Synthesis of Bioconjugate Sesterterpenoids with Phospholipids and Polyunsaturated Fatty Acids

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.


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.

Results and Discussion
The bioconjugates synthesized in this work are displayed in Figure 2, namely: 1-O-alkylglycerols 5-8, 2-O-alkylglycerols 9-12 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.

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.

Results and Discussion
The bioconjugates synthesized in this work are displayed in Figure 2, namely: 1-O-alkyl-glycerols 5-8, 2-O-alkylglycerols 9-12 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.

Synthesis of Bioconjugates 5, 6, 7 and 8
Reaction of R-solketal, 15, (Scheme 1) with bromooctadecane in the presence of NaNH 2 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 1 -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]  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.

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. 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 C 66 H 106 O 9 , thus confirming these structures.

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 NaNH 2 , led to a nearly quantitative yield of the alkylderivative 24. Deprotection of 24 with p-TsOH gave diol 25 in excellent yield. 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 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 POCl 3 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 C 51 H 90 NO 9 P, 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 C 66 H 106 O 9 in agreement with the proposed structure for compound 11.

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.
Molecules 2016, 21, 47 6 (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.

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.  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 C 66 H 106 O 9 , corroborating in this manner the structure proposed for compound, 12.

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. 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.

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.

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

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/CO 2 (19:1) the XTT assay was performed as previously described [57].
Measurements were done in triplicate, and the IC 50 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. 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.

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). 1 H-and 13 C-NMR spectra were recorded in CDCl 3 and referenced to the residual peak of CHCl 3 at δ 7.26 ppm and δ 77.0 ppm, for 1 H and 13 C, 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. (16) To a solution of (R)-(´)-solketal 15 (2.6 g, 19.7 mmol) in toluene (39 mL), NaNH 2 (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 NH 4 Cl were added and it was extracted with Et 2 O. The organic layer was washed with H 2 O and brine. After drying over anhydrous Na 2 SO 4 , 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%).  (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 H 2 O was added, and the reaction mixture was extracted with Et 2 O and washed with 6% NaHCO 3 and H 2 O. The organic layer was dried over anhydrous Na 2 SO 4 , filtered and evaporated to give 17 (3.9 g, 11.3 mmol, 93%).   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 CH 2 Cl 2 (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 (CH 2 Cl 2 /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%).  (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 CH 2 Cl 2 (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 (CH 2 Cl 2 /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%).  (7) Rose Bengal (1 mg) was added to a solution of 5 (6.4 mg, 6.3ˆ10´3 mmol) and DIPEA (11 µL, 0.06 mmol) in dry CH 2 Cl 2 (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 H 2 O and extracted with Et 2 O. The combined organic extracts were washed with H 2 O and dried over anhydrous Na 2 SO 4 . After filtration, the solvent was evaporated to give a residue that was purified by silica gel column chromatography to yield 7 (6 mg, 86%).

Preparation of 1-O-Octadecyl-2-O-[25-hydroxy-18-nor-ent-isodysidiola-2,9,19-trien-1,25-olide-4Ryloxycarbonyl]-3-eicosapentaenoyl-sn-glycerol (8)
Rose Bengal (1 mg) was added to a solution of 6 (7.6 mg, 7.5ˆ10´3 mmol) and DIPEA (13 µL, 0.075 mmol) in dry CH 2 Cl 2 (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 H 2 O and extracted with Et 2 O. The combined organic extracts were washed with H 2 O and dried over anhydrous Na 2 SO 4 . After filtration, the solvent was evaporated to give a residue which was purified by silica gel column chromatography to yield 8 (7 mg, 90%).

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 H 2 O was added, the mixture was extracted with Et 2 O and washed with 6% NaHCO 3 and H 2 O. The organic layer was dried over Na 2 SO 4 , filtered and evaporated. 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 H 2 O. It was extracted with Et 2 O and the organic layer washed with H 2 O. After drying over anhydrous Na 2 SO 4 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%).  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 CH 2 Cl 2 (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 (CH 2 Cl 2 /EtOAc 9:1 as eluent). Then the solvent was removed and the crude was purified by column chromatography (Hex/EtOAc 98:2) providing 32 ( 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 CH 2 Cl 2 (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 H 2 O and extracted with CH 2 Cl 2 . The combined organic extracts were washed with H 2 O and brine, and dried over anhydrous Na 2 SO 4 . The solvent was evaporated to give a residue that was purified by silica gel column chromatography (Hex/EtOAc 9:1) to yield 11 (

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 NH 4 Cl added. The aqueous layer was extracted with EtOAc and the organic layer was washed with H 2 O and brine, dried over anhydrous Na 2 SO 4 , filtered and evaporated. The bulk reaction mixture was purified by column chromatography on silica gel (EtOAc) to obtain 35 (3.6 g, 90%). 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 H 2 O was added, then it was extracted with EtOAc and washed with 2 M HCl, 6% NaHCO 3 and brine, dried over anhydrous Na 2 SO 4 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%).

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. 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.