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Molecules 2016, 21(1), 47; doi:10.3390/molecules21010047

Article
Synthesis of Bioconjugate Sesterterpenoids with Phospholipids and Polyunsaturated Fatty Acids
1
Departamento de Química Orgánica, Universidad de Salamanca, Plaza de los Caídos 1-5, 37008 Salamanca, Spain
2
Instituto de Biología Molecular y Celular del Cáncer, Centro de Investigación del Cáncer, CSIC-Universidad de Salamanca, Campus Miguel de Unamuno, 37007 Salamanca, Spain
3
Laboratory of Cell Death and Cancer Therapy, Department of Cellular and Molecular Medicine, Centro de Investigaciones Biológicas, CSIC, C/Ramiro de Maeztu 9, 28040 Madrid, Spain
*
Author to whom correspondence should be addressed.
Academic Editors: Jean Jacques Vanden Eynde and Annie Mayence
Received: 3 December 2015 / Accepted: 22 December 2015 / Published: 30 December 2015

Abstract

: A series of sesterterpenoid bioconjugates with phospholipids and polyunsaturated fatty acids (PUFAs) have been synthesized for biological activity testing as antiproliferative agents in several cancer cell lines. Different substitution analogues of the original lipidic ether edelfosine (1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine) are obtained varying the sesterterpenoid in position 1 or 2 of the glycerol or a phosphocholine or PUFA unit in position 3. Simple bioconjugates of sesterterpenoids and eicosapentaenoic acid (EPA) have been obtained too. All synthetic derivatives were tested against the human tumour cell lines HeLa (cervix) and MCF-7 (breast). Some compounds showed good IC50 (0.3 and 0.2 μM) values against these cell lines.
Keywords:
antitumoural; bioconjugates; ether lipidics; edelfosine; sesterterpenolides; PUFAs

1. Introduction

There is a growing interest in medicinal chemistry in the synthesis of bioconjugate compounds [1,2,3,4,5]. Bioconjugate molecules have been described as antitumour agents and as analgesics, showing a synergistic effect due to conjugation. Most known bioconjugates are oligonucleotides [6] with lipids, aminoacids with hydrophilic or lipophilic vitamins [7], lipids with sugars [8], that have a synergistic effect due to the conjugation.

Bioconjugates made by direct esterification of paclitaxel (Taxol®) with polyunsaturated fatty acids (PUFAs) give good antitumour therapy results as the docosahexaenoic acid (DHA)-paclitaxel bioconjugate is less toxic and stable enough in plasma to have a slow release at the tumour [9,10]. Some of the most studied bioconjugates are alkylglycerol derivatives with different biological active molecules [11,12,13,14,15,16,17,18,19,20]. In many cases, these hybrids are considered prodrugs [10,11].

In this work, we report the synthesis and biological evaluation of several biological active sesterterpenes derived from dysidiolide [21,22,23,24] (Figure 1) and bioconjugated with phospholipids such as the synthetic ether lipid edelfosine (1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine) [25,26,27,28,29,30,31] and PUFAs [32,33,34,35,36,37,38,39,40,41,42,43], which separately show antitumour activity and together might have a synergistic effect. Edelfosine is most widely studied antitumoural alkyl lipidic ether as it inhibits the cell growth of several tumour cell lines [26,44,45].

The terpenoids used in this work as starting materials for the bioconjugate synthesis were the bioactive nor-sesterterpenoid 3 and 4 analogues of dysidiolide [21,22,23,24], (Figure 1) and the furosesterterpene intermediates 1 and 2. These compounds have been synthesized previously by our group, starting with ent-halimic acid [46], and have a considerable antitumour activity, similar to that of dysidiolide, showing cellular proliferation inhibition (IC50 ≈ 4.8–5.4 μM) on several solid tumour cellular lines and leukemia [46].

Figure 1. Dysidiolide, edelfosine, PUFAs and sesterterpenoid compounds used for bioconjugation.
Figure 1. Dysidiolide, edelfosine, PUFAs and sesterterpenoid compounds used for bioconjugation.
Molecules 21 00047 g001 1024

2. Results and Discussion

The bioconjugates synthesized in this work are displayed in Figure 2, namely: 1-O-alkyl-glycerols 58, 2-O-alkylglycerols 912 and 13, 14. Compounds 5, 6, 7 and 8, synthesized from R-solketal, are lipidic ethers (LE) that have in the sn2 position of glycerol, a sesterterpenoid joined by a carbonate link, being the glycerol sn3 position esterified with an eicosapentaenoic acid (EPA) unit. Compounds 9, 10, 11, 12 show the sesterterpenoid unit in the glycerol sn1 position. Bioconjugates 9 and 10 with a phosphocholine unit in sn3 of the glycerol and bioconjugates 11 and 12 change the phosphocholine unit for an EPA substituent. Compounds 9, 10 and 11 were synthesized from racemic solketal and 12 from S-solketal. Other bioconjugates, such as 13 and 14 that appear in Figure 2 result from the union of a sesterterpenoid with EPA. The syntheses of all these compounds are described below. We have observed in previous work [46] that the configurational change at C-4 of the sesterterpenoid unit, as in 3 and 4, does not influence the biological activity, so some bioconjugate compounds have been synthesized and tested without separation of the C-4 epimers. In the same manner, racemic glycerol was used as starting material in the synthesis of several bioconjugates obtained in this work, as the chirality of the glycerol unit did not influence the activity in previous studies on edelfosine derivatives [45,47], so several bioconjugates obtained in this work were prepared using racemic glycerol derivative starting materials.

2.1. Synthesis of Bioconjugates 5, 6, 7 and 8

Reaction of R-solketal, 15, (Scheme 1) with bromooctadecane in the presence of NaNH2 gives 16, that by deprotection with p-TsOH led to the ether 17. Regioselective protection of the glycol unit of 17 in the sn3 position as the corresponding p-methoxybenzyl ether is achieved in good yield, using dibutyltin(IV)oxide and cesium fluoride through a O-stannylene acetal intermediate to give 18 [48,49]. This compound reacts with trichloromethyl chloroformate (diphosgene), leading to chlorocarbonate 19.

The desired carbonate 20, is obtained by reaction of 19 with the furo-nor-sesterterpenes 1/2 in the presence of 4-dimethylaminopyridine (DMAP), and N,N-diisopropylethylamine (DIPEA). Deprotection of the p-methoxybenzyl group of 20 was tried under different conditions (CAN [50], DDQ [51]), achieving the best results when DDQ was used. The obtained hydroxyl derivatives 21 and 22 were separated by column chromatography (CC).

Reaction of 21 and 22 with eicosapentaenoic acid (EPA) [20] (Scheme 1) in the presence of N-(3-dimethylaminopropil)-N′-ethyl carbodiimide (EDAC) and DMAP, led to 5 and 6, respectively. These structures were established by studying their NMR spectra. The assignments were corroborated by the [M + Na]+ molecular ions observed at 1033.7860 and 1033.7854 for 5 and 6, respectively, corresponding both to a formula C66H106O7, in agreement with the proposed structures.

Figure 2. Alkyl glycerol sesterterpenoids bioconjugate compounds 512 and sesterterpenoid-PUFAs 1314, synthesized in this work.
Figure 2. Alkyl glycerol sesterterpenoids bioconjugate compounds 512 and sesterterpenoid-PUFAs 1314, synthesized in this work.
Molecules 21 00047 g002 1024
Scheme 1. Synthesis of bioconjugates 5, 6, 7 and 8. Reagents and conditions: (a) Bromooctadecane, NaNH2, toluene, 92%; (b) p-TsOH, MeOH, 40 °C, 93%; (c) 1. n-Bu2SnO, toluene, 2. CsF, PMBCl, DMF, 80%; (d) trichloromethylchloroformate, N,N-dimethylaniline, THF, 83%; (e) DMAP, DIPEA, toluene, 60%; (f) DDQ, DCM/H2O, 21: 32%, 22: 65%; (g) EPA, EDAC, DMAP, DCM, rt, 5: 87%, 6: 82%; (h) 1O2, Rose Bengal, DIPEA, DCM, 7: 86%, 8; 90%.
Scheme 1. Synthesis of bioconjugates 5, 6, 7 and 8. Reagents and conditions: (a) Bromooctadecane, NaNH2, toluene, 92%; (b) p-TsOH, MeOH, 40 °C, 93%; (c) 1. n-Bu2SnO, toluene, 2. CsF, PMBCl, DMF, 80%; (d) trichloromethylchloroformate, N,N-dimethylaniline, THF, 83%; (e) DMAP, DIPEA, toluene, 60%; (f) DDQ, DCM/H2O, 21: 32%, 22: 65%; (g) EPA, EDAC, DMAP, DCM, rt, 5: 87%, 6: 82%; (h) 1O2, Rose Bengal, DIPEA, DCM, 7: 86%, 8; 90%.
Molecules 21 00047 g003 1024

Oxidation of 5 and 6 following Faulkner’s methodology [52] (singlet oxygen in the presence of Rose Bengal and DIPEA), gave the γ-hydroxybutenolides 7 and 8 in excellent yield (Scheme 1). The mass spectra of these compounds show molecular ions at 1065.7775 and 1065.7766, which correspond to the formula C66H106O9, thus confirming these structures.

2.2. Synthesis of 9, 10 and 11

The synthesis of 9, 10 and 11 was carried out starting from the protected glycerol 23 as shown in Scheme 2. Williamson reaction of the 1,3-O-benzylidene glycerol 23 with bromooctadecane and NaNH2, led to a nearly quantitative yield of the alkylderivative 24. Deprotection of 24 with p-TsOH gave diol 25 in excellent yield.

Scheme 2. Synthesis of 9, 10 and 11. Reagents and conditions: (a) Bromooctadecane, NaNH2, toluene, 98%; (b) p-TsOH, MeOH, 40 °C, 90%; (c) TBDMSCl, imidazole, DMF, rt, 26: 41%, 27: 16%, 25: 42%; (d) trichloromethylchloroformate, N,N-dimethylaniline, THF, 71%; (e) DMAP, DIPEA, toluene, 56%; (f) TBAF, THF, rt, 89%; (g) POCl3, pyridine, THF, 0 °C, 97%; (h) choline tetraphenylborate, TPS, pyridine, 35%; (i) 1O2, rose bengal, DIPEA, CH2Cl2, 30%; (j) EPA, EDAC, DMAP, CH2Cl2,rt, 64%; (k) 1O2, Rose Bengal, DIPEA, CH2Cl2, 53%.
Scheme 2. Synthesis of 9, 10 and 11. Reagents and conditions: (a) Bromooctadecane, NaNH2, toluene, 98%; (b) p-TsOH, MeOH, 40 °C, 90%; (c) TBDMSCl, imidazole, DMF, rt, 26: 41%, 27: 16%, 25: 42%; (d) trichloromethylchloroformate, N,N-dimethylaniline, THF, 71%; (e) DMAP, DIPEA, toluene, 56%; (f) TBAF, THF, rt, 89%; (g) POCl3, pyridine, THF, 0 °C, 97%; (h) choline tetraphenylborate, TPS, pyridine, 35%; (i) 1O2, rose bengal, DIPEA, CH2Cl2, 30%; (j) EPA, EDAC, DMAP, CH2Cl2,rt, 64%; (k) 1O2, Rose Bengal, DIPEA, CH2Cl2, 53%.
Molecules 21 00047 g004 1024

Reaction of lipidic ether 25 with tert-butyldimethylsilyl chloride (TBDMSCl) and imidazole, rendered a mixture of the starting diol and the monoprotected and diprotected derivatives 26 and 27, respectively, which were separated by CC. Treatment of 26 with diphosgene in the presence of N,N-dimethylaniline gave 28 (Scheme 2). Reaction of 28 with the furo-nor-sesterterpenes 1/2 in the presence of DMAP and DIPEA lead to 29. Deprotection of 29 was done with tetrabutylammonium fluoride (TBAF), to obtain the hydroxyderivative 30, which is the key intermediate in the synthesis of the glycerophosphocholine derivatives 9 and 10 and the bioconjugate 11. Phosphorylation of 30 was carried out with POCl3 in pyridine, affording the phosphatidic acid 31 [18] quantitatively, that was made to react with choline tetraphenylborate [53] and 2,4,6 triisopropylbenzene sulfonyl chloride (TPS) to give 9. The structure of this compound was established by its NMR spectra. The mass spectrum of 9 shows a [M + Na]+ molecular ion at 914.6229 corresponding to C51H90NO9P, corroborating in this manner the structure of the bioconjugate phospholipid. The γ-hydroxy-butenolide 10 was obtained from the furyl derivative 9 by oxidation with singlet oxygen in the presence of Rose Bengal and DIPEA.

Esterification of 30 with EPA, EDAC and DMAP gives the furyl derivative 32 (Scheme 2). Treatment of 32 with singlet oxygen in the presence of Rose Bengal and DIPEA lead to 11, whose structure was established by its NMR spectra. The mass spectrum of these compounds shows a [M + Na]+ molecular ion at 1065.7 corresponding to C66H106O9 in agreement with the proposed structure for compound 11.

2.3. Synthesis of 12

In order to test the chirality effect, a chiral glycerol was used to obtain compound 12, the stereoisomer of 11 (Scheme 3). Reaction of S-solketal 33 with PMBCl and NaH [54] leads to the p-methoxybenzyl derivative 34, that by chromatography on silica gel is transformed into 35.

Scheme 3. Synthesis of 12. Reagents and conditions: (a) PMBCl, NaH, 99%; (b) SiO2, 90%; (c) TrCl, pyridine, 92%; (d) bromooctadecane, NaNH2, toluene, 97%; (e) p-TsOH, MeOH, 40 °C, 82%; (f) trichloromethylchloroformate, N,N-dimethylaniline, THF, 82%; (g) DMAP, DIPEA, toluene, 58%; (h) DDQ, DCM/H2O, rt, 71%; (i) EPA, EDAC, DMAP, DCM, rt, 63%; (j) 1O2, rose bengal, DIPEA, DCM, 54%.
Scheme 3. Synthesis of 12. Reagents and conditions: (a) PMBCl, NaH, 99%; (b) SiO2, 90%; (c) TrCl, pyridine, 92%; (d) bromooctadecane, NaNH2, toluene, 97%; (e) p-TsOH, MeOH, 40 °C, 82%; (f) trichloromethylchloroformate, N,N-dimethylaniline, THF, 82%; (g) DMAP, DIPEA, toluene, 58%; (h) DDQ, DCM/H2O, rt, 71%; (i) EPA, EDAC, DMAP, DCM, rt, 63%; (j) 1O2, rose bengal, DIPEA, DCM, 54%.
Molecules 21 00047 g005 1024

Regioselective protection of 35 is carried out with trityl chloride (TrCl) and pyridine to obtain 36 in an excellent global yield of 83% from S-solketal. Alkylation of 36 with bromooctadecane, in the presence of NaNH2, gives 37 in good yield. Treatment of 37 with p-TsOH [55,56] gives the desired partially deprotected compound 38. Reaction of 38 with diphosgene in the presence of N,N-dimethyl-aniline led to chlorocarbonylderivative 39. Reaction of 39 with the furo-nor-sesterterpenes 1/2 mixture in presence of DMAP and DIPEA lead to 40. Deprotection of 40 was carried out by reaction with DDQ giving 41 (Scheme 3). Esterification of 41 with eicosapentaenoic acid (EPA), in the presence of EDAC and DMAP gives bioconjugate 42. Oxidation of 42 with singlet oxygen and DIPEA led to compound 12. The structure of this compound was established by study of its NMR spectra. The mass spectrum of this compound shows a [M + Na]+ molecular ion a 1065.7725 corresponding to the molecular formula C66H106O9, corroborating in this manner the structure proposed for compound, 12.

2.4. Synthesis of 13 and 14

Due to the complexity of the synthesis describe above, the synthesis of simpler bioconjugates, such as 13 and 14 (Scheme 4), was planned in order to obtain more bioconjugate compounds, enabling us to thus do SAR studies. Compound 13 was obtained by direct esterification of 1/2 with eicopentaenoic acid (EPA). Reaction of 1/2 with eicosapentaenoic acid (EPA) in the presence of EDAC and DMAP leads to compound 13, that by treatment with singlet oxygen, in the presence of Rose Bengal and DIPEA gives 14.

Scheme 4. Synthesis of 13 and 14. Reagents and conditions: (a) EPA, EDAC, DMAP, DCM, rt, 63%; (b) 1O2, rose bengal, DIPEA, DCM, 54%.
Scheme 4. Synthesis of 13 and 14. Reagents and conditions: (a) EPA, EDAC, DMAP, DCM, rt, 63%; (b) 1O2, rose bengal, DIPEA, DCM, 54%.
Molecules 21 00047 g006 1024

3. Antitumour Activity of the Bioconjugate Compounds

The in vitro antitumour activity for these compounds was determined by measurement of their cytostatic and cytotoxic properties in human tumour cell lines by the XTT assay (Table 1). The cell lines used were HeLa (human epitheloid cervix carcinoma), and MCF7 (human breast carcinoma). Cells were incubated in DMEM (HeLa) or RPMI-1640 (MCF-7) culture medium containing 10% heat-inactivated foetal bovine serum in the absence and in the presence of the indicated compounds at a concentration range of 10−4 to 10−8 M in a 96-well plate, and following 72 h of incubation at 37 °C in a humidified atmosphere of air/CO2 (19:1) the XTT assay was performed as previously described [57].

Measurements were done in triplicate, and the IC50 value, defined as the drug concentration required to cause 50% inhibition in the cellular proliferation with respect to the untreated controls, was determined for each compound.

The proliferation inhibition data showed a significant antitumour activity of several compounds as shown in Table 1. When tested compounds 1 and 2 showed less activity against HeLa and MCF7 cells than their γ-hydroxybutenolide counterparts 3 and 4 [46]. This behaviour tells us that the change of a furan fragment for a γ-hydroxybutenolide unit increases the activity, as previously observed by us [46,58,59]. Secondly bioconjugates 5 and 6 are more active than the non-conjugates 1 and 2, in the same manner bioconjugates 7 and 8 have a better behaviour than 3 and 4 showing than conjugation increases the activity against HeLa and MCF7. These compounds 7 and 8 are more active that edelfosine against HeLa tumour cells and several times better than edelfosine against MCF-7 cells. When changing the sesterterpenoid substitution position on the glycerol unit from secondary as in 7 and 8 to primary as in 9, 10, 11 and 12 a light decrease or the same biological activity can be observed in both cell lines. It is remarkable that the activity of these compounds, especially 11 and 12, duplicates that of the free sesterterpenolides 3 and 4, so bioconjugation increases the biological activity. When comparing edelfosine against several γ-hydroxybutenolide bioconjugate compounds such as 10, 11 and 12 on the MCF7 tumour cell line, it can be observed that edelfosine is more active than the phospholipidic ester 10, while on the contrary, the activity of the γ-hydroxybutenolides 11 and 12 is 6-fold higher than that of edelfosine.

Table 1. IC50 values of the synthesized compounds against HeLa (human cervix cancer) and MCF7 (human breast cancer) cells. Some natural analogues were included for comparison. Distinct fragments, substitutions or units involved in the structure were included for reference.
Table 1. IC50 values of the synthesized compounds against HeLa (human cervix cancer) and MCF7 (human breast cancer) cells. Some natural analogues were included for comparison. Distinct fragments, substitutions or units involved in the structure were included for reference.
CompoundHeLa IC50 (μM)MCF-7 IC50 (μM)Structural CharacteristicSesterterpene PositionSn3 Substitution
1/230.2 ± 1.932.1 ± 1.1Furan
34.8 ± 0.75.1 ± 0.9α-Hydroxybutenolide
45.4 ± 0.65.2 ± 1.8α-Hydroxybutenolide
5/613.1 ± 9.5NDFuransn2EPA
71.1 ± 0.10.6 ± 0.1α-Hydroxybutenolidesn2EPA
81.1 ± 0.10.5·± 0.2α-Hydroxy-butenolidesn2EPA
925.0 ± 10NDFuran1Phosphocholine
103.3 ± 0.96.5 ± 0.8α-Hydroxybutenolide1Phosphocholine
112.5 ± 0.10.6 ± 0.2α-Hydroxybutenolide1EPA
122.5 ± 0.10.5·± 0.1α-Hydroxybutenolidesn1EPA
1310.2 ± 3.512.3 ± 3.7FuranEicosapentanoylsesterterpene
140.3 ± 0.10.2 ± 0.1α-HydroxybutenolideEicosapentanoylsesterterpene
Edelfosine2.5 ± 0.73.1·± 0.9

Simple bioconjugates 13 and 14 are more biologically active than alkylglycerols 9 and 10 respectively, while in this respect compound 14 is 17 and 26 times more active than γ-hydroxylactones 3 and 4 against HeLa and MCF7 cells, respectively, and more than 40 times more active than eicosapentaenoic acid against HeLa cells [45,46]. Compound 14 is 8 and 15 times more active than edelfosine against HeLa and MCF-7 cells, respectively, making it an interesting starting material for analogue synthesis. In summary the presence of a γ-hydroxybutenolide and simple bioconjugation could be a route to better activity.

4. Materials and Methods

4.1. General Information

Unless otherwise stated, all chemicals were purchased as the highest purity commercially available and were used without further purification. IR spectra were recorded on an AVATAR 370 FT-IR spectrophotometer (Thermo Nicolet, Salamanca, Spain). 1H- and 13C-NMR spectra were recorded in CDCl3 and referenced to the residual peak of CHCl3 at δ 7.26 ppm and δ 77.0 ppm, for 1H and 13C, respectively, using 200 VX (Varian, Salamanca, Spain) and DRX 400 (Bruker, Salamanca, Spain) instruments. Chemical shifts are reported in δ parts per million and coupling constants (J) are given in hertz. MS were recorded using a VG TS 250 spectrometer at 70 eV ionising voltage (Fisons, Salamanca, Spain). Data are presented as m/z (% rel. int.). HRMS were recorded on a VG Platform spectrometer using the chemical ionization (ammonia as gas) or fast atom bombardment (FAB) techniques. For some of the samples, a QSTAR XL spectrometer (Evisa, Salamanca, Spain) was employed for electrospray ionization (ESI). Optical rotations were determined on a 241 polarimeter (Perkin-Elmer, Salamanca, Spain) in 1 dm cells. Diethyl ether and THF were distilled from sodium, and dichloromethane was distilled from calcium hydride under argon atmosphere.

4.2. Preparation of 1-O-Octadecyl-2,3-isopropyliden-sn-glycerol (16)

To a solution of (R)-(−)-solketal 15 (2.6 g, 19.7 mmol) in toluene (39 mL), NaNH2 (768 mg, 19.7 mmol) was added, and the mixture was heated at 111 °C under an argon atmosphere for 1 h. Then it was cooled to rt and a solution of bromooctadecane (6.5 g, 19.7 mmol) in toluene (5 mL) was added, before heating at 111 °C for 3 h. After that time, the reaction mixture was cooled at 0 °C, crushed ice and saturated NH4Cl were added and it was extracted with Et2O. The organic layer was washed with H2O and brine. After drying over anhydrous Na2SO4, the organic layer was filtered and evaporated. The obtained residue was purified by column chromatography (Hex/EtOAc 9:1) to yield 16 (6.9 g, 92%). [ α ] D 22 −8.27 (c 1.6, CHCl3); IR (film, cm−1): 2985, 2924, 2854, 1465, 1369, 1255, 1118, 1057, 849; 1H-NMR (400 MHz, CDCl3, δ ppm): 4.26 (1H, quin., J = 6.0 Hz, H-sn2), 4.06 (1H, dd, J = 8.2, 6.0 Hz, HA-sn3), 3.73 (1H, dd, J = 8.2, 6.0 Hz, HB-sn3), 3.51 (1H, dd, J = 9.9, 6.0 Hz, HA-sn1), 3.47 (2H, t, J = 6.8 Hz, H-1′), 3.41 (1H, dd, J = 9.9, 5.6 Hz, HB-sn1), 1.56 (2H, m, H-2′), 1.42, 1.36 (3H, s, each, Me2C-), 1.25 (30H, m, H-3′-17′), 0.88 (3H, t, J = 6.8 Hz, H-18′); 13C-NMR (100 MHz, CDCl3, δ ppm): 109.3 (Me2C-), 74.7 (C-sn2), 71.9 (C-1′), 71.8 (C-sn1), 66.9 (C-sn3), 31.9 (C-16′), 29.4 (C-2′), 29.4 (C-4′-15′), 26.7, 25.4 (Me2C-), 26.0 (C-3′), 22.6 (C-17′), 14.0 (C-18′); EIHRMS: calcd. for C24H48O3 [M + H]+: 385.3676, found: 385.3680.

4.3. Preparation of 1-O-Octadecyl-sn-glycerol (17)

To a solution of 16 (4.7 g, 12.24 mmol) in MeOH (36 mL), p-TsOH (2.3 g, 12.24 mmol) was added and stirred at 35 °C for 8 h. Then H2O was added, and the reaction mixture was extracted with Et2O and washed with 6% NaHCO3 and H2O. The organic layer was dried over anhydrous Na2SO4, filtered and evaporated to give 17 (3.9 g, 11.3 mmol, 93%). [ α ] D 22 +0.95 (c 0.84, CHCl3); IR (film, cm−1): 3325, 2918, 2849, 1470 1119, 1063; 1H-NMR (400 MHz, CDCl3, δ ppm): 3.85 (1H, dddd, J = 6.0, 6.0, 4.0, 4.0 Hz, H-sn2), 3.71 (1H, dd, J = 11.4, 4.0 Hz, HA-sn3), 3.63 (1H, dd, J = 11.4, 6.0 Hz, HB-sn3), 3.53 (1H, dd, J = 9.6, 4.0 Hz, HA-sn1), 3.49 (1H, dd, J = 9.6, 6.0 Hz, HB-sn1), 3.47 (1H, ddd, J = 9.3, 6.7, 6.7 Hz, HA-1′), 3.44 (1H, ddd, J = 9.3, 6.7, 6.7 Hz, HB-1′), 1.56 (2H, m, H-2′), 1.25 (30H, m, H-3′-17′), 0.87 (3H, t, J = 6.8 Hz, H-18′); 13C-NMR (100 MHz, CDCl3, δ ppm): 72.4 (C-sn1), 71.8 (C-1′), 70.4 (C-sn2), 64.2 (C-sn3), 31.8 (C-16′), 29.5 (C-2′), 29.5 (C-4′-15′), 26.0 (C-3′), 22.6 (C-17′), 14.0 (C-18′); EIHRMS: calcd. for C21H44O3 [M + Na]+: 367.3183, found: 367.3194.

4.4. Preparation of 1-O-Octadecyl-3-O-p-methoxybenzyl-sn-glycerol (18)

To a solution of 17 (200 mg, 0.58 mmol) in toluene (3.4 mL), dibutyl tin (IV) oxide (144 mg, 0.58 mmol) was added and it was heated up to reflux for 2 h in a Dean-Stark apparatus. After this time, the solvent was evaporated to give a white solid, and CsF (167 mg, 1.1 mmol) was added to this solid. The solid mixture was dried for 1 h 30 min under high vacuum. It was then diluted in DMF (3.4 mL) and PMBCl (258 mg, 1.65 mmol) added and stirred overnight under an argon atmosphere. Then H2O (1 mL) and EtOAc (3 mL) were added, the reaction mixture was stirred vigorously for 15 min and filtered through a pad of silica gel to remove the dibutyl tin oxide. The filtrate was washed with H2O and brine. Removal of the solvents gave a residue that was purified by column chromatography (Hex/EtOAc 96:4) to obtain 18 (215 mg, 80%). [ α ] D 22 +1.2 (c 0.11, CHCl3); IR (film, cm−1): 3485, 3404, 2916, 2846, 1470, 1031; 1H-NMR (400 MHz, CDCl3, δ ppm): 7.27 (2H, d, J = 7.4 Hz, H-2′′′, H-6′′′), 6.89 (2H, d, J = 7.4 Hz, H-3′′′, H-5′′ ), 4.50 (2H, s, –OCH2Ar), 3.98 (1H, quin, J = 4.8 Hz, H-sn2), 3.82 (3H, s, –OMe), 3.65–3.55 (6H, m, H-sn1, 1′, sn3), 1.56 (2H, m, H-2′), 1.28 (30H, m, H-3′-17′), 0.89 (3H, t, J = 6.2 Hz, H-18′); 13C-NMR (100 MHz, CDCl3, δ ppm): 159.2 (C-4′′′), 130.1 (C-1′′′), 129.3 (C-2′′′), 129.3 (C-6′′′), 113.8 (C-3′′′), 113.8 (C-5′′′), 73.1 (–OCH2Ar), 71.7 (C-1′), 71.6 (C-sn1), 71.0 (C-sn3), 69.5 (C-sn2), 55.2 (C–OMe), 31.9 (C-16′), 29.5 (C-2′), 29.5 (C-4′-15′), 26.1 (C-3′), 22.7 (C-17′), 14.1 (C-18′); EIHRMS: calcd. for C29H52O4 [M + Na]+: 487.3758, found: 487.3773.

4.5. Preparation of 1-O-Octadecyl-2-chlorocarbonyl-3-O-p-methoxybenzyl-sn-glycerol (19)

To an ice cooled solution of 18 (377 mg, 0.81 mmol) in THF (1.6 mL), trichloromethyl chloroformate (diphosgene, 160 mg, 0.81 mmol) and N,N-dimethylaniline (98 mg, 0.81 mmol) were added. The mixture was stirred at 0 °C for 10 min and then at rt overnight. Then Et2O was added and the white precipitate formed was filtered. The solution washed with 0.2 M HCl, 0.2 M NaOH and H2O, then dried over anhydrous Na2SO4 and evaporated to give 19 (353 mg, 0.67 mmol, 83%). IR (film, cm−1): 2924, 2852, 1780, 1166; 1H-NMR (200 MHz, CDCl3, δ ppm): 7.24 (1H, d, J = 7.0 Hz, each, H-2′′′, H-6′′′), 6.89 (1H, d, J = 7.4 Hz, each, H-3′′′, H-5′′′), 5.16 (1H, quin, J = 4.8 Hz, H-sn2), 4.49 (2H, s, –OCH2Ar), 3.81 (3H, s, –OMe), 3.64–3.59 (4H, m, H-sn1, sn3), 3.44–3.38 (2H, m, H-1′), 1.54 (2H, m, H-2′), 1.25 (30H, m, H-3′-17′), 0.89 (3H, t, J = 6.2 Hz, H-18′); 13C-NMR (50 MHz, CDCl3, δ ppm): 159.2 (C-4′′′), 154.9 (–O–CO–Cl), 129.8 (C-1′′′), 129.5 (C-2′′′, 6′′′), 114.1 (C-3′′′, 5′′′), 80.5 (C-sn2), 73.3 (–OCH2Ar), 72.1 (C-1′), 68.9 (C-sn1), 67.9 (C-sn3), 55.5 (C–OMe), 32.2 (C-16′), 29.8 (C-2′), 29.8 (C-4′-15′), 26.2 (C-3′), 22.9 (C-17′), 14.3 (C-18′); EIHRMS: in MeOH, calcd. for methyl ester, C31H54O6 [M + Na]+: 545.3813, found: 545.3808.

4.6. Preparation of 1-O-Octadecyl-2-O-[1,25-epoxy-18-nor-ent-isodysidiola-1,3(25),9,19-tetraen-4R/S-yloxycarbonyl]-3-p-methoxybenzyl-sn-glycerol (20)

To a solution of 1/2 (153 mg, 0.43 mmol), N,N-diisopropylethylamine (DIPEA, 71 mg, 0.55 mmol), 4-(dimethylamino) pyridine (DMAP, 26 mg, 0.21 mmol) in toluene (2.1 mL), a solution of 19 (172 mg, 0.33 mmol) in toluene (1.65 mL) was added dropwise at 0 °C . The reaction mixture was stirred at 0 °C under an argon atmosphere for 15 min and then at rt overnight. After this time the solvent was removed and the residue was purified by column chromatography (Hex/EtOAc 99:1) to obtain 20 (167 mg, 60%). IR (film, cm−1): 2924, 2853, 1744, 1514, 1464, 1258, 1115; 1H-NMR (400 MHz, CDCl3, δ ppm): 7.42/7.41 (1H, s, H-25′′), 7.34/7.33 (1H, s, H-1′′), 7.24/7.17 (2H, d, J = 8.8 Hz, H-2′′′, H-6′′′), 6.86/6.83 (2H, d, J = 8.8 Hz, H-3′′′, H-5′′′), 6.39 (1H, s, H-2′′), 5.81/5.79 (1H, dd, J = 5.6, 3.2 Hz, H-4′′), 5.35/5.33 (1H, t, J = 3.2 Hz, H-9′′), 4.98 (1H, m, H-sn2), 4.65–4.62 (2H, m, H-20′′), 4.49/4.45 (1H, d, J = 11.6 Hz, –OCH2Ar), 4.44/4.39 (1H, d, J = 11.6 Hz, –OCH2Ar), 3.80 (3H, s, –OMe), 3.64–3.50 (2H, m, H-sn1), 3.64–3.50 (2H, m, H-sn3), 3.47–3.31 (2H, m, H-1′), 2.20–1.40 (16H, m, H-5′′, 7′′, 8′′, 11′′, 12′′, 13′′, 14′′, 16′′, 17′′), 1.70/1.67 (3H, s, Me-21′′), 1.56 (2H, m, H-2′), 1.26 (30H, m, H-3′-17′), 0.91 (3H, s, Me-22′′), 0.89 (3H, t, J = 6.2 Hz, Me-18′), 0.88 (3H, s, Me-24′′), 0.81/0.80 (3H, d, J = 7.0 Hz, Me-23′′); 13C-NMR (100 MHz, CDCl3, δ ppm): 159.2 (C-4′′′), 154.2 (–O–CO–O–), 147.0/146.9 (C-19′′), 143.1 (C-1′′), 141.4/141.0 (C-10′′), 140.0/139.9 (C-25′′), 130.0 (C-1′′′), 129.2 (C-2′′′, 6′′′), 126.2 (C-3′′), 120.0/119.7 (C-9′′), 113.7 (C-3′′′, 5′′′), 109.1/109.0 (C-20′′), 108.8/108.7 (C-2′′), 75.5/75.4 (C-sn2), 72.9 (–OCH2Ar), 71.7/71.6 (C-1′), 70.0 (C-4′′), 69.2/69.1 (C-sn1), 68.5/68.3 (C-sn3), 55.2 (–OMe), 44.3 (C-5′′), 42.9 (C-15′′), 42.6 (C-11′′), 38.9/38.7 (C-14′′), 37.4/37.2 (C-16′′), 34.1/34.0 (C-6′′), 32.4/32.3 (C-17′′), 31.9 (C-16′), 29.6–28.9 (C-2′), 29.6–28.9 (C-4′-15′), 29.6–28.9 (C-7′′, 13′′), 26.0/25.9 (C-3′), 23.2 (C-8′′, 12′′), 22.8 (C-24′′), 22.6 (C-17′), 22.3 (C-21′′), 22.2 (C-22′′), 15.6 (C-23′′), 14.0 (C-18′); EIHRMS: calcd. for C54H86O7 [M + Na]+: 869.6266, found: 869.6233.

4.7. Reaction of Compound 20 with DDQ: Preparation of 1-O-Octadecyl-2-O-[1,25-epoxy-18-nor-ent-isodysidiola-1,3(25),9,19-tetraen-4S-yloxycarbonyl]-sn-glycerol (21) and 1-O-Octadecyl-2-O-[1,25-epoxy-18-nor-ent-isodysidiola-1,3(25),9,19-tetraen-4R-yloxycarbonyl]-sn-glycerol (22)

To a solution of 20 (170 mg, 0.2 mmol) in CH2Cl2/H2O 18:1 (2.2 mL), DDQ (54 mg, 0.24 mmol) was added. The reaction mixture was stirred at rt under an argon atmosphere for 1 h 15 min, quenched with 6% NaHCO3 and extracted with CH2Cl2. The organic layer was washed with 6% NaHCO3 and brine and dried over anhydrous Na2SO4. Removal of the solvent gave the crude product which was purified by column chromatography on silica gel to obtain 21 (46 mg, 32%, Hex/EtOAc 97:3 as eluent) and 22 (94 mg, 65%, Hex/EtOAc 95:5 as eluent).

Compound 21: [ α ] D 22 +42.2 (c 0.46, CHCl3); IR (film, cm−1): 3464, 2924, 2853, 1744, 1260; 1H-NMR (400 MHz, CDCl3, δ ppm): 7.43 (1H, s, H-25′′), 7.35 (1H, s, H-1′′), 6.40 (1H, s, H-2′′), 5.79 (1H, dd, J = 8.5, 3.6 Hz, H-4′′), 5.33 (1H, t, J = 4.8 Hz, H-9′′), 4.81 (1H, quin, J = 5.0 Hz, H-sn2), 4.65 and 4.64 (1H, s, each, H-20′′), 3.84 (1H, dd, J = 12.0, 5.0 Hz, HA-sn3), 3.79 (1H, dd, J = 12.0, 5.0 Hz, HB-sn3), 3.60 (1H, dd, J = 10.8, 5.0 Hz, HA-sn1), 3.56 (1H, dd, J = 10.8, 5.0 Hz, HB-sn1), 3.39 (2H, m, H-1′), 2.05–1.4 (16H, m, H-5′′, 7′′, 8′′, 11′′, 12′′, 13′′ 14′′, 16′′, 17′′), 1.70 (3H, s, Me-21′′), 1.56 (2H, m, H-2′), 1.25 (30H, m, H-3′-17′), 0.90 (3H, s, Me-22′′), 0.89 (3H, s, Me-24′′), 0.88 (3H, t, J = 7.2 Hz, Me-18′), 0.80 (3H, d, J = 7.0 Hz, Me-23′′); 13C-NMR (100 MHz, CDCl3, δ ppm): 154.2 (–O–CO–O–), 147.1 (C-19′′), 143.2 (C-1′′), 141.4 (C-10′′), 140.0 (C-25′′), 126.0 (C-3′′), 119.7 (C-9′′), 109.1 (C-20′′), 108.6 (C-2′′), 76.6 (C-sn2), 71.9 (C-1′), 70.4 (C-4′′), 69.5 (C-sn1), 62.6 (C-sn3), 44.2 (C-5′′), 42.9 (C-15′′), 42.4 (C-11′′), 38.7 (C-14′′), 37.4 (C-16′′), 34.0 (C-6′′), 32.4 (C-17′′), 31.9 (C-16′), 31.0 (C-7′′), 29.6–29.3 (C-2′), 29.6–29.3 (C-4′-15′), 28.8 (C-13′′), 25.9 (C-3′), 22.8 (C-12′′), 22.7 (C-24′′), 22.6 (C-8′′), 22.6 (C-17′), 22.5 (C-21′′), 22.3 (C-22′′), 15.6 (C-23′′), 14.1 (C-18′); EIHRMS: calcd. for C46H78O6 [M + Na]+: 749.5691, found: 749.5706.

Compound 22: [ α ] D 22 +3.5 (c 0.40, CHCl3); IR (film, cm−1): 3477, 2924, 2853, 1742, 1261; 1H-NMR (400 MHz, CDCl3, δ ppm): 7.43 (1H, s, H-25′′), 7.35 (1H, s, H-1′′), 6.40 (1H, s, H-2′′), 5.80 (1H, dd, J = 8.4, 3.4 Hz, H-4′′), 5.36 (1H, t, J = 3.4 Hz, H-9′′), 4.83 (1H, quin, J = 5.0 Hz, H-sn2), 4.65, 4.61 (1H, s, each, H-20′′), 3.79 (2H, m, H-sn3), 3.62 (1H, dd, J = 10.6, 5.0 Hz, HA-sn1), 3.60 (1H, dd, J = 10.6, 5.0 Hz, HB-sn1), 3.44 (2H, m, H-1′), 2.15–1.4 (16H, m, H-5′′, 7′′, 8′′, 11′′, 12′′, 13′′ 14′′, 16′′, 17′′), 1.67 (3H, s, Me-21′′), 1.56 (2H, m, H-2′), 1.25 (30H, m, H-3′-17′), 0.91 (3H, s, Me-22′′), 0.88 (3H, s, Me-24′′), 0.86 (3H, t, J = 6.2 Hz, Me-18′), 0.81 (3H, d, J = 7.0 Hz, Me-23′′); 13C-NMR (100 MHz, CDCl3, δ ppm): 154.2 (–O–CO–O–), 147.0 (C-19′′), 143.2 (C-1′′), 141.0 (C-10′′), 140.2 (C-25′′), 125.9 (C-3′′), 120.0 (C-9′′), 109.0 (C-20′′), 108.7 (C-2′′), 76.9 (C-sn2), 71.9 (C-1′), 70.3 (C-4′′), 69.5 (C-sn1), 62.6 (C-sn3), 44.2 (C-5′′), 42.9 (C-15′′), 42.3 (C-11′′), 38.8 (C-14′′), 37.2 (C-16′′), 34.1 (C-6′′), 32.3 (C-7′′), 32.3 (C-17′′), 31.9 (C-16′), 29.6–29.3 (C-2′), 29.6–29.3 (C-4′-15′), 28.9 (C-13′′), 26.0 (C-3′), 23.2 (C-12′′), 22.8 (C-24′′), 22.6 (C-8′′), 22.6 (C-17′), 22.2 (C-21′′), 22.2 (C-22′′), 15.6 (C-23′′), 14.1 (C-18′); EIHRMS: calcd. for C46H78O6 [M + Na]+: 749.5691, found: 749.5666.

4.8. Preparaion of 1-O-Octadecyl-2-O-[1,25-epoxy-18-nor-ent-isodysidiola-1,3(25),9,19-tetraen-4S-yloxycarbonyl]-3-eicosapentaenoyl-sn-glycerol (5)

To a solution of 21 (10 mg, 0.01 mmol), DMAP (3 mg, 0.02 mmol) and EDAC (3.5 mg, 0.02 mmol) in dry CH2Cl2 (0.14 mL), EPA (4.2 mg, 0.01 mmol) was added under an argon atmosphere. After stirring at rt for 12 h, the reaction mixture was passed through a short silica gel column (CH2Cl2/EtOAc 9:1 as eluent). Then the solvent was removed and the crude oil was purified by column chromatography (Hex/EtOAc 98:2) providing 5 (12 mg, 87%). [ α ] D 22 +7.5 (c 0.20, CHCl3); IR (film, cm−1): 2957, 2926, 2855, 1745, 1462, 1261; 1H-NMR (400 MHz, CDCl3, δ ppm): 7.42 (1H, s, H-25′′), 7.35 (1H, s, H-1′′), 6.39 (1H, s, H-2′′), 5.79 (1H, dd, J = 8.7, 3.1 Hz, H-4′′), 5.41–5.32 (10H, m, =CH), 5.32 (1H, m, H-9′′), 5.01 (1H, m, H-sn2), 4.65, 4.63 (1H, s, each, H-20′′), 4.36 (1H, dd, J = 12.0, 3.4 Hz, HA-sn3), 4.15 (1H, dd, J = 12.0, 6.7 Hz, HB-sn3), 3.51 (2H, d, J = 5.4 Hz, H-sn1), 3.40–3.34 (2H, m, H-1′), 2.85–2.80 (8H, m, =CCH2C=), 2.31 (2H, t, J = 7.3 Hz, H-2′′′), 2.09–2.04 (4H, m, H-4′′′, 19′′′), 2.05–1.40 (16H, m, H-5′′, 7′′, 8′′, 11′′, 12′′, 13′′, 14′′, 16′′, 17′′), 1.76–1.72 (2H, m, H-3′′′), 1.70 (3H, s, Me-21′′), 1.56 (2H, m, H-2′), 1.25 (30H, m, H-3′-17′), 0.97 (3H, t, J = 7.5 Hz, H-20′′′), 0.90 (3H, s, Me-22′′), 0.89 (3H, t, J = 6.8 Hz, Me-18′), 0.88 (3H, s, Me-24′′), 0.80 (3H, d, J = 6.8 Hz, Me-23′′); 13C-NMR (100 MHz, CDCl3, δ ppm): 173.4 (C-1′′′), 154.2 (–O–CO–O–), 147.3 (C-19′′), 143.5 (C-1′′), 141.7 (C-10′′), 140.0 (C-25′′), 132.3 (C-18′′′), 129.1–127.2 (=CH) × 9, 126.2 (C-3′′), 119.9 (C-9′′), 109.4 (C-20′′), 108.9 (C-2′′), 74.3 (C-sn2), 72.1 (C-1′), 70.5 (C-4′′), 68.9 (C-sn1), 63.1 (C-sn3), 44.4 (C-5′′), 43.1 (C-15′′), 42.9 (C-11′′), 39.1 (C-14′′), 37.6 (C-16′′), 34.3 (C-6′′), 33.7 (C-2′′′), 32.7 (C-17′′), 32.1 (C-16′), 31.1 (C-7′′), 29.9–29.3 (C-2′), 29.9–29.3 (C-4′-15′), 29.1 (C-13′′), 28.7–20.8 (3′′′, 4′′′, 7’’’, 10’’’, 13’’’, 16’’’, 19′′′), 26.0 (C-3′), 24.0 (C-24′′), 22.9 (C-12′′), 22.9 (C-21′′), 22.6 (C-8′′), 22.6 (C-17′), 22.5 (C-22′′), 15.9 (C-23′′), 14.5 (C-20′′′), 14.3 (C-18′); EIHRMS: calcd. for C66H106O7 [M + Na]+: 1033.7831, found: 1033.7860.

4.9. Preparation of 1-O-Octadecyl-2-O-[1,25-epoxy-18-nor-ent-isodysidiola-1,3(25),9,19-tetraen-4R-yloxycarbonyl]-3-eicosapentaenoyl-sn-glycerol (6)

To a solution of 22 (12.5 mg, 0.02 mmol), DMAP (3 mg, 0.02 mmol) and EDAC (4 mg, 0.02 mmol) in dry CH2Cl2 (0.2 mL), EPA (5.2 mg, 0.02 mmol) was added under an argon atmosphere. After stirring at rt for 12 h, the reaction mixture was passed through a short silica gel column (CH2Cl2/EtOAc 9:1 as eluent). Then the solvent was removed and the crude was purified by column chromatography (Hex/EtOAc 99:1) providing 6 (14 mg, 82%). [ α ] D 22 +2.4 (c 0.33, CHCl3); IR (film, cm−1): 2959, 2924, 2855, 1744, 1263; 1H-NMR (400 MHz, CDCl3, δ ppm): 7.42 (1H, s, H-25′′), 7.33 (1H, s, H-1′′), 6.39 (1H, s, H-2′′), 5.79 (1H, dd, J = 8.5, 3.0 Hz, H-4′′), 5.40–5.34 (10H, m, =CH), 5.34 (1H, m, H-9′′), 5.04–5.00 (1H, m, H-sn2), 4.65 and 4.61 (1H, s, each, H-20′′), 4.30 (1H, dd, J = 12.1, 3.6 Hz, HA-sn3), 4.15 (1H, dd, J = 12.1, 7.0 Hz, HB-sn3), 3.56 (1H, dd, J = 12.2, 5.4 Hz, HA-sn1), 3.52 (1H, dd, J = 14.2, 5.4 Hz, HB-sn1), 3.40 (2H, m, H-1′), 2.85–2.78 (8H, m, =CCH2C=), 2.21 (2H, t, J = 7.4 Hz, H-2′′′), 2.13–2.04 (4H, m, H-4′′′, 19′′′), 2.05–1.40 (16H, m, H-5′′, 7′′, 8′′, 11′′, 12′′, 13′′, 14′′, 16′′, 17′′), 1.76–1.72 (2H, m, H-3′′′), 1.68 (3H, s, Me-21′′), 1.56 (2H, m, H-2′), 1.25 (30H, m, H-3′-17′), 0.97 (3H, t, J = 7.5 Hz, H-20′′′), 0.90 (3H, t, J = 6.8 Hz, Me-18′), 0.88 (3H, s, Me-22′′), 0.86 (3H, s, Me-24′′), 0.81 (3H, d, J = 7.0 Hz, Me-23′′); 13C-NMR (100 MHz, CDCl3, δ ppm): 173.1 (C-1′′′), 154.2 (–O–CO–O–), 146.9 (C-19′′), 143.1 (C-1′′), 141.0 (C-10′′), 140.1 (C-25′′), 132.0 (C-18′′′), 128.9–127.0 (=CH) × 9, 126.1 (C-3′′), 120.0 (C-9′′), 109.0 (C-20′′), 108.7 (C-2′′), 74.0 (C-sn2), 71.8 (C-1′), 70.3 (C-4′′), 68.8 (C-sn1), 62.7 (C-sn3), 44.3 (C-5′′), 42.9 (C-15′′), 42.3 (C-11′′), 38.8 (C-14′′), 37.6 (C-16′′), 34.1 (C-6′′), 33.3 (C-2′′′), 32.2 (C-17′′), 31.9 (C-16′), 31.2 (C-7′′), 29.6–29.3 (C-2′), 29.6–29.3 (C-4′-15′), 28.8 (C-13′′), 28.4–22.6 (3′′′, 4′′′, 7′′′, 10′′′, 13′′′, 16′′′, 19′′′), 26.0 (C-3′), 23.7 (C-24′′), 23.2 (C-12′′), 22.8 (C-21′′), 22.6 (C-8′′), 22.6 (C-17′), 22.2 (C-22′′), 15.6 (C-23′′), 14.2 (C-20′′′), 14.0 (C-18′); EIHRMS: calcd. for C66H106O7 [M + Na]+: 1033.7831, found: 1033.7854.

4.10. Preparation of 1-O-Octadecyl-2-O-[25-hydroxy-18-nor-ent-isodysidiola-2,9,19-trien-1,25-olide-4S-yloxycarbonyl]-3-eicosapentaenoyl-sn-glycerol (7)

Rose Bengal (1 mg) was added to a solution of 5 (6.4 mg, 6.3 × 10−3 mmol) and DIPEA (11 μL, 0.06 mmol) in dry CH2Cl2 (2 mL) at rt. Anhydrous oxygen was bubbled in for 2 min and after that, the solution was placed under an oxygen atmosphere at −78 °C and irradiated with a 200 W lamp. After 4 h irradiation was stopped, the pink solution was allowed to warm to rt, and saturated aqueous oxalic acid solution (1 mL) added. After a few minutes of vigorous stirring, the mixture was diluted with H2O and extracted with Et2O. The combined organic extracts were washed with H2O and dried over anhydrous Na2SO4. After filtration, the solvent was evaporated to give a residue that was purified by silica gel column chromatography to yield 7 (6 mg, 86%). [ α ] D 22 +2.6 (c 0.2, CHCl3); IR (film, cm−1): 3427, 2924, 1747, 1259; 1H-NMR (400 MHz, CDCl3, δ ppm): 6.19/5.97 (1H, m, H-25′′), 6.01/6.00 (1H, m, H-2′′), 5.60/5.48 (1H, m, H-4′′), 5.45–5.33 (10H, m, =CH), 5.45–5.33 (1H, m, H-9′′), 5.03/4.95 (1H, m, H-sn2), 4.68–4.62 (2H, m, H-20′′), 4.35/4.18 (2H, m, H-sn3), 3.56 (2H, m, H-sn1), 3.43 (2H, m, H-1′), 2.82 (8H, m, =CCH2C=), 2.30 (2H, m, H-2′′′), 2.09 (4H, m, H-4′′′, 19′′′), 2.00–1.53 (18H, m, H-5′′, 7′′, 8′′, 11′′, 12′′, 13′′, 14′′, 16′′, 17′′, 3′′′), 1.69 (3H, s, Me-21′′), 1.54 (2H, m, H-2′), 1.25 (30H, m, H-3′-17′), 0.97 (3H, t, J = 7.6 Hz, H-20′′′), 0.91 (3H, s, Me-22′′), 0.88 (3H, t, J = 6.8 Hz, Me-18′), 0.88 (3H, s, Me-24′′), 0.81 (3H, d, J = 6.8 Hz, Me-23′′); 13C-NMR (50 MHz, CDCl3, δ ppm): 173.7 (C-1′′′), 169.4 (C-1′′), 168.2 (C-3′′), 154.4 (–O–CO–O–), 147.5 (C-19′′), 141.8 (C-10′′), 132.3 (C-18′′′), 129.2–127.2 (=CH) × 9, 120.0 (C-9′′), 117.9 (C-2′′), 109.4 (C-20′′), 97.6 (C-25′′), 74.3 (C-sn2), 72.1 (C-1′), 71.2 (C-4′′), 68.9 (C-sn1), 63.0 (C-sn3), 43.1 (C-5′′), 42.9 (C-15′′), 42.9 (C-11′′), 38.9 (C-14′′), 37.6 (C-16′′), 34.4 (C-6′′), 33.7 (C-2′′′), 32.7 (C-17′′), 32.1 (C-16′), 31.1 (C-7′′), 29.9–29.3 (C-2′), 29.9–29.3 (C-4′-15′), 29.1 (C-13′′), 28.6–20.7 (3′′′, 4′′′, 7′′′, 10′′′, 13′′′, 16′′′, 19′′′), 25.9 (C-3′), 24.0 (C-24′′), 22.9 (C-12′′), 22.9 (C-21′′), 22.6 (C-8′′), 22.6 (C-17′), 22.5 (C-22′′), 15.9 (C-23′′), 14.5 (C-20′′′), 14.3 (C-18′); EIHRMS: calcd. for C66H106O9 [M + Na]+: 1065.7729, found: 1065.7775.

4.11. Preparation of 1-O-Octadecyl-2-O-[25-hydroxy-18-nor-ent-isodysidiola-2,9,19-trien-1,25-olide-4R-yloxycarbonyl]-3-eicosapentaenoyl-sn-glycerol (8)

Rose Bengal (1 mg) was added to a solution of 6 (7.6 mg, 7.5 × 10−3 mmol) and DIPEA (13 μL, 0.075 mmol) in dry CH2Cl2 (2 mL) at rt. Anhydrous oxygen was bubbled in for 2 min, then the solution was placed under an oxygen atmosphere at −78 °C and irradiated with a 200 W lamp. After 4 h irradiation was stopped, the pink solution allowed to warm to rt, and saturated aqueous oxalic acid solution (1 mL) added. After a few minutes of vigorous stirring, the mixture was diluted with H2O and extracted with Et2O. The combined organic extracts were washed with H2O and dried over anhydrous Na2SO4. After filtration, the solvent was evaporated to give a residue which was purified by silica gel column chromatography to yield 8 (7 mg, 90%). [ α ] D 22 −3.0 (c 0.3, CHCl3); ); IR (film, cm−1): 3427, 2924, 1747, 1259; 1H-NMR (400 MHz, CDCl3, δ ppm): 6.13/5.96 (1H, m, H-25′′), 6.06/6.05 (1H, s, H-2′′), 5.69/5.46 (1H, m, H-4′′), 5.40–5.34 (10H, m, =CH), 5.40–5.34 (1H, m, H-9′′), 5.01/4.94 (1H, m, H-sn2), 4.67–4.63 (2H, m, H-20′′), 4.13 (1H, dd, J = 6.4, 12.4 Hz, HA-sn3), 4.01 (1H, dd, J = 4.0, 12.4 Hz, HB-sn3), 3.57 (2H, d, J = 5.2 Hz, H-sn1), 3.47–3.39 (2H, m, H-1′), 2.86–2.80 (8H, m, =CCH2C=), 2.34–2.30 (2H, m, H-2′′′), 2.13–2.04 (4H, m, H-4′′′, 19′′′), 2.00–1.52 (18H, m, H-5′′, 7′′, 8′′, 11′′, 12′′, 13′′, 14′′, 16′′, 17′′, 3′′′), 1.69 (3H, s, Me-21′′), 1.54 (2H, m, H-2′), 1.25 (30H, m, H-3′-17′), 0.97 (3H, t, J = 7.6 Hz, H-20′′′), 0.91 (3H, s, Me-22′′), 0.88 (3H, t, J = 6.8 Hz, Me-18′), 0.88 (3H, s, Me-24′′), 0.81 (3H, d, J = 6.8 Hz, Me-23′′); 13C-NMR (100 MHz, CDCl3, δ ppm): 173.4 (C-1′′′), 169.3 (C-1′′), 168.1 (C-3′′), 154.4 (–O–CO–O–), 147.1 (C-19′′), 141.1 (C-10′′), 132.0 (C-18′′′), 128.8–127.0 (=CH) × 9, 120.0 (C-9′′), 118.0 (C-2′′), 109.0 (C-20′′), 97.5 (C-25′′), 74.0 (C-sn2), 71.8 (C-1′), 71.1 (C-4′′), 68.8 (C-sn1), 62.6 (C-sn3), 42.9 (C-5′′), 42.9 (C-15′′), 42.3 (C-11′′), 38.8 (C-14′′), 37.6 (C-16′′), 34.1 (C-6′′), 33.3 (C-2′′′), 32.2 (C-17′′), 31.9 (C-16′), 31.2 (C-7′′), 29.7–29.3 (C-2′), 29.7–29.3 (C-4′-15′), 28.8 (C-13′′), 28.3–22.6 (3′′′, 4′′′, 7′′′, 10′′′, 13′′′, 16′′′, 19′′′), 26.0 (C-3′), 23.7 (C-24′′), 23.2 (C-12′′), 22.8 (C-21′′), 22.6 (C-8′′), 22.6 (C-17′), 22.2 (C-22′′), 15.7 (C-23′′), 14.2 (C-20′′′), 14.0 (C-18′); EIHRMS: calcd. for C66H106O9 [M + Na]+: 1065.7729, found: 1065.7766.

4.12. Preparation of 1,3-Benzyliden-2-O-octadecylglycerol (24)

To a solution of 1,3-O-benzylidene glycerol 23 (3.7 g, 20.5 mmol) in toluene (21 mL), NaNH2 (800 mg, 20.5 mmol) was added and heated at 111 °C under an argon atmosphere for 1 h. The mixture was cooled to rt and a solution of bromooctadecane (6.8 g, 20.5 mmol) in toluene (20 mL) was added, then it was heated at 111 °C for 4 h. The reaction was cooled at 0 °C, crushed ice and saturated NH4Cl were added and then it was extracted with Et2O. The organic layer was washed with H2O and brine, dried over anhydrous Na2SO4, filtered and evaporated to yield 24 (8.7 g, 98%). IR (film, cm−1): 2916, 2849, 1471, 1385, 1152, 1103, 1010, 743, 695; 1H-NMR (200 MHz, CDCl3, δ ppm): 7.54–7.49 (2H, m, H-2′, 6′), 7.40–7.32 (3H, m, H-3′, 4′, 5′), 5.55 (1H, s, O–CH–O), 4.33 (2H, d, J = 12.6 Hz, HA-1, HA-3), 4.15 (1H, m, H-2), 4.06 (2H, d, J = 12.6 Hz, HB-1, HB-3), 3.55 (2H, t, J = 6.8 Hz, H-1′′), 1.65 (2H, m, H-2′′), 1.26 (30H, m, H-3′′-17′′), 0.88 (3H, t, J = 6.4 Hz, H-18′′); 13C-NMR (50 MHz, CDCl3, δ ppm): 138.5 (C-1′), 129.0 (C-4′), 128.5 (C-2′, 6′), 126.5 (C-3′, 5′), 101.5 (O–CH–O), 72.5 (C-1′′), 70.9 (C-2), 69.2 (C-1, 3), 32.2 (C-16′′), 30.1–29.2 (C-2′′, 4′′-15′′), 26.4 (C-3′′), 23.0 (C-17′′), 14.4 (C-18′′). EIHRMS: calcd. for C28H48O3 [M + Na]+: 455.3496, found: 455.3514.

4.13. Preparation of 2-O-Octadecylglycerol (25)

To a solution of 24 (8.7 g, 20 mmol) in MeOH (40 mL), p-TsOH (3.8 g, 20 mmol) was added and it was stirred at 35–40 °C for 6 h. Then H2O was added, the mixture was extracted with Et2O and washed with 6% NaHCO3 and H2O. The organic layer was dried over Na2SO4, filtered and evaporated. The residue was purified by column chromatography (EtOAc) to yield 25 (6.2 g, 90%). IR (film, cm−1): 3326, 2918, 2850, 1468, 1114, 1078, 1058, 975, 718; 1H-NMR (400 MHz, CDCl3, δ ppm): 3.78–3.66 (4H, m, H-1, H-3), 3.57 (2H, t, J = 6.7 Hz, H-1′′), 3.46 (1H, quin, J = 4.6 Hz, H-2), 1.59 (2H, m, H-2′′), 1.25 (30H, m, H-3′′-17′′), 0.88 (3H, t, J = 6.6 Hz, H-18′′); 13C-NMR (50 MHz, CDCl3, δ ppm): 79.9 (C-2), 70.4 (C-1′′), 62.1 (C-1), 62.1 (C-3), 32.1 (C-16′′), 30.2 (C-2′′), 29.7 (C-4′′-15′′), 26.3 (C-3′′), 22.9 (C-17′′), 14.3 (C-18′′). EIHRMS: calcd. for C21H44O3 [M + Na]+: 367.3183, found: 367.3187.

4.14. Reaction of 25 with TBDMSCl: Preparation of 1-O-tert-Butyldimethylsilyl-2-O-octadecyl-glycerol (26) and 1,3-O-di-tert-Butyldimethylsilyl-2-O-octadecylglycerol (27):

To an ice-cooled solution of 25 (3.4 g, 9.9 mmol) in DMF (99 mL), TBDMSCl (1.49 g, 9.9 mmol) and imidazole (673 mg, 9.9 mmol) were added. It was stirred overnight at rt under an argon atmosphere; the reaction mixture was cooled at 0 °C and quenched with H2O. It was extracted with Et2O and the organic layer washed with H2O. After drying over anhydrous Na2SO4 the solvent was evaporated. The crude purified by column chromatography (Hex/EtOAc 97:3) to give 26 (1.85 g, 41%); 27 (910 mg, 16%) and 25 (1.43 g, 42%).

Compound 26: IR (film, cm−1): 3450, 2925, 2854, 1475, 1100, 837; 1H-NMR (400 MHz, CDCl3, δ ppm): 3.73 (1H, dd, J = 10.2, 4.0 Hz, HA-1), 3.72 (1H, dd, J = 12.0, 5.2 Hz, HA-3), 3.62 (1H, dd, J = 12.0, 5.2 Hz, HB-3), 3.61 (1H, dd, J = 10.2, 6.8 Hz, HB-1), 3.58 (1H, ddd, J = 9.2, 6.8, 6.8 Hz, HA-1′′), 3.52 (1H, ddd, J = 9.2, 6.8, 6.8 Hz, HB-1′′), 3.42 (1H, dddd, J = 6.8, 5.2, 5.2, 4.0 Hz, H-2), 2.15 (1H, m,–OH), 1.55 (2H, m, H-2′′), 1.25 (30H, m, H-3′′-17′′), 0.89 (9H, s, Me3CSi-), 0.89 (3H, t, J = 6.8 Hz, H-18′′), 0.06 (6H, s, Me2Si-); 13C-NMR (100 MHz, CDCl3, δ ppm):80.1 (C-2), 70.7 (C-1′′), 63.2 (C-3), 62.9 (C-1), 32.1 (C-16′′), 30.3 (C-2′′), 29.7 (C-4′′-15′′), 26.3 (C-3′′), 26.0 (Me3CSi-), 22.9 (C-17′′), 18.4 (Me3CSi-), 14.3 (C-18′′), −5.3 (Me2Si-). EIHRMS: calcd. for C27H58O3Si [M + Na]+: 481.4047, found: 481.4025.

Compound 27: IR (film, cm−1): 2926, 2855, 1475, 1257, 1106, 836; 1H-NMR (200 MHz, CDCl3, δ ppm): 3.72–3.53 (4H, m, H-1, H-3), 3.56 (2H, t, J = 6.6 Hz, H-1′′), 3.34 (1H, quin, J = 5.4 Hz, H-2), 1.55 (2H, m, H-2′′), 1.26 (30H, m, H-3′′-17′′), 0.89 (2·9H, s, Me3CSi-), 0.89 (3H, t, J = 6.8 Hz, H-18′′), 0.05 (2·6H, s, Me2Si-); 13C-NMR (50 MHz, CDCl3, δ ppm): 81.4 (C-2), 70.9 (C-1′′), 63.1 (C-3), 63.1 (C-1), 32.2 (C-16′′), 30.4 (C-2′′), 29.9–29.6 (C-4′′-15′′), 26.4 (C-3′′), 26.1 (2·Me3CSi-), 22.9 (C-17′′), 18.5 (2·Me3CSi-), 14.3 (C-18′′), −5.2 (2·Me2Si-). EIHRMS: calcd. for C33H72O3Si2 [M + Na]+: 595.4912, found: 595.4927.

4.15. Preparation of 1-Chlorocarbonyl-2-O-octadecyl-3-O-tert-butyldimethylsilylglycerol (28)

To an ice cooled solution of 26 (500 mg, 1 mmol) in THF (2 mL), trichloromethyl chloroformate (diphosgene, 0.12 mL, 1 mmol) and N,N-dimethylaniline (0.13 mL, 1 mmol) were slowly added. The mixture was stirred at 0 °C for 10 min and then at rt for 4 h. Then Et2O was added and the solution washed with 0.2 M HCl, 0.2 M NaOH and H2O, dried over anhydrous Na2SO4 and evaporated. The reaction bulk was purified by column chromatography to separate 28 (370 mg, 71%). IR (film, cm−1): 2925, 2854, 1781, 1462, 1254, 1164, 838, 780; 1H-NMR (200 MHz, CDCl3, δ ppm): 4.49 (1H, dd, J = 11.4, 3.4 Hz, HA-1), 4.33 (1H, dd, J = 11.4, 5.0 Hz, HB-1), 3.83–3.60 (2H, m, H-3), 3.60–3.50 (1H, m, H-2), 3.54 (2H, t, J = 6.6 Hz, H-1′′), 1.55 (2H, m, H-2′′), 1.26 (30H, m, H-3′′-17′′), 0.89 (9H, s, Me3CSi-), 0.88 (3H, t, J = 6.8 Hz, H-18′′), 0.06 (6H, s, Me2Si-); 13C-NMR (50 MHz, CDCl3, δ ppm): 150.9 (O–CO–Cl), 77.6 (C-2), 71.2 (C-1′′), 71.0 (C-1), 61.9 (C-3), 32.2 (C-16′′), 30.3 (C-2′′), 29.9–29.6 (C-4′′-15′′), 26.2 (C-3′′), 26.0 (Me3CSi-), 22.9 (C-17′′), 18.4 (Me3CSi-), 14.3 (C-18′′), −5.3 (Me2Si-). EIHRMS: in MeOH, calcd. for the methyl ester, C29H60O5Si [M + Na]+: 539.4102, found: 539.4122.

4.16. Preparation of 1-O-[1,25-Epoxy-18-nor-ent-isodysidiola-1,3(25),9,19-tetraen-4R/S-yloxycarbonyl]-2-O-octadecyl-3-O-tert-butyldimethylsilylglycerol (29)

To a solution of 1/2 (324 mg, 0.91 mmol), N,N-diisopropylethylamine (DIPEA, 0.22 mL, 1.27 mmol), 4-(dimethylamino) pyridine (DMAP, 56 mg, 0.46 mmol) in toluene (4.6 mL), a solution of 28 (364 mg, 0.7 mmol) in toluene (3.5 mL) was added dropwise at 0 °C . The reaction mixture was stirred at 0 °C under an argon atmosphere for 15 min and then at rt overnight. After this time the solvent was removed and the residue was purified by column chromatography (Hex/Et2O 99.9:0.1) to obtain 29 (330 mg, 56%). IR (film, cm−1): 2926, 2855, 1745, 1464, 1256, 1107, 837, 777; 1H-NMR (400 MHz, CDCl3, δ ppm): 7.43/7.42 (1H, s, H-25′), 7.33 (1H, s, H-1′), 6.39 (1H, s, H-2′), 5.79–5.76 (1H, m, H-4′), 5.35–5.32 (1H, m, H-9′), 4.66–4.60 (2H, m, H-20′), 4.32–4.06 (2H, m, H-1), 3.64–3.57 (2H, m, H-3), 3.54–3.49 (1H, m, H-2), 3.54–3.49 (2H, m, H-1′′), 2.20–1.40 (16H, m, H-5′, 7′, 8′, 11′, 12′, 13′, 14′, 16′, 17′), 1.70/1.66 (3H, s, H-21′), 1.54 (2H, m, H-2′′), 1.25 (30H, m, H-3′′-17′′), 0.89 (3H, s, H-22′), 0.89 (3H, s, H-24′), 0.88 (3H, t, J = 6.8 Hz, H-18′′), 0.87 (9H, s, Me3CSi-), 0.81 (3H, d, J = 6.9 Hz, H-23′), 0.04 (6H, s, Me2Si-); 13C-NMR (50 MHz, CDCl3, δ ppm): 154.6/154.5 (O–CO–O), 147.0 (C-19′), 143.2/143.1 (C-1′), 142.6 (C-10′), 140.1 (C-25′), 126.1 (C-3′), 120.0/119.7 (C-9′), 109.1/109.0 (C-20′), 108.7/108.6 (C-2′), 78.0 (C-2), 70.7 (C-1′′), 70.0/69.9 (C-4′), 67.1/66.8 (C-1), 62.2 (C-3), 44.2 (C-5′), 42.9 (C-15′), 42.4 (C-11′), 38.8 (C-14′), 37.4 (C-16′), 34.1/34.0 (C-6′), 32.4/32.3 (C-17′), 31.9 (C-16′′), 30.0–29.3 (C-2′′, 4′′-15′′, 7′, 13′), 26.0 (C-3′′), 25.8 (Me3CSi-), 22.8 (C-12′), 22.6 (C-8′), 22.6 (C-17′′), 22.4 (C-24′), 22.2 (C-21′), 22.2 (C-22′), 18.2 (Me3CSi-), 15.6 (C-23′), 14.1 (C-18′′), −5.2 (Me2Si-). EIHRMS: calcd. for C52H92O6Si [M + Na]+: 863.6555, found: 863.6542.

4.17. Preparation of 1-O-[1,25-Epoxy-18-nor-ent-isodysidiola-1,3(25),9,19-tetraen-4R/S-yloxycarbonyl]-2-O-octadecylglycerol (30)

To a solution of 29 (126 mg, 0.15 mmol) in THF (1.7 mL), 1 M TBAF in THF (0.22 mL, 0.22 mmol) was added under an argon atmosphere. The mixture was stirred for 2 h at rt, then the reaction was quenched with H2O and extracted with EtOAc. The organic layer was washed with H2O and brine, dried over anhydrous Na2SO4, filtered and removed the solvent. The residue was purified by column chromatography (EtOAc) providing 30 (97 mg, 89%). IR (film, cm−1): 3335, 2922, 2853, 1744, 1466, 1258, 1078, 1059, 1024; 1H-NMR (400 MHz, CDCl3, δ ppm): 7.42 (1H, s, H-25′), 7.34 (1H, s, H-1′), 6.39 (1H, s, H-2′), 5.78 (1H, m, H-4′), 5.35–5.32 (1H, m, H-9′), 4.66–4.61 (2H, m, H-20′), 4.22–4.14 (2H, m, H-1), 3.75–3.67 (2H, m, H-3), 3.56 (2H, t, J = 6.4 Hz, H-1′′), 3.49–3.43 (1H, m, H-2), 2.20–1.40 (16H, m, H-5′, 7′, 8′, 11′, 12′, 13′, 14′, 16′, 17′), 1.69/1.66 (3H, s, H-21′), 1.55 (2H, m, H-2′′), 1.25 (30H, m, H-3′′-17′′), 0.89 (3H, s, H-22′), 0.88 (3H, t, J = 6.8 Hz, H-18′′), 0.87 (3H, s, H-24′), 0.80/0.79 (3H, d, J = 6.8 Hz, H-23′); 13C-NMR (50 MHz, CDCl3, δ ppm): 154.8 (O–CO–O), 147.3 (C-19′), 143.5 (C-1′), 141.6/141.3 (C-10′), 140.4 (C-25′), 126.2 (C-3′), 120.2/120.0 (C-9′), 109.4/109.3 (C-20′), 108.9/108.8 (C-2′), 79.8 (C-2), 70.9/70.4 (C-1′′), 70.5 (C-4′), 66.5/66.4 (C-1), 62.4/62.1 (C-3), 44.4 (C-5′), 43.1 (C-15′), 42.7/42.6 (C-11′), 39.1 (C-14′), 37.6/37.4 (C-16′), 34.4/34.3 (C-6′), 32.7/32.6 (C-17′), 32.1 (C-16′′), 31.5 (C-7′), 30.3–29.6 (C-2′′, 4′′-15′′), 29.1 (C-13′), 26.3/26.2 (C-3′′), 23.4 (C-24′), 23.1 (C-21′), 22.9 (C-8′), 22.9 (C-12′), 22.9 (C-17′′), 22.5 (C-22′), 15.9 (C-23′), 14.3 (C-18′′). EIHRMS: calcd. for C46H78O6 [M + Na]+: 749.5691, found: 749.5665.

4.18. Preparation of 1-O-[1,25-Epoxy-18-nor-ent-isodysidiola-1,3(25),9,19-tetraen-4R/S-yloxycarbonyl]-2-O-octadecyl-glycero-3-phosphate (31)

To a solution of 30 (37 mg, 0.05 mmol) and anhydrous pyridine (8 μL) in THF (0.3 mL), POCl3 (5 μL, 0.06 mmol) was added dropwise under an argon atmosphere with stirring at 0 °C for 5 h. Then 6% NaHCO3 was added and the mixture stirred for an additional 15 min. at 0 °C. After that time, crushed ice was added, the mixture was acidified with 2 M HCl to pH = 2 and extracted with EtOAc. The organic layer was washed with H2O and the solvent removed to give 31 (39 mg, 97%). IR (film, cm−1): 2924, 2853, 1744, 1466, 1258 1H-NMR (200 MHz, CDCl3, δ ppm): 7.39 (1H, broad s, H-25′), 7.3 (1H, broad s, H-1′), 6.37 (1H, broad s, H-2′), 5.76 (1H, m, H-4′), 5.33 (1H, m, H-9′), 4.64–4.61 (2H, m, H-20′), 4.34–3.83 (4H, m, H-1, H-3), 3.64–3.31 (3H, m, , H-1′′, H-2), 2.10–1.40 (16H, m, H-5′, 7′, 8′, 11′, 12′, 13′, 14′, 16′, 17′), 1.69/1.65 (3H, s, H-21′), 1.55 (2H, m, H-2′′), 1.25 (30H, m, H-3′′-17′′), 0.89 (3H, s, H-22′), 0.88 (3H, t, J = 6.8 Hz, H-18′′), 0.87 (3H, s, H-24′), 0.80/0.79 (3H, d, J = 6.8 Hz, H-23′).

4.19. Preparation of 1-O-[1,25-Epoxy-18-nor-ent-isodysidiola-1,3(25),9,19-tetraen-4R/S-yloxycarbonyl]-2-O-octadecyl-glycero-3-phosphocoline (9)

Compound 31 (39 mg, 0.05 mmol), choline tetraphenyl borate (21 mg, 0.05 mmol) and TPS (18 mg, 0.06 mmol) were dissolved in anhydrous pyridine (0.4 mL). The mixture was heated at 70 °C for 1 h and then it was stirred at rt overnight. After addition of H2O (0.1 mL), the solvents were removed by rotary evaporation. The crude mixture was dissolved in Et2O and stirred for a few minutes. The solid formed was eliminated by filtration. The organic solution was evaporated and the reaction bulk purified by column chromatography (CHCl3/MeOH/NH4OH 65:30:5) to yield 9 (16 mg, 35%). IR (film, cm−1): 2920, 2851, 1738, 1467; 1H-NMR (400 MHz, CDCl3, δ ppm):7.42 (1H, s, H-25′), 7.37 (1H, s, H-1′), 6.39 (1H, s, H-2′), 5.75 (1H, m, H-4′), 5.36–5.34 (1H, m, H-9′), 4.66–4.61 (2H, m, H-20′), 4.37 (2H, m, H-1′′′), 4.33–4.10 (2H, m, H-1), 4.00–3.80 (2H, m, H-3), 3.98–3.75 (2H, m, H-2′′′), 3.65 (1H, m, H-2), 3.51 (2H, m, H-1′′), 3.35 (9H, s, Me3N–), 2.05–1.40 (16H, m, H-5′, 7′, 8′, 11′, 12′, 13′, 14′, 16′, 17′), 1.70/1.67 (3H, s, H-21′), 1.54 (2H, m, H-2′′), 1.26 (30H, m, H-3′′-17′′), 0.90 (3H, s, H-22′), 0.88 (3H, t, J = 6.8 Hz, H-18′′), 0.87 (3H, s, H-24′), 0.82/0.81 (3H, d, J = 6.9 Hz, H-23′); 13C-NMR (100 MHz, CDCl3, δ ppm): 154.5 (O–CO–O), 146.9 (C-19′), 143.3 (C-1′), 141.4/141.0 (C-10′), 140.0 (C-25′), 126.2 (C-3′), 120.0/119.7 (C-9′), 109.1/109.0 (C-20′), 108.7 (C-2′), 76.2 (C-2), 70.8/70.6 (C-1′′), 70.2 (C-4′), 66.9 (C-1), 66.2 (C-2′′′), 64.4 (C-3), 59.7 (C-1′′′), 54.4 (Me3N-), 44.3 (C-5′), 42.9 (C-15′), 42.6/42.5 (C-11′), 38.8/38.7 (C-14′), 37.3/37.2 (C-16′), 34.1 (C-6′), 32.4/32.3 (C-17′), 31.9 (C-16′′), 31.2 (C-7′), 29.9–29.3 (C-2′′, 4′′-15′′), 28.9/28.8 (C-13′), 25.9 (C-3′′), 23.2 (C-12′), 22.8 (C-24′), 22.7 (C-21′), 22.6 (C-8′), 22.6 (C-17′′), 22.2 (C-22′), 15.6 (C-23′), 14.0 (C-18′′). EIHRMS: calcd. for C51H90NO9P [M + Na]+: 914.6245, found: 914.6229.

4.20. Preparation of 1-O-[25-Hydroxy-18-nor-ent-isodysidiola-2,9,19-trien-1,25-olide-4R/S-yloxycarbonyl]-2-O-octadecyl-glycero-3-phosphocoline (10)

Rose Bengal (4 mg) was added to a solution of 9 (20 mg, 0.02 mmol) and DIPEA (38 μL, 0.22 mmol) in dry CH2Cl2 (10 mL) at rt. Anhydrous oxygen was bubbled in for 10 min and the solution was placed under an oxygen atmosphere at −78 °C and irradiated with a 200 W lamp. After 4 h irradiation was stopped, the pink solution allowed to warm to rt, and saturated aqueous oxalic acid solution (1.7 mL) added. After 30 min of vigorous stirring, the mixture was diluted with H2O and extracted with Et2O. The combined organic extracts were washed with H2O and brine. The solvent was evaporated to give a residue which was purified by silica gel column chromatography (CHCl3/MeOH/H2O 65:35:1) to yield 10 (6 mg, 30%). 1H-NMR (200 MHz, CDCl3, δ ppm): 6.28/6.10 (1H, s, H-25′), 5.91/5.83 (1H, m, H-2′), 5.65 (1H, m, H-4′), 5.36 (1H, m, H-9′), 4.63 (2H, m, H-20′), 4.33 (2H, m, H-1′′′), 4.32–4.10 (2H, m, H-1), 4.05–3.95 (2H, m, H-3), 4.00–3.80 (2H, m, H-2′′′), 3.65–3.55 (1H, m, H-2), 3.51 (2H, m, H-1′′), 3.34 (9H, s, Me3N–), 2.05–1.40 (16H, m, H-5′, 7′, 8′, 11′, 12′, 13′, 14′, 16′, 17′), 1.69 (3H, s, H-21′), 1.54 (2H, m, H-2′′), 1.25 (30H, m, H-3′′-17′′), 0.87 (6H, s, H-22′, 24′), 0.88 (3H, t, J = 7.0 Hz, H-18′′), 0.81 (3H, d, J = 6.9 Hz, H-23′); EIMS found for C51H90 NO11P [M + Na]+: 946.7.

4.21. Preparation of 1-O-[1,25-Epoxy-18-nor-ent-isodysidiola-1,3(25),9,19-tetraen-4R/S-yloxycarbonyl]-2-O-octadecyl-3-eicosa-pentaenoylglycerol (32)

To a solution of 30 (23 mg, 0.03 mmol), DMAP (5 mg, 0.04 mmol) and EDAC (8 mg, 0.04 mmol) in dry CH2Cl2 (0.3 mL), EPA (9.6 μL, 0.03 mmol) was added under an argon atmosphere. After stirring at rt for 13 h, the reaction mixture was passed through a short silica gel column (CH2Cl2/EtOAc 9:1 as eluent). Then the solvent was removed and the crude was purified by column chromatography (Hex/EtOAc 98:2) providing 32 (13 mg, 64%). IR (film, cm−1): 2959, 2926, 1740, 1560, 1383, 1261; 1H-NMR (400 MHz, CDCl3, δ ppm): 7.43/7.42 (1H, s, H-25′), 7.35/7.34 (1H, s, H-1′), 6.39 (1H, s, H-2′), 5.77 (1H, m, H-4′), 5.42–5.30 (10H, m, =CH), 5.42–5.30 (1H, m, H-9′), 4.64–4.61 (2H, m, H-20′), 4.21–4.07 (2H, m, H-1), 4.21–4.07 (2H, m, H-3), 3.67 (1H, m, H-2), 3.57–3.49 (2H, m, H-1′′), 2.84–2.80 (8H, m, =CCH2C=), 2.35–2.30 (2H, m, H-2′′′), 2.14–2.04 (4H, m, H-4′′′, H-19′′′), 2.00–1.40 (16H, m, H-5′, 7′, 8′, 11′, 12′, 13′, 14′, 16′, 17′), 1.74 (2H, m, H-3′′′), 1.70/1.67 (3H, s, H-21′), 1.54 (2H, m, H-2′′), 1.25 (30H, m, H-3′′-17′′), 0.97 (3H, t, J = 7.5 Hz, H-20′′′), 0.90 (3H, s, H-22′), 0.88 (3H, t, J = 7.0 Hz, H-18′′), 0.88 (3H, s, H-24′), 0.81/0.80 (3H, d, J = 6.9 Hz, H-23′); 13C-NMR (100 MHz, CDCl3, δ ppm): 173.1 (C-1′′′), 154.4 (O–CO–O), 147.0 (C-19′), 143.2 (C-1′), 141.0 (C-10′), 140.1 (C-25′), 132.0 (C-18′′′), 128.8–127.0 (=CH) × 9, 126.0 (C-3′), 120.0 (C-9′), 109.1/109.0 (C-20′), 108.7/108.6 (C-2′), 75.1/75.0 (C-2), 70.8/70.7 (C-1′′), 70.2 (C-4′), 66.7/66.4 (C-1), 63.0/62.8 (C-3), 44.2 (C-5′), 42.9 (C-15′), 42.5/42.3 (C-11′), 38.8 (C-14′), 37.3/37.2 (C-16′), 34.1/34.0 (C-6′), 33.5 (C-2′′′), 32.4/32.3 (C-17′), 31.9 (C-16′′), 31.2 (C-7′), 29.8–28.8 (C-2′′, 4′′-15′′), 29.9–29.8 (C-13′), 26.5–20.5 (3′′′, 4′′′, 7′′′, 10′′′, 13′′′, 16′′′, 19′′′), 25.9 (C-3′′), 22.8 (C-12′), 22.6 (C-8′), 22.6 (C-17′′), 22.3 (C-24′), 22.2 (C-21′), 22.2 (C-22′), 15.6 (C-23′), 14.2 (C-20′′′), 14.0 (C-18′′); EIHRMS: calcd. for C66H106O7 [M + Na]+: 1033.7831, found: 1033.7865.

4.22. Preparation of 1-O-[25-Hydroxy-18-nor-ent-isodysidiola-2,9,19-trien-1,25-olide-4R/S-yloxycarbonyl]-2-O-octadecyl-3-eicosapentaenoylglycerol (11)

Rose Bengal (1 mg) was added to a solution of 32 (9 mg, 0.009 mmol) and DIPEA (16 μL, 0.09 mmol) in dry CH2Cl2 (0.7 mL) at rt. Anhydrous oxygen was bubbled in for 10 min, the solution placed under oxygen atmosphere at −78 °C and irradiated with a 200 W lamp. After 4 h irradiation was stopped, the pink solution allowed to warm to rt, and saturated aqueous oxalic acid solution (0.7 mL) added. After 30 min of vigorous stirring, the mixture was diluted with H2O and extracted with CH2Cl2. The combined organic extracts were washed with H2O and brine, and dried over anhydrous Na2SO4. The solvent was evaporated to give a residue that was purified by silica gel column chromatography (Hex/EtOAc 9:1) to yield 11 (5 mg, 53%). IR (film, cm−1): 3387, 2924, 2855, 1747, 1454, 1258, 1134; 1H-NMR (400 MHz, CDCl3, δ ppm): 6.21/5.96 (1H, m, H-25′), 6.03/6.02 (1H, s, H-2′), 5.63 (1H, m, H-4′), 5.38–5.36 (10H, m, =CH), 5.38–5.36 (1H, m, H-9′), 4.67–4.61 (2H, m, H-20′), 4.33–4.06 (2H, m, H-1), 4.33–4.06 (2H, m, H-3), 3.70–3.67 (1H, m, H-2), 3.52 (2H, t, J = 6.7 Hz, H-1′′), 2.84–2.78 (8H, m, =CCH2C=), 2.34 (2H, t, J = 7.5 Hz, H-2′′′), 2.14–2.04 (4H, m, H-4′′′, H-19′′′), 2.00–1.40 (16H, m, H-5′, 7′, 8′, 11′, 12′, 13′, 14′, 16′, 17′), 1.74 (2H, m, H-3′′′), 1.70/1.69 (3H, s, H-21′), 1.54 (2H, m, H-2′′), 1.25 (30H, m, H-3′′-17′′), 0.97 (3H, t, J = 7.5 Hz, H-20′′′), 0.91 (3H, s, H-22′), 0.87 (3H, t, J = 6.8 Hz, H-18′′), 0.88 (3H, s, H-24′), 0.81 (3H, d, J = 6.9 Hz, H-23′); 13C-NMR (100 MHz, CDCl3, δ ppm): 173.4 (C-1′′′), 169.3 (C-1′), 168.2 (C-3′), 154.2 (O–CO–O), 147.2 (C-19′), 141.0 (C-10′), 132.0 (C-18′′′), 129.0–127.0 (=CH) × 9, 119.4 (C-9′), 118.4 (C-2′), 109.3/109.2 (C-20′), 97.4 (C-25′), 75.0 (C-2), 70.9 (C-1′′), 70.8 (C-4′), 66.9 (C-1), 62.3 (C-3), 43.1 (C-5′), 42.8 (C-15′), 42.6 (C-11′), 38.7 (C-14′), 37.4 (C-16′), 34.6 (C-6′), 33.5 (C-2′′′), 32.4 (C-17′), 31.9 (C-16′′), 29.6–29.4 (C-2′′, 4′′-15′′), 28.9 (C-13′), 29.3 (C-7′), 26.5–20.5 (3′′′, 4′′′, 7′′′, 10′′′, 13′′′, 16′′′, 19′′′), 25.9 (C-3′′), 22.9 (C-12′), 22.9 (C-8′), 22.8 (C-24′), 22.7 (C-21′),22.6 (C-17′′), 22.5 (C-22′), 15.6 (C-23′), 14.2 (C-20′′′), 14.1 (C-18′′). EIMS found for C66H106O9 [M + Na]+: 1065.7.

4.23. Preparation of 3-O-p-Methoxybenzyl-sn-glycerol (35)

To an ice cooled solution of (S)-(+)-solketal 33 (2.5 g, 18.9 mmol) in THF (94 mL), 60% NaH (756 mg, 31.5 mmol) and PMBCl (2.56 mL, 18.9 mmol) were added. The mixture was stirred at 0 °C for 10 min and at rt for 1 h. Then it was refluxed overnight, cooled to rt, and crushed ice and saturated NH4Cl added. The aqueous layer was extracted with EtOAc and the organic layer was washed with H2O and brine, dried over anhydrous Na2SO4, filtered and evaporated. The bulk reaction mixture was purified by column chromatography on silica gel (EtOAc) to obtain 35 (3.6 g, 90%). [ α ] D 22 −2.43 (c 0.7, CHCl3); IR (film, cm−1): 3395, 2934, 2866, 1612, 1514, 1248, 1082, 1034; 1H-NMR (400 MHz, CDCl3, δ ppm): 7.25 (2H, d, J = 7.2 Hz, H-2′, H-6′), 6.89 (2H, d, J = 8.6 Hz, H-3′, H-5′), 4.48 (2H, s, –OCH2Ar), 3.88 (1H, m, H-sn2), 3.81 (3H, s, –OCH3), 3.70 (1H, dd, J = 11.4, 3.9 Hz, HA-sn1), 3.63 (1H, dd, J = 11.4, 5.3 Hz, HB-sn1), 3.56 (1H, dd, J = 9.6, 3.9 Hz, HA-sn3), 3.52 (1H, dd, J = 9.6, 6.2 Hz, HB-sn3); 13C-NMR (50 MHz, CDCl3, δ ppm): 159.6 (C-4′′′), 130.0 (C-1′′′), 129.7 (C-2′′′, C-6′′′), 114.1 (C-3′′′, C-5′′′), 73.4 (–OCH2Ar), 71.6 (C-sn3), 71.0 (C-sn2), 64.2 (C-sn1), 55.5 (–OCH3). EIHRMS: calcd. for C11H16O4 [M + Na]+: 235.0941, found: 235.0946.

4.24. Preparation of 3-O-p-Methoxybenzyl-1-O-trityl-sn-glycerol (36)

To a solution of 35 (2.4 g, 11 mmol) in pyridine (23 mL), TrCl (3.1 g, 11 mmol) was added and the mixture was heated to boiling for 15 h. The reaction mixture was allowed to cool to rt and H2O was added, then it was extracted with EtOAc and washed with 2 M HCl, 6% NaHCO3 and brine, dried over anhydrous Na2SO4 and filtered. The removal of the solvent led to a crude which was purified by column chromatography (Hex/EtOAc 9:1) to obtain 36 (4.6 g, 92%). [ α ] D 22 −0.7 (c 0.8, CHCl3); IR (film, cm−1) 3449, 2932, 2870, 1512, 1491, 1448, 1248, 1076, 1034, 765, 706, 633; 1H-NMR (400 MHz, CDCl3) δ 7.46–7.42 (6H, m, H-2′, 6′), 7.32–7.24 (9H, m, H-3′-5′), 7.22 (2H, d, J = 8.6 Hz, H-2′′′, H-6′′′), 6.87 (2H, d, J = 8.6 Hz, H-3′′′, H-5′′′), 4.48 (2H, s, –OCH2Ar), 3.99 (1H, m, H-sn2), 3.81 (3H, s, –OCH3), 3.59 (1H, dd, J = 9.7, 4.3 Hz, HA-sn3), 3.54 (1H, dd, J = 9.7, 6.2 Hz, HB-sn3) 3.25 (1H, dd, J = 9.4, 5.7 Hz, HA-sn1), 3.21 (1H, dd, J = 9.4, 5.7 Hz, HB-sn1); 13C-NMR (100 MHz, CDCl3) δ 159.2 (C-4′′′), 143.8 (C-1′), 130.1 (C-1′′′), 129.3 (C-2′′′, C-6′′′), 128.6 (C-3′, C-5′), 127.8 (C-2′, C-6′), 127.0 (C-4′), 113.8 (C-3′′′, C-5′′′), 86.6 (–CPh3), 73.0 (–OCH2Ar), 71.2 (C-sn3), 69.9 (C-sn2), 64.4 (C-sn1), 55.2 (–OCH3). EIHRMS: calcd. for C30H30O4 [M + Na]+ 477.2036, found [M + Na]+ 477.2022.

4.25. Preparation of 3-O-p-Methoxybenzyl-2-O-octadecyl-1-O-trityl-sn-glycerol (37)

To a solution of 36 (570 mg, 1.26 mmol) in toluene (2.5 mL), NaNH2 (491 mg, 12.6 mmol) was added, it was heated at 111 °C under an argon atmosphere for 1 h. The mixture was cooled to rt and a solution of bromooctadecane (1.7 g, 5 mmol) in toluene (2 mL) added, then heated at 111 °C overnight. The reaction was allowed to cool to 0 °C, crushed ice and saturated NH4Cl added and extracted with EtOAc. The organic layer was washed with H2O and brine. After drying over anhydrous Na2SO4, filtering and evaporating, the reaction mixture was purified by column chromatography (Hex/EtOAc 98:2) to yield 37 (865 mg, 97%). [ α ] D 22 −3.6 (c 1.2, CHCl3); 1H-NMR (400 MHz, CDCl3, δ ppm): 7.47–7.44 (6H, m, H-2′, 6′), 7.31–7.24 (9H, m, H-3′-5′), 7.19 (1H, d, each, J = 8.6 Hz, H-2′′′, H-6′′′), 6.84 (1H, d, each, J = 8.6 Hz, H-3′′′, H-5′′′), 4.48, 4.43 (1H, d, each, J = 11.7 Hz, –OCH2Ar), 3.80 (3H, s, –OCH3), 3.61–3.54 (1H, m, H-sn2), 3.61–3.54 (2H, m, H-sn3), 3.53 (2H, t, J = 6.6 Hz, H-1′′), 3.21 (2H, d, J = 4.6 Hz, H-sn1), 1.56 (2H, m, H-2′′), 1.27 (30H, m, H-3′′-17′′), 0.89 (3H, t, J = 6.8 Hz, H-18′′); 13C-NMR (50 MHz, CDCl3, δ ppm): 159.3 (C-4′′′), 144.4 (3·C-1′), 130.8 (C-1′′′), 129.4 (C-2′′′, C-6′′′), 129.0/128.2 (3·C-3′, 3·C-5′), 128.0/127.5 (3·C-2′, 3·C-6′), 127.1 (3·C-4′), 113.9 (C-3′′′, C-5′′′), 86.8 (–CPh3), 78.6 (C-sn2), 73.1 (–OCH2Ar), 70.9 (C-1′′), 70.4 (C-sn3), 63.7 (C-sn1), 55.5 (–OCH3), 32.2 (C-16′′), 30.4–29.6 (C-2′′, 4′′-15′′), 26.4 (C-3′′), 22.9 (C-17′′), 14.4 (C-18′′); EIHRMS: calcd. for C48H66O4 [M + Na]+: 729.4853, found: 729.4854.

4.26. Preparation of 3-O-p-Methoxybenzyl-2-O-octadecyl-sn-glycerol (38)

To a mixture of 37 (865 mg, 1.22 mmol) in MeOH (12 mL) and CHCl3 (1 mL), p-TsOH (232 mg, 1.22 mmol) was added, and the mixture was stirred under an argon atmosphere at rt for 2 h 30 min., H2O added and the mixture extracted with EtOAc. The organic layer washed with 6% NaHCO3 and H2O, dried over anhydrous Na2SO4, filtered and evaporated. The residue was purified by column chromatography (Hex/EtOAc 95:5) to yield 38 (464 mg, 82%). [ α ] D 22 +8.0 (c 0.9, CHCl3); IR (film, cm−1): 3451, 2922, 2853, 1514, 1248, 1094, 1040; 1H-NMR (400 MHz, CDCl3, δ ppm): 7.25 (2H, d, J = 8.6 Hz, H-2′′′, H-6′′′), 6.88 (2H, d, J = 8.6 Hz, H-3′′′, H-5′′′), 4.49, 4.45 (1H, d, each, J = 11.9 Hz, –OCH2Ar), 3.80 (3H, s, –OCH3), 3.74–3.55 (2H, m, H-sn1), 3.55–3.47 (1H, m, H-sn2), 3.55–3.47 (2H, m, H-sn3), 3.53 (2H, t, J = 4.4 Hz, H-1′′), 1.56 (2H, m, H-2′′), 1.26 (30H, m, H-3′′-17′′), 0.88 (3H, t, J = 6.8 Hz, H-18′′); 13C-NMR (100 MHz, CDCl3, δ ppm): 159.5 (C-4′′′), 130.3 (C-1′′′), 129.5 (C-2′′′, C-6′′′), 114.0 (C-3′′′, C-5′′′), 78.6 (C-sn2), 73.4 (–OCH2Ar), 70.6 (C-1′′), 69.9 (C-sn3), 63.2 (C-sn1), 55.5 (–OCH3), 32.2 (C-16′′), 30.3–29.6 (C-2′′, 4′′-15′′), 26.3 (C-3′′), 22.9 (C-17′′), 14.4 (C-18′′); EIHRMS: calcd. for C29H52O4 [M + Na]+: 487.3758, found: 487.3755.

4.27. Preparation of 1-Chlorocarbonyl-3-O-p-methoxybenzyl-2-O-octadecyl-sn-glycerol (39)

To an ice cooled solution of 38 (261 mg, 0.56 mmol) in THF (1.1 mL), trichloromethyl chloroformate (diphosgene, 67 μL, 0.56 mmol) and N,N-dimethylaniline (71 μL, 0.56 mmol) were slowly added. The mixture was stirred at 0 °C for 15 min and then at rt for 2 h. Then Et2O was added and the white precipitate filtered. The solution washed with 0.2 M HCl, 0.2 M NaOH and H2O, dried over anhydrous Na2SO4 and evaporated to give 39 (244 mg, 82%). IR (film, cm−1): 2924, 2853, 1774, 1248, 1167, 1101; 1H-NMR (400 MHz, CDCl3, δ ppm): 7.24 (2H, d, J = 8.4 Hz, H-2′′′, H-6′′′), 6.88 (2H, d, J = 8.4 Hz, H-3′′′, H-5′′′), 4.47 (2H, s, –OCH2Ar), 4.45 (1H, dd, J = 11.2, 3.7 Hz, HA-sn1), 4.36 (1H, dd, J = 11.2, 6.2 Hz, HB-sn1), 3.80 (3H, s, –OCH3), 3.68 (1H, m, H-sn2), 3.56–3.52 (2H, m, H-1′′), 3.55–3.51 (1H, dd, J = 10.0, 4.7 Hz, HA-sn3), 3.49–3.45 (1H, dd, J = 10.0, 6.2 Hz, HB-sn3) 1.54 (2H, m, H-2′′), 1.26 (30H, m, H-3′′-17′′), 0.88 (3H, t, J = 6.8 Hz, H-18′′); 13C-NMR (100 MHz, CDCl3, δ ppm): 159.3 (C-4′′′), 150.7 (–O–CO–Cl), 129.8 (C-1′′′), 129.3 (C-2′′′, C-6′′′), 113.8 (C-3′′′, C-5′′′), 75.8 (C-sn2), 73.1 (–OCH2Ar), 71.1 (C-sn1), 70.8 (C-1′′), 68.2 (C-sn3), 55.2 (–OCH3), 31.9 (C-16′′), 29.8–29.3 (C-2′′, 4′′-15′′), 25.9 (C-3′′), 22.6 (C-17′′), 14.1 (C-18′′); EIHRMS: in MeOH, calcd. for methyl ester, C31H54O6 [M + Na]+: 545.3813, found: 545.3794.

4.28. Preparation of 1-O-[1,25-Epoxy-18-nor-ent-isodysidiola-1,3(25),9,19-tetraen-4R/S-yloxycarbonyl]-2-O-octadecyl-3-O-p-methoxybenzyl-sn-glycerol (40)

To a solution of 1/2 (176 mg, 0.49 mmol), N,N-diisopropylethylamine (DIPEA, 0.14 mL, 0.79 mmol), 4-(dimethylamino) pyridine (DMAP) (30 mg, 0.25 mmol) in toluene (2.5 mL), a solution of 39 (244 mg, 0.46 mmol) in toluene (2.3 mL) was added dropwise at 0 °C . The reaction mixture was stirred at 0 °C under an argon atmosphere for 15 min and then at rt for 20 h. Then the solvent was removed and the residue was purified by column chromatography (Hex/EtOAc 98:2) to obtain 40 (226 mg, 58%). [ α ] D 22 −7.1 (c 0.8, CHCl3); IR (film, cm−1): 2924, 2853, 1745, 1514, 1250; 1H-NMR (400 MHz, CDCl3, δ ppm): 7.42/7.41 (1H, broad s, H-25′), 7.34/7.33 (1H, broad s, H-1′), 7.24/7.22 (2H, d, J = 8.6 Hz, H-2′′′, H-6′′′), 6.87/6.86 (2H, d, J = 8.6 Hz, H-3′′′, H-5′′′), 6.39 (1H, s, H-2′), 5.79/5.77 (1H, dd, J = 5.4, 3.3 Hz, H-4′), 5.35/5.33 (1H, t, J = 4.0 Hz, H-9′), 4.65–4.60 (2H, m, H-20′), 4.47/4.44 (2H, s, –OCH2Ar), 4.38/4.37 (1H, dd, each, J = 11.2, 4.0 Hz, HA-sn1), 4.30–4.25/4.20–4.10 (1H, m, each, HB-sn1), 3.80 (3H, s, –OMe), 3.70–3.60 (1H, m, H-sn2), 3.56–3.41 (2H, m, H-sn3), 3.48 (2H, t, J = 5.0 Hz, H-1′′), 2.00–1.49 (16H, m, H-5′, 7′, 8′, 11′, 12′, 13′, 14′, 16′, 17′), 1.70/1.66 (3H, s, Me-21′), 1.56 (2H, m, H-2′′), 1.25 (30H, m, H-3′′-17′′), 0.91 (3H, s, Me-22′), 0.89 (3H, t, J = 7.0 Hz, Me-18′′), 0.88 (3H, s, Me-24′), 0.81/0.80 (3H, d, J = 7.0 Hz, Me-23′); 13C-NMR (50 MHz, CDCl3, δ ppm): 159.5/159.4 (C-4′′′), 154.8 (–O–CO–O–), 147.3 (C-19′), 143.5 (C-1′), 141.6/141.3 (C-10′), 140.3 (C-25′), 130.4/130.2 (C-1′′′), 129.5 (C-2′′′, 6′′′), 126.3 (C-3′), 120.3/120.0 (C-9′), 114.0 (C-3′′′, 5′′′), 109.4/109.3 (C-20′), 109.0/108.9 (C-2′), 76.6 (C-sn2), 73.3 (–OCH2Ar), 71.0 (C-sn1), 70.3 (C-4′), 69.3/69.0 (C-1′′), 68.5 (C-sn3), 55.5 (–OMe), 44.4 (C-5′), 43.1 (C-15′), 42.8/42.6 (C-11′), 39.1 (C-14′), 37.6/37.4 (C-16′), 34.4/34.3 (C-6′), 32.6 (C-17′), 32.2 (C-16′′), 30.2–29.0 (C-2′′), 29.6–28.9 (C-4′′-15′′), 29.6–28.9 (C-7′, 13′), 26.2/26.1 (C-3′′), 23.1 (C-8′, 12′), 24.2 (C-24′), 23.0 (C-21′), 22.9 (C-17′′), 22.5 (C-22′), 15.9 (C-23′), 14.4 (C-18′′); EIHRMS: calcd. for C54H86O7 [M + Na]+: 869.6266, found: 869.6260.

4.29. Preparation of 1-O-[1,25-Epoxy-18-nor-ent-isodysidiola-1,3(25),9,19-tetraen-4R/S-yloxycarbonyl]-2-O-octadecyl-sn-glycerol (41)

To a solution of 40 (167 mg, 0.20 mmol) in CH2Cl2/H2O 18:1 (2.2 mL), DDQ (10 mg, 0.045 mmol) was added. It was stirred at rt under an argon atmosphere for 7 h, then quenched with 6% NaHCO3 and extracted with CH2Cl2. The organic layer was washed with 6% NaHCO3 and brine and dried over anhydrous Na2SO4. Removal of the solvent gave a crude product which was purified by column chromatography (Hex/EtOAc 97:3) on silica gel to obtain 41 (103 mg, 71%). [ α ] D 22 +15.2 (c 1, CHCl3); IR (film, cm−1): 3506, 2924, 2853, 1746, 1258; 1H-NMR (400 MHz, CDCl3, δ ppm): 7.42 (1H, s, H-25′), 7.34 (1H, s, H-1′), 6.39 (1H, s, H-2′), 5.80/5.75 (1H, m, H-4′), 5.36/5.33 (1H, t, J = 3.4 Hz, H-9′), 4.65–4.61 (2H, m, H-20′), 4.21/4.20 (1H, dd, each, J = 11.4, 4.8 Hz, HA-sn3), 4.16/4.13 (1H, dd, each, J = 11.4, 4.8 Hz, HB-sn3) 3.68–3.46 (3H, m, H-sn1, H-sn2), 3.56 (2H, t, J = 6.6 Hz, H-1′′), 2.20–1.32 (16H, m, H-5′, 7′, 8′, 11′, 12′, 13′, 14′, 16′, 17′), 1.70/1.67 (3H, s, Me-21′), 1.54 (2H, m, H-2′′), 1.25 (30H, m, H-3′′-17′′), 0.90 (3H, s, Me-22′), 0.88 (3H, t, J = 7.0 Hz, Me-18′′), 0.88 (3H, s, Me-24′), 0.81/0.80 (3H, d, J = 7.0 Hz, Me-23′); 13C-NMR (100 MHz, CDCl3, δ ppm): 154.6 (–O–CO–O–), 147.0 (C-19′), 143.2 (C-1′), 141.4/141.1 (C-10′), 140.2/140.1 (C-25′), 125.9 (C-3′), 120.0/119.7 (C-9′), 109.1/109.0 (C-20′), 108.6 (C-2′), 77.5/77.4 (C-sn2), 70.6 (C-1′′), 70.3 (C-4′), 66.2/66.1 (C-sn1), 61.8 (C-sn3), 44.2 (C-5′), 42.9 (C-15′), 42.4/42.3 (C-11′), 38.8 (C-14′), 37.3/37.2 (C-16′), 34.1/34.0 (C-6′), 32.4/32.3 (C-17′), 31.9 (C-16′′), 29.9–28.8 (C-2′′), 29.9–28.8 (C-4′′-15′′), 29.9–28.8 (C-7′, 13′), 26.0 (C-3′′), 22.8 (C-24′), 22.7 (C-8′, 12′), 22.6 (C-17′′), 22.2 (C-21′), 22.2 (C-22′), 15.6 (C-23′), 14.0 (C-18′′); EIHRMS: calcd. for C46H78O6 [M + Na]+: 749.5691, found: 749.5718.

4.30. Preparation of 1-O-[1,25-Epoxy-18-nor-ent-isodysidiola-1,3(25),9,19-tetraen-4R/S-yloxycarbonyl]-2-O-octadecyl-3-eicosapentaenoyl-sn-glycerol (42)

To a solution of 41 (18 mg, 0.025 mmol), DMAP (4 mg, 0.032 mmol) and EDAC (6 mg, 0.032 mmol) in dry CH2Cl2 (0.24 mL), EPA (8 mg, 0.026 mmol) was added under an argon atmosphere. After stirring at rt for 14 h, the reaction mixture was passed through a short silica gel column (CH2Cl2/EtOAc 9:1 as eluent), the solvent removed and the crude purified by column chromatography (Hex/EtOAc 99:1) providing 42 (16 mg, 63%). IR (film, cm−1): 2959, 2924, 2853, 1744, 1258; 1H-NMR (400 MHz, CDCl3, δ ppm): 7.42 (1H, broad s, H-25′), 7.34 (1H, broad s, H-1′), 6.39 (1H, s, H-2′), 5.79–5.76 (1H, m, H-4′), 5.40–5.30 (10H, m, =CH), 5.40–5.30 (1H, m, H-9′), 4.65/4.61 (2H, m, H-20′), 4.23–4.06 (2H, m, H-sn3), 4.23–4.06 (2H, m, H-sn1), 3.66 (1H, m, H-sn2), 3.50 (2H, t, J = 6.6 Hz, H-1′′), 2.84–2.80 (8H, m, =CCH2C=), 2.32 (2H, t, J = 7.5 Hz, H-2′′′), 2.13–2.06 (4H, m, H-4′′′, 19′′′), 2.04–1.40 (16H, m, H-5′, 7′, 8′, 11′, 12′, 13′, 14′, 16′, 17′), 1.82–1.79 (2H, m, H-3′′′), 1.69/1.66 (3H, s, Me-21′), 1.56 (2H, m, H-2′′), 1.25 (30H, m, H-3′′-17′′), 0.97 (3H, t, J = 7.6 Hz, H-20′′′), 0.89 (3H, s, Me-22′), 0.88 (3H, t, J = 7.0 Hz, Me-18′′), 0.88 (3H, s, Me-24′), 0.80 (3H, d, J = 6.8 Hz, Me-23′); 13C-NMR (50 MHz, CDCl3, δ ppm): 173.4 (C-1′′′), 154.7 (–O–CO–O–), 147.3 (C-19′), 143.5 (C-1′), 141.6/141.3 (C-10′), 140.4 (C-25′), 132.3 (C-18′′′), 129.1–127.2 (=CH) × 9, 126.2 (C-3′), 120.3/120.0 (C-9′), 109.4/109.3 (C-20′), 108.9 (C-2′), 75.2 (C-sn2), 71.0 (C-1′′), 70.5 (C-4′), 66.9/66.7 (C-sn3), 63.1 (C-sn1), 44.4 (C-5′), 43.1 (C-15′), 42.8/42.6 (C-11′), 39.1/38.9 (C-14′), 37.6/37.4 (C-16′), 34.4/34.3 (C-6′), 33.7 (C-2′′′), 32.7/32.6 (C-17′), 32.2 (C-16′′), 30.6 (C-7′), 30.1–29.2 (C-2′′), 30.1–29.2 (C-4′′-15′′), 30.1–29.2 (C-13′), 26.8–20.8 (3′′′, 4′′′, 7′′′, 10′′′, 13′′′, 16′′′, 19′′′, 17′′, 8′, 12′), 25.8 (C-3′′), 23.9 (C-24′), 22.9 (C-21′), 22.5 (C-22′), 15.9 (C-23′), 14.5 (C-20′′′), 14.3 (C-18′′); EIHRMS: calcd. for C66H106O7 [M + Na]+: 1033.7831, found: 1033.7866.

4.31. Preparation of 1-O-[25-Hydroxy-18-nor-ent-isodysidiola-2,9,19-trien-1,25-olide-4R/S-yloxycarbonyl]--2-O-octadecyl-3-eicosapentaenoyl-sn-glycerol (12)

Rose Bengal (2 mg) was added to a solution of 42 (16 mg, 0.016 mmol) and DIPEA (28 μL, 0.16 mmol) in dry CH2Cl2 (2 mL) at rt. Anhydrous oxygen was bubbled in for 5 min and, the solution placed under an oxygen atmosphere at −78 °C and irradiated with a 200 W lamp. After 4 h irradiation was stopped, the pink solution allowed to warm to rt, and saturated aqueous oxalic acid solution (2.5 mL) added. After a few minutes of vigorous stirring, the mixture was diluted with H2O (2 mL) and extracted with Et2O. The combined organic extracts were washed with H2O and dried over anhydrous Na2SO4. After filtration, the solvent was evaporated to give a residue which was purified by silica gel column chromatography to yield 12 (9 mg, 54%). IR (film, cm−1): 3402, 2959, 2924, 2853, 1751, 1256, 1136, 1126; 1H-NMR (400 MHz, CDCl3, δ ppm): 6.19/5.95 (1H, m, H-25′), 6.02/6.01 (1H, s, H-2′), 5.64–5.58 (1H, m, H-4′), 5.38–5.34 (10H, m, =CH), 5.38–5.34 (1H, m, H-9′), 4.67–4.63 (2H, m, H-20′), 4.34–3.98 (4H, m, H-sn1, H-sn3), 3.70–3.66 (1H, m, H-sn2), 3.53 (2H, t, J = 6.4 Hz, H-1′′), 2.85–2.78 (8H, m, =CCH2C=), 2.34 (2H, t, J = 7.2 Hz, H-2′′′), 2.13–2.04 (4H, m, H-4′′′, 19′′′), 2.00–1.49 (16H, m, H-5′, 7′, 8′, 11′, 12′, 13′, 14′, 16′, 17′), 1.84 (2H, m, H-3′′′), 1.70/1.69 (3H, s, Me-21′), 1.54 (2H, m, H-2′′), 1.25 (30H, m, H-3′′-17′′), 0.97 (3H, t, J = 7.6 Hz, H-20′′′), 0.91 (3H, s, Me-22′), 0.87 (3H, t, J = 6.8 Hz, Me-18′′), 0.87 (3H, s, Me-24′), 0.80 (3H, d, J = 6.9 Hz, Me-23′); 13C-NMR (100 MHz, CDCl3, δ ppm): 173.3 (C-1′′′), 169.0 (C-1′), 167.5 (C-3′), 154.2 (O–CO–O), 147.2 (C-19′), 141.4 (C-10′), 132.0 (C-18′′′), 128.9–127.0 (=CH) × 9, 120.0 (C-9′), 118.4 (C-2′), 109.2 (C-20′), 97.4 (C-25′), 76.5 (C-sn2), 70.8 (C-1′′), 70.8 (C-4′), 66.5 (C-sn1), 62.3 (C-sn3), 43.1 (C-5′), 42.8 (C-15′), 42.3 (C-11′), 38.4 (C-14′), 37.2 (C-16′), 34.6 (C-6′), 33.5 (C-2′′′), 32.4 (C-17′), 31.9 (C-16′′), 29.8–29.3 (C-2′′, 4′′-15′′), 28.9 (C-13′), 29.3 (C-7′), 26.5–20.5 (3′′′, 4′′′, 7′′′, 10′′′, 13′′′, 16′′′, 19′′′, 3′′), 22.8 (C-12′), 22.8 (C-8′), 22.7 (C-24′), 22.7 (C-21′), 22.6 (C-17′′), 22.5 (C-22′), 15.6 (C-23′), 14.2 (C-20′′′), 14.0 (C-18′′); EIHRMS: calcd. for C66H106O9 [M + Na]+: 1065.7729, found: 1065.7725.

4.32. Preparation of 1,25-Epoxy-18-nor-ent-isodysidiola-1,3(25),9,19-tetraen-4R/S-yl eicosapentaenoate (13)

To a solution of 1/2 (36 mg, 0.1 mmol), DMAP (16 mg, 0.13 mmol) and EDAC (25 mg, 0.13 mmol) in dry CH2Cl2 (1 mL), EPA (30 mg, 0.1 mmol) was added under an argon atmosphere. After stirring at rt for 20 h, the reaction mixture was passed through a short silica gel column ( CH2Cl2/EtOAc/ 9:1 as eluent), the solvent removed and the crude product purified by column chromatography to give 13 (40 mg, 63%). 1H-NMR (400 MHz, CDCl3, δ ppm): 7.38 (1H, s, H-25), 7.32 (1H, s, H-1), 6.35 (1H, s, H-2), 5.98/5.96 (1H, dd, J = 5.1, 3.4 Hz, H-4), 5.39–5.32 (10H, m, =CH), 5.39–5.32 (1H, m, H-9), 4.64 (1H, broad s, HA-20), 4.61 (1H, broad s, HB-20), 2.85–2.75 (8H, m, =CCH2C=), 2.25 (2H, t, J = 7.6 Hz, H-2′), 2.12–2.03 (4H, m, H-4′, 19′), 2.03–1.30 (16H, m, H-5, 7, 8, 11, 12, 13, 14, 16, 17), 1.87–1.78 (2H, m, H-3′), 1.70/1.67 (3H, s, Me-21), 0.97 (3H, t, J = 7.5 Hz, H-20′), 0.90 (3H, s, Me-22), 0.88 (3H, s, Me-24), 0.81/0.80 (3H, d, J = 6.9 Hz, Me-23); 13C-NMR (100 MHz, CDCl3, δ ppm): 172.8 (C-1′), 147.0 (C-19), 143.0 (C-1), 141.3 (C-10), 140.0 (C-25), 132.0 (C-18′), 129.0–127.0 (=CH) × 9, 126.7 (C-3), 119.9/119.8 (C-9), 109.0 (C-20), 108.8 (C-2), 65.4 (C-4), 44.0 (C-5), 42.9 (C-15), 42.4 (C-11), 38.9/38.8 (C-14), 37.2 (C-16), 34.2/34.1 (C-6), 34.0 (C-2′), 32.3 (C-17), 29.7 (C-7), 29.6 (C-13), 28.9–20.5 (3′, 4′, 7′, 10′, 13′, 16′, 19′), 22.8 (C-24), 22.7 (C-8, 12), 22.3 (C-21), 22.3 (C-22), 15.6 (C-23), 14.2 (C-20′); EIHRMS: calcd. for C44H64O3 [M + Na]+: 663.4748, found: 663.4747.

4.33. Preparation of 4-Eicosapentaenoyl-25-hydroxy-18-nor-ent-isodysidiola-2,9,19-trien-1,25-olide (14)

Rose Bengal (2 mg) was added to a solution of 13 (18 mg, 0.028 mmol) and DIPEA (36 mg, 0.28 mmol) in dry CH2Cl2 (4 mL) at rt. Anhydrous oxygen was bubbled in for 5 min., the solution placed under an oxygen atmosphere at −78 °C and irradiated with a 200 W lamp. After 4 h irradiation was stopped, the pink solution allowed to warm to rt, and saturated aqueous oxalic acid solution (3 mL) added. After a few minutes of vigorous stirring, the mixture was diluted with H2O (3 mL) and extracted with Et2O. The combined organic extracts were washed with H2O and dried over anhydrous Na2SO4. After filtration, the solvent was evaporated to give a residue which was purified by silica gel column chromatography (Hex/EtOAc 95:5) to yield 14 (10 mg, 54%). IR (film, cm−1): 3389, 2961, 2926, 2872, 1744, 1142; 1H-NMR (400 MHz, CDCl3, δ ppm): 6.19 (1H, m, H-25)/5.96 (1H, s, H-25), 5.99/5.93 (1H, s, H-2), 5.58 (1H, d, J = 9.0 Hz, H-4), 5.44–5.32 (10H, m, =CH), 5.44–5.32 (1H, m, H-9), 4.66 (1H, broad s, HA-20), 4.61 (1H, broad s, HB-20), 2.86–2.78 (8H, m, =CCH2C=), 2.37–2.30 (2H, m, H-2′), 2.12–2.06 (4H, m, H-4′, 19′), 2.06–1.30 (16H, m, H-5, 7, 8, 11, 12, 13, 14, 16, 17), 1.89–1.83 (2H, m, H-3′), 1.70 (3H, s, Me-21), 0.97 (3H, t, J = 7.5 Hz, H-20′), 0.92 (3H, s, Me-22), 0.89 (3H, s, Me-24), 0.81 (3H, d, J = 6.9 Hz, Me-23); 13C-NMR (50 MHz, CDCl3, δ ppm): 173.3 (C-1′), 169.5 (C-1), 168.7 (C-3), 147.3 (C-19), 141.4 (C-10), 132.3 (C-18′), 129.5–127.2 (=CH) × 9, 120.1 (C-9), 118.3 (C-2), 109.5 (C-20), 97.9 (C-25), 67.2 (C-4), 43.1 (C-15), 42.9 (C-11), 42.8 (C-5), 39.0/38.6 (C-14), 37.6 (C-16), 34.9/34.6 (C-6), 33.9 (C-2′), 32.7 (C-17), 31.6 (C-7), 28.9 (C-13), 26.7–20.8 (3′, 4′, 7′, 10′, 13′, 16′, 19′, 8, 12), 23.0 (C-24), 22.4 (C-21), 22.4 (C-22), 15.8 (C-23), 14.5 (C-20′); EIHRMS: calcd. for C44H64O5 [M + Na]+: 695.4646, found: 695.4669.

5. Conclusions

In summary, we have synthesized several bioconjugate compounds combining sesterterpenoids, alkyl glycerol chains and PUFAs. The in vitro antitumour activity of these compounds was studied against the HeLa and MCF-7 tumour cell lines. From the results reported here, several conclusions could be deduced: (a) the change of a furan for a γ-hydroxybutenolide unit increases the biological antitumour activity; (b) bioconjugation of γ-hydroxybutenolide sesterterpenes with glycerol derivatives and PUFAs increase the activity with respect to the sesterterpenoids in the edelfosine range; (c) simple bioconjugates of a sesterterpenoid and EPA, as γ-hydroxybutenolide 14, show the best biological activity for the tumour cell lines tested. In this respect, compounds 11 and 12 are in the range of edelfosine for HeLa cells and slightly better for MCF-7 cells. The remarkable activity of compound 14 makes of it a very interesting molecule for further studies and shows the synergy of bioconjugation of sesterterpenolides and PUFAs. Additional experiments are needed to establish the scope and limitations of this behaviour.

Acknowledgments

This work was supported by grants from the Spanish Ministry of Economy and Competitiveness (SAF2011-30518 and SAF2014-59716-R). Junta de Castilla y León BIO/SA59/15. The excellent technical assistance of Ana C. Bento at the early stages of the study is gratefully acknowledged. The authors gratefully acknowledge the help of A. Lithgow (NMR) and C. Raposo (MS) of Universidad de Salamanca.

Author Contributions

A.G.-M., performed experiments and collected data. A.M.R., I.E.T. collected data. P.B., D.D. and I.S.M. were responsible for the design of the synthesis and F.M. for the biological activities. All authors contributed to the paper and approved the manuscript.

Conflicts of Interest

The authors declare no conflict of interest.

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  • Sample Availability: Samples of the compounds 1214 are available from the authors.
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