Synthesis of bioconjugate sesterterpenoids with phospholipids and polyunsaturated fatty acids

Article abstract of DOI:10.3390/molecules21010047

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 or

Full text of DOI:10.3390/molecules21010047

Article
Synthesis of Bioconjugate Sesterterpenoids with
Phospholipids and Polyunsaturated Fatty Acids
Ana Gil-Mesón 1, Alejandro M. Roncero 1, Ignacio E. Tobal 1, Pilar Basabe 1, David Díez 1,
Faustino Mollinedo 2,3 and Isidro S. Marcos 1,
*
Received: 3 December 2015 ; Accepted: 22 December 2015 ; Published: 30 December 2015
Academic Editors: Jean Jacques Vanden Eynde and Annie Mayence
1
Departamento de Química Orgánica, Universidad de Salamanca, Plaza de los Caídos 1-5,
37008 Salamanca, Spain; anagm@usal.es (A.G.-M.); alexmaron@usal.es (A.M.R.);
ignaciotobal@usal.es (I.E.T.); pbb@usal.es (P.B.); ddm@usal.es (D.D.)
Instituto de Biología Molecular y Celular del Cáncer, Centro de Investigación del Cáncer,
2
CSIC-Universidad de Salamanca, Campus Miguel de Unamuno, 37007 Salamanca, Spain; fmollin@usal.es
Laboratory of Cell Death and Cancer Therapy, Department of Cellular and Molecular Medicine,
3
Centro de Investigaciones Biológicas, CSIC, C/Ramiro de Maeztu 9, 28040 Madrid, Spain
*
Correspondence: ismarcos@usal.es; Tel.: +34-923-294-474; Fax: +34-923-294-574
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 [1–5]
.
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 [
effect due to the conjugation.
7], lipids with sugars [8], that have a synergistic
Bioconjugates made by direct esterification of paclitaxel (Taxolr) 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 [11–20]. 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
as the synthetic ether lipid edelfosine (1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine) [25–31]
and PUFAs [32 43], which separately show antitumour activity and together might have a synergistic
–24] (Figure 1) and bioconjugated with phospholipids such
–
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 [21–24], (Figure 1) and the
Molecules 2016, 21, 47; doi:10.3390/molecules21010047
www.mdpi.com/journal/molecules

Molecules 2016, 21, 47
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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
cellular lines and leukemia [46].
«
4.8–5.4 µM) on several solid tumour
Figure 1. Dysidiolide, edelfosine, PUFAs and sesterterpenoid compounds used for bioconjugation.
2. 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
phosphocholine unit in sn3 of the glycerol and bioconjugates 11 and 12 change the phosphocholine
unit for an EPA substituent. Compounds 10 and 11 were synthesized from racemic solketal and
, 11, 12 show the sesterterpenoid unit in the glycerol sn1 position. Bioconjugates 9 and 10 with a
9,
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
.

Molecules 2016, 21, 47
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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)-N1-ethyl carbodiimide (EDAC) and DMAP, led to
5
and
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
and , respectively,
corresponding both to a formula C66H106O7, in agreement with the proposed structures.
6, respectively.
5
6
O
R
O
R
H
O
H
O
O
O
O
O
O
O
O
O
16
16
5
6
O
O
H
H
O
O
O
O
O
O
O
O
O
O
O
O
16
HO
HO
16
O
O
R
R
7
8
O
O
O
H
O
O
OH
O
O
C17H35
C17H35
O
O
O
O
H
O
O
O
O
O
O
N
N
P
O
P
O
O
O
10
9
O
H
O
O
O
OH
O
OH
O
C17H35
C17H35
O
O
O
O
H
O
O
O
R
O
O
R
O
11
12
O
O
O
OH
O
O
O
R
O
R
H
H
R
13
14
Figure 2. Alkyl glycerol sesterterpenoids bioconjugate compounds
13–14, synthesized in this work.
5–12 and sesterterpenoid-PUFAs

Molecules 2016, 21, 47
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Scheme 1. Synthesis of bioconjugates
NaNH2, toluene, 92%; (
) p-TsOH, MeOH, 40 ˝C, 93%; (
80%; ( ) trichloromethylchloroformate, N,N-dimethylaniline, THF, 83%; (
60%; ( ) DDQ, DCM/H2O, 21: 32%, 22: 65%; ( ) EPA, EDAC, DMAP, DCM, rt,
Rose Bengal, DIPEA, DCM, 7: 86%, 8; 90%.
5
,
6
,
7
and
8
. Reagents and conditions: (
) 1. n-Bu2SnO, toluene, 2. CsF, PMBCl, DMF,
) DMAP, DIPEA, toluene,
: 87%, : 82%; (
) 1O2,
a) Bromooctadecane,
b
c
d
f
e
g
5
6
h
Oxidation of
Rose Bengal and DIPEA), gave the
5
and
6
following Faulkner’s methodology [52] (singlet oxygen in the presence of
-hydroxybutenolides and in excellent yield (Scheme 1).
γ
7
8
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.

Molecules 2016, 21, 47
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Scheme 2. Synthesis of
98%; (
) p-TsOH, MeOH, 40 ˝C, 90%; (
) trichloromethylchloroformate, N,N-dimethylaniline, THF, 71%; (
) TBAF, THF, rt, 89%; ( ) choline tetraphenylborate, TPS,
) POCl3, pyridine, THF, 0 ˝C, 97%; (
) O2, rose bengal, DIPEA, CH2Cl2, 30%; ( ) EPA, EDAC, DMAP, CH2Cl2,rt, 64%;
(k) 1O2, Rose Bengal, DIPEA, CH2Cl2, 53%.
9,
10 and 11. Reagents and conditions: (
) TBDMSCl, imidazole, DMF, rt, 26: 41%, 27: 16%, 25: 42%;
) DMAP, DIPEA, toluene, 56%;
a) Bromooctadecane, NaNH2, toluene,
b
c
(
d
e
(
f
g
h
1
pyridine, 35%; (
i
j
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 in the
,
1/2
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

Molecules 2016, 21, 47
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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 . The structure of this compound was established by its NMR spectra. The mass spectrum
of
shows a [M + Na]+ molecular ion at 914.6229 corresponding to C51H90NO9P, corroborating
in this manner the structure of the bioconjugate phospholipid. The -hydroxy-butenolide 10 was
by oxidation with singlet oxygen in the presence of Rose Bengal
9
9
γ
obtained from the furyl derivative
and DIPEA.
9
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: (
pyridine, 92%; ( ) bromooctadecane, NaNH2, toluene, 97%; (
) trichloromethylchloroformate, N,N-dimethylaniline, THF, 82%; (
a
) PMBCl, NaH, 99%; (
b) SiO2, 90%; (c) TrCl,
d
e
) p-TsOH, MeOH, 40 ˝C, 82%;
(
(
f
g
) DMAP, DIPEA, toluene, 58%;
1
h) DDQ, DCM/H2O, rt, 71%; (i) EPA, EDAC, DMAP, DCM, rt, 63%; (
j
) O2, rose bengal, DIPEA,
DCM, 54%.
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

Molecules 2016, 21, 47
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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 mixture in
1/2
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 with eicosapentaenoic acid (EPA) in the presence of EDAC and DMAP
1/2
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: (
(b) 1O2, rose bengal, DIPEA, DCM, 54%.
a) EPA, EDAC, DMAP, DCM, rt, 63%;
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
cells than their -hydroxybutenolide counterparts
of a furan fragment for a -hydroxybutenolide unit increases the activity, as previously observed by
us [46 58 59]. Secondly bioconjugates and are more active than the non-conjugates and , in the
same manner bioconjugates and have a better behaviour than and showing than conjugation
increases the activity against HeLa and MCF7. These compounds and are more active that edelfosine
1
and
2
showed less activity against HeLa and MCF7
γ
3
and
4 [46]. This behaviour tells us that the change
γ
,
,
5
6
1
2
7
8
3
4
7
8

Molecules 2016, 21, 47
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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 and to primary
as in 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 and , so bioconjugation increases the biological activity. When comparing
edelfosine against several -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
7
8
9,
,
3
4
γ
,
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.
HeLa IC50
(µM)
MCF-7
IC50 (µM)
Structural
Characteristic
Sesterterpene
Position
Sn3
Substitution
Compound
1/2
3
4
5/6
7
8
9
10
11
12
13
14
30.2 ˘ 1.9
4.8 ˘ 0.7
5.4 ˘ 0.6
13.1 ˘ 9.5
1.1 ˘ 0.1
1.1 ˘ 0.1
25.0 ˘ 10
3.3 ˘ 0.9
2.5 ˘ 0.1
2.5 ˘ 0.1
10.2 ˘ 3.5
0.3 ˘ 0.1
2.5 ˘ 0.7
32.1 ˘ 1.1
5.1 ˘ 0.9
5.2 ˘ 1.8
ND
0.6 ˘ 0.1
0.5¨ ˘ 0.2
ND
6.5 ˘ 0.8
0.6 ˘ 0.2
0.5¨ ˘ 0.1
12.3 ˘ 3.7
0.2 ˘ 0.1
3.1¨ ˘ 0.9
Furan
α
-Hydroxybutenolide
-Hydroxybutenolide
Furan
-Hydroxybutenolide
-Hydroxy-butenolide
Furan
-Hydroxybutenolide
-Hydroxybutenolide
-Hydroxybutenolide
Furan
α
sn2
sn2
sn2
1
1
1
EPA
EPA
EPA
α
α
Phosphocholine
Phosphocholine
EPA
α
α
α
sn1
EPA
Eicosapentanoylsesterterpene
α
-Hydroxybutenolide Eicosapentanoylsesterterpene
Edelfosine
Simple bioconjugates 13 and 14 are more biologically active than alkylglycerols
respectively, while in this respect compound 14 is 17 and 26 times more active than -hydroxylactones
and 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
9 and 10
γ
3
4
,
analogue synthesis. In summary the presence of a
could be a route to better activity.
γ-hydroxybutenolide and simple bioconjugation
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
1
spectrophotometer (Thermo Nicolet, Salamanca, Spain). H- and 13C-NMR spectra were recorded
in CDCl3 and referenced to the residual peak of CHCl3 at
13C, respectively, using 200 VX (Varian, Salamanca, Spain) and DRX 400 (Bruker, Salamanca, Spain)
instruments. Chemical shifts are reported in parts per million and coupling constants (J) are given in
δ 7.26 ppm and δ
77.0 ppm, for 1H and
δ
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.

Molecules 2016, 21, 47
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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%).
22
rαsD
´
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),
1
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-21), 1.42, 1.36 (3H, s, each, Me2C-), 1.25 (30H, m,
H-31-171), 0.88 (3H, t, J = 6.8 Hz, H-181); 13C-NMR (1100 MHz, CDCl3,
δ
pp1m):1 109.3 (Me2
C-), 74.7
1
1
(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.
1
1
1
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
22
and evaporated to give 17 (3.9 g, 11.3 mmol, 93%). rαsD +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-11), 3.44
(1H, ddd, J = 9.3, 6.7, 6.7 Hz, HB-11), 1.56 (2H, m, H-21), 1.25 (30H, m, H-31-171), 0.87 (3H, t, J = 6.8 Hz,
,
H-181); 13C-NMR (100 MHz, CDCl3, ppm): 72.4 (C-sn1), 71.8 (C-11), 70.4 (C-sn2), 64.2 (C-sn3), 31.8
δ
(C-161), 29.5 (C-21), 29.5 (C-41-151), 26.0 (C-31), 22.6 (C-171), 14.0 (C-181); 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
22
(Hex/EtOAc 96:4) to obtain 18 (215 mg, 80%). rαsD +1.2 (c 0.11, CHCl3); IR (film, cm´1): 3485, 3404,
111 111
δ ppm): 7.27 (2H, d, J = 7.4 Hz, H-2 , H-6 ), 6.89
2916, 2846, 1470, 1031; 1H-NMR (400 MHz, CDCl3,
(2H, d, J = 7.4 Hz, H-3111, H-511 ), 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, 11, sn3), 1.56 (2H, m, H-21), 111.128 (30H, m, H-31-171), 0.89 (3H, t,
1
111 111
δ ppm): 159.2 (C-4 ), 130.1 (C-1 ), 129.3 (C-2 ), 129.3
J = 6.2 Hz, H-18 ); 13C-NMR (100 MHz, CDCl3,
111
111
111
1
(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-161), 29.5 (C-21), 29.5 (C-41-151), 26.1 (C-31), 22.7 (C-171), 14.1 (C-181); EIHRMS:
calcd. for C29H52O4 [M + Na]+: 487.3758, found: 487.3773.

Molecules 2016, 21, 47
10 of 25
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,
111
111
111
111
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,
1 1
H2Ar), 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 ),
–OC
1.25 (30H, m, H-3 -17 ), 0.89 (3H, t, J = 6.2 Hz, H-18 ); 13C-NMR (50 MHz, CDCl3,
154.9 (–O–
O–Cl), 129.8 (C-1111), 129.5 (C-2111, 6111), 114.1 (C-3111, 5111), 80.5 (C-sn2), 73.3 (–O
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-171), 14.3 (C-181); EIHRMS: in MeOH, calcd. for methyl ester, C31H54O6 [M + Na]+: 545.3813,
found: 545.3808.
1
1
1
111
δ
ppm): 159.2 (C-4 ),
H2Ar),
C
C
1
1
1
1
1
1
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-2511), 7.34/7.33 (1H, s, H-111),171 .24/7.17 (2H, d, J = 8.8 Hz, H-2111, H-6111),
111
111
11
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-911), 4.98 (1H, m, H-sn2), 4.65–4.62 (2H, m, H-2011), 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-11), 2.20–1.40 (16H, m, H-511, 711, 811, 1111,
1211, 1311, 1411, 1611, 1711), 1.70/1.67 (3H, s, Me-2111), 1.56 (2H, m, H-21), 1.26 (30H, m, H-31-171), 0.91
(3H, s, Me-2211), 0.89 (3H, t, J = 6.2 Hz, Me-181), 0.88 (3H, s, Me-2411), 0.81/0.80 (3H, d, J = 7.0 Hz,
Me-2311); 13C-NMR (100 MHz, CDCl3, ppm): 159.2 (C-4111), 154.2 (–O– O–O–), 147.0/146.9 (C-1911),
δ C
143.1 (C-111), 141.4/141.0 (C-1011), 140.0/139.9 (C-2511), 130.0 (C-1111), 129.2 (C-2111, 6111), 126.2 (C-311),
120.0/119.7 (C-911), 113.7 (C-3111, 5111), 11109.1/109.0 (C-2011), 108.8/108.7 (C-211), 75.5/75.4 (C-sn2), 72.9
1 11
(–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-1511), 42.6 (C-1111), 38.9/38.7 (C-1411), 37.4/37.2 (C-1611), 34.1/34.0 (C-611), 32.4/32.3 (C-1711),
31.9 (C-161), 29.6–28.9 (C-21), 29.6–28.9 (C-41-151), 29.6–28.9 (C-711, 1311), 26.0/25.9 (C-31), 23.2 (C-811,
1211), 22.8 (C-2411), 22.6 (C-171), 22.3 (C-2111), 22.2 (C-2211), 15.6 (C-2311), 14.0 (C-181); 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).

Molecules 2016, 21, 47
11 of 25
22
Compound 21: rαsD +42.2 (c 0.46, CHCl3); IR (film, cm´1): 3464, 2924, 2853, 1744, 1260; 1H-NMR (400 MHz
,
11
11
11
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-411), 5.33 (1H, t, J = 4.8 Hz, H-911), 4.81 (1H, quin, J = 5.0 Hz, H-sn2), 4.65 and 4.64 (1H, s, each,
H-2011), 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-11), 2.05–1.4 (16H,
m, H-5111, 711, 811, 1111, 1211, 1311 1411, 1611, 1711), 1.70 (3H, s, Me-2111), 1.56 (2H, m, H-21), 1.25 (30H, m,
1
11
11
1
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-2311); 13C-NMR (100 MHz, CDCl3,
δ
ppm)1:1154.2 (–O–
C
O–O–), 147.1 (C-1911), 143.2 (C-111), 141.4
(C-1011), 140.0 (C-2511), 126.0 (C-311), 119.7 (C-911), 109.1 (C-2011), 108.6 (C-211), 76.6 (C-sn2), 71.9 (C-11),
11
11
11
11
11
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-611), 32.4 (C-1711), 31.9 (C-161), 31.0 (C-711), 29.6–29.3 (C-21), 29.6–29.3 (C-41-151), 28.8 (C-1311),
25.9 (C-31), 22.8 (C-1211), 22.7 (C-2411), 22.6 (C-811), 22.6 (C-171), 22.5 (C-2111), 22.3 (C-2211), 15.6 (C-2311),
14.1 (C-18 ); EIHRMS: calcd. for C46H78O6 [M + Na]+: 749.5691, found: 749.5706.
1
22
1
Compound 22
:
rαsD +3.5 (c 0.40, CHCl3); IR (film, cm´1): 3477, 2924, 2853, 1742, 1261; H-NMR
(400 MHz, CDCl3,
δ
ppm): 7.43 (1H, s, H-2511), 7.35 (1H, s, H-111), 6.40 (1H, s, H-211), 5.80 (1H, dd,
J = 8.4, 3.4 Hz, H-411), 5.36 (1H, t, J = 3.4 Hz, H-911), 4.83 (1H, quin, J = 5.0 Hz, H-sn2), 4.65, 4.61 (1H,
s, each, H-2011), 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-11), 2.15–1.4 (16H, m, H-511, 711, 811, 1111, 1211, 1311 1411, 1611, 1711), 1.67
(3H, s, Me-2111), 1.56 (2H, m, H-21), 1.25 (30H, m, H-31-171), 0.91 (3H, s, Me-2211), 0.88 (3H, s, Me-2411),
0.86 (3H, t, J = 6.2 Hz, Me-181),101 .81 (3H, d, J = 7.0 Hz, Me-2311); 13C-NMR (100 MHz, CDCl3,
11 11 11 11 11
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 ),
δ ppm):
109.0 (C-2011), 108.7 (C-12111), 76.9 (C-sn2), 71.9 (C-1111), 70.3 (C-411), 69.5 (C-sn1), 62.6 (C-sn3), 44.2 (C-5111),
11
11
11
11
11
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 ),
1
1
1
11
1
11
11
11
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-171), 22.2 (C-2111), 22.2 (C-2211), 15.6 (C-2311), 14.1 (C-181); 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
22
(Hex/EtOAc 98:2) providing
5
(12 mg, 87%). rαsD +7.5 (c 0.20, CHCl3); IR (film, cm´1): 2957, 2926,
11 11
δ ppm): 7.42 (1H, s, H-25 ), 7.35 (1H, s, H-1 ), 6.39
2855, 1745, 1462, 1261; 1H-NMR (400 MHz, CDCl3,
11
11
11
(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,
11
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, 1d11, J = 5.4 Hz, H-sn1), 3.40–3.3411(12H, m, H-11), 2.85–2.80 (8H, m, =CCH2C=),
111
11 11 11
11
11
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 ,
1311, 1411, 1611, 1711), 1.76–1.72 (2H, m, H-3111), 1.70 (3H, s, Me-2111), 1.56 (2H, m, H-21), 1.25 (30H, m,
H-31-171), 0.97 (3H, t, J = 7.5 Hz, H-20111), 0.90 (3H, s, Me-2211), 0.89 (3H, t, J = 6.8 Hz, Me-181), 0.88
(3H, s, Me-2411), 0.80 (3H, d, J = 6.8 Hz, Me-2311); 13C-NMR (100 MHz, CDCl3, ppm): 173.4 (C-1111),
δ
11
11
11
11
111
154.2 (–O–
(=CH)
C
O–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
9, 126.2 (C-311), 119.9 (C-911), 109.4 (C-2011), 108.9 (C-211), 74.3 (C-sn2), 72.1 (C-11), 70.5 (C-4 ),
11
11
ˆ
11
11
11
11
11
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-2111), 32.7 (C-1711), 32.1 (C-161), 31.1 (C-711), 29.9–29.3 (C-21), 29.9–29.3 (C-41-151), 29.1 (C-1311),
28.7–20.8 (3111, 4111, 7”’, 10”’, 13”’, 16”’, 19111), 26.0 (C-311)1,124.0 (C-2411), 22.9 (C-1211), 22.9 (C-2111), 22.6
11
1
11
11
1
(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.

Molecules 2016, 21, 47
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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
22
(Hex/EtOAc 99:1) providing
6
(14 mg, 82%). rαsD +2.4 (c 0.33, CHCl3); IR (film, cm´1): 2959, 2924,
11 11
δ ppm): 7.42 (1H, s, H-25 ), 7.33 (1H, s, H-1 ), 6.39 (1H, s,
2855, 1744, 1263; 1H-NMR (400 MHz, CDCl3,
H-211), 5.79 (1H, dd, J = 8.5, 3.0 Hz, H-411), 5.40–5.34 (10H, m, =C ), 5.34 (1H, m, H-911), 5.04–5.00
H
(1H, m, H-sn2), 4.65 and 4.61 (1H, s, each, H-2011), 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,
1
111
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,
111
111
11 11 11
11
11
11
11
11
11
111
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-2111), 1.56 (2H, m, H-21), 1.25 (30H, m, H-31-171), 0.97 (3H, t, J = 7.5 Hz, H-20111), 0.1910
(3H, t, J = 6.8 Hz, Me-181), 0.88 (3H, s, Me-2211), 0.86 (3H, s, Me-2411), 0.81 (3H, d, J = 7.0 Hz, Me-23 );
13C-NMR (100 MHz, CDCl3,
δ
ppm): 173.1 (C-1111), 154.2 (–O–
C
ˆ
O–O–), 146.9 (C-1911), 143.1 (C-111),
9, 126.1 (C-311), 120.0 (C-911), 109.0
141.0 (C-1011), 140.1 (C-2511), 132.0 (C-18111),1128.9–127.0 (=CH)
(C-2011), 108.7 (C-211), 74.0 (C-sn2), 71.8 (C-11)1, 70.3 (C-411), 68.8 (C-sn1), 62.7 (C-sn131), 44.3 (C-511), 42.9
11
11
11
11
11
111
1
(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
1
1
1
11
111 111 111
111
111
111
111
(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-31), 23.7 (C-2411), 23.2 (C-1211), 22.8 (C-2111), 22.6 (C-811), 22.6 (C-171), 22.2 (C-2211), 15.6 (C-2311),
14.2 (C-20 ), 14.0 (C-18 ); EIHRMS: calcd. for C66H106O7 [M + Na]+: 1033.7831, found: 1033.7854.
111
1
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
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
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
5
(6.4 mg, 6.3
ˆ
10´3 mmol) and DIPEA (11
µL,
˝
´
78 C and irradiated with a 200 W lamp. After
anhydrous Na2SO4. After filtration, the solvent was evaporated to give a residue that was purified by
22
silica gel column chromatography to yield
7
(6 mg, 86%). rαsD +2.6 (c 0.2, CHCl3); IR (film, cm´1):
11
δ ppm): 6.19/5.97 (1H, m, H-25 ), 6.01/6.00 (1H, m,
H
H-211), 5.60/5.48 (1H, m, H-411), 5.45–5.33 (10H, m, =C ), 5.45–5.33 (1H, m, H-911), 5.03/4.95 (1H, m,
3427, 2924, 1747, 1259; 1H-NMR (400 MHz, CDCl3,
11
1
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
111
111
111
11 11 11
11
(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 ,
11
11
11
11
11 111
11
1
1
1
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,
111
11
1
11
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-2311); 13C-NMR (50 MHz, CDCl3,
δ
ppm): 173.7 (C-1111), 169.4 (C-111), 168.2 (C-311),
154.4 (–O–
C
O–O–), 147.5 (C-1911), 141.8 (C-1011), 132.3 (C-18111), 129.2–12171.2 (=CH)
ˆ
9, 120.0 (C-911),
11
11
11
1
11
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),
11
11
11
11
11
111
11
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 ),
1
11
1
1
1
11
111 111 111
111
11
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 ,
111
111
111
1
11
11
11
11
1
13 , 16 , 19 ), 25.9 (C-3 ), 24.0 (C1-24 ), 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.
11
111

Molecules 2016, 21, 47
13 of 25
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
0.075 mmol) in dry CH2Cl2 (2 mL) at rt. Anhydrous oxygen was bubbled in for 2 min, then the solution
6
(7.6 mg, 7.5
ˆ
10´3 mmol) and DIPEA (13
µL,
˝
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
22
gel column chromatography to yield
8
(7 mg, 90%). rαsD
´
3.0 (c 0.3, CHCl3); ); IR (film, cm´1): 3427,
11
11
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, =C
), 5.40–5.34 (1H, m, H-911), 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),
11
11
H
1
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,
111
111
111
11 11 11
11
11
11
11
11
11 111
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-2111), 1.54 (2H, m, H-21), 1.25 (30H, m, H-31-171), 0.97 (3H, t, J = 7.6 Hz, H-20111), 0.91
(3H, s, Me-2211), 0.88 (3H, t, J = 6.8 Hz, Me-181), 0.88 (3H, s, Me-2411), 0.811(13H, d, J = 6.8 Hz, Me-2311);
111 11
δ ppm): 173.4 (C-1 ), 169.3 (C-1 ), 168.1 (C-3 ), 154.4 (–O–CO–O–), 147.1
13C-NMR (100 MHz, CDCl3,
(C-1911), 141.1 (C-1011), 132.0 (C-18111), 128.8–127.0 (=CH)
ˆ
9, 120.0 (C-911), 118.0 (C-211), 109.0 (C-2011),
97.5 (C-2511), 74.0 (C-sn2), 71.8 (C-11), 71.1 (C-411), 68.8 (C-sn1), 62.6 (C-sn3), 42.9 (C-511)1, 42.9 (C-1511),
42.3 (C-1111), 38.8 (C-1411), 37.6 (C-1611), 34.1 (C-611), 33.3 (C-2111), 32.2 (C-1711), 31.9 (C-16 ), 31.2 (C-711),
29.7–29.3 (C-21), 29.7–29.3 (C-41-151), 28.8 (C-1311), 28.3–22.6 (3111, 4111, 7111, 10111, 13111, 16111, 19111), 26.0
(C-31), 23.7 (C-2411), 23.2 (C-1211), 22.8 (C-2111), 22.6 (C-811), 22.6 (C-171), 22.2 (C-2211), 15.7 (C-2311), 14.2
(C-20 ), 14.0 (C-18 ); EIHRMS: calcd. for C66H106O9 [M + Na]+: 1065.7729, found: 1065.7766.
111
1
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 ,
1
1
1
1
1
6 ), 7.40–7.32 (3H, m, H-3 , 4 , 5 ), 5.55 (1H, s, O–C
H–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-111), 1.65 (2H, m, H-211), 1.26
(30H, m, H-311-1711), 0.88 1(3H, t, J = 6.4 Hz, H-1811); 13C-NMR (50 MHz, CDCl3,
δ
ppm): 138.5 (C-11),
129.01(1C-41), 128.5 (C-21, 6 1)1, 12161.5 (C-31, 51), 101.5 (O–
C
H–O), 72.5 (C1-1111), 70.9 (C-2), 69.2 (C-1, 3), 32.2
11
11
11
(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,
11
11
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,
11
11
11
11
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-1611), 30.2 (C-211), 29.7 (C-411-1511), 26.3 (C-311), 22.9 (C-1711), 14.3 (C-1811).
EIHRMS: calcd. for C21H44O3 [M + Na]+: 367.3183, found: 367.3187.

Molecules 2016, 21, 47
14 of 25
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-111), 3.52 (1H, ddd,
J = 9.2, 6.8, 6.8 Hz, HB-111), 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-211), 1.25 (30H, m, H-311-1711), 0.89 (9H, s, Me3CSi-), 0.89 (3H, t, J = 6.8 Hz, H-1811), 0.06 (6H, s,
11 11
δ ppm):80.1 (C-2), 70.7 (C-1 ), 63.2 (C-3), 62.9 (C-1), 32.1 (C-16 ),
Me2Si-); 13C-NMR (100 MHz, CDCl3,
11
11
11
11
11
11
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 ),
(Me2Si-). EIHRMS: calcd. for C27H58O3Si [M + Na]+: 481.4047, found: 481.4025.
´5.3
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-211), 1.26 (30H, m, H-311-1711), 0.89 (2 9H, s, Me3CSi-), 0.89 (3H, t, J = 6.8 Hz, H-1811), 0.05 (2
6H, s,
Me2Si-); 13C-NMR (50 MHz, CDCl3,
11
¨
¨
11 11
ppm): 81.4 (C-2), 70.9 (C-1 ), 63.1 (C-3), 63.1 (C-1), 32.2 (C-16 ),
δ
30.4 (C-211), 29.9–29.6 (C-411-1511), 26.4 (C-311), 26.1 (2¨ Me3CSi-), 22.9 (C-1711), 18.5 (2
¨
Me3CSi-), 14.3
11
(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-111), 1.55 (2H, m, H-211), 1.26 (30H, m, H-311-1711), 0.89 (9H, s, Me3CSi-), 0.88 (3H,
t, J = 6.8 Hz, H1-11811), 0.06 (6H, s, Me2Si-); 13C-NMR (50 MHz, CDCl3,
δ
ppm): 1501.19 (O–CO–Cl), 77.6
11
11
11
11
11
(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
11
(
Me3CSi-), 22.9 (C-17 ), 18.4 (Me3
C
Si-), 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,
,
1 1 1 1
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
δ
(1H, m, H-91), 4.66–4.60 (2H, m, H-201), 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-111), 2.20–1.40 (16H, m, H-51, 71, 81, 111, 121, 131, 141, 161, 171), 1.70/1.66

Molecules 2016, 21, 47
15 of 25
1
11
11
11
1
1
(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-1811), 0.87 (9H, s, Me3CSi-), 0.81 (3H, d, J = 6.9 Hz, H-231), 0.04 (6H, s, Me2Si-);
13C-NMR (50 MHz, CDCl3,
δ
ppm): 154.6/154.5 1(O–
C
O–O), 147.0 (C-191), 143.2/143.1 1(C-11), 142.6
1
1
1
1
(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-111), 70.0/69.9 (C-41), 67.1/66.8 (C-1), 62.2 (C-3), 44.2 (C-51), 42.9 (C-151), 42.4 (C-111), 38.8
(C-141), 37.4 (C-161), 34.1/34.0 (C-61),132.4/32.3 (C-171), 31.9 (1C1 -1611), 30.0–29.3 (C-211, 411-1511, 71, 1311),
11
1
1
1
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 (Me3
C
Si-), 15.6 (C-231), 14.1 (C-1811),
´
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, ppm): 7.42 (1H, s, H-251), 7.34 (1H, s, H-11), 6.39 (1H,
δ
s, H-21), 5.78 (1H, m, H-41), 5.35–5.32 (1H, m, H-91), 4.66–4.61 (2H, m, H-201), 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-111), 3.49–3.43 (1H, m, H-2), 2.20–1.40 (16H, m, H-51,
71, 81, 111, 121, 131, 141, 161, 171), 1.69/1.66 (3H, s, H-211), 1.55 (2H, m, H-211), 1.25 (30H, m, H-311-1711),
0.89 (3H, s, H-221), 0.88 (3H, t, J = 6.8 Hz, H-1811), 0.87 (3H, s, H-241), 0.80/0.79 (3H, d, J = 6.8 Hz,
H-2311); 13C-NMR (50 MHz, CDCl3,
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-141), 37.6/37.4 (C-161), 34.4/34.3 (C-61), 32.7/32.6 (C-171), 32.1 (C-1611), 31.5 (C-71), 30.3–29.6
(C-211, 411-1511), 29.1 (C-131), 26.3/26.2 (C-311), 23.4 (C-241), 23.1 (C-211), 22.9 (C-81), 22.9 (C-121), 22.9
(C-1711), 22.5 (C-221), 15.9 (C-231), 14.3 (C-1811). EIHRMS: calcd. for C46H78O6 [M + Na]+: 749.5691,
found: 749.5665.
δ
ppm): 154.81(O–
C
O–O), 147.3 (C-191), 143.5 (C-11),1141.6/141.3
1
1
1
(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),
11
1
1
1
1
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-NM1R (200 MHz, CDCl3,
δ
ppm): 7.39 (1H, broad s, H-251), 7.3 (1H,
1
1
1
1
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-111, H-2), 2.10–1.40 (16H, m, H-51, 71, 81, 111, 121, 131,
141, 161, 171), 1.69/1.65 (3H,11s, H-211), 1.55 (2H,1m, H-211), 1.25 (30H, m, H-311-1711), 0.89 (3H, s, H-221),
1
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

Molecules 2016, 21, 47
16 of 25
purified by column chromatography (CHCl3/MeOH/NH4OH 65:30:5) to yield
9
(16 mg, 35%). IR
1
δ ppm):7.42 (1H, s, H-25 ), 7.37 (1H, s,
(film, cm´1): 2920, 2851, 1738, 1467; 1H-NMR (400 MHz, CDCl3,
H-11), 6.39 (1H, s, H-21), 5.75 (1H, m, H-41), 5.36–5.34 (1H, m, H-91), 4.66–4.61 (2H, m, H-201), 4.37
(2H, m, H-1111), 4.33–4.10 (2H, m, H-1), 4.00–3.80 (2H, m, H-3), 3.98–3.75 (2H, m, H-2111), 3.65 (1H, m,
H-2), 3.51 (2H, m, H-111), 3.35 (9H, s, Me3N–), 2.05–1.40 (16H, m, H-51, 71, 81, 111, 121, 131, 141, 161, 171),
1.70/1.67 (3H, s, H-211), 1.54 (2H, m, H-211), 1.26 (30H, m, H-311-1711), 0.90 (3H, s, H-221), 0.88 (3H,
t, J = 6.8 Hz, H-1811), 0.87 (3H, s, H-241), 0.82/0.81 (3H, d, J = 6.9 Hz, H-231); 13C-NMR (100 MHz,
CDCl3,
δ
ppm): 154.5 (O–CO–O), 146.9 (C-191), 143.3 (C-11), 141.4/141.0 (C-101), 140.0 (C-251)1, 126.2
1
1
1
1
11
(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-2111), 64.4 (C-3), 59.7 (C-1111), 54.4 (Me3N-), 44.3 (C-51), 42.9 (C-151), 42.6/42.5 (C-111),
38.8/38.7 (C-141), 37.3/37.2 (C-161), 34.1 (C-61), 32.4/32.3 (C-171), 31.9 (C-1611), 31.2 (C-71), 29.9–29.3
(C-211, 411-1511), 28.19/28.8 (C-131), 25.9 (C-311), 23.2 (C-121), 22.8 (C-241), 22.7 (C-211), 22.6 (C-81), 22.6
11
1
11
(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
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
µL, 0.22 mmol)
´
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
1
yield 10 (6 mg, 30%). H-NMR (200 MHz1, CDCl3,
δ
ppm): 6.28/6.10 (1H, s, H-251), 5.91/5.83 (1H, m,
1
1
1
111
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,
111
11
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-51, 71, 81, 111, 121, 131, 141, 161, 171), 1.69 (3H, s, H-211), 1.54 (2H,
m, H-211), 1.25 (30H, m, H-311-1711), 0.87 (6H, s, H-221, 241), 0.88 (3H, t, J = 7.0 Hz, H-1811), 0.81 (3H, d,
J = 6.9 Hz, H-23 ); EIMS found for C51H90 NO11P [M + Na]+: 946.7.
1
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, 17410, 1560, 1383, 1261;
1 1
δ ppm): 7.43/7.42 (1H, s, H-25 ), 7.35/7.34 (1H, s, H-1 ), 6.39 (1H, s, H-2 ),
1H-NMR (400 MHz, CDCl3,
1
1
1
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-111), 2.84–2.80 (8H, m,
=CCH2C=), 2.35–2.30 (2H, m, H-2111), 2.14–2.04 (4H, m, H-4111, H-119111), 2.00–1.40 (16H, m, H-51, 71, 81,
1
1
1
1
1
1
111
11
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,
11
11
111
1
11
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-241), 0.81/0.80 (3H, 1d, J = 6.9 Hz, H-231); 13C-NMR (100 MHz, CDCl3,
δ
ppm): 173.1 (C-1111), 154.4
1
1
1
1
111
(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) ˆ 9
,
1
1
1
11
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
1
1
1
1
1
(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
1
1
111
1
11
1
11 11
11
1
111 111 111
111
111
111
111
11
1
1

Molecules 2016, 21, 47
17 of 25
(C-1711), 22.3 (C-241), 22.2 (C-211), 22.2 (C-221), 15.6 (C-231), 14.2 (C-20111), 14.0 (C-1811); 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-251), 6.03/6.02 (1H, s, H-21), 5.63 (1H, m,
H-41), 5.38–5.36 (10H, m, =CH), 5.38–5.36 (1H, m, H-91), 4.67–4.61 (2H, m, H-201), 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,1t1,1 J = 6.7 Hz, H-111), 2.84–2.78 (8H, m,
111
111
1
1
=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 ,
1
1
1
1
1
1
1
111
1
11
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-311-17111), 0.97 (3H, t, J = 7.5 Hz, H-20111), 0.91 (3H, s, H-221), 0.87 (3H, t, J = 6.8 Hz, H-1811), 0.88
111
δ ppm): 173.4 (C-1 ), 169.3
(3H, s, H-24 ), 0.81 (3H, d, J = 6.9 Hz, H-23 ); 13C-NMR (100 MHz, CDCl3,
(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)
1
1
1
1
1
111
ˆ
9,
1
1
1
1
11
1
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 (1C-3), 43.1 (C-51), 42.8 (C-151),141 2.6 (C-111), 38.7 (C-141), 37.4 (C-161), 34.6 (C-61), 13131 .51(11C-2111), 32.4
11
11
11
1
1
111
11
111
1
111
(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 ,
111
111
11
1
1
1
1
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.
1
111
11
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
22
purified by column chromatography on silica gel (EtOAc) to obtain 35 (3.6 g, 90%). rαsD
´
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-21, H-61), 6.89 (2H, d, J = 8.6 Hz, H-31, H-51), 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,
(–O H2Ar), 71.6 (C-sn3), 71.0 (C-sn2), 64.2 (C-sn1), 55.5 (–O
[M + Na]+: 235.0941, found: 235.0946.
δ
ppm): 159.6 (C-4111), 130.0 (C-1111), 129.7 (C-2111, C-6111), 114.1 (C-3111, C-5111), 73.4
H3). EIHRMS: calcd. for C11H16O4
C
C
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
22
by column chromatography (Hex/EtOAc 9:1) to obtain 36 (4.6 g, 92%). rαsD
´
0.7 (c 0.8, CHCl3); IR
(film, cm´1) 3449, 2932, 2870, 1512, 1491, 1448, 1248, 1076, 1034, 765, 706, 633; H-NMR (400 MHz,
CDCl3) δ
7.46–7.42 (6H, m, H-21, 61), 7.32–7.24 (9H, m, H-31-51), 7.22 (2H, d, J = 8.6 Hz, H-2111, H-6111),
1

Molecules 2016, 21, 47
18 of 25
111
111
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,
111 1
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 ),
111
111
111
1
1
1
1
1
111
111
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
22
98:2) to yield 37 (865 mg, 97%). rαsD
´
3.6 (c 1.2, CHCl3); 1H-NMR (400 MHz, CDCl3,
δ ppm): 7.47–7.44
(6H, m, H-21, 61), 7.31–7.24 (9H, m, H-31-51), 7.19 (1H, d, each, J = 8.6 Hz, H-2111, H-6111), 6.84 (1H, d,
each, J = 8.6 Hz, H-3111, H-5111), 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,
11
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,
ppm): 159.3 (C-4111), 144.4 (3 C-11), 130.8 (C-1111), 129.4 (C-2111, C-6111), 129.0/128.2 (3 C-31,
C-51), 128.0/127.5 (3 C-21, 3 C-61), 127.1 (3 C-41), 113.9 (C-3111, C-5111), 86.8 (–
Ph3), 78.6 (C-sn2),
73.1 (–O
H2Ar), 70.9 (C-111), 70.4 (C-sn3), 63.7 (C-sn1), 55.5 (–O H3), 32.2 (C-1611), 30.4–29.6 (C-211,
11
11
11
11
δ
¨
¨
3
¨
¨
¨
¨
C
C
C
411-1511), 26.4 (C-311), 22.9 (C-1711), 14.4 (C-1811); 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
22
chromatography (Hex/EtOAc 95:5) to yield 38 (464 mg, 82%). rαsD +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-2111, H-6111), 6.88 (2H, d, J = 8.6 Hz, H-3111, H-5111), 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-111), 1.56 (2H, m, H-211), 1.26 (30H, m, H-311-1711), 0.88 (3H, t, J = 6.8 Hz,
H-1811); 13C-NMR (100 MHz, CDCl3,
δ
ppm): 159.5 (C-4111), 130.3 (C-1111), 129.5 (C-2111, C-6111), 114.0
111
111
11
(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
11
11 11
11
11
11
11
(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;
111
111
111
,
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-5111), 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,
11
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 ),
11
11
11

Molecules 2016, 21, 47
19 of 25
13
0.88 (3H, t, J = 6.8 Hz, H-1811); C-NMR (100 MHz, CDCl3,
δ
ppm): 159.3 (C-4111), 150.7 (–O–
C
O–Cl),
111
111
111
111
111
129.8 (C-1 ), 129.3 (C-2 , C-6 ), 113.8 (C-3 , C-5 ), 75.8 (C-sn2), 73.1 (–O
CH2Ar), 71.1 (C-sn1), 70.8
11
11 11 11 11 11 11
(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.
11
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%).
22
rαsD
´
7.1 (c 0.8, CHCl3); IR (film, cm´1): 2924, 2853, 1745, 1514, 1250; 1H-NMR (400 MHz, CDCl3,
δ
1
1
111
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 ,
111
111
111
1
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-41), 5.35/5.33 (1H, t, J = 4.0 Hz, H-91), 4.65–4.60 (2H, m, H-201), 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-111), 2.00–1.49
(16H, m, H-51, 71, 81, 111, 121, 131, 141, 161, 171), 1.70/1.66 (3H, s, Me-211), 1.56 (2H, m, H-211), 1.25 (30H,
m, H-311-1711), 0.91 (3H, s, Me-221), 0.89 (3H, t, J = 7.0 Hz, Me-1811), 0.88 (3H, s, Me-241), 0.81/0.80 (3H,
d, J = 7.0 Hz, Me-231); 13C-NMR (50 MHz, CDCl3,
δ CO–O–),
ppm): 159.5/159.4 (C-4111), 154.8 (–O–
147.3 (C-191), 143.5 (C-11), 141.6/141.3 (C-101), 140.3 (C-251), 130.4/130.2 (C-1111), 129.5 (C-2111, 6111),
126.3 (C-31), 120.3/120.0 (C-91), 114.0 (C-3111, 5111), 109.4/109.3 (C-201), 109.0/108.9 (C-21), 76.6 (C-sn2),
73.3 (–OC
H2Ar), 71.0 (C-sn1), 70.3 (C-41), 69.3/69.0 (C-111), 68.5 (C-sn3), 55.5 (–OMe), 44.4 (C-51), 43.1
(C-151), 42.8/42.6 (C-111), 39.1 (C-141), 37.6/37.4 (C-161), 34.4/34.3 (C-61), 32.6 (C-171), 32.2 (C-1611),
30.2–29.0 (C-211), 129.6–28.9 (C-411-1511), 29.16–28.9 (C-71, 131), 26.2/26.1 (C-311), 23.1 (C-81, 121), 24.2
1
11
1
11
(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%). rαs2D2 +15.2 (c 1, CHCl3);
IR (film, cm´1)1: 3506, 2924, 2853, 1746, 1258; 1H-NMR (400 MHz, CDCl3,
(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
δ
ppm): 7.42 (1H, s, H-251),
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
1
1
1
1
11
1
1
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 ,
1
1
1
1
1
1
1
1
11
11
11
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
1
11
1
1
(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 );
1 1 1
δ ppm): 154.6 (–O–CO–O–), 147.0 (C-19 ), 143.2 (C-1 ), 141.4/141.1 (C-10 ),
13C-NMR (100 MHz, CDCl3,
1
1
1
1
1
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-111), 70.3 (C-41), 66.2/66.1 (C-sn1), 61.8 (C-sn3), 44.2 (C-51), 42.9 (C-151), 42.4/42.3 (C-111), 38.8
(C-11411), 37.3/37.2 (C-161),134.1/34.0 (C-611)1, 32.4/32.3 (1C-171), 31.91(C-1611), 29.9–28.8 (C-211), 29.19–28.8
11
1
1
11
(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.
1
1
11

Molecules 2016, 21, 47
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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-251), 7.34 (1H, broad s, H-11), 6.39 (1H, s, H-21), 5.79–5.76 (1H, m,
), 5.40–5.30 (1H, m, H-91), 4.65/4.61 (2H, m, H-201), 4.23–4.06 (2H, m,
H-41), 5.40–5.30 (10H, m, =C
H
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-111), 2.84–2.80 (8H, m,
=CCH2C=), 2.32 (2H, t, J = 7.5 Hz, H-2111), 2.13–2.06 (4H, m, H-4111, 19111), 2.04–1.40 (16H, m, H-51, 71,
81, 111, 121, 131, 141, 161, 171), 1.82–1.79 (2H, m, H-3111), 1.69/1.66 (3H, s, Me-211), 1.56 (2H, m, H-211),
1.25 (30H, m, H-311-1711), 0.97 (3H, t, J = 7.6 Hz, H-20111), 0.89 (3H, s, Me-221), 0.88 (3H, t, J = 7.0 Hz,
Me-1811), 0.88 (3H, s, Me-241), 0.80 (3H, d, J = 6.8 Hz, Me-231); 13C-NMR (50 MHz, CDCl3,
δ ppm):
173.4 (C-1111), 154.7 (–O–
C
O–O–), 147.3 (C-191), 143.5 (C-11), 141.6/141.3 (C-101), 140.4 (C-251), 132.3
(C-18111), 129.1–127.2 (=CH)
ˆ
9, 126.2 (C-31), 120.3/120.0 (C-91), 109.4/109.3 (C-2011), 108.9 (C-21), 75.2
11
1
1
1
(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-141), 37.6/37.4 (C-161), 34.4/34.3 (C-61), 33.7 (C-2111), 32.7/32.6 (C-171), 32.2 1(1C1 -1611), 30.6
1
11
11
11
1
111 111 111
111
111
(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 ,
19111, 111711, 81, 121), 25.8 (C-311), 23.9 (C-241), 22.9 (C-211), 22.5 (C-221), 15.9 (C-231), 14.5 (C-20111), 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,
5.64–5.58 (1H, m, H-41), 5.38–5.34 (10H, m, =C
δ
ppm): 6.19/5.95 (1H, m, H-25 ), 6.02/6.01 (1H, s, H-2 ),
), 5.38–5.34 (1H, m, H-91), 4.67–4.63 (2H, m, H-201),
1
1
H
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-111), 2.85–2.78
(8H1, m, =CCH2C=), 2.34 (2H, t, J = 7.2 Hz, H-2111), 2.13–2.04 (4H, m, H-4111, 19111), 2.00–1.49 (16H, m,
1
1
1
1
1
1
1
1
111
1
11
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-311-1711), 0.97 (3H, t, J = 7.6 Hz, H-20111), 0.91 (3H, s, Me-221), 0.87 (3H, t, J = 6.8 Hz,
Me-1811), 0.87 (3H, s, Me-241), 0.80 (3H, d, J = 6.9 Hz, Me-231); 13C-NMR (100 MHz, CDCl3,
173.3 (C-1111), 169.0 (C-11), 167.5 (C-31), 154.2 (O–CO–O), 147.2 (C-191), 141.4 (C-101), 132.0 (C-18111),
δ ppm):
128.9–127.0 (=CH)
ˆ
9, 120.0 (C-91), 118.4 (C-211), 109.2 (C-201), 97.4 (C-251), 76.5 (C-sn2), 70.8 (C-111),
70.8 (C-41), 66.5 (C-sn1), 62.3 (C-sn3), 43.1 (C-5 ), 42.8 (C-151), 42.3 (C-111), 38.4 (C-141), 37.2 (C-161),
34.6 (C-61), 13113.5 (C-2111), 32.4 (C-171), 31.9 (C-1611), 29.8–29.3 (C-211, 4111-1511), 28.9 (C-131), 29.31(C-71),
111 111
111
111
1
111
111 11
111
1
1
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]+:
11
1
11
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

Molecules 2016, 21, 47
21 of 25
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
1
13 (40 mg, 63%). H-NMR (400 MHz, CDCl3,
δ
ppm): 7.38 (1H, s, H-25), 7.32 (1H, s, H-1), 6.35 (1H,
), 5.39–5.32 (1H, m, H-9), 4.64
s, H-2), 5.98/5.96 (1H, dd, J = 5.1, 3.4 Hz, H-4), 5.39–5.32 (10H, m, =C
H
(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-21), 2.12–2.03 (4H, m, H-41, 191), 2.03–1.30 (16H, m, H-5, 7, 8, 11, 12, 13, 14, 16, 17), 1.87–1.78 (2H, m,
H-31), 1.70/1.67 (3H, s, Me-21), 0.97 (3H, t, J = 7.5 Hz, H-201), 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-11), 147.0 (C-19),
9, 126.7 (C-3), 119.9/119.8
143.0 (C-1), 141.3 (C-10), 140.0 (C-25), 132.0 (C-181), 129.0–127.0 (=CH)
ˆ
(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-21), 32.3 (C-17), 29.7 (C-7), 29.6 (C-13), 28.9–20.5 (31, 41, 71, 101, 131, 161,
191), 22.8 (C-24), 22.7 (C-8, 12), 22.3 (C-21), 22.3 (C-22), 15.6 (C-23), 14.2 (C-201); 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,
H-2), 5.58 (1H, d, J = 9.0 Hz, H-4), 5.44–5.32 (10H, m, =C
δ
ppm): 6.19 (1H, m, H-25)/5.96 (1H, s, H-25), 5.99/5.93 (1H, s,
), 5.44–5.32 (1H, m, H-9), 4.66 (1H, broad s,
H
1
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-41, 191), 2.06–1.30 (16H, m, H-5, 7, 8, 11, 12, 13, 14, 16, 17), 1.89–1.83 (2H, m, H-31), 1.70 (3H, s,
Me-21), 0.97 (3H, t, J = 7.5 Hz, H-201), 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-11), 169.5 (C-1), 168.7 (C-3), 147.3 (C-19), 141.4
1
(C-10), 132.3 (C-18 ), 129.5–127.2 (=CH)
ˆ
9, 120.1 (C-9), 118.3 (C-2), 109.5 (C-20), 97.9 (C-25), 67.2 (C-4),
1
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 (31, 41, 71, 101, 131, 161, 191, 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.
1
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
biological antitumour activity; (b) bioconjugation of -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 -hydroxybutenolide 14, show the
γ-hydroxybutenolide unit increases the
γ
γ
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

Molecules 2016, 21, 47
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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 12–14 are available from the authors.
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