65.4 (C-15), 65.2 (C-20), 62.8 (C-4), 60.5 (C-10), 34.3 (C-3), 31.9−22.7 (C-5−C-9, C-11−C-14, C-17, C-18, C-21−C-31), 18.9 (C35), 14.1 (C-32); HRESIMS (+) m/z 697.4997 [M + H]+ (calcd for C35H60N12O3, 697.4984). (S)-5-Methyl-3-((2S,8S,13S)-2,8,13-triiodo-13-((2R,5R)-5-((S)-1iodotridecyl)tetrahydrofuran-2-yl)tridecyl)furan-2(5H)-one (4). Triphenylphosphine (420 mg, 1.6 mmol, 16 equiv) and imidazole (218 mg, 3.2 mmol, 32 equiv) were added to a reaction flask under nitrogen. Dichloromethane (2.0 mL) was added, and the reaction mixture was stirred and cooled to 0 °C. Iodine (203 mg, 1.6 mmol, 16 equiv) was then added slowly at 0 °C under a stream of nitrogen. After the disappearance of the yellow color, a solution of 1 (60 mg, 1.0 equiv) in dichloromethane (500 μL) was added in 100 μL portions dropwise over 30 min. After the addition of 1 was complete, the reaction mixture was stirred at 0 °C (30 min) and then allowed to warm to room temperature with stirring overnight. The reaction progress was monitored by TLC (hexane−ethyl acetate, 4:1). On completion, the reaction mixture was treated with iodomethane (1.71 g, 12 mmol, 750 μL) to remove the excess triphenylphosphine and then stirred for 60 min. The reaction mixture was then flushed through a silica gel pad eluting with ethyl acetate followed by concentration of the filtrate. The crude product was submitted to flash column chromatography (hexane−ethyl acetate, 5:1) to provide pure 4 as a colorless oil (25 mg, 24%); Rf 0.42 (hexane− ethyl acetate, 4:1); FTIR νmax 2923, 2853, 1756, 1075 cm−1; 1H NMR (CDCl3, 700 MHz) δ 7.23 (1H, s, H-33), 5.07 (1H, q, J = 7.0 Hz, H34), 4.31 (1H, m, H-4), 4.14 (3H, m, H-10, H-15, H-20), 3.86 (2H, m, H-16, H-19), 2.84 (2H, dd, J = 13.3, 9.8 Hz, H-3a, H-3b), 2.23 (2H, m, H-17b, H-18b), 1.69−1.87 (12H, m, H-5, H-9, H-11, H-14, H-17a, H18a, H-21), 1.46 (3H, d, J = 6.3 Hz, H-35), 1.26−1.48 (31H, m, H-6− H-8, H-12, H-13, H-22−H-31), 0.879 (3H, t, J = 7.0 Hz, H-32); 13C NMR (CDCl3, 700 MHz) δ 173.2 (C-1), 152.0 (C-33), 131.5 (C-2), 83.0 (C-16), 82.8 (C-19), 77.7 (C-34), 44.2 (C-10), 43.6 (C-15, C-20), 42.5 (C-4), 40.4−22.7 (C-3, C-5−C-9, C-11−C-14, C-17, C-18, C21−C-31), 19.0 (C-35), 14.1 (C-32); HRESIMS (+) m/z 1037.0801 [M + H]+ (calcd for C35H60O3I4, 1037.0794). (S)-5-Methyl-3-((2R,8R,13R)-2,8,13-triazido-13-((2R,5R)-5-((R)-1azidotridecyl)tetrahydrofuran-2-yl)tridecyl)furan-2(5H)-one (5). Sodium azide (13.0 mg, 0.19 mmol, 20.0 equiv) was added to 4 (10.0 mg, 0.0097 mmol, 1.0 equiv) under nitrogen. Dimethylformamide (350 μL) was added, and the reaction mixture was heated to 45 °C with stirring overnight. The reaction progress was monitored by TLC (hexane− ethyl acetate, 4:1). Removal of the DMF under high vacuum gave a crude residue that was submitted to flash column chromatography (hexane−ethyl acetate, 4:1), to provide pure 5 as a colorless oil (3.0 mg, 45%): [α]20D +11.0 (c 0.51, CHCl3); Rf 0.30 (hexane−ethyl acetate, 4:1); FTIR νmax 2926, 2864, 2101, 1758, 1257 cm−1; 1H NMR (CDCl3, 700 MHz) δ 7.22 (1H, s, H-33), 5.06 (1H, q, J = 5.6 Hz, H-34), 4.05 (2H, m, H-16, H-19), 3.61 (1H, m, H-4), 3.24 (1H, m, H-10), 3.07 (2H, m, H-15, H-20), 2.46 (2H, dd, J = 15.4, 9.1 Hz, H-3a, H-3b), 2.05 (2H, m, H-17a, H-18a), 1.81 (2H, m, H-17b, H-18b), 1.44 (3H, d, J = 6.3 Hz, H-35), 1.25−1.55 (43H, m, H-5−H-9, H-11−H-14, H-21−H31), 0.877 (3H, t, J = 6.3 Hz, H-32); 13C NMR (CDCl3, 700 MHz) δ 173.4 (C-1), 152.2 (C-33), 130.0 (C-2), 81.9 (C-16), 81.7 (C-19), 77.8 (C-34), 65.0 (C-15), 64.8 (C-20), 62.8 (C-4), 60.5 (C-10), 34.3 (C-3), 31.9−22.7 (C-5−C-9, C-11−C-14, C-17, C-18, C-21−C-31), 18.9 (C35), 14.1 (C-32); HRESIMS (+) m/z 697.4987 [M + H]+ (calcd for C35H60N12O3, 697.4984). 1908



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Note

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.8b00284.

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Experimental procedures for RAR bioassay and PAMPA assay; NMR spectra (1H and 13C) and FTIR spectra for compounds 2, 3, 4, and 5 (PDF)

AUTHOR INFORMATION

Corresponding Author

*Tel: (502) 852-7323. Fax: (502) 852-8149. E-mail: faluzz01@ louisville.edu. ORCID

Frederick A. Luzzio: 0000-0002-9903-0781 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors acknowledge the following members of the NCI Clinical Pharmacology group for performing the RAR assays: J. Strope, S. Pisle, S. Beedie, E. Harris, and Dr. W. Figg. The measurement of HRMS by Dr. M. Walla of the Mass Spectrometry Laboratory, Department of Chemistry and Biochemistry, University of South Carolina, is acknowledged.

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DEDICATION Dedicated to the memory of Professor Robert Ben Channell (1924−2001), botanist, Vanderbilt University. REFERENCES

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