Synthesis of carbohydrate analogues of the THF-acetogenin 4-Deoxyannomontacin and their cytotoxicity against human prostate cancer cell lines

Abstract

The THF containing acetogenin 4-deoxyannonmontacin (4-DAN) has attracted interest for its potent cytotoxicity against a broad range of human tumor cell lines, and relatively simple structure. Herein is described the synthesis and cytotoxicity of C-10 epimers of 4-DAN and analogues thereof comprising carbohydrate and thiophene substitutes for the THF and butenolide moieties respectively. The key synthetic ploy was the union of THF and butenolide segments or their substitutes, via an alkene cross metathesis. The different analogues showed cytotoxicity in the low micromolar to nanomolar range against the human prostate cancer cell lines LNCaP and PC3. A relatively simple mannose-linked thiophene analog was found to be similar be in activity to 4-DAN.

Graphical Abstract

1. Introduction

The tetrahydrofuran containing annonaceous acetogenins (THF-AGEs), are naturallyo ccurring, cytotoxic agents that are active against a broad range of human cancer cell lines, including multi-drug resistant (MDR) strains, often in the picomolar range and lower.^1,2,3,4 However, their equally potent toxicity to normal cells presents a hurdle to clinical applications. To this end, diverse and easily accessible analogues are of interest for a clearer understanding of their anti-tumor activity and as therapeutic leads.^1–4,5 In this context the mono-THF-AGE 4-deoxyannonomontacin (4-DAN) 1a is an attractive template for drug discovery because its potent activity, relative simple structure.⁶ In addition, the C-10 carbinol is a convenient handle for conjugation of tumor vectors or imaging agents (Figure 1).^7,8 Accordingly, we sought a scalable synthesis of analogues of 4-DAN.⁹ To simplify the synthesis, a mixture of C-10 epimers was targeted (i.e 1a/b), as our earlier studies suggested that the configuration at the analogous position in the very closely related THF-AGE 4-deoxyreticuin did not impact on cytotoxicity.¹⁰

Figure 1.

Analogues of 4-DAN

We were also interested in substituting the THF and butenolide segments in 4-DAN with carbohydrate and thiophene residues respectively. Carbohydrate moieties may act as vectors for receptors on certain tumors, and reduce overall hydrophobicity, both of which are relevant to increasing tumor selectivity. Our earlier studies supported such carbohydrate analogues, which are structurally distinct from the acetogenin-sugar conjugates that have been reported by other laboratories.^11,12 Data from the Kojima group validated the use of thiophene-carboxamide moieties as surrogates for the butenolide.¹³ However, introduction of both of sugar and thiophene substitutes on a single framework has not been examined. Herein we present a more systematic evaluation of these THF and butenolide replacements by synthesis of 1a/b - 4a/b and evaluation of their cytotoxicity against androgen dependent and androgen independent human prostate cell lines, LNCaP and PC3 respectively.

2. Results and discussion

Retrosynthesis.

Our synthesis plan followed a convergent strategy in which THF and butenolide components, (or their substitutes) were coupled via an alkene cross metathesis (CM), similar to that used in our earlier syntheses.^14,15

Synthesis of THF and carbohydrate segments.

We devised a more streamlined synthesis of the THF segment compared to our synthesis of the THF segment in 4-deoxyreticuin.¹⁰ Accordingly, AD-mix β mediated dihydroxylation on the known E-hepta-4-decen-1-ol 5^16,17, (obtainable in two steps from tridecanal), afforded the derived triol, which was transformed to the isopropylidene derivative 6. The enantiomeric purity of 6 was determined to be greater than 95% by ¹HNMR analysis of the (R)-MPTA ester. Chain extension on 6 through an alcohol oxidation-stabilized Wittig olefination-ester reduction sequence provided alkenol 7 in 26% overall yield from 6. In the key step in the synthesis, 7 was treated with NIS and TfOH in dichloromethane. In agreement with our earlier observations on the iodoetherification of related O-isopropyidene alkenes, this reaction gave a high yield (89%), of the trans-THF isomer 8.^10,18,19,20 The cis product was not observed. The stereochemistry of 8 was confirmed by X-ray analysis (Figure 2).²¹ Exposure of 8 to dimethylsulfoniuom methylide effected in situ epoxide formation followed by ring opening to provide alkene THF-alkene 9.²² The NMR data for 9 was essentially identical to that for a sample we obtained from a different synthetic route, and very similar to data from another laboratory.^12,13 Alcohol protection in 9 using MOMCl and diisopropylethylamine afforded 10.

Figure 2.

X-Ray structure of iodo-THF 8

The sugar alkene segment 13 was prepared via routine alcohol protecting group operations on 2,3-O-isopropylidene allyl α-D-mannopyranoside 11, which was obtained in three steps from D-mannose (Scheme 2).²³ Thus, selective silylation of the primary alcohol in 11 provided 12. Subsequent protection of the secondary alcohol in 12 as the methoxymethyl ether, desilylation of the product and alkylation of the resulting primary alcohol with 1-bromo-undecane afforded 13.

Scheme 2.

Synthesis of carbohydrate alkene

Synthesis of butenolide and thiophene segments.

The synthesis of the butenolide alkene and substitutes thereof for the CM reactions, started with epoxide 14, obtained from methyl 1-undecenoate (Scheme 3).²⁴ The CuI promoted reaction of 14 with 4-penten-1-ylmagnesium bromide and silylation of the resulting alcohol afforded 15. Following an established protocol, a two-step aldol-elimination sequence on 15 and aldehyde 16 afforded butenolidealkene 17.²⁵ LAH reduction of ester 15 provided alcohol 18, which was transformed to thiophene alkene 21 via a straightforward three-step sequence: (i) mesylation of 18 to 19; (ii) aminolysis of 19 to 20; (iii) EDCI mediated amidation of 20 and 2-thiophene carboxylic acid.

Scheme 3.

Synthesis of butenolide and thiophene alkenes

CM reactions.

The CM of THF-alkene 9 and 1.5 equivalents of butenolide alkene 17 was performed in CH₂Cl₂, at room temperature, in the presence of 20 mol% Grubbs II catalyst (Scheme 4). The THF-butenolide CM product 22 was obtained in 54% yield. For the THF-thiophene analog 2-R/S the MOM-protected alkene 10 performed better than 9 as the THF-alkene component. Thus, reaction of 10 and 3 equivalents of 21 provided the THF-thiophene CM product 23 in 90% yield based on recovered 10. Similarly, CMs using two equivalents of carbohydrate alkene 13 and 17 or 21 in the presence of Grubbs I catalyst at 40 °C afforded the sugar-butenolide and sugar-thiophene CM products, 24 and 25 in 74 and 70 % yield respectively.

Scheme 4.

Cross metathesis reactions

Transformation of CM products to target compounds.

Alkene reduction on 22 using diimide, followed by acid catalyzed removal of alcohol protecting groups provided a mixture of C-10 epimers of 4-DAN, 1-a/b. Not surprisingly, given the remoteness of the epimeric carbon, this mixture was chromatographically inseparable, and the NMR data for both epimers were very similar and essentially identical to the natural epimer, 4-DAN by ¹H and ¹³C NMR (Table 1). This synthesis which involves the late stage union of THF and butenolide precursors, available in nine and five steps respectively from inexpensive precursors, compares favorably to the previous total synthesis of 4-DAN that requires 17 and 10 step routes to analogous THF and butenolide precursors.⁹ For either of the individual C-10 epimers, the present synthesis can be easily modified by starting with the requisite enantiomer of 14, which can be obtained by resolution of rac-14, or asymmetric epoxidation of the alkene precursor.^10,26,27

Table 1.

Selected NMR data for naturally occurring 4-DAN (1a) and 1-a/b

Carbon #	1a		1-a/b
	1H (δ ppm)	13C (δ ppm)	1H (δ ppm)	13C (δ ppm)
1	-	174.0	-	174.2
2	-	134.3	-	134.5
3	2.26 t (7.0)	25.1	2.26 tt (1.5, 7.5)	25.3
4	1.53 (m)	27.4	1.55 (m)	27.6
10	3.58 m	71.9	3.56 m	72.1
17, 22	3.40 q (5.5)	74.0	3.40 q (6.5)	74.2
18, 21	3.80 q (7.5)	82.7, 82.6	3.79 q (6.5)	82.7, 82.8
19a,19b	1.69 m, 1.98 m	28.7	1.66 m, 1.97 m	28.9
20a,20b	1.69 m, 1.98 m	28.7	1.66 m, 1.97 m	28.9
34	0.88 t (7.5)	14.1	0.87 t (7.6)	14.4
35	6.99 t (1.5)	148.9	6.99 q (1.5, 6.5)	149.1
36	5.00 qq (7.2)	77.4	4.99 bq (6.7)	77.7
37	1.41 d (6.5)	19.2	1.42 d (6.8)	19.4

Following the two-step reaction sequence on 22, CM products 23 and 24 were transformed to 2-a/b and 3-a/b respectively. Palladium mediated hydrogenation of 25, followed by the standard protecting group removal step provided 4-a/b. The final products, as for 1-a/b were obtained as inseparable mixtures of epimers at the remote secondary carbinol.

Cytotoxicity studies.

The cytotoxicity of 1-a/b, 2-a/b, 3-a/b and 4-a/b against two human prostate cell lines, LNCaP (androgen dependent) and PC-3 (androgen independent) was determined by the XTT cell viability assay after 48 h of treatment with the test compounds (Table 2). The assays indicate androgen independent PC-3 cells were slightly more sensitive than LNCaP cells towards these compounds. The parent analog 1-a/b showed low micromolar to nanomolar range activity against both cell lines. Replacement of the butenolide segment with the thiophene moiety (cf 2-a/b), or the THF residue with the mannose residue (cf 3-a/b), led to an appreciable reduction in potency. Surprisingly, when both modifications were implemented (cf 4-a/b), the activity was very similar to that of the parent analog. These data suggests that the potency of 4-a/b may be due to a synergistic effect of these individual modifications, rather than on predominantly one of the two changes. In the absence of mechanistic data it is unclear whether the mode of action of this novel analog is the same as the naturally occurring class of THF containing acetogenins. The IC₅₀ values obtained with the test compounds are comparable to those for the commonly used chemotherapeutic Doxorubicin, which had IC₅₀ values of 0.71 and 2.3 µM in LNCaP and PC-3 cells respectively. Thus, these are interesting lead compounds for in vivo studies.

Table 2.

Cytotoxicity (IC₅₀, μM) of 1-a/b, 2-a/b, 3-a/b, 4-a/b

Test Comp.	LNCaP (μM)	PC-3 (μM)
1-R/S	0.27	0.075
2-R/S	1.9	1.5
3-R/S	3.4	4.5
4-R/S	0.48	0.061
Doxorubicin	0.71	2.3

3. Conclusion

In summary, a modular synthesis of 4-DAN, employing an alkene CM as the key segment coupling reaction was achieved. The synthetic strategy was applied to analogues of 4-DAN in which the THF and butenolide segments were replaced with mannose and thiophene moieties respectively. The final compounds were screened against LNCaP and PC-3 prostate cancer cell lines. The most active was a mannose-thiophene congener which showed very similar activity to the naturally occurring parent THF-butenolide. Given its potency and relatively simple synthesis, this mannose-thiophene is an attractive candidate for further biological studies. Mechanistic studies and tumor targeting strategies on this lead mannose derivative are in progress and will be reported in due course.

4. Experimental section

4.1. General methods

Moisture and oxygen sensitive reactions were performed under an argon atmosphere. Solvents were purified by standard procedures or used directly from commercial sources. Thin layer chromatography (TLC) was done on 0.25 mm thick precoated silica gel HF254 aluminum sheets. Chromatograms were observed under UV (short and long wavelength) light and were visualized by heating plates that were dipped in a solution of ammonium (VI) molybdate tetrahydrate (12.5 g) and cerium (IV) sulfate tetrahydrate (5.0 g) in 10 % aqueous sulfuric acid (500 mL). Flash column chromatography (FCC) was performed using silica gel 60 (230–400 mesh) and employed a stepwise solvent polarity gradient, correlated with TLC mobility. Hexanes used for FCC had a boiling point in the 40–60 °C range. ¹H and ¹³C NMR spectra were obtained on Bruker 400, 500 and 600 MHz instruments. Chemical shifts are quoted in ppm relative to tetramethysilane (δ_H 0.00) and coupling constants (J) are given in Hertz. First order approximations are employed throughout. High resolution mass spectrometry was performed on an Agilent 6520 Q-TOF instrument. X-ray diffraction data were collected on a Bruker X8 Kappa Apex II diffractometer. Crystal data, data collection and refinement parameters are summarized in Table S1.1. The structure was solved using direct methods and standard difference map techniques and was refined by full-matrix least-squares procedures on F2 with SHELXTL (Version 2014/7). The absolute configuration was established by anomalous dispersion effects.

4.2. THF-butenolide (1-a/b)

A solution of sodium acetate (0.62 g, 3 mmol) in water (12 mL) was added via a syringe pump, over 4 h, to a mixture of 22 (86 mg, 0.10 mmol), p-toluenesulfonylhydrazide (1.25 g, 3 mmol) and DME (10 mL) at reflux. After cooling to rt, the reaction mixture was concentrated under reduced pressure, diluted with water and extracted with EtOAc. The organic extract was washed with 2M HCl, and brine, dried (Na₂SO₄), filtered and concentrated under reduced pressure. FCC of the residue afforded the 15,16-dihydro derivative of 22 (74 mg, 86%) as a pale yellow oil: Rf = 0.68 (2% acetone/dichloromethane). ¹H NMR (CDCl₃, 500 MHz) δ 7.63 (m, 4H), 7.29–7.45 (m, 6H), 6.94 (s, 1H), 4.95 (m, 1H), 3.76 (m, 2H), 3.66 (m, 1H), 3.36 (m, 2H), 2.21 (m, 2H), 1.96 (m, 4H), 1.85 (br s, 2H), 1.36 (d, partially buried, J = 6.8 Hz, 3H), 1.34 (m, 10H), 1.25 (m, 36H), 1.00 (s, 9H), 0.85 (t, J = 7.1, 3H). ¹³C NMR (CDCl₃, 125 MHz) δ 174.1, 149.0, 136.1, 135.0, 134.5, 129.5, 127.5, 82.8, 82.7, 77.7, 74.2, 73.4, 36.5, 36.4, 33.7 (2 signals), 32.1, 31.8, 29.9 (2 signals), 29.8 (3 signals), 29.6, 29.4, 29.3, 28.9, 27.5, 27.3, 25.8, 25.8, 25.4, 25.0, 22.9 (2 signals), 19.6, 19.4, 14.3, 11.7. HRMS (ESI) m/z calcd for C₅₃H₈₆O₆SiNa (M + Na)⁺ 869.6091; found 869.6068.

A mixture of 5% AcCl in MeOH (0.5 mL) was added at rt to a solution of the product from the previous step (30 mg, 0.035 mmol) in CH₂Cl₂ (1 mL). The mixture was stirred at this temperature for 4 h, then adjusted to pH 6 with a saturated solution of K₂CO₃ in methanol. The mixture was filtered and concentrated in vacuo. FCC of the residue afforded 1-a/b (15 mg, 68%) as a white wax: Rf = 0.20 (60% EtOAc/hexanes). ¹H NMR (CDCl₃, 500 MHz) 6.99 (q, J = 1.5 Hz, 1H), 4.99 (qq, J = 1.5, 6.5 Hz, 1H), 3.79 (q, J = 6.5 Hz, 2H), 3.56 (m, 1H), 3.40 (q, J = 6.5 Hz, 2H), 2.26 (tt, J = 1.5, 7.5 Hz, 2H), 1.97 (m, 2H), 1.66 (m, 2H), 1.55 (m, 2H), 1.42 (d, partially buried, J = 6.8 Hz, 3H), 1.55–1.25 (m, 44H), 0.87 (t, J = 7.6 Hz, 3H). ¹³C NMR (CDCl₃, 125 MHz) δ 174.1, 149.1, 134.5, 82.8, 82.8, 77.7, 74.3, 74.2, 72.2, 37.7, 37.6, 33.7, 33.6, 32.1, 29.9 (4 signals), 29.8 (2 signals), 29.6, 29.5, 29.3, 29.0, 27.6, 25.8, 25.7, 25.4, 22.9, 19.4, 14.4. HRMS (ESI) m/z calcd for C₃₇H₆₉O₆ (M + H)⁺ 609.5094; found 609.5087.

4.3. THF-Thiophene (2-a/b)

A solution of sodium acetate (100 mg, 1.2 mmol) in water (1.3 mL) was added via a syringe pump, over 4 h, to a mixture of 23 (15 mg, 0.015 mmol) and p-toluenesulfonylhydrazide (200 mg, 1.06 mmol) in DME (1.3 mL) at reflux. The reaction was then processed as described for 1-a/b to give the dihydro derivative of 23 (14 mg, 93%) as a yellow gum: Rf = 0.45 (25% EtOAc/hexanes). ¹H NMR (CDCl₃, 400 MHz) δ 7.65 (d, J = 8.0 Hz, 4H), 7.35 (m, 8H), 7.01 (t, J = 3.8 Hz, 1H), 5.91 (br s, 1H), 4.76 (m, 2H), 4.59 (m, 2H), 3.88 (m, 2H), 3.62 (m, 1H), 3.37 (m, 4H), 3.30 (s, 3H), 3.29 (s, 3H), 1.84–1.00 (m, 60H), 0.98 (s, 9H), 0.81 (t, J = 6.5 Hz, 3H). ¹³C NMR (CDCl₃, 100 MHz) δ 162.0, 139.4, 136.1, 135.1, 129.8, 129.5, 128.0, 127.7, 127.5, 96.9, 81.7, 79.9, 73.4, 55.9, 40.3, 36.5, 32.1, 31.5, 30.0 (two peaks), 29.9 (two peaks), 29.8, 29.7, 29.6, 29.5 (two peaks), 28.7, 27.3, 27.1, 25.7, 25.1, 22.9, 19.6, 14.3.

The product from the previous step (14 mg, 0.014 mmol) was dissolved in CH₂Cl₂ (1 mL) and treated with 5% AcCl in MeOH (0.4 mL) at rt for 4 h. At that time an additional portion of 5% AcCl in MeOH (0.2 mL) was added and stirring continued for an additional 1 h. The reaction was then processed as described for 1-a/b to give 2-a/b (5.5 mg, 59%) as a white powder: Rf = 0.40 (60% EtOAc/hexanes). ¹H NMR (CDCl₃, 500 MHz) δ 7.45 (d, J = 3.7 Hz 1H), 7.42 (d, J = 4.9 Hz, 1H), 7.03 (dd, J = 3.7, 4.9 Hz, 1H), 5.96 (br s, 1H), 3.76 (m, 2H), 3.54 (m, 1H), 3.38 (m, 4H), 2.00–1.10 (m, 54H), 0.84 (t, J = 7.0 Hz, 3H). ¹³C NMR (CDCl₃, 125 MHz) δ 161.9, 139.1, 129.7, 127.8, 127.6, 82.7, 82.6, 74.1, 74.0, 72.0, 59.0, 40.1, 37.5, 33.5, 32.0, 29.7, 29.6 29.5, 29.4, 29.2, 28.8, 26.7, 25.6, 22.7, 14.2. HRMS (ESI) m/z calcd for C₃₉H₇₂NO₅S (M + H)⁺ 666.5131; found 666.5127.

4.4. Mannose-Butenolide (3-a/b)

A solution of sodium acetate (1.50 g, 18.8 mmol) in water (25 mL) was added via a syringe pump, over 4 h, to a mixture of 24 (388 mg, 0.40 mmol) and p-toluenesulfonylhydrazide (2.70 g, 14.5 mmol) in DME (25 mL) at reflux. The reaction was then processed as described for 1-a/b to give the dihydro derivative of 24 (246 mg, 63%) as a yellow gum: Rf = 0.50 (2% acetone/DCM). ¹H NMR (CDCl₃, 600 MHz) δ 7.60 (m, 4H), 7.33 (m, 6H), 6.88 (bs, 1H), 4.99 (s, 1H), 4.97 (m, 1H), 4.71 (ABq, J = 6.2 Hz, Δδ = 0.21 ppm, 2H), 4.12 (t, J = 6.0 Hz, 1H), 4.02 (m, 1H), 3.62 (m, 5H), 3.52 (m, 1H), 3.43 (m, 1H), 3.34 (m, 2H), 3.32 (s, 3H), 2.22 (m, 2H), 1.50–1.00 (m, 52H), 0.95 (s, 9H), 0.80 (t, J = 7.2 Hz, 3H). HRMS (ESI) m/z calcd for C₅₈H₉₂O₁₀SiNa (M+Na)⁺ 1001.6514, found 1001.6500.

A portion of the product from the previous step (30 mg, 0.031 mmol) was dissolved in CH₂Cl₂ (2 mL) and treated with 5% AcCl in MeOH (0.4 mL) at rt for 4 h. Additional 5% AcCl in MeOH (0.4 mL) was then introduced and stirring continued for 4 h. The reaction was then processed as described for 1-a/b to give 3-a/b (12 mg, 60%) as a white powder: Rf = 0.40 (5% MeOH/DCM). ¹H NMR (CDCl_3, 600 MHz) δ 6.96 (s, 1H), 4.97 (m, 1H), 4.80 (s, 1H), 3.93 (dd, J = 1.4, 3.2 Hz, 1H), 3.85 (dd. J = 3.0, 9.1 Hz, 1H), 3.81–3.66 (m, 5H), 3.60 (m, 1H), 3.52 (m, 1H), 3.43 (m, 1H), 2.25 (m, 2H), 2.18 (bs, 4H, OH), 1.45–1.20 (m, 42H), 1.37 (partially buried d, J = 6.8 Hz, 3H), 0.86 (t, J = 7.2 Hz, 3H); ¹³C NMR (CDCl_3, 125 MHz) 174.2, 149.2, 134.5, 99.7, 77.5, 72.4, 72.2, 71.9, 70.8, 70.7, 69.5, 68.0, 37.7, 32.1, 29.8 (4 signals), 29.7, 29.6, 29.5, 29.4, 29.3, 27.6, 26.3, 26.2, 25.8, 25.7, 25.4, 22.9, 19.4, 14.3. HRMS (ESI) m/z calcd for C₃₇H₆₉O₉ (M+H)⁺ 657.4942, found 657.4930.

4.5. Mannose-Thiophene (4-a/b)

A solution of 25 (245 mg, 0.23 mmol) in EtOH (10 mL) and Pd/C (150 mg, 10% wt) was purged with N₂ for 30 min. Then, the mixture was stirred under a hydrogen atmosphere (balloon) for 12 h. Additional Pd/C was then added (50 mg, 10% wt) and stirring continued for 24 h. The reaction was then purged with N₂ and filtered through a bed of Celite to yield the dihydro derivative of 25 (200 mg, 82%) as a clear oil: Rf = 0.53 (20% EtOAc/hexanes). ¹H NMR (CDCl₃, 600 MHz) δ 7.64 (m, 4H), 7.43 (m, 1H), 7.38 (m, 1H), 7.34 (m, 6H), 7.04 (m, 1H), 5.92 (br s, 1H), 5.00 (s, 1H), 4.86 (apparent d, J = 6.3 Hz, 1H), 4.66 (apparent d, J = 6.3 Hz, 1H), 4.18 (t, J = 5.4 Hz, 1H), 4.07 (d, J = 5.4 Hz, 1H), 3.68 (m, 6H), 3.58 (m, 1H), 3.49 (m, 1H), 3.38 (m, 7H), 1.56 (m, 6H), 1.55 (s, 3H), 1.37 (m, 4H), 1.32 (partially buried s, 3H), 1.09–1.26 (m, 36H), 1.02 (s, 9H), 0.85 (t, J = 7.2 Hz, 3H); ¹³C NMR (CDCl₃, 600 MHz) δ 162.0, 139.4, 136.2, 135.0, 129.9, 129.8, 129.6, 128.0, 127.9, 127.8, 127.6, 109.5, 97.1, 96.5, 78.7, 76.3, 73.4, 72.0, 69.9, 68.4, 67.8, 56.2, 40.3, 36.5, 32.1, 30.0, 29.9 (2 signals), 29.8 (2 signals), 29.7 (2 signals), 29.6 (2 signals), 29.5, 28.1, 27.3, 27.2, 26.8, 26.6, 26.4 (2 signals), 25.1, 22.9, 22.6, 19.6, 14.3.

A portion of the product from the previous step (42 mg, 0.041 mmol) was dissolved in CH₂Cl₂ (2 mL) and treated with 5% AcCl in MeOH (0.4 mL). Additional 5% AcCl in MeOH (0.2 mL) was then introduced and stirring continued for 8h. The reaction was then processe das described for 1-a/b to give 4-a/b (19 mg, 66%) as a white amorphous solid. ¹H NMR (CDCl₃, 600 MHz) δ 7.42 (d, J = 3.6 Hz, 1H), 7.38 (d, J = 4.9 Hz, 1H), 6.99 (dd, J = 4.9 Hz, J = 3.8 Hz, 1H), 5.94 (br s, 1H), 3.84 (dd, J = 1.5, 3.2 Hz, 1H), 3.76 (dd, J = 2.7, 9.1 Hz, 1H), 3.72–3.58 (m, 5H), 3.51 (m, 1H), 3.43 (m, 2H), 3.35 (m, 3H), 2.28 (bs, 4H, OH), 1.58 (m, 5H), 1.38–1.18 (m, 41H), 0.81 (t, J = 7.1 Hz, 3H); ¹³C NMR (CDCl₃, 600 MHz) δ 162.1, 139.3, 129.9, 128.1, 127.8, 99.8, 72.3, 72.2, 71.9, 71.8, 70.8, 70.6, 69.7, 68..0, 40.3, 37.6 (2 signals), 32.1, 29.8 (2 signals), 29.7 (2 signals), 29.6, 29.5, 29.4, 27.1, 6.3, 26.2, 25.8, 25.7, 22.9, 14.3. HRMS (ESI) m/z calcd for C₃₉H₇₁NO₈SNa (M+Na)⁺ 736.4793, found 736.4782.

4.6. 3-((4R,5R)-5-dodecyl-2,2-dimethyl-1,3-dioxolan-4-yl)propan-1-ol (6)

Methanesulfonamide (2.0 g, 45.3 mmol), AD-mix β (18.5 g, 0.023 mol) in 1:1 (v/v) water:t-BuOH (100 mL) was stirred for 1 h until the mixture was homogeneous. The mixture was cooled to 0 °C for 30 min at which time an orange precipitate was formed. A solution of 5^14,15 (6.16 g, 0.24 mmol) in t-BuOH (17 mL) was next added and the mixture was stirred at 0 °C for 48 h. The reaction was then quenched with Na₂SO₃, filtered and extracted with EtOAc. The organic phase was concentrated in vacuo and the residue recrystallized from ethyl acetate to give the derived triol (6.71 g, 96%) as a white amorphous solid: MP: 66–69 °C; Rf = 0.15 (75% EtOAc/hexanes); [α]_D²⁵ = 15.3 (c 0.23, CHCl₃). ¹H NMR (CDCl₃, 500 MHz) δ 3.72 (m, 2H), 3.47 (m, 2H), 3.08 (br s, 1H), 2.33 (br s, 1H), 2.30 (br s, 1H), 1.77 (m, 2H), 1.58–1.42 (m, 4H), 1.28 (br s, 20H), 0.90 (t, J = 7.1 Hz, 3H). ¹³C NMR (CDCl₃, 125 MHz) δ 74.9, 74.6, 63.2, 33.8, 32.1, 31.1, 29.9 (2 signals), 29.8 (2 signals), 29.6, 29.2, 25.9, 22.9, 14.3. HRMS (ESI): m/z [C₁₇H₃₆O₃Na]⁺ calcd for 311.2555; found 311.2562.

To a solution of the product from the previous step (8.55 g, 29.7 mmol) in CH₂Cl₂ (58 mL) was added 2,2-dimethoxypropane (4.32 mL, 35.1 mmol) and p-TsOH (0.56 g, 2.93 mmol). The mixture was stirred for 1 h, then quenched with saturated aqueous NaHCO₃ and extracted with CH₂Cl₂. The organic phase was dried (Na₂SO₄), filtered and concentrated under reduced pressure. FCC of the residue (10–15% EtOAc/hexanes) afforded 6 (9.23 g, 94%) as a clear oil: Rf = 0.36 (35% acetone/hexanes). The enantiomeric purity of 6 was determined to be greater than 95% by Mosher ester analysis (vide infra). ¹H NMR (CDCl₃, 500 MHz) δ 3.65 (m, 2H), 3.59 (m, 2H), 2.24 (br s, 1H), 1.71 (m, 2H), 1.56–1.20 (m, 22H), 1.37 (s, partially buried, 6H), 0.86 (t, J = 7.1 Hz, 3H). ¹³C NMR (CDCl₃, 125 MHz) δ 108.2, 81.2, 81.1, 62.9, 32.9, 32.1, 29.9 (2 signals), 29.8 (3 signals), 29.7 (2 signals), 29.5, 27.5, 27.4, 26.3, 22.8, 14.3. HRMS (ESI): m/z [C₂₀H₄₀O₃Na]⁺ calcd for 351.2875, found 351.2869.

4.7. (R)-MTPA ester of 6

To a stirred solution of 6 (35 mg, 0.11 mmol) and (R)-(+)-α-methoxy-α-(trifluoromethyl)phenylacetyl chloride (46 mg, 0.20 mmol) in CH₂Cl₂ (5 mL), DMAP (ca 2 mg) and DCC (42 mg, 0.20 mmol) were added. The reaction was stirred at rt for 1 h at which time the mixture was diluted with ethyl ether. The resulting suspension was filtered over Celite and the filtrate was washed with brine and water. The organic phase was dried (Na₂SO₄), filtered and concentrated in vacuo. The crude mixture was purified by FFC to give the 6-(R)-MTPA (50 mg, 84%) as a white solid: Rf = 0.78 (30% acetone/hexanes). ¹H NMR (C₆D₆, 500 MHz) δ 7.68 (m, 2H), 7.09–6.96 (m, 3H), 4.10 (m, 1H), 4.05 (m, 1H), 3.49 (m 1H), 3.42 (s, 3H), 3.41 (partially buried m, 1H), 1.65–1.55 (m, 6H), 1.29 (s, 3H), 1.28 (s, 3H), 1.26 (m, 20H), 0.91 (t, J = 5.7 Hz, 3H). ¹³C NMR (C₆D₆, 125 MHz) δ 166.9, 133.5, 130.1, 130.0, 129.2, 129.0, 128.8, 128.7, 127.9, 125.6, 123.7, 108.5, 81.6, 81.0, 66.4, 55.7, 33.5, 32.7, 30.6, 30.5 (2 signals), 30.4 (2 signals), 30.2, 29.5, 28.0, 27.9, 27.1, 26.1 (2 signals), 26.0, 23.5, 14.7, 1.7. HRMS (ESI): m/z [C₃₀H₄₇F₃O₅Na]⁺ calcd for 567.3268; found 567.3249.

4.8. (R)-MTPA ester of rac-6

Osmium tetroxide in t-butanol (2.5% wt, 0.35 mL, 0.03 mmol) was added to a solution of 5 (50 mg, 0.20 mmol) in a 1:1 mixture of water acetone (2 mL) at rt. Then, 50% w/w solution of N-methylmorpholine-N-oxide in water (0.04 mL, 0.20 mmol) was introduced and the reaction mixture was stirred for 2 h, at which time Na₂SO₃ (17 mg, 0.06 mmol) was added and stirring continued for an additional 1 h. The mixture was then diluted with water and extracted with ethyl acetate. The organic phase was dried (Na₂SO₄), filtered and evaporated under reduced pressure. FCC of the residue provided the triol derivative (50 mg, 87%) as a clear gum: Rf = 0.15 (75% EtOAc/hexanes). This product was transformed in two steps, as described for optically active 6, to the R-MTPA-ester of (+/−)-6: Rf = 0.78 (30% acetone/hexanes). ¹H NMR (C₆D₆, 400 MHz) δ 7.66 (m, 2H), 7.05 (m, 3H), 4.07 (m, 2H), 3.89 (m, 1H), 3.50 (m, 1H), 3.47 (m, 1H), 3.38 (m, 3H), 3.33 (m, 3H), 1.55 (m, 2H), 1.40 (br s, 16H), 0.8 (m, 2H), 0.7 (m, 2H), 0.4 (m, 3H). ¹³C NMR (C₆D₆, 100 MHz) δ 165.4, 165.3, 132.0, 128.6, 128.5, 128.5, 127.7, 127.5, 127.4, 127.1, 127.0, 126.9, 126.7, 126.6, 126.1, 124.5, 121.7, 107.0, 84.1, 83.8, 80.1, 79.5, 64.9, 64.9, 60.9, 55.7, 54.1 (2 signals), 51.1, 48.9, 32.0, 31.3, 31.2, 30.2, 29.0 (2 signals), 28.9 (3 signals), 28.6, 28.0, 26.5, 26.4, 25.5, 25.3, 25.2, 24.6 (3 signals), 24.5, 23.6 (2 signals), 21.9, 13.2, 12.6.

4.9. (E)-5-((4R,5R)-5-dodecyl-2,2-dimethyl-1,3-dioxolan-4-yl)pent-2-en-1-ol (7)

A suspension of powdered, freshly activated molecular sieves (7 g), Fluorosil (7 g), Celite (7 g), CH₃COONa (2.54 g, 31.2 mmol), PCC (6.61 g, 31 mmol) and 6 (6.84 g, 20.8 mmol) in CH₂Cl₂ (19 mL) was stirred under nitrogen for 1 h. At that time, the mixture was diluted with ether and filtered over Fluorosil. The filtrate was concentrated under reduced pressure and the residue subjected to FCC to give the derived aldehyde (4.42 g, 65%) as a yellow oil: Rf = 0.45 (15% EtOAc/hexanes). ¹H NMR (CDCl₃, 500 MHz) δ 9.74 (s, 1H), 3.54 (m, 2H), 2.57 (m, 2H), 1.90 (m, 1H), 1.68 (m, 1H), 1.46 (m, 2H), 1.34–1.19 (m, 20H), 1.34 (s, partially buried, 6H), 0.86 (t, J = 7.0 Hz, 3H). ¹³C NMR (CDCl₃, 125 MHz) δ 202.1, 108.4, 81.0, 80.0, 40.7, 33.0, 32.1, 29.9 (2 signals), 29.8, 29.7, 27.5, 27.4, 26.3, 25.3, 22.9, 14.3.

To a solution of the product from the previous step (5.06 g, 15.5 mmol) in CH₃CN (150 mL) was added methyl (triphenylphosphoranylidene) acetate (15.5 g, 46.5 mmol). The mixture was heated at reflux for 1.5 h, then filtered over Celite. The filtrate was concentrated under reduced pressure and the residue purified by FCC to give the unsaturated ester derivative (4.67 g, 79%) as a clear oil: Rf = 0.85 (15% EtOAc/hexanes). ¹H NMR (CDCl₃, 500 MHz) δ 6.94 (m, 1H), 5.81 (m, 1H), 3.68 (s, 3H), 3.55 (m, 2H), 2.36 (m, 1H), 2.25 (m, 1H), 1.76 (m, 2H), 1.45 (m, 2H), 1.32–1.10 (m, 20H), 1.32 (s, partially buried 6H), 0.82 (t, J = 7.0 Hz, 3H). ¹³C NMR (CDCl₃, 125 MHz) δ 167.2, 148.8, 121.4, 108.2, 81.0, 80.2, 51.6, 33.1, 32.1, 31.4, 29.9, 29.8 (3 signals), 29.7, 29.5, 29.0, 27.5, 27.4, 26.3, 22.9, 14.3. HRMS (ESI): m/z [C₂₃H₄₂O₄Na]⁺ calcd for 405.2981; found 405.2985.

To a portion of the product from the previous step (450 mg, 1.18 mmol) in CH₂Cl₂ (10 mL) was added a 1.2 M DIBAL-H in toluene (2.3 mL, 2.76 mmol) at −78 °C under nitrogen. The mixture was warmed to rt, stirred at this temperature for an additional 1.5 h, poured into a saturated aqueous solution of Rochelle’s salt (10 mL), and extracted with CH₂Cl₂. The organic phase was washed with brine, dried (NaSO₄) and filtered and evaporated under reduced pressure. FCC of the residue (20% EtOAc/hexanes) afforded 7 (405 mg, 97%) as a clear gum: Rf = 0.53 (25% EtOAc/hexanes). ¹H NMR (CDCl₃, 500 MHz) δ 5.66 (m, 2H), 4.06 (m, 2H), 3.57 (m, 2H), 2.23 (m, 1H), 2.10 (m, 1H), 1.69 (s, 1H), 1.56 (m, 4H), 1.32 (s, partially buried, 6H), 1.18 (m, 20H), 0.81 (t, J = 7.0 Hz, 3H). ¹³C NMR (CDCl₃, 125 MHz) δ 132.5, 129.6, 108.1, 81.2, 81.1, 63.9, 33.1, 32.6, 32.1, 30.0, 29.9, 29.8 (2 signals), 29.7, 29.5, 29.0, 27.5 (2 signals), 26.4, 22.9, 14.3. HRMS (ESI): m/z [C₂₂H₄₂O₃Na]⁺ calcd for 377.3032; found 377.3026.

4.10. (R)-1-((2R,5R)-5-((S)-2-hydroxy-1-iodoethyl)tetrahydrofuran-2-yl)tridecan-1-ol (8)

To a solution of compound 7 (390 mg, 1.10 mmol) in CH₃CN (100 mL) was added NIS (100 mg, 4.40 mmol) and AgOTf (6 mg, 0.02 mmol). The mixture was stirred at rt for 3 h, then diluted with 10% aqueous Na₂S₂O₃ and extracted with EtOAc. The organic phase was dried (Na₂SO₄), filtered and evaporated in vacuo. FCC of the residue afforded 8 (430 mg, 89%) as white needles: MP: 47–49 °C; Rf = 0.33 (25% EtOAc/hexanes). ¹H NMR (CDCl₃, 500 MHz) δ 4.23 (m, 1H), 4.11 (m, 1H), 4.00 (m, 1H), 3.95 (m, 2H), 3.46 (m, 1H), 2.85 (t, J = 13.1 Hz, 1H), 2.41 (m, 1H), 2.10 (m, 2H), 1.99–1.73 (m, 2H), 1.42 (m, 2H), 1.28 (s, 20H), 0.85 (t, J = 7.1 Hz, 3H). ¹³C NMR (CDCl₃, 125 MHz) δ 84.1, 83.1, 74.0, 68.0, 39.3, 34.3, 33.9, 32.1, 29.9 (2 signals), 29.8 (2 signals), 29.6, 28.2, 25.8, 22.9, 14.4. HRMS (ESI): m/z [C₁₉H₃₇IO₃Na]⁺ calcd for 463.1685; found 463.1681.

4.11. (R)-1-((2R,5R)-5-((R)-1-hydroxyallyl)tetrahydrofuran-2-yl)tridecan-1-ol (9)

To a solution of Me₃SI (2.23 g, 10.4 mmol) in THF (11 mL) at −78 °C was added a 2.5 M solution n-BuLi in THF (3.6 mL, 9.0 mmol) under N₂. The mixture was warmed to rt, maintained at this temperature for 1 h, then recooled to −78 °C. A solution of 8 (500 mg, 1.14 mmol) in THF (12 mL) was then introduced and the mixture stirred at −78 °C for 2 h. A second portion of sulfonium ylide was then prepared in THF (11 mL) from Me₃SI (1.88 g, 9.1 mmol) and n-BuLi (2.9 mL, 7.3 mmol) at −78 °C, then warmed to rt and added to the original reaction mixture at −78 °C. The mixture was allowed to warm to rt, and stirred for an additional 2 h at this temperature. The reaction was then quenched with saturated aqueous NH₄Cl and extracted with EtOAc. The organic phase was dried (Na₂SO₄) and evaporated under reduced pressure. FCC of the residue gave 9 (220 mg, 60%) as a clear oil: Rf = 0.57 (50% EtOAc/hexanes). ¹H NMR (CDCl₃, 500 MHz) δ 5.76 (m, 1H), 5.34 (dd, J = 1.4, 17.2 Hz, 1H), 5.17 (dd, J = 1.3, 10.5 Hz, 1H), 3.92 (t, J = 6.2 Hz, 1H), 3.82 (m, 2H), 3.38 (m, 1H), 2.93 (bs, 1H, OH), 2.67 (bs, 1H, OH), 1.93 (m, 2H), 1.64 (m, 2H), 1.42 (m, 1H), 1.34 (m, 3H), 1.21 (m, 18H), 0.74 (t, J = 6.9 Hz, 3H).¹³C NMR (CDCl₃, 125 MHz) δ 136.8, 117.4, 83.1, 82.4, 75.8, 74.2, 33.5, 32.1, 29.9 (2 signals), 29.8 (4 signals), 29.5, 28.7, 28.6, 25.8, 22.9, 14.3. HRMS (ESI): m/z [C₂₀H₃₈O₃Na]⁺ calcd for 349.2719; found 349.2715.

4.12. (2R,5R)-2-((R)-1-(methoxymethoxy)allyl)-5-((R)-1-(methoxymethoxy)tridecyl)-tetrahydrofuran (10)

MOMCl (0.14 mL, 1.84 mmol) was added to a mixture of 9 (150 mg, 0.46 mmol) and DIPEA (0.41 mL, 2.3 mmol) in CH₂Cl₂ (8 mL) at 0 °C. The solution was allowed to warm to rt, stirred at this temperature for 16 h, then poured into water and extracted with CH₂Cl₂. The organic phase was dried (Na₂SO₄), concentrated in vacuo and purified by FCC (10% EtOAc/hexanes) to afford 10 (190 mg, 95%) as a clear oil: Rf = 0.5 (20% EtOAc/hexanes). ¹H NMR (CDCl₃, 500 MHz) δ 5.71 (m, 1H), 5.28 (m, 1H), 5.24 (m, 1H), 4.82 (m, 1H), 4.65 (m, 4H), 3.99 (m, 3H), 3.37 (d, J = 2.8 Hz, 6H), 1.89 (m, 2H), 1.66 (m, 2H), 1.39 (m, 4H), 1.23 (m, 20H), 0.86 (t, J = 7.1 Hz, 3H). ¹³C NMR (CDCl₃, 125 MHz) δ 135.0, 118.9, 96.9, 94.2, 81.8, 81.1, 79.7, 79.4, 55.9, 55.5, 32.1, 31.3, 30.0, 29.9 (2 signals), 29.8, 29.6, 28.3, 28.2, 25.8, 22.9, 14.3. HRMS (ESI): m/z [C₂₄H₄₆O₅Na]⁺ calcd for 437.3243; found 437.3238.

4.13. Propen-1-yl-6-O-tert-butyldiphenylsilyl-4-O-methoxyethyl-2,3-O-isopropylidene-α-D-mannopyranoside (12)

A mixture of 11 (2.19 g, 8.41 mmol), TBDPSCl (2.37 mL, 9.25 mmol) and imidazole (1.14 g, 16.8 mmol) in anhydrous THF (80 mL) was stirred overnight at rt. The mixture was concentrated under reduced pressure, diluted with water and extracted with ether. The combined organic phase was washed with brine, dried (Na₂SO₄) and evaporated in vacuo. FCC of the residue afforded recovered 11 (630 mg) and 12 (1.7 g, 56% brsm) as a clear oil: Rf = 0.40 (20% EtOAc/hexanes). ¹H NMR (CDCl₃, 500 MHz) δ 7.72 (m, 4H), 7.44 (m, 6H), 5.90 (m, 1H), 5.28 (m, 1H), 5.22 (m, 1H), 5.06 (s, 1H), 4.18 (m, 3H), 3.99 (m, 1H), 3.95 (m, 2H), 3.91 (m, 1H), 3.88 (m, 1H), 2.75 (m, 1H), 1.53 (s, 3H), 1.27 (s, 3H), 1.08 (s, 9H); ¹³C NMR (CDCl₃, 500 MHz) δ 135.8, 135.7, 133.6, 133.0, 130.0 (2 signals), 127.9 (2 signals), 118.1, 109.7, 96.3, 78.3, 75.5, 71.0, 69.6, 68.0, 64.8, 28.1, 27.0, 26.3, 19.4. HRMS (ESI) calcd for C₂₈H₃₈O₆SiNa (M+Na)⁺ 521.2335, found 521.2329.

4.14. Propen-1-yl-3,4-O-isopropyledene-4-O-methoxymethyl-6-O-undecyl-α-D-mannopyranoside (13)

MOMCl (0.87 mL, 11.5 mmol) was added to a solution 12 (1.86 g, 3.72 mmol) and DIPEA (2.67 mL, 15.3 mmol) in anhydrous DCM (25 mL) at 0 °C. The mixture was stirred at rt for 16 h, then diluted with water and extracted with DCM. The organic phase was dried (Na₂SO₄), concentrated in vacuo. FCC of the residue afforded the 4-O-methoxymethyl derivative (1.77 mg, 88%) as a clear oil: Rf = 0.60 (20% EtOAc/hexanes). ¹H NMR (CDCl₃, 500 MHz) δ 7.80 (m, 4H), 7.40 (m, 6H), 5.93 (m, 1H), 5.30 (dd, J = 1.6, 17.2 Hz, 1H), 5.22 (dd, J = 1.2, 10.4 Hz, 1H), 5.13 (s, 1H), 4.77 (ABq, J = 6.4 Hz, Δδ = 0.21, 2H), 4.27 (m, 2H), 4.23 (m, 1H), 4.02 (dd, J = 6.4, 10.0 Hz, 1H), 3.90 (dd, J = 1.9, 9.6 Hz, 1H), 9.85 (dd, J = 5.4, 11.0 Hz, 1H), 3.72 (m, 2H), 3.22 (s, 3H), 1.55 (s, 3H), 1.38 (s, 3H), 1.08 (s, 9H).

A mixture of the product from the previous step (140 mg, 0.26 mmol), TBAF (1 M in THF, 0.60 mL, 0.60 mmol) and THF (2 mL) was stirred for 16 h at rt. The solvent was then evaporated in vacuo. FCC of the residue provided the desilylated derivative (75 mg, 95%) as a gum: Rf = 0.30 (20% EtOAc/hexanes). ¹H NMR (CDCl₃, 500 MHz) δ 5.94 (m, 1H), 5.35 (dd, J = 2.2, 17.2 Hz, 1H), 5.25 (dd, J = 1.2, 10.2 Hz, 1H), 5.12 (s, 1H), 4.84 (ABq, J = 6.9 Hz, Δδ = 0.25, 2H), 4.27 (dd, J = 5.7, 7.5 Hz, 1H), 4.17 (m, 2H), 4.03 (dd, J = 7.2, 12.8 Hz, 1H), 3.85 (m, 1H), 3.76 (dd, J = 7.4, 10.2 Hz, 1H), 3.65 (m, 1H), 3.45 (s, 3H), 2.35 (t, J = 6.9 Hz, 1H), 1.38 (s, 3H), 1.29 (s, 3H).

A 60% suspension of NaH in mineral oil (54 mg, 1.35 mmol) was added to a mixture of the material from the previous step (75 mg, 0.25 mmol) and TBAI (14 mg, 0.04 mmol) in DMF (1 mL) at 0 °C, under nitrogen. The mixture was then warmed to rt over 30 min, then cooled to 0 °C, after which undecyl bromide (0.18 mL, 0.74 mmol) was added. The reaction was stirred at rt for 16 h, then cooled to 0 °C, quenched with MeOH (0.5 mL) and diluted with water. The mixture was extracted with ethyl ether and the organic phase washed with brine, dried (Na₂SO₄) and evaporated in vacuo. FCC of the residue afforded 13 (71 mg, 62%) as a gum: Rf = 0.60 (10% EtOAc/hexanes). ¹H NMR (CDCl₃, 600 MHz) δ 5.87 (m, 1H), 5.26 (m, 1H), 5.17 (m, 1H), 5.65 (s, 1H), 4.86 (d, J = 5.3 Hz, 1H), 4.65 (d, J = 5.2 Hz, 1H), 4.19 (m, 1H), 4.11 (m, 1H), 3.98 (m, 1H), 3.66 (m, 3H), 3.57 (m, 1H), 3.48 (m, 1H), 3.42 (m, 4H), 1.55 (m, 4H), 1.50 (s, 3H), 1.31 (partially buried s, 3H), 1.30–1.22 (m, 21H), 0.86 (t, J = 5.8 Hz, 3H). ¹³C NMR (125 MHz, CDCl₃) δ 133.6, 117.8, 109.3, 96.3, 96.2, 78.4, 75.9, 73.2, 71.8, 69.7, 68.3, 67.9, 56.0, 31.9, 29.8, 29.7, 29.6, 29.5, 29.3, 27.9, 26.4, 26.2, 22.7, 14.2. HRMS (ESI) calcd for C₂₅H₄₇O₇ (M+H)⁺ 458.3244, found 458.3243.

4.15. Methyl (R/S)-10-((tert-butyldiphenylsilyl)oxy)hexadec-15-enoate (15)

An approximately 1 M solution of 4-pentenylmagnesium bromide in THF was prepared by dropwise addition of 4-pentenyl bromide (24 mL, 0.21 mol) to a suspension of magnesium (4.8 g, 0.20 mmol) in anhydrous THF (200 mL). To a suspension of CuBr (I) (3.70 g, 0.26 mol) in THF (300 mL) at 0 °C was added a portion of the 1 M solution of 4-pentenylmagnesium bromide (150 mL, 0.13 mol) over 5 min. A solution of 14²⁴ (10.6 g, 49.5 mmol) in anhydrous THF (60 mL) was then introduced, dropwise over 5 min. The reaction was stirred at 0 °C for 10 min, then saturated aqueous NH₄Cl was added and the mixture extracted with ethyl ether. The organic phase was washed with brine, dried (Na₂SO₄), filtered and evaporated under reduced pressure. FCC of the residue gave the derived alcohol (10.2 g, 72%) as a clear oil: Rf = 0.15 (10% EtOAc /hexanes). ¹H NMR (CDCl₃, 500 MHz) δ 5.77 (m, 1H), 4.92 (m, 1H), 4.91 (m, 1H), 3.63 (s, 3H), 3.53 (br s, 1H), 2.26 (m, 2H), 2.03 (m, 2H), 1.34–1.26 (m, 2H). ¹³C NMR (CDCl₃, 125 MHz) δ 174.6, 139.1, 114.6, 72.1, 51.7, 37.6, 37.5, 34.3, 29.8, 29.6, 29.4, 29.3 (2 signals), 29.1, 25.8, 25.3, 25.1.

A mixture of the product from the previous step (7.31 g, 0.025 mol), TBDPSCl (6.24 mL, 0.024 mol) and imidazole (8.33 g, 0.12 mol) in anhydrous THF (200 mL) was heated at reflux under nitrogen for 4 h. The mixture was then diluted with water, extracted with ethyl ether and washed with brine. The organic phase was dried (Na₂SO₄), filtered, and evaporated under reduced pressure. The residue was purified by FCC to afford 15 (9.37 g, 75%) as a colorless oil: Rf = 0.75 (10% EtOAc/hexanes). ¹H NMR (CDCl₃, 500 MHz) δ 7.64 (m, J = 7.6 Hz, 4H), 7.36 (m, 6H), 5.72 (m, 1H), 4.89 (m, 1H), 4.87 (m, 1H), 3.67 (m, partially buried, 1H), 3.64 (s, 3H), 2.27 (t, J = 7.5 Hz, 2H), 1.91 (m, 2H), 1.08–1.25 (m, 20H), 1.04 (s, 9H). ¹³C NMR (CDCl₃, 125 MHz) δ 174.4, 139.1, 136.0, 134.8, 129.4, 127.4, 114.2, 73.2, 51.5, 36.3, 36.1, 34.1, 33.7, 29.6, 29.4, 29.2, 29.1, 29.0, 27.1, 25.0, 24.8, 24.4, 19.4. HRMS (ESI): m/z [C₃₃H₅₁O₃Si]⁺ calcd for 523.3602, found 523.3585.

4.16. (5S)-3-((R/S)8-((tert-butyldiphenylsilyl)oxy)tetradec-13-en-1-yl)-5-methylfuran-2(5H)-one (17)

To a solution of diisopropylamine (9.8 mL, 0.07 mmol) in anhydrous THF (120 mL) at −78 °C was added n-BuLi (21.7 mL, 2.3 M in hexane, 0.05 mol). The mixture was warmed to 0 °C and stirred at this temperature for 10 min, then re-cooled to −78 °C. A solution of 15 (5.2 g, 0.01 mol) in anhydrous THF (60 mL) was then added and the mixture maintained at −78 °C for 1 h, at which time a solution of 16²² (4.7 g, 0.03 mmol) in anhydrous THF (30 mL) was added dropwise over 30 min. After stirring at −78 °C for an additional 45 min, the reaction was diluted with saturated aqueous NH₄Cl and extracted with ethyl ether. The organic phase was dried (Na₂SO₄), filtered and concentrated under reduced pressure and the residue taken up in a mixture methanol (40 mL) and 2-propanol (4 mL). Th solution was adjusted to pH 4 by addition of TsOH^.H₂O (0.25 g), stirred at rt for 20 h, then concentrated under reduced pressure. FCC of the residue afforded recovered 15 (1.5 g), and a more polar material as a yellow oil (3.6 g): Rf = 0.25 (20 % EtOAc/hexanes),

To a portion of the product (3.3 g) from the previous step in CH₂Cl₂ (100 mL) was added Et₃N (4.1 mL, 0.03 mol) and MsCl (0.9 mL, 0.01 mol) at 0 °C. After stirring at rt for 14 h, the reaction was diluted with saturated aqueous NH₄Cl and extracted with ethyl ether. The organic phase was dried (Na₂SO₄), filtered and concentrated under reduced pressure. FCC of the residue afforded 17 (2.34 g, 66% based on recovered 15) as a colorless oil: Rf = 0.45 ((20 % EtOAc/hexanes). ¹H NMR (CDCl₃, 500 MHz) δ 7.65–7.63 (m, 4H), 7.42–7.31 (m, 6H), 6.94 (s, 1H), 5.76–5.67 (m, 1H), 4.96 (m, 1H), 4.87 (m, 1H), 4.83 (m, 1H), 3.67 (m, 1H), 2.22 (m, 2H), 1.91 (m, 2H), 1.16 (m, 3H), 1.07–1.15 (m, 18H), 1.03 (s, (H). ¹³C NMR (CDCl₃, 125 MHz) δ 149.0, 139.2, 136.1, 135.0, 130.0, 129.5, 127.9, 127.5, 114.3, 77.3, 36.4, 36.3, 33.9, 29.7, 29.4, 29.3, 29.1, 27.5, 27.3, 26.7, 25.3, 25.0, 24.6, 19.6, 19.4. HRMS (ESI): m/z [C₃₅H₅₁O₃Si]⁺ calcd for 546.3529; found 546.3529.

4.17. (R/S)-10-((tert-butyldiphenylsilyl)oxy)hexadec-15-en-1-ol (18)

LAH (0.10 g, 2.7 mmol) was slowly added to a solution of 15 (0.92 g, 1.8 mmol) in CH₂Cl₂ (18 mL) at 0 °C. The mixture was warmed to rt and stirred for an additional 1 h. Then, slowly water (10 mL), NaOH (1 mL, 1M), and anhydrous Na2SO4 were sequentially added. The mixture was stirred for 30 min and the resulting suspension filtered over Celite. The filtrate was concentrated in vacuo. FCC of the residue provided 18 (0.71 g, 82%) as a clear oil: Rf = 0.20 (10% EtOAc/hexanes). ¹H NMR (CDCl₃, 600 MHz) δ 7.65 (m, 4H), 7.34 (m, 6H), 5.72 (m, 1H), 4.92 (m, 1H), 4.88 (m, 1H), 4.10 (m, 1H), 3.68 (m, 1H), 3.62 (m, 2H), 1.92 (m, 2H), 1.54 (m, 4H), 1.38–1.17 (m, 8H), 1.02 (s, 9H). HRMS (ESI): m/z [C₃₂H₅₀O₂SiNa]⁺ calcd for 517.3478; found 517.3472.

4.18. (R/S)-10-((tert-butyldiphenylsilyl)oxy)hexadec-15-en-1-yl methanesulfonate (19)

To a solution of 18 (1.00 g, 2.02 mmol) at 0 °C in CH₂Cl₂ (30 mL) was added Et₃N (0.35 mL, 2.57 mmol) and MsCl (0.19 mL, 2.57 mmol). The mixture was warmed to rt and stirred for 16 h, then diluted with water and extracted with ethyl ether. The organic phase was washed with brine, dried (Na₂SO₄), filtered and evaporated in vacuo. FCC of the residue afforded 19 (0.84 g, 72%) as a yellow oil: Rf = 0.5 (20% EtOAc/hexanes). ¹H NMR (CDCl₃, 500 MHz) δ 7.65 (m, 4H), 7.41–7.32 (m, 6H), 5.72 (m, 1H), 4.91 (m, 1H), 4.88 (m, 1H), 4.20 (t, J = 6.6 Hz, 2H), 3.67 (m, 1H), 2.98 (s, 3H), 1.91 (m, 2H), 1.72 (m, 2H), 1.40–1.06 (m, 20H), 1.02 (br s, 9H). ¹³C NMR (CDCl₃, 125 MHz) δ 139.3, 136.1, 129.6, 127.6, 114.4, 73.4, 70.4, 37.6, 36.5, 36.3, 34.0, 29.9, 29.8, 29.6, 29.5, 29.3, 29.2, 29.1, 27.3, 25.6, 25.1, 24.6, 19.6. HRMS (ESI): m/z [C₃₃H₅₂O₄SSiNa]⁺ calcd for 595.3253; found 595.3246.

4.19. (R/S)-10-((tert-butyldiphenylsilyl)oxy)hexadec-15-en-1-amine (20)

A mixture of 19 (0.65 g, 1.13 mmol) and a saturated solution of ammonia in ethanol (150 mL) in a sealed high-pressure tube was heated at 80 °C for 4 h. The volatiles were then removed in vacuo and the residue purified by FCC to give 20 (0.48 g, 86%) as a yellow oil: Rf = 0.32 (20% MeOH/CH₂Cl₂). ¹H NMR (CDCl₃, 600 MHz) δ 7.63 (m, 4H), 7.36 (m, 6H), 5.70 (m, 1H), 4.88 (m, 1H), 4.85 (m, 1H), 4.10 (m, 1H), 3.10 (m, 2H), 1.90 (m, 2H), 1.45 (m, 2H), 1.35 (m, 4H), 1.16 (m, 16H), 1.06 (s, 9H). ¹³C NMR (CDCl₃, 125 MHz) δ 139.2, 136.1, 134.9, 129.5, 127.5, 114.3, 73.3, 40.1, 39.4, 36.4, 36.3, 33.9, 29.8, 29.7, 29.6, 29.2, 29.1, 27.8, 27.2, 26.7, 25.0, 24.5, 19.6.

4.20. N-((R/S)-10-((tert-Butyldiphenylsilyl)oxy)hexadec-15-en-1-yl)thiophene-2-carboxamide (21)

EDCI (0.30 g, 1.58 mmol), Et₃N (0.29 mL, 2.10 mmol) and DMAP (13 mg, 0.10 mmol) were added sequentially to a mixture of 20 (0.26 g, 0.53 mmol) and 2-thiophene carboxylic acid (0.10 g, 0.78 mmol) in anhydrous CH₂Cl₂ (26 mL). The reaction was stirred for 16 h, then diluted with water and extracted with CH₂Cl₂. The organic phase was dried (Na₂SO₄), filtered and evaporated under reduced pressure. FCC of the residue provided 21 (0.22 g, 69%) as a yellow oil: Rf = 0.7 (20% EtOAc/hexanes). ¹H NMR (CDCl₃, 600 MHz) δ 7.65 (m, 4H), 7.45 (m, 2H), 7.42 (m, 6H), 7.04 (d, J = 6.0 Hz, 1H), 5.90 (br s, 1H), 5.71 (m, 1H), 4.90 (m, 1H), 4.88 (m, 1H), 3.67 (m, 1H), 3.40 (m, 2H), 1.90 (m, 2H), 1.57 (m, 4H), 1.36 (m, 6H), 1.15 (m, 12H), 1.08 (s, 9H). ¹³C NMR (CDCl₃, 150 MHz) δ 139.3, 136.2, 135.0, 129.8, 129.6, 128.0, 127.8, 127.6, 114.4, 73.4, 40.3, 36.5, 36.3, 33.9, 30.0, 29.9, 29.7, 29.7, 29.5, 29.2, 27.3, 27.2, 25.0, 24.6, 19.6. HRMS (ESI): m/z [C₃₇H₅₃NO₂SSiNa]⁺ calcd for 626.3458; found 626.3456.

4.21. (5S)-3-((15R)-(8R/S)-((tert-butyldiphenylsilyl)oxy)-15-hydroxy-15-((2R,5R)-5-((R)-1-hydroxytridecyl)tetrahydrofuran-2-yl)pentadec-13-en-1-yl)-5-methylfuran-2(5H)-one (22)

A mixture of 9 (0.10 g, 0.30 mmol) and 17 (0.28 g, 0.51 mmol) in CH₂Cl₂ (2 mL) was purged with nitrogen for 30 min. Grubbs 2nd generation catalyst (25 mg, 0.03 mmol) was then introduced and the mixture stirred for 16 h at rt, after which DMSO (0.05 mL) was added and stirring continued for 30 min. The mixture was then concentrated in vacuo. FCC of the residue (40% EtOAc/hexanes) afforded 22 (136 mg, 54%) as a clear oil: Rf = 0.65 (40% EtOAc/hexanes). ¹H NMR (CDCl₃, 500 MHz) δ 7.63 (m, 4H), 7.40–7.30 (m, 6H), 6.94 (s, 1H), 5.67 (m, 1H), 5.30 (m, 1H) 4.96 (m, 1H), 3.84–3.77 (m, 3H), 3.80 (m, 1H), 3.38 (m, 1H), 2.47 (br s, 1H), 2.28 (br s, partially buried, 1H), 2.21 (m, 2H), 1.90 (m, 4H), 1.63 (m, 2H), 1.45 (m, 3H), 1.33 (m, 10H), 1.22–1.14 (m, 30H), 1.00 (s, 9H), 0.85 (t, J = 6.6 Hz, 3H). ¹³C NMR (CDCl₃, 125 MHz) δ 174.1, 149.0, 136.1, 134.9, 134.5, 129.6, 128.3, 127.6, 82.9, 82.7, 77.7, 75.9, 74.2, 73.3, 36.4, 36.3, 33.8, 32.5, 32.5, 32.1, 29.9 (4 signals), 29.8 (3 signals), 29.6, 29.4, 29.3, 28.7, 27.6, 27.3, 25.8, 25.4, 25.0, 24.6, 22.9, 19.6, 19.4, 14.3. HRMS (ESI): m/z [C₅₃H₈₄O₆SiNa]⁺ calcd for 867.5935, found 867.5909.

4.22. N-((17R)-(10R/S)-((tert-butyldiphenylsilyl)oxy)-17-(methoxymethoxy)-17-((2R,5R)-5-((R)-1-(methoxymethoxy)tridecyl)tetrahydrofuran-2-yl)heptadec-15-en-1-yl)thiophene-2-carboxamide (23)

A mixture of 10 (22 mg, 0.053 mmol) and 21 (86 mg, 0.14 mmol) in CH₂Cl₂ (2 mL) was purged with nitrogen for 30 min. Grubbs 2nd generation catalyst (25 mg, 0.03 mmol) was then introduced and the mixture stirred for 3 h at rt. At that time, additional Grubbs 2nd generation catalyst (5 mg, 0.006 mmol) was added and stirring continued for 20 h at rt. The reaction was then processed as described for 22., to give unreacted 10 (15 mg) and 23 (15 mg, 90% brsm) as a yellow gum: Rf = 0.45 (25% EtOAc/hexanes). ¹H NMR (CDCl₃, 400 MHz) δ 7.60 (m, 4H), 7.45 −7.30 (m, 12H), 7.03 (t, J = 4.0 Hz, 1H), 6.04 (br s, 1H), 5.58 (m, 1H), 5.16 (m, 1H), 4.82 (apparent d, J = 6.9 Hz, 1H), 4.64 (m, 3H), 4.51 (apparent d, J = 6.6 Hz, 1H), 3.94 (m, 2H), 3.82 (m, 1H), 3.62 (m, 1H), 3.45 (m, 1H), 3.38 (m, partially buried, 2H), 3.37 (s, 3H), 3.34 (s, 3H), 1.88–1.00 (m, 50H), 0.99 (s, 9H), 0.83 (t, J = 7.0 Hz, 3H). ¹³C NMR (CDCl₃, 100 MHz) δ 162.0, 139.2, 136.7, 136.0, 134.7 (2 signals), 129.9, 129.5, 128.1, 127.9, 127.7, 127.5, 126.0, 96.8, 93.1, 81.5, 81.1, 79.6, 78.7, 73.0, 55.9, 55.3, 40.1, 36.2, 36.1, 32.5, 32.0, 31.1, 29.9, 29.8, 29.6, 29.5, 29.4, 29.2, 28.5, 28.1, 27.1, 25.8, 24.9, 24.5, 22.9, 19.5, 14.4. HRMS (ESI): m/z [C₅₉H₉₅NO₇SiNa]⁺ calcd for 1012.6490, found 1012.6489.

4.23. (5S)-3-((E)-8R/S-((tert-butyldiphenylsilyl)oxy)-15-(((4S,6R,7R,7aS)-7-(methoxymethoxy)-2,2-dimethyl-6-((undecyloxy)methyl)tetrahydro-4H-[1,3]dioxolo[4,5-c]pyran-4-yl)oxy)pentadec-13-en-1-yl)-5-methylfuran-2(5H)-one (24)

A mixture of 13 (255 mg, 0.56 mmol) and 17 (680 mg, 1.25 mmol) in CH₂Cl₂ (15 mL) was purged with nitrogen for 30 min. Grubbs I catalyst (65 mg, 0.084 mmol) was then introduced and the mixture stirred for 3h at 40 °C. The reaction was then processed as described for 22. FCC of the crude product gave recovered 13 (8 mg) and 24 (388 mg, 74% brsm, E:Z ~4:1), as a light yellow oil. Rf = 0.33 (10% acetone/hexanes ). ¹H NMR (CDCl₃, 500 MHz) δ 7.69 (m, 4H), 7.42 (m, 6H), 6.99 (s, 1H), 5.75 (m, 1H), 5.52 (m, 1H), 5.12 (s, 1H), 5.02 (m, 1H), 4.90 (apparent d, J = 6.3 Hz, 1H), 4.70 (apparent d, J = 6.3 Hz, 1H), 4.25–4.15 (m, 3H), 3.96 (m, 1H), 3.72 (m, 4H), 3.62 (m, 1H), 3.55 (m, 1H), 3.45 (m, 4H), 2.26 (m, 2H), 1.96 (m, 2H), 1.70–1.20 (m, 45H), 1.07 (s, 9H), 0.90 (t, J = 7.2 Hz, 3H). ¹³C NMR (CDCl₃, 150 MHz) δ 173.8, 136.0, 135.8, 134.8, 134.7, 134.4, 129.5, 129.3, 127.4 (2 signals), 109.3, 96.3, 95.9, 78.5, 77.4, 73.3 (2 signals) 73.2, 71.8, 69.7, 68.3, 36.3, 36.2, 32.2, 31.9, 29.8, 29.6 (2 signals), 29.3, 29.2, 29.1 (2 signals), 27.9, 27.4, 27.1 (2 signals), 26.4, 26.2, 25.2, 24.9, 24.6, 22.7, 19.4, 19.2 (2 signals), 14.1. HRMS (ESI) calcd for C₅₈H₉₃O₁₀Si (M+H)⁺ 976.6460, found 976.6454.

4.24. N-(10R/S-((tert-butyldiphenylsilyl)oxy)-17-(((4S,6R,7R,7aS)-7-(methoxymethoxy)-2,2-dimethyl-6-((undecyloxy)methyl)tetrahydro-4H-[1,3]dioxolo[4,5-c]pyran-4-yl)oxy)-2-methylheptadec-15-en-1-yl)thiophene-2-carboxamide (25)

A mixture of 13 (160 mg, 0.35 mmol) and 21 (560 mg, 0.93 mmol), in CH₂Cl₂ (10 mL) was purged with nitrogen for 30 min. Grubbs I catalyst (43 mg, 0.05 mmol) was then introduced and the mixture stirred for 6 h at 40 °C. The reaction was then processed as described for 22 to provide 25 (251 mg, 70%) as a pale yellow oil. Rf = 0.53 (20% EtOAc/hexanes). ¹H NMR (CDCl₃, 600 MHz) δ 7.64 (m, 4H), 7.40 (dd, J = 1.1, 3.7 Hz,, 1H), 7.43 (dd, J = 1.1, 5.0 Hz, 1H), 7.34 (m, 6H), 7.04 (dd, J = 3.7, 5.0 Hz, 1H), 5.92 (bs, 1H), 5.63 (m, 1H), 5.45 (m, 1H), 5.05 (br s, 1H), 4.85 (apparent d, J = 6.3 Hz, 1H), 4.65 (apparent d, J = 6.3 Hz, 1H), 4.18 (m, 1H), 4.10 (m, 2H), 3.86 (m, 1H), 3.66 (m, 3H), 3.58 (m, 1H), 3.48 (m, 1H), 3.39 (m, 6H), 1.91 (m, 2H), 1.55 (m, 4H), 1.47 (s, 3H), 1.40–1.05 (m, 37H), 1.02 (s, 9H), 0.85 (t, J = 7.1 Hz, 3H); ¹³C NMR (CDCl₃, 150 MHz) δ 162.0, 139.3, 136.1 (2 signals), 135.0, 134.9, 129.8, 129.6 (2 signals), 128.0, 127.7, 127.6, 125.1, 109.4, 96.5, 96.0, 78.6, 76.2, 73.4, 73.3, 72.0, 69.9, 68.4, 67.8, 56.1, 40.2, 36.5, 35.3, 32.4, 32.1, 29.9 (2 signals), 29.8, 29.7 (2 signals), 29.6, 29.5 (2 signals), 29.2, 28.0, 27.3, 27.1, 26.6, 26.4, 25.0, 24.7, 22.9, 19.6, 14.3. HRMS (ESI) calcd for C₆₀H₉₅NO₉SSiNa (M+Na)⁺ 1056.6389, found 1056.6389.

Cytotoxicity measurements

Cell number optimization:

Serial dilutions of cells in culture media were plated in triplicate from 100–100000 in a 96 well plate. Cells were incubated at 37 °C in a CO₂ incubator for 72 h. Activated solution of XTT reagent was made by adding 0.1ml of activation reagent to 5ml of XTT reagent. 50 uL of activated XTT reagent was added per well. The plate was incubated for 2 h and absorbance from wells was measured at 475 nM. Background was measured at 660 nM. Background subtraction was made, and absorbance was plotted against cell number. Optimum cell number was determined based on the linear range of XTT standard curve.

IC₅₀ determination:

Serial dilutions of compounds to be tested were made in culture media from 1nM-1mM concentration. Determined cell number from the standard graph were plated in 96 well plate in triplicate per each dilution of the compounds. The cells were allowed to attach overnight by incubation at 37 °C in a CO₂ incubator. The media from the well was removed and media with serial dilutions of the compounds was added to the cells. The cells were incubated with the compound for 48 h at 37 °C in CO₂ incubator. Activated XTT reagent was added to the wells and incubated for 2 h. Absorbance from the wells was measured at 475 nM and background at 660 nM. Background subtraction was made, and all the data was plotted in prism and IC₅₀ values were determined.

Scheme 1.

Synthesis of THF alkene

Scheme 5.

Transformation of CM products to target compounds