Total synthesis of desacetylumuravumbolide, umuravumbolide and their biological evaluation

Abstract

Stereoselective synthesis of naturally occurring α,β-unsaturated lactones desacetylumuravumbolide and umuravumbolide is described. Commercially available propargyl alcohol was used as the starting material. The key steps of this synthesis were alkynylation, a Noyori asymmetric reduction and Still–Gennari olefination. Additionally, the biological activity of umuravumbolides was evaluated on HeLa, MDA-MB-231, MCF7 and A549 cancer cell lines. Umuravumbolide (2) showed potent anticancer activity.

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Introduction

The 6-substituted 5,6-dihydro-α-pyrones are an interesting group of natural products widely distributed in the plant kingdom,¹ they exhibit potential biological activities such as antibacterial, antifungal, plant growth inhibitors, insect antifeedants, as well as cytotoxicity against human tumor cells.² In addition, these pyrones have been reported to inhibit HIV protease³ and also exhibit many other pharmacological properties.⁴ Desacetylumuravumbolide 1 and umuravumbolide 2⁵ (Fig. 1) are such natural pyrones, isolated from Tetradenia riparia, a plant of South African origin belonging to the Lamiaceae family. Tetradenia riparia is one of the most popular medicinal plants available in Rwanda. This plant species is used as a remedy for malaria, diarrhoea and for several kinds of fevers and aches.⁶ Thus, these pyrones have attracted much attention among synthetic chemists. Later, Davies-Coleman and Rivett determined the absolute configuration of 1 and 2 based on NMR and CD spectral studies.⁷ Untill today, only two syntheses of umuravumbolides⁸ were developed but their biological activities have not been investigated. In a continuation of our interest in the synthesis of natural lactones⁹ with potential biological activities, here we develop an alternative method for the synthesis of umuravumbolides 1 and 2 from inexpensive, commercially available propargyl alcohol in a convergent manner to evaluate their biological activities.

Fig. 1 Chemical structures of umuravumbolides.

Retrosynthetic analysis (Scheme 1) demonstrates that desacetylumuravumbolide 1 and umuravumbolide 2 can be obtained from 3, which in turn can be prepared from 4 by cis-Wittig olefination. Compound 4 can be generated from the nucleophilic addition reaction of TBS protected (S)-heptyn-3-ol 5 with the aldehyde 6 followed by Noyori asymmetric reduction. The chiral alcohol 5 can be made from propargylic alcohol.

Scheme 1 Retrosynthetic analysis for 1 and 2

Results and discussion

Synthesis of the target molecules, 1 and 2, commenced from commercially available propargyl alcohol, which was converted to the TBS protected (S)-heptyn-3-ol 5via epoxy alcohol 9. Thus, alkylation of propargyl alcohol with n-butyl bromide¹⁰ in liquid NH₃ and Li at −33 °C afforded 7 in 65% yield. Reduction of triple bond in 7 with LiAlH₄ furnished allylic alcohol as a single E-isomer 8 with 85% yield. Under Sharpless asymmetric epoxidation conditions using (+)-diisopropyl tartrate ((+)-DIPT), (iPrO)₄Ti, 5M TBHP in CH₂Cl₂, −30 °C alkenol 8 was converted to (2S,3S)-epoxy alcohol 9 with 85% yield having 96% ee (determined by chiral HPLC analysis). The chlorination of 9 using PPh₃/CCl₄ in the presence of NaHCO₃ afforded epoxy chloride 10 in 80% yield. Subsequent elimination reaction¹¹ with n-BuLi in THF furnished chiral propargylic alcohol which was without purification converted to its TBS ether 5, thereby establishing the C3′ stereocenter.

The construction of the lactone ring of 1 and 2 (Scheme 2) was achieved by the alkynylation reaction of the known aldehyde 6 with the (silyloxy)alkyne 5. To achieve (S)-configured alkynol in an optically pure form, we resorted to an oxidation–reduction protocol. The aldehyde 6 was treated with 5 in the presence of n-butyl lithium in THF at −78 °C to form the propargyl alcohol 11 as a mixture of diastereomers, which was without separation subjected to oxidation using IBX to yield the acetylenic ketone 12. Asymmetric reduction of the keto group in the presence of a Noyori (1R,2R)-catalyst yielded chiral propargyl alcohol 4 with 89% yield having 98% de. The diastereomeric purity of product 4 was determined by HPLC analysis. The chiral alcohol 4 was protected as the corresponding TBS ether 13 and then treated with DDQ in CH₂Cl₂, pH 7 buffered solution (9 : 1) to yield the free primary alcohol 14. Oxidation with 2-iodoxybenzoic acid (IBX) in dimethyl sulfoxide formed the corresponding aldehyde, which was subsequently treated with Still–Gennari reagent,¹² [(F₃CCH₂O)₂–P(O)CH₂CO₂Me] in the presence of NaH in dry THF at −78 °C to form the Z-olefinic ester 3. The deprotection of TBS groups followed by lactonisation of 3 was achieved in one-pot using PTSA in MeOH at room temperature to furnish lactone 15. Partial hydrogenation of the triple bond in 15 to the Z-olefin with Lindlar's catalyst afforded the target lactone, desacetylumuravumbolide 1 in 92% yield. Lactone 1 was subjected to acetylation under Ac₂O, Et₃N, and DMAP (cat.), CH₂Cl₂ at room temperature for 1 h to provide the natural lactone, umuravumbolide 2. The spectroscopic and physical data (¹H and ¹³C NMR, & [α]²⁵_D) of compounds 1 and 2 were identical in all respects to the data reported in the literature.¹³

Scheme 2 Reagents and conditions: a) Li, liq. NH₃, Fe (NO₃)₃·9H₂O, n-BuBr, THF, −33 °C, 8 h, 70%. b) LiAlH₄, THF, 0 °C–r.t, 6 h, 85%. c) (+)-DIPT, Ti(ⁱPrO)₄, 5 M TBHP in CH₂Cl₂, 4A° molecular sieves powder, CH₂Cl₂, −30 °C, 6 h, 85%. d) CCl₄, PPh₃, NaHCO₃, reflux, 6 h, 80%. e) (i) n-BuLi, THF, −78 °C, 3 h, (ii) TBSCl, imidazole, CH₂Cl₂, 0 °C, r.t, 2 h, (69% overall yield of two steps). f) n-BuLi, THF, −30 °C then add aldehyde 6 at −78 °C, 4 h, 70%. g) IBX, DMSO, CH₂Cl₂, 0 °C–r.t, overnight, 80%. h) (1R,2R)-Noyori catalyst, HCO₂H (10 eq), Et₃N (4eq), r.t, overnight, 89%. i) TBSCl, imidazole, CH₂Cl₂, 0 °C–r.t, 3 h, 92%. j) DDQ, CH₂Cl₂, pH 7 (10 : 1), r.t, 4 h, 75%. k) (i) IBX, DMSO, CH₂Cl₂, 0 °C–r.t, overnight, (ii) NaH, Still–Gennari reagent, 30 min at 0 °C, then addition of 14 at −78 °C, 2 h, (75% overall yield of two steps). l) PTSA, MeOH, 0 °C–r.t, overnight, 80%. m) Pd/CaCO₃, H₂, quinoline (cat.), EtOAc, rt, 6 h, 92%. n) Ac₂O, Et₃N, DMAP (cat.), CH₂Cl₂, 0 °C, r.t, 2 h, 85%.

Biological evaluation

From the data shown in Table 1, umuravumbolide 2 showed the most potent growth inhibitory effects on all the tested human tumor cell lines with IC₅₀ values ranging from 0.24–1.92 μM, while desacetylumuravumbolide 1 showed higher inhibitory activity with IC₅₀ values ranging between 1.54–8.19 μM towards A549, HeLa and MCF7 cancer cell lines at 24 h incubation. However, compound 1 did not exhibit any cytotoxicity towards MDA-MB-231 breast cancer cell line. We also observed very good growth inhibitory activities for doxorubicin (positive control) with IC-50 values ranging between 0.451–1.21; nevertheless, varied IC-50 values were reported in literature for doxorubicin against these tumor cell lines such as 3.24 μM¹⁴ and 0.07 μM¹⁵ for HeLa; 1.23 μM¹⁶ for MDA-MB-231; 2.62 μM¹⁴ and 3.22 μM¹⁷ for MCF-7 and for 1.05 μM¹⁸ and 1.99 μM¹⁹ for A549 cell line. Further, anti-bacterial and anti-Candida activities were also evaluated and showed no promising activity.²⁰

Table 1 Antitumor activity of umuravumbolide 2 and desacetylumuravumbolide 1

Test compounds	IC50 values (in μM)^a
	HeLa	MDA-MB-231	MCF-7	A549
a Average of duplicate measurements. b No activity. c Doxorubicin as positive control.
Umuravumbolide 2	0.245	0.565	1.926	0.936
Desacetylumuravumbolide 1	3.48	—^b	8.19	1.54
Doxorubicin^c	0.451	0.501	1.05	1.21

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Conclusions

In conclusion, we developed an efficient method for the asymmetric synthesis of umuravumbolides 1 and 2 by using alkynylation, Noyori reduction, Still–Gennari olefination and lactonization reactions. The cytotoxicity towards HeLa, MDA-MB-231, MCF7 and A549 cancer cells has also been disclosed.

Experimental section

General methods

All the solvents and reagents were purified by standard techniques, reactions were performed in oven-dried round bottom flask; crude products were purified by column chromatography on silica gel (60–120 mesh). Thin layer chromatography plates were visualized by exposure to ultraviolet light and/or by exposure to an methanolic acidic solution of p-anisaldehyde on a hot plate (∼250 °C). Organic solutions were concentrated on rotary evaporator at 40–45 °C. IR spectra were recorded on Perkin-Elmer 683, Thermo Nicolet Nexus 670 spectrometer. ¹H NMR and ¹³C NMR spectra were recorded in CDCl₃ solvent on a Varian Gemini 200, Bruker AV-300 and Varian Innova 500 NMR spectrometer. Chemical shifts were reported in parts per million (ppm) with respect to internal TMS. Coupling constants (J) are quoted in Hertz (Hz). s, d, dd, ddd, dt, t, q, qt and m refer to singlet, broad singlet, doublet, doublet of doublet, doublet of doublet of doublet, triplet, quartet, quintet and multiplet, respectively. The optical rotations were measured on Perkin-Elmer 343. The diastereomeric excess and enantiomeric excess of the products were measured by HPLC using Shimadzu LC-20AT series. Mass spectra were recorded on a Micro Mass VG-7070H mass spectrometer for ESI and EI are given in mass units (m/z). High-resolution mass spectra (HRMS) [ESI] were obtained using either a TOF or a double focusing spectrometer.

Spectral data for all compounds

Hept-2-yn-1-ol (7). To a stirred soltion of Li (2.25 mg) in liquid NH₃ (100 mL) under N₂ at −33 °C were added a few crystals of Fe(NO₃)₃·9H₂O, followed by finely cut Li (2.25 g, 321.43 mmol) in small portions. After the mixture turned gray, it was stirred for another 30 min. Distilled prop-2-yn-1-ol (6.0 g, 107.14 mmol) in dry THF (50 mL) was added in 30 min, followed by stirring for 90 min. n-BuBr (13.87 g, 128.56 mmol) in dry THF (50 mL) was added within 30 min. The resulting mixture was stirred for 6 h at −33 °C. Then, NH₃ was allowed to evaporate overnight. After slow addition of saturated aqueous NH₄Cl solution (50 mL), the mixture was extracted with EtOAc (3 × 75 mL), the combined organic extract washed with brine (100 mL), dried over anhydrous Na₂SO₄, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (hexane : EtOAc = 90 : 10) to afford compound 7 as a yellow coloured liquid (8.4 g, 70%). ¹H NMR (300 MHz, CDCl3): δ_H 4.19 (br s, 2H), 2.25–2.16 (m, 2H), 1.67 (br s, OH), 1.56–1.35 (m, 4H), 0.93 (t, J = 6.7 Hz, 3H).

(E)-Hept-2-en-1-ol (8). To a stirred suspension of LiAlH₄ (2.82 g, 74.10 mmol) in dry THF (20 mL) at 0 °C was added dropwise a solution of alkyne 7 (8.3 g, 74.10 mmol) in dry THF (50 mL) under nitrogen. The reaction mixture was allowed to warm to room temperature for 6 h. It was then cooled to 0 °C, diluted with ether and quenched by dropwise addition of saturated aqueous Na₂SO₄ solution (30 mL). The solid material was filtered and washed thoroughly with EtOAc several times. The combined organic layers were dried over anhydrous Na₂SO₄. Solvent was concentrated under reduced pressure and the crude product was purified by silica gel column chromatography (hexane : EtOAc = 88 : 12) to afford allyl alcohol 8 as a light yellow coloured liquid (7.18 g, 85%). ¹H NMR (500 MHz, CDCl₃): δ_H 5.67–5.54 (m, 2H), 4.02 (d, J = 4.8 Hz, 2H), 2.06–1.99 (m, 2H), 1.60 (br s, OH), 1.39–1.27 (m, 4H), 0.89 (t, J = 6.8 Hz, 3H).

((2S, 3S)-3-butyloxiran-2-yl) methanol (9). In a 100 mL two neck round bottomed flask, 15 mL of anhydrous CH₂Cl₂ was added to 4 Å powdered activated molecular sieves and the suspension mixture was cooled to −30 °C. Next, (iPrO)₄Ti (3.65 mL, 12.28 mmol) and L-(+) DIPT (2.87 g, 12.28 mmol) in anhydrous CH₂Cl₂ (15 mL) were added subsequently with stirring and the resulting mixture was stirred for 30 min at −30 °C. Compound 8 (7 g, 61.40 mmol) in anhydrous CH₂Cl₂ (10 mL) was then added and the resulting mixture was stirred for another 30 min at −30 °C followed by the addition of TBHP (5 M solution in CH₂Cl₂, 16 mL, 79.82 mmol). The resulting mixture was stirred at the same temperature for 6 h. It was then warmed to 0 °C, quenched with 1 mL of water and stirred for 1 h at room temperature. After that, a 30% aqueous NaOH solution saturated with NaCl (3 mL) was then added and the reaction mixture was stirred vigorously for another 30 min at room temperature. The resulting mixture was then filtered through Celite rinsing with CH₂Cl₂. The organic phase was separated and the aqueous phase was extracted with CH₂Cl₂. The combined organic phases were washed with brine and dried over anhydrous Na₂SO₄. The solvent was removed under reduced pressure and purified by silica gel column chromatography (hexane : EtOAc = 75 : 25) to afford 9 as a colourless liquid (6.78 g, 85%, 96 : 4 ee). The enantiomeric excess was determined by HPLC method: Waters HR C18 300 × 3.9 mm 5 μ (column), 80% MeOH in H₂O (mobile phase), flow rate 1 mL min⁻¹. tR: 2.5 and 2.9 min.. [α] _D²⁵ −11.0 (C 2.9 in CHCl₃); IR (Neat): ν_max cm⁻¹ 3422, 2930, 2865, 1462, 1383, 1222, 1099, 1024, 844, 761, 558; ¹H NMR (300 MHz, CDCl₃): δ_H 3.97–3.86 (m, 1H), 3.68–3.56 (m, 1H), 3.00–2.89 (m, 2H), 1.81 (t, J = 6.0 Hz, OH), 1.63–1.53 (m, 2H), 1.51–1.28 (m, 4H), 0.92 (t, J = 6.8 Hz, 3H); ¹³C NMR (75 MHz, CDCl₃): δ_C 61.7, 58.6, 56.0, 31.1, 27.9, 22.3, 13.8.

(2S, 3R)-2-butyl-3-(chloromethyl)oxirane (10). To a stirred solution of epoxy alcohol 9 (6.60 g, 50.76 mmol) in anhydrous CCl₄ (150 mL), TPP (19.87 g, 76.15 mmol) and NaHCO₃ (4.26 g, 50.76 mmol) were added. The reaction mixture was vigorously refluxed for 6 h and the resulting solid was filtered and washed with ether. Concentration under reduced pressure and purification of the residue with flash chromatography on silica gel (hexane : EtOAc = 90 : 10) afforded 10 as a colourless liquid (7.84 g, 80%). [α] _D²⁵ −6.3(C 1.0 in CHCl₃); IR (Neat): ν_max cm⁻¹ 2958, 2926, 2857, 1596, 1459, 1385, 1216, 1034, 761, 694, 670, 536, 472, 416; ¹H NMR (300 MHz, CDCl₃): δ_H 3.60 (dd, J = 12.0, 6.0 Hz, 1H), 3.38 (dd, J = 11.3, 6.0 Hz, 1H), 2.92 (td, J = 6.0, 2.2 Hz, 1H), 2.79 (td, J = 5.3, 2.2 Hz, 1H), 1.62–1.52 (m, 2H), 1.50–1.32 (m, 4H), 0.93 (t, J = 6.8 Hz, 3H); ¹³C NMR (75 MHz, CDCl₃): δ_C 58.9, 57.0, 44.7, 31.0, 27.8, 22.3, 13.8.

(S)-tert-Butyl(hept-1-yn-3-yloxy)dimethylsilane (5). A solution of (chloromethyl)oxirane 10 (7.70 g, 51.85 mmol) in THF (30 mL) at −78 °C, n-BuLi (51.85 mL, 129.62 mmol; 2.5 M in hexane) in THF (30 mL) was added, and the stirring was continued for 2 h at −78 °C. The reaction was quenched with saturated aqueous NH₄Cl solution (20 mL) and the mixture diluted with Et₂O. The organic layer was washed with brine (50 mL), dried over anhydrous Na₂SO₄, and concentrated to furnish the chiral propargyl alcohol and without any further purification used for the next step without characterization. To a stirred solution of chiral propargyl alcohol (4.35 g, 38.83 mmol) in CH₂Cl₂ (50 mL), imidazole (6.60 g, 97.07 mmol) was added at 0 °C and the mixture was stirred for 5 min. Then ^tBuMe₂SiCl (7 g, 46.60 mmol) was added, and the stirring was continued for 2 h at r.t. The mixture was diluted with CH₂Cl₂ (20 mL), the organic layer washed with H₂O (60 mL) and brine (10 mL), the combined organic layer concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (hexane : EtOAc = 98 : 2) to afford 5 as a colourless liquid (8.07 g, 65%). [α] _D²⁵ −24.1 (C 1.0 in CHCl₃); IR (neat): ν_maxcm⁻¹ 3131, 2958, 2930, 2858, 1725, 1632, 1463, 1400, 1255, 1217, 1095, 836, 763; ¹H NMR (300 MHz, CDCl₃): δ_H 4.33 (td, J = 6.8, 2.2 Hz, 1H), 2.38 (d, J = 2.2 Hz, 1H), 1.72–1.62 (m, 2H), 1.48–1.25 (m, 4H), 0.94–0.88 (m, 12H), 0.13 (s, 3H), 0.11 (s, 3H); ¹³C NMR (75 MHz, CDCl₃): δ_C 85.8, 71.8, 62.7, 38.3, 27.3, 25.7, 22.3, 18.2, 13.9, −4.6, −5.1; ESI-MS (m/z): C₁₃H₂₆O Si [M − H]⁺ (225).

(S)-6-(tert-Butyldimethylsilyloxy)-1-(4-methoxybenzyloxy) dec-4-yn-3-one (12). To a stirred solution of alkyne 5 (2 g, 8.84 mmol) in dry THF (10 mL) was slowly added n-BuLi (4.60 mL, 11.49 mmol, 2.5 M solution in hexanes) at −78 °C under N₂. The reaction mixture was stirred for 30 min at −78 °C, and a solution of compound 6 (1.71 g, 9.72 mmol) in dry THF (10 mL) was added dropwise with stirring. The mixture was kept at −78 °C for 2 h and then allowed to warm to rt for 2 h. The reaction was quenched with saturated aqueous NH₄Cl solution, and extracted with EtOAc, dried over anhydrous Na₂SO₄, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (hexane : EtOAc = 75 : 25) to afford alcohol 11 as a diastereomeric mixture (2.59 mg, 70%). The above obtained alcohol 11 (2 g, 4.76 mmol) in dry CH₂Cl₂ (10 mL) was added dropwise at 0 °C to an ice-cooled solution of 2-iodoxybenzoic acid (2 g, 7.14 mmol) in DMSO (1.69 mL, 23.8 mmol). The mixture was stirred at room temperature for 2 h and then filtered through a Celite pad and washed with ether. The combined organic filtrates were washed with water and brine, dried over anhydrous Na₂SO₄, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (hexane : EtOAc = 88 : 12) to afford keto 12 as a yellow coloured liquid (1.59 g, 80%). [α]²⁵_D −26.8 (C 1.9 in CHCl₃); IR (neat): ν_max cm⁻¹ 2955, 2931, 2859, 2208, 1678, 1612, 1513, 1465, 1386, 1361, 1301, 1249, 1216, 1169, 1091, 1033, 935, 837, 764, 669, 575, 465; ¹H NMR (300 MHz, CDCl₃): δ_H 7.25 (d, J = 8.1 Hz, 2H), 6.87 (d, J = 8.7 Hz, 2H), 4.52–4.44 (m, 3H), 3.86–3.74 (m, 5H), 2.84 (t, J = 6.4 Hz, 2H), 1.76–1.66 (m, 2H), 1.46–1.25 (m, 4H), 0.96–0.87 (m, 12H), 0.13 (s, 3H), 0.11 (s, 3H); ¹³C NMR (75 MHz, CDCl₃): δ_C 185.2, 159.2, 130.0, 129.3, 113.8, 93.9, 82.9, 72.9, 64.4, 62.7, 55.2, 45.6, 37.5, 27.2, 25.7, 22.2, 18.1, 13.9, −4.6, −5.1; ESI-MS (m/z): C₂₄H₃₈O₄NaSi [M+Na]⁺ (441); HRMS-ESI: m/z calcd for C₂₄H₃₈O₄NaSi [M+Na]⁺: 441.24316, found: 441.24213.

(3R,6S)-6-(tert-Butyldimethylsilyloxy)-1-(4-ethoxybenzyloxy)dec-4-yn-3-ol (4). To a mixture of propargyl ketone 12 (1.4 g, 3.34 mmol) in formic acid (1.30 mL, 33.49 mmol, 10 eq.) and triethylamine (1.86 mL, 13.39 mmol, 4 eq.) were added at r.t. an aliquot amount of the stock solution of the RuCl[N-(tosyl)-1R,2R-diphenylethylenediamine)(p-cymene)] complex 0.0337 M in CH₂Cl₂ (2.48 mL, 0.083 mmol, 0.025 eq.), prepared according to ref. 21. The reaction mixture was stirred at room temperature overnight. The reaction was quenched with saturated aqueous NaHCO₃ solution, extracted with CH₂Cl₂, dried over anhydrous Na₂SO₄, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (hexane : EtOAc = 75 : 25) to afford chiral propargyl alcohol 4 as a light yellow coloured liquid (1.25 g, 89%). The diastereomeric excess was determined by HPLC method: water XTerra RP18, H₂O : CH₃CN = 50 : 50, flow rate 1 mL min⁻¹, tR = 7.53 min. (major), t2 = 9.11 min. (minor), de = 98%. [α]²⁵_D −10.4 (C 1.65 in CHCl₃); IR (neat): ν_max cm⁻¹ 3446, 2954, 2930, 2857, 1613, 1513, 1464, 1363, 1301, 1249, 1175, 1147, 1085, 1036, 936, 836, 774, 671, 568; ¹H NMR (300 MHz, CDCl₃): δ_H 7.25 (d, J = 7.6 Hz, 2H), 6.88 (d, J = 8.3 Hz, 2H), 4.62 (dd, J = 11.3, 5.3 Hz, 1H), 4.46 (ABq, J = 15.1, 11.3 Hz, 2H), 4.40–4.33 (m, 1H), 3.87–3.78 (m, 4H), 3.69–3.60 (m, 1H), 3.00 (d, J = 6.8 Hz, OH), 2.12–2.00 (m, 1H), 1.99–1.86 (m, 1H), 1.70–1.57 (m, 2H), 1.45–1.25 (m, 4H), 0.95–0.86 (m, 12H), 0.12 (s, 3H), 0.10 (s, 3H); ¹³C NMR (75 MHz, CDCl₃): δ_C 159.2, 129.9, 129.3, 113.8, 86.6, 84.1, 73.0, 67.4, 62.9, 61.4, 55.2, 38.3, 36.7, 27.4, 25.8 (3C), 22.3, 18.2 , 14.0, −4.5, −5.0; ESI-MS (m/z): C₂₄H₄₀O₄NaSi [M+Na]⁺ (443); HRMS-ESI: m/z calcd for C₂₄H₄₀O₄NaSi [M+Na]⁺: 443.25881, found: 443. 25717.

(5S,8R)-5-Butyl-8-(2-(4-methoxybenzyloxy)ethyl)-2,2,3,3,10,10,11,11-octamethyl-4,9-dioxa-3,10-disiladodec-6-yne (13). To a stirred solution of alcohol 4 (1 g, 2.38 mmol) and imidazole (0.405 g, 5.95 mmol) in dry CH₂Cl₂ (20 mL) was added ^tBuMe₂SiCl (0.429 mg, 2.85 mmol) portion wise at 0 °C. The reaction mixture was stirred at the same temperature for 3 h. The reaction mixture was quenched with a saturated aqueous solution of NH₄Cl and extracted with CH₂Cl₂. The extract was washed with water and brine, dried over anhydrous Na₂SO₄. The solvent was removed under reduced pressure and the crude product was purified by silica gel column chromatography (hexane : EtOAc = 98 : 2) to afford pure compound 13 as a colourless liquid. (1.16 g, 92%). [α]²⁵_D −5.0 (C 1.75 in CHCl₃); IR (neat): ν_max cm⁻¹ 3426, 2933, 2858, 1614, 1513, 1465, 1354, 1300, 1249, 1090, 1044, 937, 837, 775, 669, 579, 423; ¹H NMR (300 MHz, CDCl₃): δ_H 7.25 (d, J = 8.3 Hz, 2H), 6.87 (d, J = 8.3 Hz, 2H), 4.57 (td, J = 6.8, 1.5 Hz, 1H), 4.41 (ABq, J = 13.6, 11.3 Hz, 2H), 4.36–4.30 (m, 1H), 3.80 (s, 3H), 3.63–3.50 (m, 2H), 1.99–1.90 (m, 2H), 1.68–1.56 (m, 2H), 1.44–1.24 (m, 4H), 0.94–0.85 (m, 21H), 0.12 (s, 3H), 0.11 (s, 3H), 0.09 (s, 3H), 0.08 (s, 3H); ¹³C NMR (75 MHz, CDCl₃): δ_C 159.1, 130.5, 129.2, 113.7, 85.8, 85.2, 72.7, 66.1, 62.9, 59.9, 55.2, 38.8, 38.3, 27.4, 25.8 (6C), 22.3, 18.2 (2C), 14.0, −4.4, −4.5, −5.0, −5.1; ESI-MS (m/z): C₃₀H₅₄O₄Na Si₂ [M+Na]⁺ (557); HRMS-ESI: m/z calcd for C₃₀H₅₄O₄NaSi₂ [M+Na]⁺: 557.34528, found: 557.34458.

(3R,6S)-3,6-Bis(tert-butyldimethylsilyloxy)dec-4-yn-1-ol (14). Compound 13 (1 g, 1.87 mmol) was taken in 30 mL of CH₂Cl₂, pH 7 buffered solution (9 : 1), DDQ (0.638 g, 2.80 mmol) was added to it, and the solution was stirred for 4 h at room temperature. The reaction mixture was filtered off and the filtrate was washed with 5% NaHCO₃ solution (30 mL) and brine (30 mL), and dried over anhydrous Na₂SO₄. The solvent was removed under reduced pressure and purified by silica gel column chromatography (hexane : EtOAc = 80 : 20) to afford 14 as a light yellow coloured liquid (0.620 g, 80%). [α]²⁵_D 11.4 (C 0.9 in CHCl₃); IR (neat): ν_max cm⁻¹ 3405, 2955, 2932, 2889, 2858, 1467, 1387, 1341, 1253, 1214, 1146, 1088, 937, 838, 777, 669, 569; ¹H NMR (300 MHz, CDCl₃): δ_H 4.67–4.61 (m, 1H), 4.36–4.30 (m, 1H), 3.94–3.84 (m, 1H), 3.78–3.69 (m, 1H), 2.00–1.79 (m, 2H), 1.68–1.56 (m, 2H), 1.46–1.28 (m, 4H), 1.00–0.83 (m, 21H), 0.15 (s, 3H), 0.13 (s, 3H), 0.11 (s, 3H), 0.08 (s, 3H); ¹³C NMR (75 MHz, CDCl₃): δ_C 86.9, 84.2, 62.9, 62.5, 60.3, 39.9, 38.3, 27.4, 25.8 (6C), 22.3, 18.2, 18.1, 14.0, −4.5 (2C), −5.0, −5.2; ESI-MS (m/z): C₂₂H₄₆O₃NaSi₂ [M+Na]⁺ (437); HRMS-ESI: m/z calcd for C₂₂H₄₆O₃NaSi₂ [M+Na]⁺: 437.28777, found: 437.28678.

(5R, 8S, Z)-Ethyl 5, 8-bis (tert-butyldimethylsilyloxy) dodec-2-en-6-ynoate (3). To an ice-cooled solution of 2-iodoxybenzoic acid (0.558 mg, 1.99 mmol) in DMSO (0.47 mL, 6.64 mmol) was added a solution of alcohol 14 (0.550 mg, 1.32 mmol) in dry CH₂Cl₂ (5 mL). The mixture was stirred at room temperature overnight and then filtered through a Celite pad and washed with ether. The combined organic filtrates were washed with water and brine, dried over anhydrous Na₂SO₄, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (hexane : EtOAc = 85 : 15) to afford the pure aldehyde as a light yellow coloured liquid (0.437 mg, 80%). In a 50 mL two neck round bottomed flask, NaH (0.038 g, 1.59 mmol) was taken and to it 5 mL of dry THF was added under N₂ atmosphere. After 5 min, bis-2,2,2-trifluoromethyl(methoxy carbonylmethyl)phosphonate (0.27 mL, 1.27 mmol) in 2 mL dry THF was added dropwise at 0 °C. It was allowed to stir for 30 min. The reaction mixture was cooled to −78 °C and the aldehyde (0.437 g, 1.06 mmol) in THF (5 mL) was added dropwise over a period of 15 min and the resulting mixture was stirred for 2 h at −78 °C. The reaction mixture was quenched with saturated aqueous solution of NH₄Cl and the product was extracted into ether (2 × 10 mL). The ether extracts were dried over anhydrous Na₂SO₄ and concentrated under reduced pressure and the crude product was purified by silica gel column chromatography (hexane : EtOAc = 99 : 1) to afford (Z)-olefin ester 3 as a light yellow coloured liquid (0.397 g, 80%). [α]²⁵_D −17.9 (C 0.85 in CDCl₃); IR (neat): ν_max cm⁻¹ 3450, 2955, 2931, 2858, 1727, 1641, 1466, 1404, 1253, 1213, 1171, 1083, 936, 836, 774; ¹H NMR (300 MHz, CDCl₃): δ_H 6.42–6.32 (m, 1H), 5.85 (dt, J = 11.3, 1.5 Hz, 1H), 4.53–4.46 (m, 1H), 4.35–4.28 (m, 1H), 3.70 (s, 3H), 3.04–2.95 (m, 2H), 1.68–1.57 (m, 2H), 1.44–1.24 (m, 4H), 0.95–0.84 (m, 2H), 0.12 (s, 3H), 0.10 (s, 3H), 0.09 (s, 3H), 0.08 (s, 3H); ¹³C NMR (75 MHz, CDCl₃): δ_C 166.7, 145.5, 120.9, 86.4, 84.5, 62.9, 61.9, 51.1, 38.3, 38.0, 27.4, 25.8 (6C), 22.3, 18.2 (2C), 14.0, −4.5 (2C), −5.1 (2C); ESI-MS (m/z): C₂₅H₄₈O₄NaSi₂ [M+Na]⁺ (491); HRMS-ESI: m/z calcd for C₂₅H₄₈O₄NaSi₂ [M+Na]⁺ : 491.29833, found : 491.29783.

(R)-6-((S)-3-Hydroxyhept-1-ynyl)-5, 6-dihydropyran-2-one (15). To a stirred solution of 3 (0.340 mg, 0.72 mmol) in MeOH (5 mL) was added a catalytic amount of PTSA under N₂ atmosphere. The mixture was stirred at room temperature for overnight and then the reaction mixture was quenched by addition of solid NaHCO₃, filtered and the solvent was removed under reduced pressure. The crude product was purified by silica gel column chromatography (hexane : EtOAc = 70 : 30) to afford 15 as a light yellow coloured liquid (0.120 g, 80%). [α]²⁵_D 15.0 (C 0.85 in CHCl₃); IR (neat): ν_max cm⁻¹ 3447, 2925, 2854, 1724, 1646, 1459, 1381, 1034, 760; ¹H NMR (300 MHz, CDCl₃): δ_H 6.93–6.84 (m, 1H), 6.08 (dt, J = 9.8, 1.9 Hz, 1H), 5.27–5.20 (m, 1H), 4.41 (t, J = 6.2 Hz, 1H), 2.70–2.63 (m, 2H), 1.87 (br s, OH), 1.76–1.66 (m, 2H), 1.48–1.30 (m, 4H), 0.92 (t, J = 6.8 Hz, 3H); ¹³C NMR (75 MHz, CDCl₃): δ_C 162.6, 144.1, 121.4, 88.0, 80.2, 67.1, 62.2, 37.1, 30.2, 27.1, 22.2, 13.9; ESI-MS (m/z): C₁₂H₁₆O₃Na [M+Na]⁺ (231); HRMS-ESI: m/z calcd for C₁₂H₁₆O₃Na [M+Na]⁺: 231.25364, found: 231.23985.

(R)-6-((S,Z)-3-Hydroxyhept-1-enyl)-5,6-dihydropyran-2-one (desacetylumuravumbolide) (1). A suspension of compound 15 (0.080 mg, 0.38 mmol), Lindlar catalyst (0.020 mg, 300 wt%), and quinoline (catalytic amount) in EtOAc (3 mL) was stirred at room temperature under hydrogen atmosphere (1 atm) until partially reduced product appeared on TLC. The reaction mixture was filtered through a pad of Celite with EtOAc (5 mL). The filtrate was concentrated under reduced pressure and the crude product was purified by silica gel column chromatography (hexane : EtOAc 70 : 30) to afford lactone 1 as a colourless liquid (0.080 g, 85%). [α]²⁵_D −6.0 (C 0.35, CHCl₃); ¹H NMR (300 MHz, CDCl₃): δ_H 6.90 (ddd, J = 9.8, 5.3, 3.0 Hz, 1H), 6.06 (dd, J = 9.8, 2.0 Hz, 1H), 5.73–5.60 (m, 2H), 5.38–5.29 (m, 1H), 4.47–4.39 (m, 1H), 2.53–2.30 (m, 2H), 1.88 (br s, OH), 1.74–1.57 (m, 2H), 1.41–1.20 (m, 4H), 0.91 (t, J = 6.8 Hz, 3H); ¹³C NMR (75 MHz, CDCl₃) : δ_C 163.7, 144.7, 137.9, 127.6, 121.6, 73.7, 67.9, 36.8, 29.9, 27.5, 22.6, 14.0; ESI-MS (m/z): C₁₂H₁₈O₃Na [M+Na]⁺ (233); HRMS-ESI: m/z calcd for C₁₂H₁₈O₃Na [M+Na]⁺: 233.11482, found: 233.11503.

(S,Z)-1-((R)-6-Oxo-3,6-dihydro-2H-pyran-2-yl)hept-1-en-3-yl acetate (umuravumbolide) (2). Anhydrous Et₃N (0.053 mL, 0.38 mmol), Ac₂O (0.022 mL, 0.22 mmol), and catalytic amount of DMAP were added to a solution of compound 1 (0.040 mg, 0.19 mmol), in dry CH₂Cl₂ (5 mL), under N₂ atmosphere at room temperature. The mixture was stirred at room temperature for 2 h. The solvent was removed under reduced pressure, and then the crude product was purified by silica gel column chromatography (hexane : EtOAc = 85 : 15) to afford the pure compound 2 as a yellow coloured liquid (0.040 g, 85%). [α]²⁵_D 28.6 (C 0.9 in CHCl₃); IR (neat): ν_max cm⁻¹ 2926, 2859, 1731, 1375, 1239, 963, 867, 814, 759, 608, 402; ¹H NMR (300 MHz, CDCl₃): δ_H 6.88 (ddd, J = 9.8, 5.3, 2.2 Hz, 1H), 6.06 (dd, J = 9.8, 3.7 Hz, 1H), 5.74 (dd, J = 11.3, 8.3 Hz, 1H), 5.55 (dd, J = 10.5, 9.0 Hz, 1H), 5.47–5.37 (m, 2H), 2.54–2.40 (m, 1H), 2.35–2.22 (m, 1H), 2.04 (s, 3H), 1.77–1.45 (m, 2H), 1.38–1.18 (m, 4H), 0.90 (t, J = 6.8 Hz, 3H); ¹³C NMR (75 MHz, CDCl₃): δ_C 170.2, 163.5, 144.3, 131.6, 130.0, 121.5, 74.0, 69.4, ,34.2, 29.9, 27.1, 22.4, 21.1, 13.9; ESI-MS (m/z): C₁₄H₂₀O₄Na [M+Na]⁺ (275); HRMS-ESI: m/z calcd for C₁₄H₂₀O₄Na [M+Na]⁺: 275.12538, found: 275.12509.

Cytotoxicity assay

Cytotoxicity of the compounds 1 and 2 was determined on the basis of measurement of in vitro growth inhibition of tumor cell lines in 96 well plates by cell-mediated reduction of tetrazolium salt to water insoluble formazan crystals using doxorubicin as a standard. The cytotoxicity as assessed against a panel of four different human tumor cell lines: A549 derived from human alveolar adenocarcinoma epithelial cells (ATCC No. CCL-185), HeLa derived from human cervical cancer cells (ATCC No. CCL-2), MDA-MB-231 derived from human breast adenocarcinoma cells (ATCC No. HTB-26) and MCF7 derived from human breast adenocarcinoma cells (ATCC No. HTB-22) using the MTT assay.²² The IC₅₀ values (50% inhibitory concentration) were calculated from the plotted absorbance data for the dose-response curves. IC₅₀ values (in μM) are expressed as the average of two independent experiments.

Acknowledgements

DVR, SSSR and PS thank the Council of Scientific and Industrial Research (CSIR), New Delhi for research fellowships.

References

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Footnote

† Electronic supplementary information (ESI) available: ¹H and ¹³C NMR spectra of all compounds. See DOI: 10.1039/c2ra20830j

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