Synthesis of a simplified triazole analogue of pateamine A.

Europe PMC Funders Group Author Manuscript Org Biomol Chem. Author manuscript; available in PMC 2017 June 14. Published in final edited form as: Org Biomol Chem. 2016 June 14; 14(22): 5117–5127. doi:10.1039/c6ob00086j.

Europe PMC Funders Author Manuscripts

Synthesis of a Simplified Triazole Analogue of Pateamine A A. Hemi Cumminga, Sarah L. Browna,b, Xu Taoa, Claire Cuyamendousa, Jessica J. Fieldb, John H. Millerb, Joanne E. Harveya, and Paul H. Teesdale-Spittleb aSchool

of Chemical and Physical Sciences, Victoria University of Wellington, Wellington, NZ

bSchool

of Biological Sciences, Victoria University of Wellington, Wellington, NZ

Abstract Pateamine A is a naturally occurring metabolite extracted from the marine sponge Mycale hentscheli. It exhibits potent cytotoxicity towards cancer cell lines and has been shown to target protein translation initiation via inhibition of the function of eukaryotic initiation factor 4A proteins. We have synthesised a simplified analogue of pateamine A, consisting of the skeletal core of the natural product but with the thiazole heterocycle replaced by a triazole. The convergent design of the synthesis features a base-induced opening of a δ-valerolactone to access the Z,Edienoate moiety, Julia-Kocienski olefination and copper-catalysed azide-alkyne cycloaddition. Bioactivity testing of the simplified pateamine A analogue (3) indicated a significant reduction in cytotoxicity, compared to natural pateamine A. We propose that this reduced activity is due mainly to the substitution of the thiazole for the triazole heterocycle. This supports the hypothesis that the thiazole of pateamine A is important for binding to its biological target.


Introduction Naturally derived chemicals are a rich source of potent biologically active compounds with pharmaceutical applications.1 Whilst these chemical species are often found in only small amounts in their natural source, total synthesis provides an alternative supply. Because natural products are typically complex, preparation of simplified analogues that retain the necessary components for desired activity offers a beneficial solution for large-scale synthesis.2 Pateamine A (1) was isolated from a marine sponge, Mycale hentscheli, located off the coast of New Zealand and was found to have potent cytotoxicity towards the P388 cancer cell line (IC50 0.27 nM).3,4 The key biological targets of pateamine A were found to be the eukaryotic initiation factor 4A (eIF4A) isoforms, namely eIF4A I, II and III.5,6 Pateamine A increases the binding interaction between eIF4A and its substrate RNA.7 With regards to eIF4AI/II, this prevents the formation of the eIF4F complex necessary for the ribosome recruitment step of cap-dependent translation initiation.5 This results in the stalling of translation, and consequently might be the cause of the programmed cell death (apoptosis) induced by pateamine A.8 Through its interaction with eIF4A proteins, pateamine A induces stress granule formation by a novel pathway,9 and more recently, has been shown to prevent iNOS-induced muscle wasting and promote muscle fibre formation.10

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Two total syntheses of pateamine A have been reported,11,12 and a range of analogues have been prepared, focusing on alterations to the side chain and the Z,E-diene within the macrocycle.13,14 Of interest, an analogue of pateamine A devoid of the C5-methyl and C3amino groups (DMDA-pateamine A, 2) was found to have comparable cytotoxicity and eIF4A inhibitory activity to natural pateamine A.13,15The retained activity indicates that, at least to some extent, groups pendant to the core structure of pateamine A are not be required for activity. The key challenges in developing pateamine analogues are to retain activity against the eIF4A isoforms over the other targets of pateamine16and to probe the ability to differentiate binding between the eIF4A I, II and III isoforms. The mode of pateamine A binding to these eIF4A isoforms has not been established. In addition, no analogues that examine the role of the thiazole ring have been prepared. We were therefore interested in preparing simplified pateamine A analogues that would allow investigation of the role of the thiazole ring, particularly the impact of changes to the heterocycle on selectivity towards the known biochemical targets. In this way, the mode of binding of pateamine A to eIF4A might be revealed. In this paper, we describe the synthesis of a simplified, racemic triazole analogue of pateamine A and preliminary characterisation of its biological activity.

Results and discussion


The retrosynthetic strategy identified for the synthesis of the simplified triazole analogue of pateamine A (3) is shown in scheme 1. We envisioned that 3 could be accessed from δlactone-containing precursor 4 by base-induced ring opening of the δ-valerolactone moiety to afford the Z,E-dienoic acid, followed by macrolactonisation and reductive amination. The full skeleton 4 would be constructed from 6-heptynoic acid, δ-valerolactone 5 and trienal 6. Key steps would include a copper-catalysed azide-alkyne cycloaddition (CuAAC) reaction and an esterification reaction. δ-Valerolactone5 could be prepared from 1,3-propanediol, while triene6 could be prepared by Julia-Kocienski reaction of sulfone7 and aldehyde 8, which can themselves be prepared from epichlorohydrin and furan, respectively. The synthesis of sulfone7 began with the efficient ring opening of a racemic mixture of epichlorohydrin with 1-phenyl-1H-tetrazole-5-thiol (PTSH), without the need for solvent or catalyst, providing chloroalkylthioether9 (Scheme 2). Subsequent tetrabutylammonium iodide (TBAI)-assisted substitution of the chloride with sodium azide to give azido compound 10 followed by silylation afforded the TBS-protected azidothioether 11. Ammonium heptamolybdate-mediated oxidation of the thioether with peroxide afforded sulfone7.17Although this procedure generated racemic 7, use of enantiopure epichlorohydrin would be expected to deliver a fragment with the (S) configuration found in the natural product. Dienal8 was prepared in four steps from furan under Achmatowicz-like conditions (Scheme 3). Ring-opening of furan with bromine in anhydrous methanol afforded fumaraldehyde bis(dimethyl acetal) 12.18 Mono-deprotection was conveniently achieved with water and the

Org Biomol Chem. Author manuscript; available in PMC 2017 June 14.

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solid-supported acid catalyst, Amberlyst® 15, to give aldehyde 13.19 Wittig reaction of 13 with (carbethoxymethylene)-triphenylphosphorane afforded ethyl ester 14, and subsequent acetal cleavage with Amberlyst® 15 and water provided aldehyde 8.


Coupling of sulfone 7 and aldehyde 8 was achieved using a Julia-Kocienski olefination reaction (Scheme 4).20 Subsequent reduction of ester 15 with diisobutylaluminium hydride (DIBAL-H) provided alcohol 16 and Parikh-Doering oxidation afforded key aldehyde fragment 6.21 Dess-Martin periodinane oxidation can also be used for this oxidation in comparable yield.22 Sulfone 7 is a persistent contaminant through this sequence, and is most readily purified from aldehyde 6. Preparation of the triazole precursor, alkyne 23 began with the formation of cyclic acetal 17 using para-anisaldehyde and catalytic para-toluenesulfonic acid (p-TSA) (Scheme 5). Treatment of acetal 17 with DIBAL-H afforded the monoalcohol 18, which was subjected to Swern oxidation to provide aldehyde 19. The reaction of the resultant aldehyde with allylmagnesium bromide followed by acryloyl chloride in the same reaction pot afforded diene 20. Ring closing metathesis using Grubbs’ 2nd generation catalyst provided lactone 21. Removal of the PMB protecting group with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) provided alcohol 22. This was then coupled to 6-heptynoic acid by esterification using EDCI with catalytic DMAP to afford the major fragment 23.Aldehyde 6 was then coupled to alkyne 23 in a copper-catalysed azide-alkyne cycloaddition (CuAAC) reaction to afford triazole 4 (Scheme 6).


We attempted to take advantage of the dual reactivity of TBAF to facilitate the next two reactions simultaneously, namely desilylationto remove the TBS group of 4 and baseinduced ring opening of the δ-valerolactone moiety.23 Disappointingly, treatment of compound 4 with TBAF afforded an inseparable mixture of the desired seco-acid 24 and Z,E-dienoic acid by-products that had degraded at the side chain (based on 1H NMR analysis). Due to the apparent incompatibility of the conjugated triene moiety of 4 to treatment with TBAF, it was decided to ring-open the δ-valerolactone moiety prior to coupling with the triene component. Thus, compound 23 was treated with TBAF to afford the Z,E-dieneoic acid 25 (Scheme 7). Triene 6 was desilylated with HCl in methanol to afford alcohol 26. Coupling of compounds 25 and 26 in a CuAAC reaction gave seco-acid 24. Subsequent macrolactonisation using Yamaguchi’s methodology provided the macrocycle 27 in 13% yield.24 The poor yield of the macrolactonisation is likely a result of the reaction conditions adopted to prevent isomerisation of the Z,E-diene, as previously reported.25 Finally, reductive amination of 27 with dimethylamine and tetramethylammonium triacetoxyborohydride afforded the target pateamine A analogue 3.26 Whilst there is substantial opportunity to optimise the conditions for these final two reactions, sufficient product was obtained to permit biological evaluation after which further optimisation was not warranted.


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Biological testing


To measure the growth inhibitory activity against cancer cells, the simplified, racemic pateamine A analogue 3 and the precursor aldehyde 27 were assayed in an MTT assay8 against two cancer cell lines, namely HL-60 (human promyelocytic leukemia cells) and 1A9 (human ovarian carcinoma cells). The IC50 of 3 containing the terminal dimethylamine showed at least a 3-fold greater potency than the aldehyde containing analogue 27 in both cell lines assayed (Table 1). Although analogue 3 showed reasonable activity (low micromolar), it was drastically less potent than its natural counterpart, with pateamine A having an IC50 of 1.43 ± 0.06 nM in HL-60 cells.16

Conclusions In conclusion, the synthesis of a simplified analogue of pateamine A containing a triazole in place of the thiazole of the natural product has been described. This synthesis, which utilises easily accessible starting materials and reagents, represents a convergent strategy with 11 steps in the longest linear sequence.


Bioactivity testing of the racemic analogue 3 indicated a greater than a thousand-fold reduction in activity, compared to natural pateamine A. Whilst the removal of key methyl substituents may be responsible for some of the loss of activity, we propose that the substitution of the thiazole for the triazole heterocycle is a major contributor to this reduction in activity. Whilst the potential for alternative heterocyles, such as oxazoles or imidazoles remains an avenue for structure-activity analysis, this study supports Romo’s hypothesis that the thiazole is important for binding to its biological target.11 The synthesis of other analogues that include the thiazole heterocycle are currently underway to further probe the structure-activity relationship of simplified pateamine A analogues and examine the mode of binding to eIF4A isoforms.

Experimental (±)-3-Chloro-1-(1-phenyl-1H-1,2,3,4-tetrazol-5-ylthio)-2-propanol, (±)-9 1-Phenyl-1H-tetrazole-5-thiol (391 mg, 2.2 mmol) was dissolved in epichlorohydrin (2.5 mL) and the mixture stirred overnight at room temperature. Excess epichlorohydrin was then removed under reduced pressure. To assist in the removal of the final traces of epichlorohydrin, the material was re-dissolved in toluene (1 mL) and the solvent removed under reduced pressure. This was repeated twice, providing alkyl chloride (±)-9 (593 mg, quant.) as a colourless oil. Rf: 0.26 (2:1 pet. ether:EtOAc). 1H NMR (500 MHz, CDCl3): δ 7.59 (s, 5H), 4.39–4.31 (m, 1H), 3.91 (d, J = 5.0 Hz, 1H), 3.80-3.70 (m, 2H), 3.69 (dd, J =14.6, 3.6 Hz, 1H), 3.55 (dd,J = 7.0,14.5 Hz, 1H).13C NMR (125 MHz, CDCl3): δ 154.4, 133.4, 130.4, 129.9, 123.8, 70.4, 47.3, 37.0. IR (film): 3400 (broad), 1596, 1499, 1411, 1387, 1243, 1074, 1042, 1015, 759, 729, 685 cm-1. HRMS-ESI (m/z) calculated for C10H12ClN4OS+ [M + H]+: 271.0415; found 271.0403.


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(±)-3-Azido-1-(1-phenyl-1H-1,2,3,4-tetrazol-5-ylthio)-2-propanol, (±)-10


To a solution of alkyl chloride (±)-9 (610.7 mg, 2.26 mmol) in DMF (15 mL) at room temperature was added sodium azide (297.1 mg, 4.57mmol) and TBAI (167.9 mg, 0.5mmol) and the reaction mixture heated to 80 °C overnight. The mixture was carefully concentrated to 5 mL under reduced pressure then diluted with water (30 mL), extracted with DCM (3 x 30 mL) and washed with saturated brine. The combined organic fractions were dried (MgSO4), filtered, and solvent removed under reduced pressure. Silica gel flash chromatography (3:1 pet. ether:EtOAc) provided alcohol (±)-10 (468 mg, 75%) as a colourless oil. Rf: 0.26 (2:1 pet. ether:EtOAc). 1H NMR (500 MHz, CDCl3): δ 7.59 (br. s, 5H), 4.28–4.25 (m, 1H), 3.60–3.47 (m, 5H). 13C NMR (125 MHz, CDCl3): δ 154.3, 133.4, 130.4, 129.9, 123.8, 69.9, 55.1, 37.1. IR (film): 3400 (broad), 2099, 1662, 1596, 1499, 1411, 1387, 1279, 1243, 1091, 1075, 981, 731, 686 cm-1. HRMS-ESI (m/z) calculated for C10H12N7OS+ [M + H]+: 278.0819; found 278.0806. (±)-5-(3-Chloro-2-(tert-butyldimethylsilyloxy)propylthio)-1-phenyl-1H-1,2,3,4-tetrazole, (±)-11


To a solution of alcohol (±)-10 (210 mg, 0.76 mmol) in DMF (4 mL) at room temperature was added sequentially imidazole (210 mg, 3.08 mmol) , TBAI (31 mg, 0.08 mmol) and TBSCl (357 mg, 2.37 mmol). The reaction mixture was stirred overnight at room temperature then diluted with ethyl acetate (10 ml) and washed with water (3 x 10 mL). The aqueous phase was extracted with ethyl acetate (3 x 10 mL). The combined organic fractions were dried (Na2SO4), filtered and the solvent removed under reduced pressure. Gradient silica gel flash chromatography (5:1 to 2:1 pet. ether:EtOAc) provided thioether (±)-11 (287 mg, 97%) as a colourless oil that became semi-crystalline during storage in the freezer. Rf: 0.74 in 2:1 (pet. ether:EtOAc). 1H NMR (500 MHz, CDCl3): δ 7.59–7.56 (m, 5H), 4.33– 4.30 (m, 1H), 3.55 (d, J = 5.7 Hz, 2H), 3.49 (dd, J = 4.4, 12.7 Hz, 1H), 3.34 (dd, J = 4.4, 12.7 Hz, 1H), 0.90 (s, 9H), 0.14 (s, 3H), 0.12 (s, 3H). 13C NMR (125 MHz, CDCl3): δ 154.0, 133.5, 130.2, 129.9, 123.8, 69.8, 55.3, 37.0, 25.7, 18.0, -4.67, -4.69. Melting point: 49-51 °C. IR (film): 2930, 2857, 2360, 2101, 1597, 1500, 1463, 1388, 1281, 1252, 1106, 961, 837, 807, 778, 760, 694 cm-1. HRMS-ESI (m/z) calculated for C16H25N7OSSi+ [M + H]+: 392.1683; found 392.1678. (±)-5-(3-Chloro-2-(tert-butyldimethylsilyloxy)propylsulfonyl)-1-phenyl-1H-1,2,3,4-tetrazole, (±)-7 To a solution of thioether (±)-11 (8.09 g, 20.0 mmol) in absolute ethanol (100 mL) was added ammonium heptamolybdate (3.8 g, 3.1 mmol). The reaction mixture was then cooled to 0 °C prior to addition of H2O2 (30% aqueous solution, 1.3 mL, approximately 12.0 mmol) and the reaction stirred at room temperature. The reaction progress was followed by TLC, and formation of a slower running spot was observed, attributed to sulfoxide (Rf: 0.53 in 2:1 (pet. ether:EtOAc)), followed by the loss of this spot as it converted to the desired sulfone. (Although possessing the same Rf, sulfide and sulfone could be distinguished as only the sulfone reacted instantly with a KMnO4 dip). The reaction mixture was stirred overnight or until reaction was complete by TLC with addition of extra H2O2 solution if needed. When complete, peroxides were quenched with saturated aqueous Na2SO3 (15 mL), ethanol removed under reduced pressure, and the mixture diluted with distilled water (80 mL) and


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extracted with DCM (3 x 60 mL). The combined organic fractions were dried (MgSO4), filtered and the solvent removed under reduced pressure to provide sulfone (±)-7 (7.6 g, 90%) as a colourless oil that slowly crystallised during storage in freezer. Rf: 0.74 in 2:1 (pet. ether:EtOAc). 1H NMR (500 MHz, CDCl3): δ 7.69–7.62 (m, 5H), 4.62–4.58 (m, 1H), 4.07 (dd, J = 5.9,14.9 Hz, 1H), 4.01 (dd, J = 5.4, 14.9 Hz, 1H), 3.57 (dd, J = 4.30, 12.8 Hz, 1H), 3.41 (dd, J =4.2, 12.8 Hz, 1H), 0.88 (s, 9H), 0.15 (s, 3H), 0.12 (s, 3H). 13C NMR (125 MHz, CDCl3): δ 153.9, 132.9, 131.6, 129.8, 125.0, 66.4, 59.6, 55.8, 25.6, 17.8, -4.7, -4.9. Melting point: 40-42°C. IR (film): 2931, 2858, 2105, 1498, 1463, 1343, 1287, 1255, 1154, 1104, 961, 839, 808, 780, 762, 731, 688 cm-1. HRMS-ESI (m/z) calculated for C16H26N7O3SSi+ [M + H]+: 424.1582; found 424.1571. Fumaraldehydebis(dimethyl acetal), 12 To a solution of furan (9.40 mL, 129 mmol) in dry methanol (60 mL) at -45 °C (dry ice/ acetone bath) was added dropwise a solution of bromine (6.8 mL, 133 mmol) in dry methanol (60 mL). The reaction was then stirred for 3 hours maintaining the temperature between -40 and -10 °C (dry ice/acetone bath). Anhydrous sodium carbonate (40 g) was then added portionwise over about 30 minutes and stirring continued overnight at room temperature. The mixture was filtered and the solvent was removed under reduced pressure. Crude material was then purified by distillation in a kugelrohr apparatus (65-70 °C, 1 mmHg), giving acetal (12) (18.02 g, 79%) as a colourless liquid. Rf: 0.52 (2:1 pet. ether:EtOAc). 1H NMR (500 MHz, CDCl3): δ 5.81 (s, 2H), 4.82 (s, 2H), 3.31 (s, 12H). 13C NMR (125 MHz, CDCl3): δ 130.9, 101.9, 52.7. These characterisation data match those in the literature.27 (E)-4,4-Dimethoxybut-2-enal, 13


To a solution of fumaraldehydebis(dimethyl acetal) 12 (3.81 g, 21.6 mmol) in acetone at room temperature was added Amberlyst-15 (800 mg) and water (1.2 mL, 64.9 mmol). The reaction was stirred vigorously for 10 minutes before removal of the acidic resin by filtration. Solvent was removed under reduced pressure and purified by silica gel flash chromatography (4:1 pet. ether:diethyl ether) to provide aldehyde 13 (1.95g, 69%) as a colourless oil. Rf: 0.48 (2:1 pet. ether:EtOAc). 1H NMR (500 MHz, CDCl3): δ 9.62 (d, J = 7.9 Hz, 1H), 6.63 (dd, J = 3.9, 16.0 Hz, 1H), 6.37 (dd, J = 7.9, 16.0 Hz, 1H), 5.07 (d, J = 3.9 Hz, 1H), 3.36 (s, 6H). 13C NMR (125 MHz, CDCl3): δ 193.2, 150.4, 134.2, 100.4, 53.0. These characterisation data match those in the literature.28 Ethyl (2E,4E)-6,6-dimethoxy-2,4-hexadienoate, 14 To a solution of aldehyde 13 (2.27 g, 17.5 mmol) in DCM (40 mL) at room temperature was added ethyl (triphenylphosphoranylidene)acetate (6.0 g, 17.5 mmol) and the mixture stirred overnight. Solvent was removed under reduced pressure and the crude material was passed through a short silica plug (5:1 pet. ether:EtOAc) to remove triphenylphosphine oxide byproduct. Solvent was then removed under reduced pressure and the residue was used directly in the subsequent reaction. A small quantity was purified by silica gel flash chromatography (5:1 pet. ether:EtOAc) to provide a sample for characterisation. Rf: 0.61 (2:1 pet. ether:EtOAc). 1H NMR (500 MHz, CDCl3): δ 7.28 (dd, J = 11.2, 15.6 Hz, 1H), 6.49 (dd, J =


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11.2, 15.6 Hz, 1H), 6.00 (dd, J = 4.1, 15.6 Hz, 1H), 5.95 (d, J = 15.6 Hz, 1H), 4.92 (d, J = 4.1 Hz, 1H), 4.22 (q, J = 7.1 Hz, 2H), 3.33 (s, 6H), 1.30 (t, J = 7.1 Hz, 3H). 13C NMR (125 MHz, CDCl3): δ 166.7, 142.9, 137.3, 131.1, 123.2, 101.3, 60.5, 52.7, 14.3. These characterisation data match those in the literature.29


Ethyl (2E,4E)-6-oxo-2,4-hexadienoate, 8 To a solution of dimethyl acetal 14 (direct from previous reaction) in acetone at room temperature was added Amberlyst 15 (1 g) and water (1.3 mL, 70 mmol). The reaction was stirred vigorously for 1 hour at 60 °C. After removal of the acidic resin by filtration, solvent was removed under reduced pressure and purified by silica gel flash chromatography (4:1 pet. ether:diethyl ether) to provide ester 8 (1.95 g, 69% from 13) as a colourless oil. Rf: 0.61 (2:1 pet. ether:EtOAc). Rf: 0.61 (2:1 pet. ether:EtOAc). 1H NMR (500 MHz, CDCl3): δ 9.67 (d, J = 7.9 Hz, 1H), 7.42 (dd, J = 11.3, 15.4 Hz, 1H), 7.17 (dd, J = 11.3, 15.3 Hz, 1H), 6.42 (dd, J = 7.9, 15.3 Hz, 1H), 6.31 (d, J = 15.4 Hz, 1H), 4.26 (q, J = 7.2 Hz, 2H), 1.32 (t, J = 7.2 Hz, 3H). 13C NMR (125 MHz, CDCl3): δ 192.9, 165.4, 147.2, 140.3, 136.9, 129.9, 61.1, 14.2. These characterisation data match those in the literature.30 Ethyl (2E,4E,6E)-9-azido-8-(tert-butyldimethylsilyloxy)-2,4,6-nonatrienoate, (±)-15


To a solution of sulfone (±)-7 (5.69 g, 13.4 mmol) in THF (80 mL) at -78 °C (dry ice/ acetone bath) was added a solution of KHMDS in toluene (0.5 M, 40.2 mL, 20.1 mmol) and the reaction stirred for 15 minutes. Aldehyde 8 (2.28 g, 14.8 mmol) in THF (30 mL) was then added slowly to the reaction mixture and allowed to stir for 1 hour at -78 °C, before slowly warming to room temperature over 30 minutes. The reaction was then quenched with saturated aqueous ammonium chloride (80 mL) and extracted with EtOAc (3 x 50 mL). The combined organic fractions were washed with water, then saturated brine, dried (MgSO4), filtered and the solvent removed under reduced pressure. Silica gel flash chromatography (5:1 pet. ether:EtOAc) provided ester (±)-15 (3.05 g as a colourless oil, that was accompanied by inseparable residual starting material (±)-7 that could be harmlessly carried through subsequent reactions and most readily removed during purification of aldehyde (±)-6. Rf: 0.77 (2:1 pet. ether:EtOAc). 1H NMR (500 MHz, CDCl3): δ 7.30 (dd, J = 11.4, 15.5 Hz, 1H), 6.54 (dd, J = 11.0, 14.8 Hz, 1H), 6.29–6.40 (m, overlapping peaks, 2H), 5.90 (d, J = 15.5 Hz, 1H), 5.86 (dd, J = 6.0, 15.3 Hz, 1H), 4.40–4.36 (m, 1H), 4.21 (q, J = 7.1 Hz, 2H), 3.24–3.15 (m, 2H), 1.30 (t, J = 7.1 Hz, 3H), 0.93 (s, 9H), 0.12 (s, 4H), 0.07 (s, 3H). 13C NMR (125 MHz, CDCl3): δ 166.9, 144.0, 139.2, 137.2, 130.61, 130.59, 121.5, 72.6, 60.3, 56.9, 25.7, 18.1, 14.3, -4.6, -5.0. NMR spectra include additional peaks derived from compound 7. IR (film): 2955, 2930, 2858, 2099, 1708, 1620, 1254, 1112, 1005, 956, 836, 778 cm-1. HRMS-ESI (m/z) calculated for C17H30N3O3Si+ [M + H]+: 352.2051; found 352.2044. (2E,4E,6E)-9-Azido-8-(tert-butyldimethylsilyloxy)-2,4,6-nonatrien-1-ol, (±)-16 To a solution of ethyl ester (±)-15 (701 mg, approx. 2.0 mmol) in THF (30 mL) at -78°C (dry ice/acetone bath) was added a solution of DIBAL-H in hexane (1 M, 4.0 mL, 4.0 mmol). The reaction mixture was allowed to warm to room temperature over 2 hours, before the reaction was quenched by slow addition of saturated potassium sodium tartrate


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(Rochelle’s salt, 30 mL) and the mixture stirred for 2 hours. The aqueous fraction was then extracted with EtOAc (3 x 30 mL), and the combined organic fractions were dried (MgSO4), filtered, then concentrated under reduced pressure, providing alcohol (±)-16 (595 mg) as a pale yellow oil. 1H NMR spectroscopy indicated material contained residual (±)-7, but was sufficiently pure for subsequent reaction without further purification. A small sample was purified for characterisation purified by silica gel flash chromatography (5:1 to 4:1 pet. ether:EtOAc, but with poor mass recovery (~15%) from pure fractions. Rf: 0.45 (2:1 pet. ether:EtOAc). 1H NMR (500 MHz, CDCl3): δ 6.35–6.18 (m, overlapping peaks, 4H), 5.92– 5.87 (m, 1H), 5.67 (dd, J = 6.3, 14.8 Hz, 1H), 4.36–4.32 (m, 1H), 4.22 (d, J = 5.7 Hz, 2H), 3.18 (d, J = 5.3 Hz, 2H), 0.93 (s, 9H), 0.12 (s, 3H), 0.07 (s, 3H). 13C NMR (125 MHz, CDCl3): δ 133.2, 132.8, 132.6, 131.9, 131.4, 131.1, 73.0, 63.3, 57.2, 25.8, 18.1, -4.5, -5.0. IR (film): 3413, 2930, 2858, 2100, 1707, 1620, 1256, 1101, 999, 837, 779 cm-1. HRMS-ESI (m/z) calculated for C15H27N3NaO2Si+ [M + Na]+: 332.1765; found 332.1763. (2E,4E,6E)-9-Azido-8-(tert-butyldimethylsilyloxy)-2,4,6-nonatrienal, (±)-6 (Parikh Doering oxidation)


To a solution of alcohol (±)-16 (89 mg, approx. 0.29 mmol) in DCM (10 mL) at room temperature was added DMSO (206 μL, 2.9 mmol) and triethylamine (202 μL, 1.45 mmol). The reaction mixture was cooled to 0 °C, SO3.Py (137 mg, 0.86 mmol) added and the reaction stirred for 1 hour before warming to room temperature and stirring for a further hour. The reaction was quenched with saturated aqueous ammonium chloride (10 mL) and extracted with DCM (3 x 10 mL). The combined organic fractions were washed with distilled H2O (10 mL) then saturated brine (10 mL), dried (MgSO4), filtered and concentrated under reduced pressure. The crude material was purified by silica gel flash chromatography (5:1 pet. ether:EtOAc) to provide aldehyde (±)-6 (73 mg, 51% over 3 steps) as a pale yellow oil. Rf: 0.79 (2:1 pet. ether:EtOAc). 1H NMR (500 MHz, CDCl3): δ 9.58 (d, J = 7.8 Hz, 1H), 7.13 (dd, J = 11.2, 15.4 Hz, 1H), 6.67 (dd, J = 11.1, 14.6 Hz, 1H), 6.39– 6.50 (m, overlapping peaks, 2H), 6.19 (dd, J = 7.8, 15.4 Hz, 1H), 5.97 (dd, J = 5.8, 15.2 Hz, 1H), 4.42–4.39 (m, 1H), 3.27–3.17 (m, 2H), 0.94 (s, 9H), 0.14 (s, 3H), 0.08 (s, 3H). 13C NMR (125 MHz, CDCl3): δ 193.5, 151.4, 141.1, 139.1, 131.8, 130.5, 130.3, 72.5, 56.9, 25.7, 18.1, -4.6, -5.0. IR (film): 2930, 2858, 2101, 1679, 1616, 1346, 1254, 1157, 1109, 993, 835, 778, 688 cm-1. HRMS-ESI (m/z) calculated for C15H26N3O2Si+ [M + H]+: 308.1789; found 308.1769. (2E,4E,6E)-9-Azido-8-(tert-butyldimethylsilyloxy)-2,4,6-nonatrienal, (±)-6 (DMP oxidation) To a solution of alcohol (±)-16 (5.6 mg, 0.018 mmol) in DCM (1 mL) at room temperature was added Dess-Martin periodinane (15 mg, 0.035 mmol) and sodium bicarbonate (6 mg, 0.071 mmol). The reaction mixture was stirred for 2 hours. Then the reaction was quenched with a 1:1 mixture of saturated aqueous sodium bicarbonate and saturated aqueous sodium thiosulfate (2 mL) and extracted with DCM (3 × 5 mL). The combined organic fractions were washed with saturated brine (3 mL), dried (Na2SO4), filtered and concentrated under reduced pressure. The crude material was purified by silica gel flash chromatography (5:1 pet. ether:EtOAc) to provide aldehyde (±)-6 (4.5 mg, 81%) as a colorless oil. Rf: 0.50 (3:1 pet. ether:EtOAc). The product was spectroscopically indistinguishable from that obtained through Parikh Doering oxidation. Org Biomol Chem. Author manuscript; available in PMC 2017 June 14.

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2-(4-Methoxyphenyl)-1,3-dioxane, 17


A solution of 1,3-propanediol (6.64 g, 87.3 mmol) and p-anisaldehyde (9.90 g, 72.7 mmol) in toluene (50 mL) was treated with a catalytic amount of p-toluenesulfonic acid (100 mg, 0.6 mmol) and then heated at reflux in a Dean-Stark apparatus overnight. The reaction mixture was allowed to cool to room temperature, quenched with saturated aqueous sodium bicarbonate (2 mL) and washed with distilled water (50 mL). The aqueous fraction was then re-extracted with diethyl ether (50 mL), and the combined organic fractions were dried (MgSO4), filtered and the solvent removed under reduced pressure to provide acetal17 (13.53 g, 96%) as a brown crystalline solid which was used without further purification. Rf: 0.73 (1:1 pet. ether:EtOAc). Melting point: 39-42°C 1H NMR (500 MHz, CDCl3): δ 7.42 (d, J = 8.7 Hz, 2H), 6.90 (d, J = 8.7 Hz, 2H), 5.47 (s, 1H), 4.27-4.24 (m, 2H), 4.00-3.95 (m, 2H), 3.81 (s, 3H), 2.26–2.17 (m, 1H), 1.43 (d, J = 13.4 Hz, 1H). 13C NMR (125 MHz, CDCl3): δ 159.9, 131.3, 127.2, 113.6, 101.6, 67.4, 55.3, 25.7. Spectral data matched those reported in the literature.31 3-[(4-Methoxybenzyl)oxy]-1-propanol, 18


To a solution of acetal 17 (2.56 g, 13.16 mmol) in DCM (50 mL) at -78 °C was added a solution of DIBAL-H in hexane (1M, 48 mL, 48 mmol). The reaction mixture was allowed to warm to room temperature over 2 hours, then the reaction was quenched by slow addition of saturated aqueous potassium sodium tartrate (Rochelle’s salt, 50 mL) and the mixture stirred for 2 hours. The aqueous fraction was extracted with EtOAc (3 × 30 mL). The combined organic fractions were dried (MgSO4), filtered, and the solvent removed under reduced pressure providing alcohol 18 (2.58 g, quant.) as a colourless oil. 1H NMR indicated that the product was sufficiently pure for the subsequent steps so no further purification was undertaken. Rf: 0.36 (1:1 pet. ether:EtOAc). 1H NMR (500 MHz, CDCl3): δ 7.26 (d, J = 8.7 Hz, 2H), 6.89 (d, J = 8.7 Hz, 2H), 4.46 (s, 2H), 3.81 (s, 3H), 3.78 (apparent q, J = 5.6 Hz, 2H), 3.65 (t, J = 5.6 Hz, 2H), 2.28 (br s, 1H), 1.86 (quin., J = 5.6 Hz, 2H). 13C NMR (125 MHz, CDCl3): δ 159.2, 130.1, 129.3, 113.8, 72.9, 69.2, 62.1, 55.2, 32.0. These characterisation data match those in the literature.31 3-[(4-Methoxybenzyl)oxy]propanal, 19 To a solution of DMSO (1.16 mL, 16.4 mmol) in DCM (50 mL) at -78 °C (dry ice/acetone bath) was added oxalyl chloride (714 mL, 8.2 mmol) and the reaction stirred for 15 minutes. A solution of alcohol 18 (803 mg, 4.1 mmol) in DCM (5 mL) was then added and the mixture stirred for 15 minutes. Following addition of triethylamine (2.28 mL, 16.4 mmol) the reaction was stirred for 30 minutes at room temperature then quenched with saturated aqueous ammonium chloride (40 mL) and extracted with DCM (3 × 40 mL). The combined organic fractions were washed with water and saturated brine, then dried (MgSO4), filtered and the solvent removed under reduced pressure. The sample was then passed through a silica plug to provide colourless oil and used directly in the next reaction without further purification. Rf: 0.43 (1:1 pet. ether:EtOAc). 1H NMR (500 MHz, CDCl3): δ 9.79 (s, 1H), 7.29 (d, J = 8.5 Hz, 2H), 6.91 (d, J = 8.5 Hz, 2H), 4.49 (s, 2H), 3.83 (s, 3H), 3.81 (t, J = 6.1 Hz, 2H), 2.71 (td, J = 1.7, 6.1 Hz, 2H). 13C NMR (125 MHz, CDCl3): δ 201.3, 159.3, 129.9,


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129.3, 113.8, 72.9, 63.5, 55.3, 43.8. These characterisation data match those in the literature. 32 (±)-1-[(4-Methoxybenzyl)oxy]hex-5-en-3-yl acrylate, (±)-20


To a solution of aldehyde 19 (803 mg, 4.09 mmol) in THF (25 mL) at room temperature was added a solution of allylmagnesium bromide in diethyl ether (1 M, 4.91 mL, 4.91 mmol) and the mixture was stirred overnight. Diisopropylethylamine (697 µL, 517 mg, 4.09 mmol) was added and the mixture cooled to 0 °C, before addition of acryloyl chloride (498 µL, 555 mg, 6.14 mmol) and DMAP (40 mg, 0.33 mmol). The reaction mixture was stirred overnight at room temperature then quenched with saturated aqueous ammonium chloride (30 mL), and extracted with EtOAc (3 × 30 mL). The combined organic fractions were washed with water and saturated brine, then dried (MgSO4), filtered and the solvent removed under reduced pressure. The crude material was purified by silica gel flash chromatography (5:1 pet. ether:EtOAc) to provide ester (±)-20 (1.05 g, 88% over 3 steps from 18) as a colourless oil. Rf: 0.64 (2:1 pet. ether:EtOAc). 1H NMR (500 MHz, CDCl3): δ 7.25 (d, J = 8.6 Hz, 2H), 6.87 (d, J = 8.6 Hz, 2H), 6.38 (d, J = 17.1 Hz, 1H), 6.11-6.06 (m, 1H), 5.81-5.79 (m, 1H), 5.76-5.72 (m, 1H), 5.18-5.14 (m, 1H), 5.09-5.05 (m, 2H), 4.41 (s, 2H), 3.81 (s, 3H), 3.52-3.43 (m, 2H), 2.41-2.34 (m, 2H), 1.92-1.87 (m, 2H). 13C NMR (125 MHz, CDCl3): δ 165.7, 159.1, 133.4, 130.5, 130.4, 129.3, 128.7, 118.0, 113.7, 72.7, 71.0, 66.2, 55.3, 38.8, 33.8 cm-1. These characterisation data match those in the literature.33 (±)-6-{2-[(4-Methoxybenzyl)oxy]ethyl}-5,6-dihydro-2H-pyran-2-one, (±)-21


To a solution of diene (±)-20 (340 mg, 1.18mmol) in DCM (50 mL) at room temperature was added (in two portions over 14 hours) Grubbs second generation catalyst (50 mg, 0.06 mmol, 5 mol%). The reaction mixture was then refluxed for 3 hours, and the solvent was removed under reduced pressure. The crude material was purified by gradient silica gel flash chromatography (9:1 to1:1 pet. ether:EtOAc) to provide lactone (±)-21 (240 mg, 78%) as a pale brown oil. Rf: 0.32 (1:1 pet. ether:EtOAc). 1H NMR (500 MHz, CDCl3): δ 7.25 (d, J = 8.3 Hz, 2H), 6.88–6.85 (m, overlapping peaks, 3H), 6.03 (d, J = 9.8 Hz, 1H), 4.64 (td, J = 3.3, 7.9 Hz, 1H), 4.46 (d, J = 11.5 Hz, 1H), 4.42 (d, J = 11.5 Hz, 1H), 3.81 (s, 3H), 3.70– 3.65 (m, 2H), 2.37–2.35 (m, 2H), 2.10–2.03 (m, 1H), 1.97–1.91 (m, 1H). 13C NMR (125 MHz, CDCl3): δ 164.4, 159.2, 145.2, 130.2, 129.4, 121.4, 113.8, 75.3, 72.9, 65.3, 55.3, 35.1, 29.6. IR (film): 2932, 2863, 1716, 1612, 1512, 1244, 1174, 1087, 1031, 815 cm-1. HRMSESI (m/z) calculated for C15H22NO4+ [M + NH4]+: 280.1549; found 280.1549. (±)-6-(2-Hydroxyethyl)-5,6-dihydro-2H-pyran-2-one, (±)-22 To a solution of lactone (±)-21 (160 mg, 0.61 mmol) in 9:1 DCM:H2O (15 mL) at room temperature was added 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (152 mg, 0.67 mmol) and the mixture stirred overnight. The reaction mixture was then filtered (filter paper) and extracted with DCM (3 × 15 mL). The crude material was purified by gradient silica gel flash chromatography (1:1 pet.ether:EtOAc to EtOAc) to provide lactone (±)-22 (73 mg, 84%) as a colourless oil. Rf: 0.26 (EtOAc). 1H NMR (500 MHz, CDCl3): δ 6.91 (dt, J = 4.3, 9.8 Hz, 1H), 6.02 (d, J = 9.8 Hz, 1H), 4.70-4.65 (m, 1H), 3.94-3.89 (m, 1H), 3.87-3.82 (m, 1H), 2.42-2.39 (m, 2H), 2.27 (br. s, 1H), 2.08-2.01 (m, 1H), 1.95-1.89 (m, 1H). 13C NMR


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(125 MHz, CDCl3): δ 164.4, 145.4, 121.2, 75.6, 58.3, 37.3, 29.5. These characterisation data match those in the literature.34 (±)-2-(6-Oxo-3,6-dihydro-2H-pyran-2-yl)ethyl hept-6-ynoate, (±)-23


To a solution of alcohol (±)-22 (7.8 mg, 0.06 mmol) and 6-heptynoic acid (8.3 mg, 0.07 mmol) in DCM (2.1 mL) at 0 °C was added EDCI (16.8 mg, 0.09 mmol) followed by DMAP (4.2 mg, 0.03 mmol). The reaction mixture was stirred overnight at room temperature, then quenched with saturated aqueous ammonium chloride (3 mL). The aqueous layer was extracted with DCM (4 x 5 mL), and washed with saturated aqueous NaHCO3 and saturated brine. The combined organic fractions were dried (MgSO4), filtered and concentrated under reduced pressure. The crude material was purified by gradient silica gel flash chromatography (5:1 to 1:1 pet. ether:EtOAc) to provide ester (±)-23 (11.9 mg, 86%) as a colourless liquid. Rf: 0.33 (1:1 pet. ether:EtOAc). 1H NMR (500 MHz, CDCl3): δ 6.89 (dd, J = 3.7, 9.4 Hz, 1H), 6.02 (d, J = 9.4 Hz, 1H), 4.60–4.54 (m, 1H), 4.33–4.29 (m, 1H), 4.27-4.22 (m, 1H), 2.40–2.37 (m, 2H), 2.34 (t, J = 7.6 Hz, 2H), 2.21 (td, J = 1.4, 7.2Hz, 2H), 2.15–2.09 (m, 1H), 2.03–1.97 (m, 1H), 1.95 (t, J = 2.6 Hz, 1H), 1.78-1.71 (m, 2H), 1.59-1.53 (m, 2H). 13C NMR (125 MHz, CDCl3): δ 173.2, 163.9, 144.7, 121.5, 83.9, 74.7, 68.6, 59.9, 34.0, 33.6, 29.4, 27.8, 23.9, 18.1. IR (film): 3277, 2935, 1771, 1720, 1389, 1246, 1170, 1037, 818, 638 cm-1. HRMS-ESI (m/z) calculated for C14H19O4+ [M + H]+: 251.1278; found 251.1282. (±)-2-(6-Oxo-3,6-dihydro-2H-pyran-2-yl)ethyl 5-{1-[(3E,5E,7E)-9-oxo-2-(tertbutyldimethylsilyloxy)-3,5,7-nonatrienyl]-1H-1,2,3-triazol-4-yl}valerate, (±)-4


An aqueous solution (2 mL) of CuSO4.5H2O (3 mg, 0.01 mmol) and ascorbic acid (11 mg, 0.06 mmol), was neutralised to pH 7 with aqueous NaOH. A solution of alkyne (±)-23 (31 mg, 0.12 mmol, 1 equiv) and azide 6 (35 mg, 0.12 mmol, 1 equiv) in THF (2 mL) was added and the reaction mixture stirred overnight at room temperature, gradually turning yellow/ green. The mixture was then extracted with EtOAc (3 × 5 mL), and the combined organic fractions were dried (MgSO4), filtered and concentrated under reduced pressure. The crude material was purified by silica gel flash chromatography (1:1 pet. ether:EtOAc) to provide triazole (±)-4 (36 mg, 54%) as a pale yellow oil. Rf: 0.14 (1:1 pet. ether:EtOAc). 1H NMR (500 MHz, CDCl3): δ 9.57 (d, J = 8.0 Hz, 1H), 7.34 (s, 1H), 7.11 (dd, J = 11.2, 15.2 Hz, 1H), 6.96–6.85 (m, 1H), 6.64 (dd, J = 11.1, 14.8 Hz, 1H), 6.51–6.33 (m, overlapping peaks, 2H), 6.18 (dd, J = 8.0, 15.2 Hz, 1H), 6.03 (d, J = 9.8 Hz, 1H), 5.91 (dd, J = 6.0, 15.1 Hz, 1H), 4.66–4.63 (m, 1H), 4.58–4.54 (m, 1H), 4.42 (dd, J = 3.3, 13.8 Hz 1H), 4.34–4.16 (m, 3H), 2.75–2.70 (m, 2H), 2.43–2.37(m, 2H), 2.37–2.29 (m, 2H), 2.05–2.15 (m, 1H), 1.94– 2.04 (m, 1H), 1.72–1.67 (m, overlapping peaks, 4H), 0.84 (s, 9H), -0.06 (s, 3H), -0.18 (s, 3H). 13C NMR (125 MHz, CDCl3): δ 193.5, 173.2, 163.9, 151.1, 147.1, 144.8, 140.7, 137.7, 135.6, 132.0, 130.9, 122.6, 121.4, 74.7, 72.0, 59.9, 56.0, 33.9, 29.4, 28.8, 25.7, 25.6, 25.1, 24.3, 18.0, -4.7, -5.4. IR (film): 2952, 2930, 2857, 1724, 1674, 1614, 1250, 1160, 1110, 1043, 1014, 940, 836, 779, 730cm-1. HRMS-ESI (m/z) calculated for C29H44N3O6Si+[M + H]+: 558.2994; found 558.3014.


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(2Z,4E)-7-(5-Hexynylcarbonyloxy)-2,4-heptadienoic acid, 25


To a solution of lactone (±)-23 (55 mg, 0.22 mmol) in THF (4 mL) at room temperature was added dropwise a solution of tetrabutylammonium fluoride in THF (1 M, 0.88 mL, 0.88 mmol) and the reaction stirred for 2 hours. The reaction mixture was diluted with EtOAc (20 mL), and washed with dilute aqueous sulfuric acid (2 vol%, 3 × 20 mL) to remove tetrabutylammonium salts. The organic fraction was then dried (MgSO4), filtered, and concentrated under reduced pressure to provide carboxylic acid 25 (52 mg, 95%) as a colourless oil. Rf: 0.54 (1:1 pet. ether:EtOAc). 1H NMR (500 MHz, CDCl3): δ 7.42 (dd, J = 11.6, 15.0 Hz, 1H), 6.66 (dd, J = 11.4, 11.6 Hz, 1H), 6.08 (td, J = 6.9, 15.0 Hz, 1H), 5.65 (d, J = 11.4 Hz, 1H), 4.19 (t, J = 6.5 Hz, 2H), 2.56 (dt, J = 6.6, 6.9 Hz, 2H), 2.35 (t, J = 7.4 Hz, 2H), 2.21 (dt, J = 2.4, 7.4 Hz, 2H), 1.96 (t, J = 2.4 Hz, 1H), 1.75 (quin, J = 7.5 Hz, 2H), 1.56 (quin, J = 7.5 Hz, 2H). 13C NMR (125 MHz, CDCl3): δ 173.3, 171.2, 146.7, 140.9, 129.1, 115.8, 83.9, 68.6, 62.8, 33.7, 32.4, 27.8, 24.0, 18.1. IR (film): 3302, 2951, 2867, 1728, 1687, 1441, 1227, 1172, 1146,910, 731, 646 cm-1. HRMS-ESI (m/z) calculated for C14H17O4- [M - H]-: 249.1132; found 249.1137. (±)-(2E,4E,6E)-9-Azido-8-(tert-butyldimethylsilyloxy)-2,4,6-nonatrienal, (±)-26


To a solution of compound (±)-6 (412 mg, 1.34 mmol) in MeOH (20 mL) at room temperature was added a few drops of conc. HCl. The reaction mixture was stirred for 2 hours, then quenched with saturated aqueous sodium carbonate (1 mL) and solvent removed under reduced pressure. (The reaction was halted before completion based on TLC because the product was expected to be susceptible to elimination.) The crude material was purified by silica gel flash chromatography (2:1 pet. ether:EtOAc) to provide aldehyde (±)-26 (57 mg, 22% [84% BRSM]) as a yellow oil. Rf: 0.21 (2:1 pet. ether:EtOAc). 1H NMR (500 MHz, CDCl3): δ 9.57 (d, J = 7.9 Hz, 1H), 7.12 (dd, J = 11.2. 15.0 Hz, 1H), 6.66 (dd, J = 11.1, 14.8 Hz, 1H), 6.36–6.58 (m, overlapping peaks, 2H), 6.18 (dd, J = 7.9, 15.0 Hz, 1H), 5.97 (dd, J = 4.9, 15.1 Hz, 1H), 4.49–4.45 (m, 1H), 3.43 (d, J = 11.8 Hz, 1H), 3.39–3.25 (m, 1H). 13C NMR (125 MHz, CDCl3): δ 193.5, 151.2, 140.8, 137.3, 132.0, 131.1, 131.0, 71.1, 56.3. IR (film): 3381, 2924, 2834, 2097, 1667, 1610, 1283, 1159, 1115, 912, 843, 730 cm-1. HRMS-ESI (m/z) calculated for C9H12N3O2+ [M + H]+: 194.0924; found 194.0916. (±)-(2Z,4E)-7-(4-{1-[(3E,5E,7E)-9-Oxo-2-hydroxy-3,5,7-nonatrienyl]-1H-1,2,3-triazol-4yl}valeroxy)-2,4-heptadienoic acid, (±)-24 An aqueous solution (5 mL) of CuSO4.5H2O (10 mg, 0.04 mmol) and ascorbic acid (30 mg, 0.17 mmol), was neutralised to pH 7 with aqueous NaOH. A solution of alkyne 25 (103 mg, 0.41 mmol) and azide (±)-26 (66 mg, 0.34 mmol) in THF (5 mL) was added and the reaction mixture was stirred overnight at room temperature, gradually turning yellow/green. The mixture was then extracted with EtOAc (3 x 15 mL), and the combined organic fractions were dried (MgSO4), filtered and concentrated under reduced pressure. The crude material was purified by silica gel flash chromatography (EtOAc) to provide seco acid (±)-24 (114 mg, 75%) as a pale yellow oil. Rf: 0.40 (4:1 DCM:MeOH). 1H NMR (500 MHz, CDCl3)35: δ 9.56 (d, J = 7.8 Hz, 1H, H-16), 7.46 (s, 1H, H-7), 7.38 (dd, J = 11.8, 14.9 Hz, 1H, H-20), 7.12 (dd, J = 11.2, 15.2 Hz, 1H, H-14), 6.56–6.67 (m, overlapping peaks, 2H, H-11 and 13), 6.41–6.56 (m, overlapping peaks, 2H, H-19 and 12), 6.17 (dd, J = 7.8, 15.2 Hz, 1H, H-15),


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6.09–5.95 (m, overlapping peaks, 2H, H-10 and 21), 5.62 (d, J = 11.3 Hz, 1H, H-18), 4.82-75 (m, 1H, H-9), 4.50 (dd, J = 2.9, 13.8 Hz, 1H, H-8a), 4.28 (dd, J = 8.0, 13.8 Hz, 1H, H-8b), 4.19 (t, J = 5.8 Hz, 2H, H-23), 2.66–2.72 (m, 2H, H-5), 2.52 (apparent q, J = 5.8 Hz, 2H, H-22), 2.29–2.32 (m, 2H, H-2), 1.55–1.70 (m, overlapping peaks, 4H, H-3 and 4). 13C NMR (125 MHz, CDCl3): δ 193.6, 173.5, 169.6, 151.3, 147.3, 145.4, 145.3, 140.8, 140.0, 136.7, 132.0, 131.2, 131.1, 129.1, 116.6, 70.5, 62.5, 55.4, 33.9, 32.0, 28.6, 24.9, 24.2. IR (film): 2947, 2865, 1722, 1673, 1640, 1609, 1429, 1365, 1161, 1117, 1014, 997, 967, 913, 822, 730 cm-1. HRMS-ESI (m/z) calculated for C23H30N3O6+ [M + H]+: 444.2129; found 444.2106. (±)-(2E,4E,6E)-7-{(6Z,8E)-5,13-Dioxo-4.12-dioxa-1.19.20triazabicyclo[16.2.1]henicosa-6,8,18(21),19-tetraen-3-yl}-2,4,6-heptatrienal, (±)-27


To a solution of seco acid (±)-24 (60 mg, 0.14 mmol) in THF (3 mL) at room temperature was added triethylamine (39 μL, 0.28 mmol) followed by 2,4,6-trichlorobenzoyl chloride (33 μL, 0.21 mmol). After stirring for 2 hours, the reaction mixture was diluted with DCM (40 mL), cooled to -78 °C (dry ice/acetone bath) and DMAP (5 mg, 0.042 mmol, 0.3 equiv) added. The reaction mixture was stirred for 2 hours and then allowed to gradually warm to room temperature while stirring overnight. The reaction was quenched with saturated aqueous ammonium chloride (10 mL). The aqueous layer was extracted with EtOAc (3 x 15 mL), and the combined organic fractions were dried (MgSO4), filtered and concentrated under reduced pressure. The crude material was purified by silica gel flash chromatography (9:1 DCM:MeOH) to provide the lactone (±)-27 (8 mg, 13%) as a yellow oil. Rf: 0.66 (4:1 DCM:MeOH). 1H NMR (600 MHz, CDCl3)32: δ 9.61–9.55 (d, J = 7.9 Hz, 1H, H-16), 7.27 (s, 1H, H-7), 7.05–7.16 (m, overlapping peaks, 2H, H-14 and 20), 6.66 (dd, J = 10.8, 14.9 Hz, 1H, H-10), 6.59 (t, J = 11.4 Hz, 1H, H-19), 6.49–6.56 (m, overlapping peaks, 2H, H-12 and 13), 6.21 (dd, J = 7.9, 15.2 Hz, 1H, H-15), 5.91–6.07 (m, overlapping peaks, 3H, H-9, 11 and 21), 5.56 (d, J = 11.4 Hz, 1H, H-18), 4.71–4.65 (m, 1H, H-8a), 4.41 (dd, J = 10.1, 14.1 Hz, 1H, H-8b), 4.37–4.30 (m, 1H, H-23a), 4.19 (ddd, J = 3.5, 5.0, 11.1 Hz, 1H, H-23b), 2.76 (td, J = 7.0, 14.6 Hz, 1H, H-5a), 2.64 (td, J = 7.4, 14.8 Hz, 1H, H-5b), 2.59–2.49 (m, 1H, H-22a), 2.49–2.41 (m, 1H, H-22b), 2.38–2.21 (m, 2H, H-2), 1.77–1.63 (m, 2H, H-4), 1.60–1.51 (m, 2H, H-3).13C NMR (151 MHz, CDCl3): δ 193.4, 173.3, 164.0, 150.6, 146.8, 142.5, 139.9, 132.9, 132.6, 132.2, 131.6, 128.2, 128.0, 121.4, 114.3, 70.5, 61.8, 53.3, 34.7, 33.2, 28.6, 25.1, 24.3. IR (film): 2929, 2856, 2361, 1721, 1673, 1615, 1222, 1157, 1116, 996, 731 cm-1. HRMS-ESI (m/z) calculated for C23H28N3O5+ [M + H]+: 426.2023; found 426.2010. (6Z,8E)-3-[(1E,3E,5E)-7-(Dimethylamino)-1,3,5-heptatrienyl]-4.12-dioxa-1.19.20triazabicyclo[16.2.1]henicosa-6,8,18(21),19-tetraene-5,13-dione, (±)-3 To a solution of aldehyde (±)-27 (8 mg, 0.02 mmol, 1 equiv) in THF (2 mL) at 0 °C (ice bath) was added HCl.HNMe2 (6 mg, 0.08 mmol) followed by NMe4BH(OAc)3 (10 mg, 0.04 mmol) and triethylamine (11 μL, 0.08 mmol). The reaction mixture was stirred overnight on ice, in a closed system (to prevent loss of dimethylamine with a bp = 7-9 °C), quenched with aqueous NaOH (0.5 M, 2 mL), and extracted with EtOAc (3 x 5 mL). The combined organic fractions were dried (MgSO4), filtered, then concentrated under reduced pressure. The crude material was purified by silica gel flash chromatography (90:10:1 DCM:MeOH:saturated Org Biomol Chem. Author manuscript; available in PMC 2017 June 14.

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aqueous NH4OH) to provide (±)-3 (5 mg, 56%) as a pale orange oil. Rf: 0.67 (80:20:1 DCM:MeOH:saturated aqueous NH4OH). 1H NMR (500 MHz, CDCl3)34: δ 7.24 (s, 1H, H-7), 7.18–7.09 (m, 1H, H-20), 6.56 (t, J = 11.4 Hz, 1H, H-19), 6.47–6.40 (m, 1H, H-11), 6.34–6.18 (m, 3H, H-12, 13 and 14), 6.01 (d, J = 9.2 Hz, 1H, H-21), 5.92–5.84 (m, overlapping peaks, 2H, H-9 and 15), 5.74 (dd, J = 6.9, 15.2 Hz, 1H, H-10), 5.55 (d, J = 11.4 Hz, 1H, H-18), 4.62 (d, J = 14.3 Hz, 1H, H-8a), 4.39 (dd, J = 10.2, 14.3 Hz, 1H, H-8b), 4.32 (d, J = 9.7 Hz, 1H, H-23a), 4.23–4.16 (m, 1H, H-23b), 3.30–3.24 (m, 2H, H-16), 2.78–2.67 (m, 1H, H-5a), 2.67–2.56 (m, 1H, H-5b), 2.56–2.46 (m, 2H, H-22, partially obscured by an ammonium ion signal), 2.29–2.33 (m, 2H, H-2), 2.17 (s, 6H, H-24), 1.76–1.64 (m, 2H, H-4), 1.60–1.50 (m, 2H, H-3).13CNMR (151 MHz, CDCl3): δ 173.3, 164.1, 146.5, 142.2, 139.8, 134.6, 133.3, 132.2, 130.4, 128.9, 128.3, 121.2, 120.7, 114.5, 70.7, 61.8, 53.3, 45.7, 41.9, 34.7, 33.2, 28.6, 25.2, 24.4. IR (film): 2934, 2862, 1717, 1640, 1612, 1513, 1246, 1159, 1090, 1033, 1001, 912, 817, 728 cm-1. HRMS-ESI (m/z) calculated for C25H35N4O4+ [M + H]+: 455.2653; found 455.2650. Small samples were subjected to reverse phase HPLC purification immediately prior to biological testing as previously reported.36

Supplementary Material Refer to Web version on PubMed Central for supplementary material.

Acknowledgements This work was financially supported by a NgāPae o te Māramatanga bridging grant and a Victoria PhD scholarship to HC (Victoria University of Wellington). The authors thank Ian Vorster for his help with NMR and HRMS, Jingjing Wang for running HRMS samples, A/Prof Peter Northcote for purification advice and Dr Jonathan Singh for help with NMR and HPLC purification.

Notes and references Europe PMC Funders Author Manuscripts

1. Newman DJ, Cragg GM. J Nat Prod. 2012; 75:311–335. [PubMed: 22316239] 2. Wender PA, Verma VA, Paxton TJ, Pillow TH. Acc Chem Res. 2008; 41:40–49. [PubMed: 18159936] 3. Northcote PT, Blunt JW, Munro MHG. Tetrahedron Lett. 1991; 32:6411–6414. 4. Page M, West L, Northcote P, Battershill C, Kelly M. J Chem Ecol. 2005; 31:1161–1174. [PubMed: 16124239] 5. Bordeleau ME, Matthews J, Wojnar JM, Lindqvist L, Novac O, Jankowsky E, Sonenberg N, Northcote P, Teesdale-Spittle P, Pelletier J. Proc Natl Acad Sci USA. 2005; 102:10460–10465. [PubMed: 16030146] 6. Low WK, Dang Y, Schneider-Poetsch T, Shi Z, Choi NS, Merrick WC, Romo D, Liu JO. Mol Cell. 2005; 20:709–722. [PubMed: 16337595] 7. Bordeleau ME, Cencic R, Lindqvist L, Oberer M, Northcote P, Wagner G, Pelletier J. Chem Biol. 2006; 13:1287–1295. [PubMed: 17185224] 8. Hood KA, West LM, Northcote PT, Berridge MV, Miller JH. Apoptosis. 2001; 6:207–219. [PubMed: 11388670] 9. Dang Y, Kedersha N, Low WK, Romo D, Gorospe M, Kaufman R, Anderson P, Liu JO. J Biol Chem. 2006; 281:32870–32878. [PubMed: 16951406] 10. Di Marco S, Cammas A, Lian XJ, Kovacs EN, Ma JF, Hall DT, Mazroui R, Richardson J, Pelletier J, Gallouzi IE. Nat Commun. 2012; 3:896. [PubMed: 22692539] 11. Romo D, Rzasa RM, Shea HA, Park K, Langenhan JM, Sun L, Akhiezer A, Liu JO. J Am Chem Soc. 1998; 120:12237–12254.


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12. Remuinan MJ, Pattenden G. Tetrahedron Lett. 2000; 41:7367–7371. 13. Romo D, Choi NS, Li S, Buchler I, Shi Z, Liu JO. J Am Chem Soc. 2004; 126:10582–10588. [PubMed: 15327314] 14. Low W-K, Li J, Zhu M, Kommaraju SS, Shah-Mittal J, Hull K, Liu JO, Romo D. Bioorg Med Chem. 2014; 22:116–125. [PubMed: 24359706] 15. Kuznetsov G, Xu Q, Rudolph-Owen L, TenDyke K, Liu J, Towle M, Zhao N, Marsh J, Agoulnik S, Twine N, Parent L, et al. Mol Cancer Ther. 2009; 8:1250–1260. [PubMed: 19417157] 16. Matthews JH, Maass DR, Northcote PT, Atkinson PH, Teesdale-Spittle PH. Z Naturforsch C Bio Sci. 2013; 68:406–415. 17. Jeyakumar K, Chakravarthy RD, Chand DK. Catal Commun. 2009; 10:1948–1951. 18. Grée R, Tourbah H, Carrié R. Tetrahedron Lett. 1986; 27:4983–4986. 19. Coppola GM. Synthesis. 1984:1021–1023. 20. Blakemore PR, Cole WJ, Kocieński PJ, Morley A. Synlett. 1998:26–28. 21. Parikh JR, Doering WvE. J Am Chem Soc. 1967; 89:5505–5507. 22. Dess DB, Martin JC. J Org Chem. 1983; 48:4155–4156. 23. Nakata T, Hata N, Oishi T. Heterocycles. 1990; 30:333–334. 24. Zanato C, Pignataro L, Ambrosi A, Hao Z, Trigili C, Diaz JF, Barasoain I, Gennari C. Eur J Org Chem. 2011:2643–2661. 25. Zhu W, Jimenez M, Jung WH, Camarco DP, Balachandran R, Vogt A, Day BW, Curran DP. J Am Chem Soc. 2010; 132:9175–9187. [PubMed: 20545347] 26. Abdel-Magid AF, Mehrman SJ. Org Process Res Dev. 2006; 10:971–1031. 27. Frederico D, Donate PM, Constantino MG, Bronze ES, Sairre MI. J Org Chem. 2003; 68:9126– 9128. [PubMed: 14604394] 28. Trost BM, Bartlett MJ. Org Lett. 2012; 14:1322–1325. [PubMed: 22356535] 29. Schoning K-U, Wittenberg R, Kirschning A. Synlett. 1999:1624–1626. 30. Kashin D, Meyer A, Wittenberg R, Schoning K-U, Kamlage S, Kirschning A. Synthesis. 2007:304–319. 31. Arikan F, Li J, Menche D. Org Lett. 2008; 10:3521–3524. [PubMed: 18630925] 32. Zampella A, Sepe V, D'Orsi R, Bifulco G, Bassarello C, D'Auria MV. Tetrahedron: Asymmetry. 2003; 14:1787–1798. 33. Mohapatra DK, Das PP, Sai Reddy D, Yadav JS. Tetrahedron Lett. 2009; 50:5941–5944. 34. Bose D, Fernandez E, Pietruszka J. J Org Chem. 2011; 76:3463–3469. [PubMed: 21434691] 35. See proton-NMR spectra in Supplementary Material for position numbering in assignment of signals. 36. Singh AJ, Razzak M, Teesdale-Spittle P, Gaitanos TN, Wilmes A, Paterson I, Goodman JM, Miller JH, Northcote PT. Org Biomol Chem. 2011; 9:4456–4466. [PubMed: 21512693]


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Figure 1.

Structures of pateamine A (1), DMDA-pateamine A (2) and a simplified triazole analogue of pateamine A (3).


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Scheme 1.

Retrosynthesis of simplified triazole analogue of pateamine A. (PT=1-phenyl-1H-tetrazole)

Europe PMC Funders Author Manuscripts Org Biomol Chem. Author manuscript; available in PMC 2017 June 14.

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Scheme 2.

Synthesis of fragment 7.


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Scheme 3.

Synthesis of fragment 8.


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Scheme 4.

Synthesis of fragment 6. (KHMDS = potassium hexamethyldisilazide, Py = pyridine)


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Europe PMC Funders Author Manuscripts Scheme 5.

Synthesis of key fragment 23.


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Scheme 6.

Attempted dual reaction with tetrabutylammonium fluoride (TBAF).


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Europe PMC Funders Author Manuscripts Scheme 7.

Completion of pateamineA analogue 3. (BRSM = based on recovered starting material, TCBC = trichlorobenzoyl chloride).


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Table 1

IC50 values based on MTT assay.


Compound

HL-60

1A9

Aldehyde 27

100 µM

>200 µM

Target Analogue 3

34.6 µM

51.1 µM


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