Synthesis and evaluation of polyunsaturated fatty acid-phenol conjugates as anti-carbonyl-stress lipophenols

Article abstract of DOI:10.1002/ejoc.201402282

Carbonyl and oxidative stress play a substantial role in various neurodegenerative diseases such as Alzheimer's Disease, Parkinsonism, and Age-related Macular Degeneration (AMD). In retinal pathologies, both mechanisms are involved in the transformation of all-trans-retinal (AtR, reactive aldehyde) into bis-retinoid A2E. Since an accumulation of AtR and A2E contributes to photoreceptor apoptosis, we designed and synthesized a series of O-alkylated resorcinol derivatives featuring enhanced anti-carbonyl-stress properties. Additionally, these phenolic structures are linked to a polyunsaturated fatty acid such as docosahexaenoic acid (C22:6 n-3; DHA) or to a lysophosphatidylcholine-DHA conjugate, in order to specifically increase their bioavailability, and thus, to target the retina. Selective syntheses of phloroglucinol-DHA, resveratrol-DHA, and phloroglucinol-DHA-PC (PC = phosphatidylcholine) conjugates using silyl protecting group strategies are presented, along with results of testing that demonstrate their ability to lower AtR toxicity in ARPE-19 cell lines. In order to use resorcinol derivatives to fight against the carbonyl-stress mechanism observed in retina pathologies, new lipophenol conjugates were synthesized with the aim of increasing bioavailability and reaching retina tissue. Strategies to link polyunsaturated fatty acids to polyphenol backbones are presented, along with the results of testing against retinal pigment epithelial cell lines. Copyright

Full text of DOI:10.1002/ejoc.201402282

FULL PAPER
DOI: 10.1002/ejoc.201402282
Synthesis and Evaluation of Polyunsaturated Fatty Acid–Phenol Conjugates as
Anti-Carbonyl-Stress Lipophenols
Céline Crauste,[a] Claire Vigor,[a] Philippe Brabet,[b] Madeleine Picq,[c] Michel Lagarde,[c]
Christian Hamel,[b] Thierry Durand,[a] and Joseph Vercauteren*[a]
Keywords: Medicinal chemistry / Drug design / Fatty acids / Lipids / Polyphenols / Carbonyl stress
Carbonyl and oxidative stress play a substantial role in vari-
ous neurodegenerative diseases such as Alzheimer’s Disease,
Parkinsonism, and Age-related Macular Degeneration
(AMD). In retinal pathologies, both mechanisms are involved
in the transformation of all-trans-retinal (AtR, reactive alde-
hyde) into bis-retinoid A2E. Since an accumulation of AtR
and A2E contributes to photoreceptor apoptosis, we de-
signed and synthesized a series of O-alkylated resorcinol de-
rivatives featuring enhanced anti-carbonyl-stress properties.
Additionally, these phenolic structures are linked to a poly-
unsaturated fatty acid such as docosahexaenoic acid (C22:6
n-3; DHA) or to a lysophosphatidylcholine–DHA conjugate,
in order to specifically increase their bioavailability, and thus,
to target the retina. Selective syntheses of phloroglucinol–
DHA, resveratrol–DHA, and phloroglucinol–DHA–PC (PC =
phosphatidylcholine) conjugates using silyl protecting group
strategies are presented, along with results of testing that
demonstrate their ability to lower AtR toxicity in ARPE-19
cell lines.
thesis occurs when molecules of all-trans-retinal (AtR),
rather than undergoing detoxification to give retinol, ac-
cumulate and react with phosphatidylethanolamine (PE) by
a dual mechanism involving a combination of carbonyl and
oxidative stress (COS; Scheme 1).[8,9] This reactive aldehyde
may itself also contribute to retinal dystrophy through a
direct toxic effect.[10,11] Thus, anti-COS derivatives, capable
of detoxifying the main carbonyl stressor, AtR, may reduce
A2E formation and slow down the pace of lipofuscin depo-
sition.
Recent literature has addressed the ability of (poly)phen-
ols, which are known to be potent antioxidants,[12,13] to trap
reactive toxic electrophilic carbonyl entities, showing them
to be potent anti-carbonyl stressors.[14–16] One such com-
pound is phloroglucinol, a monomer of the phlorotannin
pigments abundant in brown algae; this agent has some
clinical application as a spasmolytic agent, and it can re-
duce oxidative-stress damage in cultured cells.[17,18] An im-
portant role has also been attributed to phloroglucinol as a
scavenger under physiological conditions of common carb-
onyl stressors: α,β-unsaturated aldehydes [such as 4-HNE
(4-hydroxynonenal) or acrolein][19] and α-dicarbonyl com-
pounds (methylglyoxal).[20] A major disadvantage of such a
compound for the treatment of retinal disorders is its poor
lipid solubility and low bioavailability.
Introduction
Reactive carbonyl species, such as sugars, α-dicarbonyls,
or metabolites derived from lipid oxidation, are involved in
glycation and cross-linking reactions of nucleophiles, and
thus they affect cellular viability. Aging-associated disorders
like age-related macular degeneration (AMD),[1,2] but also
other neurodegenerative diseases, such as Alzheimer’s Dis-
ease and Parkinsonism,[3] result from carbonyl and oxidat-
ive stress. Therefore, the discovery of agents that are dual
scavengers of carbonyl compounds and oxidative stressors
(anti-COS) may lead to promising therapeutic strategies.
Retinal pathologies (such as AMD) are a major public
health issue in the world. Suggestive evidence gathered over
a number of years has implicated the retinal pigment epi-
thelial (RPE) product lipofuscin in the etiology of atrophic
AMD and genetic macular degeneration (e.g., Stargardt
disease).[4–6] Major constituents of RPE lipofuscin are the
bisretinoid conjugate A2E, its photoisomers, and oxidized
metabolites (epoxides, furanoid oxides, cyclic peroxides, 4-
hydroxy-trans-2-nonenal, etc.).[7] Pathological A2E biosyn-
[a] Institute of Biomolecules Max Mousseron (IBMM),
UMR5247-CNRS-UM1-UM2-ENSCM, Faculty of Pharmacy,
15 av. C. Flahault 34093 Montpellier, France
E-mail: jvercauteren@univ-montp1.fr
http://www.ibmm.univ-montp1.fr
[b] Institute for Neurosciences of Montpellier, INSERM U1051-
UM1 – UM2,
Similarly to several other research groups working on
natural polyphenols,[15,16,21–23] our strategy to improve the
lipophilicity and enhance the cell penetration, relies on two
chemical modifications of the resorcinol core: etherification
of the phenolic functional group(s) in order to modulate
their nucleophilicity, and attachment of a lipid moiety. We
34295 Montpellier, France
[c] Université de Lyon, INSERM UMR 1060 (CarMeN), INSA-
Lyon, IMBL,
69621 Villeurbanne, France
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201402282.
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Polyunsaturated Fatty Acid–Phenol Conjugates
Scheme 1. How anti-COS could be active to reduce macular degeneration.
chose to use docosahexaenoic acid (C22:6 n-3, DHA) as carbonyl stress. These cultured-cell experiments led to the
the lipid part. This has commonly been used in the design identification of a potent derivative that can significantly
of new conjugated drugs.[23–27] In our case, we propose that increase cell survival compared to the unmodified phloro-
it will improve selectivity for the retina, since the retinal glucinol nucleus.
content of this polyunsaturated fatty acid (PUFA) is dispro-
portionately high.[28]
Results and Discussion
According to the results of several physiological studies,
a lysophosphatidylcholine–DHA conjugate [1-lyso-2-DHA-
PC, glycerophosphatidylcholine, in which only one hydroxy
group at the 2-position (sn-2) of the glycerol backbone is
Phloroglucinol as a Carbonyl Scavenger
We started by checking the assumption that phloroglu-
acylated by DHA] may facilitate transport of DHA to the cinol could trap the type of carbonyl stressors involved in
brain or retina.[29,30] Therefore this lysophospholipid conju- retinal disorders. We found that a chromene adduct A
gate will also be considered as a lipid vector. In this paper, (Scheme 2) could be formed when phloroglucinol is treated
we describe the first synthesis of these new lipophenols, and with an equimolar amount of AtR. This 2H-chromene A is
evaluate their ability to protect ARPE-19 cell lines from clearly the result of first, a 1,2-C-addition of the free carbon
Scheme 2. Formation of chromene A by reaction of phloroglucinol with trans-retinal.
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atom of the resorcinol framework onto the carbonyl group bone linked to DHA, to evaluate a possible protective effect
of AtR, followed by an intramolecular O-addition onto the against AtR-induced carbonyl stress in RPE cells.
double bond of the allylic benzyl alcohol. The alternative
4H-chromene regioisomer is not seen, probably due to the
hindered β-carbon of the double bond that precludes
Michael-type 1,4-addition. Furthermore, when 2H-
Synthesis of DHA–Phloroglucinol Conjugates
chromene A is formed, theoretically, it could react further
The DHA used in the following synthesis was extracted
to scavenge at least one more molecule of AtR. However, from cod liver oil using a process developed to concentrate
neither the symmetrical nor the unsymmetrical diadducts PUFAs starting from fish or algal oil.[31] The process was
could be seen. Presumably, this is related to the incorpora- carried out in a single step: fish liver oil was saponified in
tion of the polyisoprenic side-chain, which must make it the presence of NaOH, and free fatty acids were extracted
too hydrophobic to remain in the ethanolic phase.
using liquid–liquid extraction, and separated from the unsa-
With this background information, we synthesized vari- ponifiable material. Subsequently, monounsaturated fatty
ous polyphenol conjugates containing a resorcinol back- acids were removed by urea complexation,[32] based on the
Scheme 3. Synthesis of mono- 6, di- 7, and tri-8 DHA–phloroglucinol conjugates.
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Polyunsaturated Fatty Acid–Phenol Conjugates
difference in the spatial configuration of fatty acids accord- explained as being due to steric hindrance, or to the lower
ing to their degree of unsaturation. This process led to a reactivity of phloroglucinol compared to its silylated deriva-
crude mixture enriched in DHA and EPA (eicosapentaenoic tives.
acid, C20:5 n-3), which after purification on reverse
phase[33] yielded 5% of DHA (starting from cod liver oil)
with a purity of 85% [the impurities consisted of monoun-
saturated fatty acids such as palmitoleic (C16:1 n-7) and
Synthesis of Alkylated DHA–Phloroglucinol Conjugates
oleic (C18:1 n-9) acids]. Direct coupling of phloroglucinol
Alkylation, such as methylation, is a major metabolic
with DHA using the classical coupling reagent dicyclohex- route for dietary (poly)phenols after ingestion.[36] Therefore,
ylcarbodiimide (DCC), led to a mixture of mono-, di-, and it is of interest to evaluate the influence of O-alkylation of
trisubstituted conjugates, because of the increasing reactiv- the phenol group on the efficiency of carbonyl trapping. In
ity of the three hydroxy groups upon esterification. The particular, in the reaction to form chromene from phloro-
yield of the monoacylation was very poor (Ͻ10%), despite glucinol and trans-retinal, C-alkylation may be influenced
using 1 equiv. of DHA. Since this single-step strategy could by the introduction of an alkyl substituent. We hypothesize
not be used for the efficient preparation of lipophenol struc- that by increasing the electron density of one of the oxygen
tures (with minimal consumption of DHA), we developed atoms, due to an additional inductive electronic effect, the
a strategy based on protection/deprotection of the hydroxy electron density in the ring could be enhanced, due to the
groups of phloroglucinol.
mesomeric effect, leading to improved C-alkylation. To
To obtain phloroglucinol–DHA conjugates in acceptable assess this effect on reactivity, we prepared a series of meth-
yield, a silyl protecting group, triisopropylsilyl (TIPS), was ylated and isopropylated phloroglucinol–DHA conjugates.
selected as it can be efficiently deprotected under mild con-
The synthesis of monoalkylated and monoprotected
ditions in the presence of ester linkages.[34] Diprotected phloroglucinol 11 was designed to allow the introduction of
phloroglucinol 2 (Scheme 3) was prepared in an original the lipid moiety at the last step of the synthesis (Scheme 4).
way, starting with the total protection of phloroglucinol 1 The difficulty was to perform a selective monoalkylation
using TIPS-OTf (3 equiv.) in THF, followed by a slow and/or monosilylation of the symmetrical phloroglucinol in
monodesilylation using triethylamine trihydrofluoride the presence of phenol groups with identical reactivities.
(Et3N·3HF; 3 equiv.) for 4 h. This process allowed us to Using a solution of MeOH[37] (for 9a) or iPrOH (for 9b)
prepare intermediate 2 in 59% overall yield over two steps saturated with HCl gas, the monoalkylation could be car-
(only 30% could be obtained by a direct disilylation pro- ried out in an acceptable yield starting from phloroglucinol.
cess).[35] When the reaction time was increased (from 4 h to The yield was optimised by lowering the proportion of dial-
8 h), using the same amount of Et3N·3HF, monoprotected kylated derivatives formed using a typical O-alkylation rea-
derivative 3 was obtained in 62%. An attempted controlled gent such as dimethylsulfate or 2-bromopropane.[38] Total
monosilylation (1 equiv. TIPS-OTf, 0 °C) gave no more protection of 9 with the TIPS protecting group (to give 10),
than 38% yield. Next, the coupling reactions were carried followed by monodeprotection, gave the desired monome-
out using classical DCC/DMAP (4-dimethylaminopyridine) thylated/monoisopropylated and protected phloroglucinols
reagents, and gave DHA conjugates 4 and 5 in 74 and 66% 11 in two steps in yields of 58% for the methylated com-
yields, respectively. Deprotection of the TIPS groups was pound and 54% for the isopropylated compound. Less
carried out using Et3N·3HF in THF at room temperature, Et3N·3HF than was used for deprotection of 1 was needed
and gave lipophenols 6 and 7 in 78 and 86% yields, respec- to obtain Ͼ60% yield, starting from alkylated phenolics 10.
tively, without cleavage of the ester linkage or degradation It should be noted that a direct one-step monosilylation of
of the polyunsaturated moiety.
9a could be achieved using TIPS-OTf at 0 °C, but the yield
Tri-DHA conjugate 8 was obtained in a much better was only 25%.
yield starting from di-DHA–phloroglucinol conjugate 7
Using the same coupling conditions (DCC/DMAP) as
(73%) than from direct coupling of phloroglucinol with for lipophenol 6, monoalkyl phloroglucinols 11 were cou-
DHA (3 equiv.) (only 8%). The low coupling yield can be pled to DHA (Scheme 5). The alkylated phloroglucinol–
Scheme 4. Synthesis of monomethylated and monoprotected phloroglucinols 11.
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Scheme 5. Synthesis of alkylated DHA–phloroglucinol conjugates 13 and 15.
DHA conjugates (i.e., 13) were produced in excellent to subjected to TIPS deprotection to give resveratrol–DHA
moderate yields, after removal of TIPS groups in the pres- conjugate 20 in 20% overall yield.
ence of Et3N·3HF. Dialkylated derivatives 15 were obtained
in one step starting from commercially available 14a and
diisopropyl-phloroglucinol 14b.[38]
Synthesis of DHA–Resveratrol Conjugate
As shown in Scheme 2, the trapping mechanism of a
carbonyl stressor (AtR) by phloroglucinol requires the pres-
ence of a resorcinol pattern on the phenolic backbone. Such
a framework is found in many naturally occurring poly-
phenols, including flavonoids, stilbenoids, and, especially,
resveratrol (Scheme 6). The latter highly conjugated com-
pound is a vinyl homologue of phloroglucinol, and is well-
suited for our purpose. Because of the importance of the
reactivity of resorcinol for scavenging AtR, we decided to
link the DHA moiety to the hydroxy group at the 4Ј-posi-
tion of resveratrol, and leave the 3- and 5-hydroxy groups
free to form the chromene derivative. Compared to the
phloroglucinol series, the strategy envisaged for the synthe-
sis of the stilbenoid presents additional difficulties; it re-
quires selective protection of the phenol at the 3- and 5-
positions, but the three hydroxy groups of resveratrol have
similar reactivities. To overcome this drawback, resveratrol
was regioselectively acylated using Candida antartica lipase
B (CALB or Novozyme 435), which catalyses esterification
at the 4Ј-position with high selectivity.[39,40] By carrying out
Scheme 6. Synthesis of resveratrol–DHA conjugate 20.
Synthesis of 1-Phloroglucinol-2-DHA–
Glycerophosphatidylcholine Conjugate
DHA could accumulate in the retina and the brain
this enzymatic reaction in the presence of vinyl acetate, we through the specific uptake of DHA-containing lysophos-
were able to isolate a single protected product, 4Ј-O-acetyl- phatidylcholine [1-Lyso-2-DHA-PC (PC = phosphatidyl-
resveratrol 16, in 57% yield. The hydroxy groups at the 3- choline) or LysoPCDHA 21].[29,30] Since the DHA at the sn-
and 5-positions were then protected using TIPS-OTf to give 2 position of LysoPCDHA is considered to be the physio-
17, which was subjected to acetyl deprotection in the pres- logical form of this polyunsaturated LysoPC, the coupling
ence of a methanolic solution of NaOMe. The deacetylated reaction of the phenolic moiety was carried out at the sn-1
product was obtained in excellent yield. 4Ј-Deprotected position. The challenge in the synthesis of such phospholip-
resveratrol 18 was then coupled to DHA (to give 19), simi- ids is in avoiding internal transesterification from the sn-2
larly to the phloroglucinol series. Compound 19 was then to the sn-1 position when the sn-1 position remains free.
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Polyunsaturated Fatty Acid–Phenol Conjugates
The chemical approach to obtaining a phloroglucinol–PC– crude material revealed that under these conditions, only a
DHA conjugate was based on the synthesis of sn-1-substi- slight migration was observed after a reaction time of 29 h
tuted LysoPCDHA 21. This lysolecithin appears to be a (see Supporting Information). Moreover, HMBC NMR
suitable intermediate to access the desired lipophenol by spectroscopic analysis of isolated 21 confirmed the location
chemical coupling with a phloroglucinol bearing an appro- of the DHA moiety at the sn-2 position by the presence of
priate acid linker (22 or 23, Scheme 7). In previous research, a coupling between the carbon of the carboxylic group of
we observed that the immobilized enzyme, lipozyme, from DHA and the CH proton of the glycerol moiety. With
Mucor miehei, was able to regioselectively hydrolyse fatty LysoPC-DHA 21 in hand, we attempted to link succinyl
acids at the sn-1 position of various phosphatidylcholines. phloroglucinol 22 at the sn-1 position using DCC and
This lipase, which acts specifically at the sn-1 and sn-3 posi- DMAP. A short succinyl linker was chosen to link the li-
tions, was tested to quantitatively obtain compound 21 pidic and phenolic parts, so as to retain the hydrophilic
from commercially available PC-16:0/DHA, a phosphatid- properties at the sn-1 position, to limit the lipophilicity, and
ylcholine having DHA at the sn-2 position and palmitic to mimic as far as possible the physiological vector
acid at the sn-1 position (Scheme 7). The enzymatic hydrol- LysoPCDHA. Unfortunately, the coupling reaction led to
ysis was optimised by replacing the initially tested solvent the unexpected cleavage of the succinyl ester bond, and only
(toluene) by a mixture of EtOH and H2O (95:5). Using traces of succinyl-DHA–phosphatidylcholine devoid of the
200% (w/w) of enzyme and a reaction time of 40 h, the phenol moiety were identified by mass spectrometry. The
reaction led to a 50% yield of purified LysoPC-DHA 21. lability of the succinyl linker, which regenerated succinic an-
When another portion of enzyme was added after 9 h, and hydride under acidic or basic conditions, prompted the use
the reaction time was decreased to 29 h, the yield of of a glutaryl linker. This linker was introduced onto the
LysoPC-DHA increased to 85%. It appears that a longer phloroglucinol backbone using glutaric anhydride in the
reaction time facilitates the migration of the DHA to the presence of DMAP (to give 23, Scheme 7). The coupling
sn-1 position to form 1-DHA-2-LysoPC, which, in turn, be- step gave phospholipid conjugate 24 (65% yield), whose
comes a substrate for the lipozyme, resulting in the regener- TIPS groups were then deprotected. This convergent se-
ation of glycerophosphocholine. HPLC monitoring of the quence of reactions led to the desired 1-glutaryl-phloroglu-
Scheme 7. Synthesis of phloroglucinol–DHA–PC conjugate 25.
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cinol-2-DHA-PC 25 in an overall yield of 20% (six steps), as % cell survival in treated (lipophenol/all-trans-retinal) vs.
starting from commercially available compounds.
untreated cells (taken as 100%). In this bioassay, phloroglu-
cinol showed moderate to weak activity, with a 10% in-
crease of cell survival at 40 μm (Table 1, entry 2), while the
resveratrol analogues were inactive. Introduction of one
DHA or one Lyso-PC-DHA onto the phloroglucinol ring
structure did not increase the cell survival rate. However,
increasing the number of DHA chains led to active deriva-
tives capable of enhancing cell survival to 50%, as observed
for 8 (with three DHA moieties). Mono-DHA-alkylated
lipophenol 13a showed comparable activity to 8. The great-
est enhancement of viability (70% survival) was observed
with 13b (monoisopropyl derivative), which showed dose-
dependent cytoprotection (Figure 2). Lower activities were
obtained with two alkyl substituents. To validate the ac-
tivity of lipophenol conjugate 13b, a mixture of DHA and
monoisopropyl phloroglucinol 9b was evaluated under the
same conditions (pretreatment design), and this mixture
was found to cause toxicity comparable to that observed
with DHA alone (Table 1, entries 13 and 14). This result
lends strong support to the concept that one can obtain
potent cytoprotective derivatives by introduction of a
PUFA moiety onto the O-alkylated polyphenol backbone.
Effect of Lipophenol on all-trans-Retinal Toxicity in
Cultured ARPE-19 Cells
A possible protective effect of the synthesized derivatives
against all-trans-retinal toxicity[11] was evaluated in retinal
pigment epithelium cell cultures (ARPE-19 cell lines). The
bioassay method was based on the measurement of cell sur-
vival following challenge with the carbonyl stressor AtR at
a concentration of 25 μm. In control cell cultures, this con-
centration of AtR was shown to reduce cell survival by 60–
70%. The effects on cell viability of pre-treatment of the
cultured cells with lipophenols at 10 μm and 40 μm are
shown in Table 1 and Figure 1. The results are expressed
Table 1. ARPE-19 cell viability in the presence of lipophenol and
AtR.
Survival [%]
n
Untreated cells
AtR (25 μm)
100
29.1Ϯ5.4
7
Entry Compounds
Survival at
Survival at
n
n
10 μm [%][a]
40 μm [%][a]
1
2
3
4
5
6
7
8
phloroglucinol
35.5Ϯ3.3
25.8Ϯ3.7
32.1Ϯ7.1
33.3Ϯ4.1
28.8Ϯ5.8
30.1Ϯ4.5
32.6Ϯ3.7
26.5Ϯ4.5
43.2Ϯ7.5
31.9Ϯ1.3
28.2Ϯ2.4
30.6Ϯ1.2
23.5Ϯ2.7
29.0Ϯ1.7
41.6Ϯ9.2
21.9Ϯ3.8
37.4Ϯ2.4
47.7Ϯ3.2
31.0Ϯ9.4
32.1Ϯ7.3
44.9Ϯ1.9
26.2Ϯ2.5
66.8Ϯ8.6
45.7Ϯ3.3
20.1Ϯ2.0
32.7Ϯ1.8
7.3Ϯ5.0
3
3
3
3
4
4
3
3
4
3
3
3
3
3
6
7
8
resveratrol
20
13a
15a
9
13b
10
11
12
13
14
15b
25
9b
Figure 2. Dose-response activity of compound 13b; ARPE-19 cell
survival in the presence of AtR (25 μm).
DHA
DHA + 9b
10Ϯ2.6
To explain the protection conferred by 13b compared to
the phloroglucinol-monoisopropyl compound (Table 1, en-
tries 9 and 12), one could argue that the increased lipophil-
icity facilitates membrane penetration and/or that the DHA
[a] Incubation of lipophenols 1 h followed by AtR incubation at
25 μm for 4 h. Cell viability is measured by MTT (dimethylthiazo-
lyldiphenyltetrazolium bromide) assay after 16–20 h. Each experi-
ment was carried out in triplicate.
Figure 1. Protective effect of lipophenols on ARPE-19 cell line in the presence of AtR (25 μm).
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Polyunsaturated Fatty Acid–Phenol Conjugates
fatty acyl chain may help to orientate the phloroglucinol carbonyl scavenging activity in the same molecule may have
moiety and bring it into close proximity to retinal, promot- therapeutic applications in the treatment of retinal and neu-
ing the formation of the chromene adduct. However, this rodegenerative disorders.
increase in reactivity is not observed with the addition of a
single DHA chain on the phloroglucinol derivative (Table 1,
entries 1 and 2). Moreover, although DHA–phloroglucinol
conjugate 8 confers significant protection, this compound
Experimental Section
Synthesis of Lipophenols
would require at least one intracellular hydrolysis to take
place before the chromene adduct can be formed. Thus, the
details of the cytoprotection mechanism need further study.
Whether the protection involves a direct reaction with
trans-retinal or indirect influences on cellular enzymatic
systems remains an open question.
Obtaining DHA from Cod Liver Oil: Commercially sourced cod
liver oil (5 g, Cooper, France) was dissolved in a mixture of ethanol
and water (95:5; 35 mL) in the presence of NaOH (1.50 g) under
an argon atmosphere. The mixture was protected from the light
with foil, and heated at 82 °C for 2 h. The ethanolic fraction was
evaporated, and the residue was dissolved in hexane (30 mL) after
heating. Then water (25 mL) was added to the organic layer, and
unsaponifiable material was removed by repeated hexane extraction
(4ϫ 30 mL) of the aqueous phase. The aqueous phase containing
the soaps was acidified to pH 2 using HCl solution (50%). The
fatty acids were extracted with hexane (4ϫ 25 mL). The organic
phase was concentrated under reduced pressure to give the crude
fatty acids (4.62 g) as an oil. Urea (13.86 g) and ethanol (95%;
55 mL) were added to the crude residue. The mixture was heated
at 60–70 °C, protected from the light, until it turned into a clear
homogeneous solution. The mixture was then returned to room
temperature, and then cooled to 4 °C for 24 h. The resulting crys-
tals were separated from the liquid by filtration. The filtrate was
diluted with water (35 mL), and acidified to pH 4–5 with HCl solu-
tion (6 n). Hexane (70 mL) was added, and the solution was stirred
thoroughly for 1 h. The hexane layer containing the liberated fatty
acids was separated from the aqueous layer, and washed with water
(3ϫ 40 mL). The organic phase was dried with Na2SO4, and con-
centrated under reduced pressure to give a crude mixture of PUFAs
(820 mg), which was purified by preparative HPLC [column Atlan-
tis Prep OBDTM 10 μm (19ϫ250 mm), H2O/MeOH, 13:87 iso-
cratic, detection 217 nm] to give pure DHA (266 mg, 5% w/w). 1H
NMR (500 MHz, CDCl3): δ = 5.43–5.30 (m, 12 H, CH=CH), 2.85–
2.80 (m, 10 H, CH2 bis-allylic), 2.42–2.40 (m, 4 H, CH2-C=O, CH2
allylic), 2.07 (quint, J = 7.5 Hz, 2 H, CH2 allylic), 0.98 (t, J =
7.5 Hz, 3 H, CH3) ppm. MS (ESI): m/z = 327 [M – H]–.
Conclusions
We have developed efficient synthetic routes to conju-
gates of phenolic derivatives with DHA, an n-3 PUFA. The
phloroglucinol–DHA series of eight new compounds relies
on an original selective protection process using TIPS pro-
tecting groups. The synthesis of lipophenols bearing the
resveratrol motif was achieved through chemical protection
as well as enzymatic means and control of reaction condi-
tions, which allowed the introduction of DHA specifically
at the 4Ј-position. Finally, the introduction of phloroglu-
cinol at the sn-1 position of a DHA–glycerophosphocholine
conjugate was realized for the first time, using an efficient
enzymatic route to LysoPC-DHA and the selection of an
appropriate glutaryl linker.
The protective effect of the lipophenol derivatives was
evaluated in cultured retinal epithelial cells challenged with
a toxic concentration of trans-retinal. One of the com-
pounds showed a significant cytoprotective effect, increas-
ing cell survival by up to 40% compared to untreated cells
(13b at 40 μm). The importance of the lipid portion for the
activity of this derivative was confirmed by comparison 2-Methyl-2-[(1E,3E,5E)-4-methyl-6-(2,6,6-trimethylcyclohex-1-
enyl)-hexa-1,3,5-trienyl]-2H-chromene-5,7-diol (Chromene A): trans-
Retinal (200 mg, 0.35 mmol) was dissolved in ethanol (8 mL), and
phloroglucinol (48.28 mg, 0.35 mmol) and acetic acid (40 μL,
0.35 mmol) were added to the stirred solution. The reaction mix-
ture was stirred at room temperature for 48 h, protected from light
with foil. The solvent was removed under reduced pressure, the
residue was dissolved in EtOAc (20 mL), and this solution was
washed with water (10 mL). The organic layer was dried with
MgSO4, and concentrated under reduced pressure. The residue was
purified by chromatography on silica gel (pentane/EtOAc, 90:10 to
with similar compounds lacking the lipid component. These
results are promising, especially in light of the work by
Maeda et al., who reported that 5-ASA (5-aminosalicylic
acid; 300 μm) increased cell survival by 43% in a similar
bioassay with ARPE-19 cells exposed to AtR.[41]
The mechanism of the cytoprotective effect of adding
DHA chains to the phloroglucinol ring structure remains
to be determined. The modification may enhance cell pene-
tration (by passive diffusion or active transport), or another
mechanism could be involved. This is currently under inves- 85:15) to give Chromene A (112.4 mg, 41%) as a solid contami-
nated with a by-product (13%). Chromene A was isolated after
purification by preparative HPLC to allow full characterization
(gradient of hexane/EtOAc, t0Ј = 100/0, t15Ј = 90/10, t45Ј = 80/20,
t7 5 Ј = 70/30; 15 mL/min, column Luna 5μ Silica 100A
250ϫ21.20 mm, detection 254 nm). Rf (CH2Cl2/MeOH, 95:5): 0.4.
1H NMR (500 MHz, [D4]methanol): δ = 6.63 (dd, J = 11.5, J =
15.5 Hz, 1 H, 11-H), 6.63 (d, J = 10.0 Hz, 1 H, 15-H), 6.14 (d, J =
16.5 Hz, 1 H, 7-H), 6.03 (d, J = 16.5 Hz, 1 H, 8-H), 5.98 (d, J =
11.0 Hz, 1 H, 10-H), 5.85 and 5.81 (d, J = 2.0 Hz, 1 H, and d, J =
2.5 Hz, 1 H, 18-H and 20-H), 5.75 (d, J = 15.0 Hz, 1 H, 12-H),
tigation in our laboratory, and the findings will be reported
in due course.
This work paves the way for the synthesis of new polyun-
saturated fatty acyl derivatives ester-linked to appropriate
phenolic ring structures. The results should be of interest to
researchers studying the role of oxidative and carbonyl
stress in various pathological conditions. According to a re-
cent report, dietary supplementation with high dose DHA
appeared to significantly improve the visual acuity of an
AMD patient.[42] New lipophenols that combine PUFA and 5.39 (d, J = 10.0 Hz, 1 H, 14-H), 2.02–2.00 [m, 2 H, 4-H (CH2)],
Eur. J. Org. Chem. 2014, 4548–4561
© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
4555

J. Vercauteren et al.
FULL PAPER
1.86 [s, 3 H, 25-H (CH3)], 1.68 [s, 3 H, 24-H (CH3)], 1.67–1.60 [m, as a white solid. Data for 2: Rf (pentane/EtOAc, 80:20): 0.8. 1H
2 H, 3-H (CH2)], 1.49 [s, 3 H, 26-H (CH3)], 1.48–1.46 [m, 2 H, 2-H NMR (500 MHz, CDCl3): δ = 6.03 (t, J = 2.0 Hz, 1 H, CHaro),
(CH2)], 1.00 [s, 6 H, 22-H, 23-H (CH3)] ppm. 13C NMR (125 MHz,
CDCl3): δ = 159.7 (C-19), 156.4 (C-21/C-17), 139.1 (C-8), 139.0 (C-
6), 137.1 (C-12), 137.0 (C-9), 130.4 (C-10), 129.9 (C-5), 127.6 (C-
7), 126.3 (C-15), 123.1 (C-14), 119.1 (C-11), 103.9 (C-16), 96.3 (C-
20/C-18), 78.5 (C-13), 40.7 (C-2), 35.1 (C-1), 33.9 (C-4), 29.3 (C-
22/C-23), 27.6 (C-26), 21.8 (C-24), 20.3 (C-3), 12.6 (C-25) ppm.
HRMS (ESI-TOF): calcd. for C26H31O3 [M – H]– 391.2278; found
391.2272. HPLC [Atlantis C18 5 μm (4.6ϫ250 mm), H2O (0.1%
CF3COOH)/MeCN, t0Ј = 25/75, t25Ј = 20/80, t28Ј = 0/100, t33Ј = 0/
100, detection 298 nm]: tR = 11.26 min.
6.01 (d, J = 2.0 Hz, 2 H, CHaro), 1.27–1.19 (m, 6 H, CH-Si), 1.09
[d, J = 7.3 Hz, 36 H, (CH3)2C] ppm. 13C NMR (125 MHz, CDCl3):
δ = 157.8, 157.0, 105.1, 101.1, 18.1, 12.8 ppm. HRMS (ESI-TOF):
calcd. for C24H45O3Si2 [M – H]– 437.2907; found 437.2911.
5-(Triisopropylsilyloxy)benzene-1,3-diol (3): Triprotected phloroglu-
cinol 1 (200 mg, 0.33 mmol) was dissolved in dry THF (12 mL).
Et3N·3HF (164 μL mL, 1 mmol) was added dropwise. The reaction
was followed by TLC, and it was stopped so as to limit as much
as possible the formation of the monodeprotected derivative. The
mixture was stirred for 6 h at room temperature, then further
Et3N·3HF (1 equiv.) was added. The reaction was stopped after
8 h. EtOAc (15 mL) was added to the mixture, and the organic
layer was washed with water (10 mL) and brine (10 mL). The or-
General Procedure for Coupling of DHA with Polyphenolic Deriva-
tives: DHA (1.1 equiv., 0.30 mmol) and each of the phenolic deriva-
tives (1.0 equiv., 0.27 mmol) were dissolved in dry CH2Cl2 (6 mL).
DCC (1.1 equiv., 0.30 mmol) and DMAP (0.1 equiv., 0.03 mmol) ganic phase was dried (MgSO4), and concentrated under vacuum.
were added, and the reaction mixture was stirred at room tempera-
ture for 5 h under nitrogen. The mixture was left at 4 °C for 2 h to
induce crystallization of dicyclohexylurea. The urea residue was
then removed by filtration, and the filtrate was washed with water
and brine. The organic layer was dried with MgSO4, and concen-
trated under reduced pressure. Purification of the crude material
by chromatography on silica gel gave the desired lipophenol.
The residue was purified by chromatography on silica gel (pentane/
EtOAc, 95:5) to give monoprotected phloroglucinol 3 (59 mg, 62%)
as a white solid. Diprotected derivative 2 was isolated (20 mg, 13%)
as a yellow oil. Data for 3: Rf (hexane/EtOAc, 70:30): 0.34. 1H
NMR (500 MHz, CDCl3): δ = 5.99 (d, J = 2.0 Hz, 2 H, CHaro),
6.01 (t, J = 2.0 Hz, 1 H, CHaro), 1.27–1.18 (m, 3 H, CH-Si), 1.08
[d, J = 7.5 Hz, 18 H, (CH3)2C] ppm. 13C NMR (125 MHz, CDCl3):
δ = 158.2, 157.3, 100.5, 96.5, 18.0, 12.8 ppm. HRMS (ESI-TOF):
calcd. for C15H25O3Si [M – H]– 281.1573; found 281.1570.
General Procedure for Deprotection of the TIPS Protecting Group
from DHA–Polyphenol Derivatives: The protected DHA–pol-
yphenol (1.0 equiv., 0.19 mmol) was dissolved in anhydrous THF
(13 mL), and triethylammonium trihydrofluoride (Et3N·3HF;
3 equiv., 0.57 mmol for monoprotected compounds, or 6 equiv.,
1.14 mmol for diprotected compounds) was added dropwise. The
mixture was stirred at room temperature for 4 to 6 h, until the
reaction was complete. EtOAc (40 mL) was added to the mixture,
and the organic layer was washed with water (15 mL) and brine
(15 mL). The organic phase was dried (MgSO4) and concentrated
under vacuum. The residue was purified by chromatography on
silica gel to give the deprotected lipophenol.
(4,7,10,13,16,19Z)-3,5-Bis(triisopropylsilyloxy)phenyl-docosa-
4,7,10,13,16,19-hexaenoate (4): Coupling of di-TIPS-phloroglu-
cinol 2 (130 mg, 0.29 mmol) and DHA (104 mg, 0.32 mmol) was
carried out according to the general procedure to give 4 (164 mg,
74%) as a colourless oil after purification by silica gel chromatog-
raphy (hexane/EtOAc, 99.7:0.3 to 99:1). Rf (hexane/EtOAc, 99:1):
0.23. 1H NMR (500 MHz, CDCl3): δ = 6.29–6.27 (m, 1 H, CHaro),
6.25–6.24 (m, 2 H, CHaro), 5.45–5.30 (m, 12 H, CH=CH), 2.87–
2.81 (m, 10 H, CH2 bis-allylic), 2.57 (t, J = 7.3 Hz, 2 H, CH2-
C=O), 2.52–2.48 (m, 2 H, CH2 allylic), 2.07 (quint, J = 7.5 Hz, 2
H, CH2 allylic), 1.26–1.18 (m, 6 H, CH-Si), 1.08 [d, J = 7.5 Hz, 36
H, (CH3)2C], 0.97 (t, J = 7.5 Hz, 3 H, CH3) ppm. 13C NMR
(125 MHz, CDCl3): δ = 171.1, 157.1, 151.8, 132.0, 129.6, 128.6,
128.3, 128.3, 128.2, 128.1, 128.1, 128.0, 127.9, 127.0, 109.2, 106.9,
34.4, 25.6, 25.5, 22.7, 20.6, 17.8, 14.2, 12.7 ppm. HRMS (ESI-
TOF): calcd. for C46H77O4Si2 [M + H]+ 749.5354; found 749.5363.
1,3,5-Tris(triisopropylsilyloxy)benzene (1): Phloroglucinol (1 g,
7.90 mmol) was dissolved in dry THF (60 mL), and triethylamine
(3.68 mL, 23 mmol) and triisopropylsilyl trifluoromethanesulfonate
(TIPS-OTf; 7 mL, 23 mmol) were added dropwise. The reaction
mixture was stirred at room temperature for 2 h. EtOAc (60 mL)
was added to the mixture, and the organic layer was washed with
water (40 mL) and brine (40 mL). The organic phase was dried
(MgSO4), and concentrated under vacuum. The residue was puri-
fied by chromatography on silica gel (hexane/EtOAc, 99:1) to give
triprotected phloroglucinol 1 (4.64 g, 98%) as a yellow oil. Rf (pent-
(4,4Ј,7,7Ј,10,10Ј,13,13Ј,16,16Ј,19,19ЈZ)-5-(Triisopropylsilyloxy)-1,3-
phenylene-didocosa-4,7,10,13,16,19-hexaenoate (5): Coupling of
mono-TIPS-phloroglucinol 3 (86 mg, 0.30 mmol) and DHA
(200 mg, 0.60 mmol) was carried out according to the general pro-
cedure to give 5 (183 mg, 66%) as a colourless oil after purification
1
ane): 0.28. H NMR (500 MHz, CDCl3): δ = 6.07 (s, 3 H, CHaro),
1.27–1.18 (m, 9 H, CH-Si), 1.09 [d, J = 7.3 Hz, 54 H, (CH3)2C] by silica gel chromatography (pentane/EtOAc, 98:2). Rf (pentane/
ppm. 13C NMR (125 MHz, CDCl3): δ = 157.4, 105.8, 18.1,
12.8 ppm. HRMS (ESI-TOF): calcd. for C33H67O3Si3 [M + H]+
595.4392; found 595.4395.
EtOAc, 98:2): 0.34. 1H NMR (500 MHz, CDCl3): δ = 6.51 (s, 3 H,
CHaro), 5.47–5.28 (m, 24 H, CH=CH), 2.87–2.80 (m, 20 H, CH2
bis-allylic), 2.60–2.57 (m, 4 H, CH2-C=O), 2.51–2.47 (m, 4 H, CH2
allylic), 2.07 (quint, J = 7.5 Hz, 4 H, CH2 allylic), 1.27–1.22 (m, 3
H, CH-Si), 1.08 [d, J = 7.5 Hz, 18 H, (CH3)2C], 0.97 (t, J = 7.5 Hz,
6 H, CH3) ppm. 13C NMR (125 MHz, CDCl3): δ = 170.9, 157.0,
151.4, 132.0, 129.7, 128.5, 128.3, 128.2, 128.2, 128.1, 128.0, 127.9,
127.8, 127.4, 127.0, 110.9, 108.0, 34.2, 25.6, 25.6, 25.5, 22.6, 20.5,
17.8, 14.2, 12.5 ppm. HRMS (ESI-TOF): calcd. for C59H87O5Si
[M + H]+ 903.6317; found 903.6321.
3,5-Bis(triisopropylsilyloxy)phenol (2): Triprotected phloroglucinol
1 (5.01 g, 8.43 mmol) was dissolved in dry THF (200 mL).
Et3N·3HF (2.90 mL, 17.70 mmol) was added dropwise, and the
mixture was stirred at room temperature for 4 h. The reaction was
followed by TLC, and it was stopped so as to avoid as much as
possible the formation of the dideprotected compound. EtOAc
(200 mL) was added to the mixture, and the organic layer was
washed with water (200 mL) and brine (100 mL). The organic
phase was dried (MgSO4) and concentrated under vacuum. The
residue was purified by chromatography on silica gel (pentane/
EtOAc, 95:5) to give diprotected phloroglucinol 2 (2.25 g, 60%) as
a yellow oil. Monoprotected derivative 3 was isolated (0.45 g, 18%)
(4,7,10,13,16,19Z)-3,5-Dihydroxyphenyl-docosa-4,7,10,13,16,19-
hexaenoate (6): Deprotection of protected DHA–phloroglucinol 4
(50 mg, 0.07 mmol) was carried out according to the general pro-
cedure to give 6 (23 mg, 78%) as a colourless oil after purification
by silica gel chromatography (hexane/EtOAc, 9:1 to 75:25). Rf (hex-
4556
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© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2014, 4548–4561

Polyunsaturated Fatty Acid–Phenol Conjugates
ane/EtOAc, 70:30): 0.36. 1H NMR (500 MHz, CDCl3): δ = 6.06 (s,
(0.27 mg, 51%) as a white solid. Dialkylated derivative 14b[38] was
3 H, CHaro), 5.48–5.28 (m, 12 H, CH=CH), 2.87–2.79 (m, 10 H, also isolated (13%). Data for 9b: Rf (CH2Cl2/MeOH, 95:5): 0.4. 1H
CH2 bis-allylic), 2.63 (t, J = 7.0 Hz, 2 H, CH2-C=O), 2.53–2.48 (m,
2 H, CH2 allylic), 2.06 (quint, J = 7.5 Hz, 2 H, CH2 allylic), 0.96
(t, J = 7.5 Hz, 3 H, CH3) ppm. 13C NMR (125 MHz, CDCl3): δ =
172.7, 157.3, 151.8, 132.0, 129.9, 128.6, 128.4, 128.3, 128.3, 128.1,
128.0, 127.8, 127.8, 127.2, 127.0, 101.9, 101.1, 34.3, 25.6, 25.6, 25.6,
25.5, 22.7, 20.5, 14.2 ppm. HRMS (ESI-TOF): calcd. for C28H35O4
[M – H]– 435.2535; found 435.2538.
NMR (500 MHz, CDCl3): δ = 5.99 (d, J = 1.9 Hz, 2 H, CHaro),
5.96 (t, J = 1.9 Hz, 1 H, CHaro), 5.70 (br., 2 H, OH), 4.45 (quint,
J = 7.5 Hz, 1 H, CH), 1.31 (d, J = 6.0 Hz, 6 H, CH3) ppm. 13C
NMR (125 MHz, CDCl3): δ = 160.0, 158.1, 95.8, 95.5, 70.0,
22.0 ppm. HRMS (ESI-TOF): calcd. for C9H11O3 [M – H]–
167.0708; found 167.0710.
5-Methoxy-1,3-bis(triisopropylsilyloxy)benzene (10a): Phloroglu-
cinol-OMe 9a (100 mg, 0.71 mmol) was dissolved in dry CH2Cl2
(6 mL) and dry THF (600 μL). Diisopropylethylamine (DIPEA;
257 μL, 1.50 mmol) and TIPS-OTf (403 μL, 1.50 mmol) were
added dropwise to the solution, and the reaction mixture was
stirred at room temperature for 6 h. Further DIPEA (61.20 µL,
0.35 mmol) and TIPS-OTf (95.9 µL, 0.35 mmol) were added to
drive the reaction to completion. After 6 h, EtOAc (15 mL) was
added to the mixture, and the organic layer was washed with water
(10 mL) and brine (10 mL). The organic phase was dried (MgSO4),
and concentrated under vacuum. The residue was purified by
chromatography on silica gel (hexane/EtOAc, 99:1) to give dipro-
tected phloroglucinol-OMe 10a (311 mg, 96%) as a colourless oil.
(4,4Ј,7,7Ј,10,10Ј,13,13Ј,16,16Ј,19,19ЈZ)-5-Hydroxy-1,3-phenylene
Didocosa-4,7,10,13,16,19-hexaenoate (7): Deprotection of protected
DHA–phloroglucinol 5 (168 mg, 0.19 mmol) was carried out ac-
cording to the general procedure to give 7 (119 mg, 86%) as a
colourless oil after purification by silica gel chromatography (hex-
ane/EtOAc, 90:10). Rf (hexane/EtOAc, 90:10): 0.19. 1H NMR
(500 MHz, CDCl3): δ = 6.48 (s, 3 H, CHaro), 5.48–5.29 (m, 24 H,
CH=CH), 2.88–2.80 (m, 20 H, CH2 bis-allylic), 2.60 (t, J = 7.1 Hz,
4 H, CH2-C=O), 2.52–2.48 (m, 4 H, CH2 allylic), 2.08 (quint, J =
7.5 Hz, 4 H, CH2 allylic), 0.98 (t, J = 7.5 Hz, 6 H, CH3) ppm. 13C
NMR (125 MHz, CDCl3): δ = 171.1, 156.7, 151.6, 132.0, 129.8,
128.5, 128.3, 128.3, 128.2, 128.0, 128.0, 127.9, 127.8, 127.3, 127.0,
107.6, 106.7, 34.2, 25.6, 25.6, 25.6, 25.5, 22.6, 20.5, 14.2 ppm.
HRMS (ESI-TOF): calcd. for C50H67O5 [M + H]+ 747.4989; found
747.4994.
1
Rf (hexane/EtOAc, 95:5): 0.80. H NMR (500 MHz, CDCl3): δ =
6.09–6.08 (m, 2 H, CHaro), 6.07–6.06 (m, 1 H, CHaro), 3.73 (s, 3
H, CH3O), 1.27–1.21 (m, 6 H, CH-Si), 1.10 [d, J = 7.0 Hz, 36 H,
(CH3)2C] ppm. 13C NMR (125 MHz, CDCl3): δ = 161.3, 157.8,
105.0, 99.7, 55.5, 18.2, 13.0 ppm. HRMS (ESI-TOF): calcd. for
C25H49O3Si2 [M + H]+ 453.3214; found 453.3226.
(4,4Ј,4ЈЈ,7,7Ј,7ЈЈ,10,10Ј,10ЈЈ,13,13Ј,13ЈЈ,16,16Ј,16ЈЈ,19,19Ј,19ЈЈZ)-
Benzene-1,3,5-triyl Tridocosa-4,7,10,13,16,19-hexaenoate (8): Cou-
pling of di-DHA–phloroglucinol 7 (108 mg, 0.14 mmol) and DHA
(46 mg, 0.15 mmol) was carried out according to the general pro-
cedure to give 8 (108 mg, 73%) as a colourless oil after purification
by silica gel chromatography (hexane/EtOAc, 98:2). Rf (hexane/
EtOAc, 95:5): 0.29. 1H NMR (500 MHz, CDCl3): δ = 6.82 (s, 3 H,
CHaro), 5.48–5.28 (m, 36 H, CH=CH), 2.87–2.79 (m, 30 H, CH2
bis-allylic), 2.59 (t, J = 7.5 Hz, 6 H, CH2-C=O), 2.46–2.50 (m, 6
H, CH2 allylic), 2.07 (quint, J = 7.5 Hz, 6 H, CH2 allylic), 0.97 (t,
J = 7.5 Hz, 9 H, CH3) ppm. 13C NMR (125 MHz, CDCl3): δ =
170.9, 151.4, 132.3, 130.1, 128.8, 128.6, 128.5, 128.5, 128.3, 128.3,
128.2, 128.1, 127.6, 127.3, 112.9, 34.5, 25.9, 25.9, 25.8, 22.8, 20.8,
14.5 ppm. HRMS (ESI-TOF-ASAP+): calcd. for C72H97O6 [M +
H]+ 1057.7280; found 1057.7285.
5-Isopropoxy-1,3-bis(triisopropylsilyloxy)benzene (10b): Phloroglu-
cinol-OiPr 9b (231 mg, 1.37 mmol) was dissolved in dry CH2Cl2
(24 mL). Diisopropylethylamine (617 μL, 3.60 mmol) and TIPS-
OTf (969 μL, 3.60 mmol) were added dropwise to the solution, and
the reaction mixture was stirred at room temperature for 6 h.
EtOAc (30 mL) was added to the mixture, and the organic layer
was washed with water (15 mL) and brine (15 mL). The organic
phase was dried (MgSO4) and concentrated under vacuum. The
residue was purified by chromatography on silica gel (hexane/
EtOAc, 99.5:0.5) to give the diprotected phloroglucinol-OiPr 10b
(573 mg, 87%) as a colourless oil. Rf (hexane/EtOAc, 95:5): 0.88.
1H NMR (500 MHz, CDCl3): δ = 6.07–6.06 (m, 2 H, CHaro), 6.04–
6.02 (m, 1 H, CHaro), 4.42 (quint, J = 6.0 Hz, 1 H, CHip), 1.29 [d,
J = 6.0 Hz, (CH3)2Cip], 1.26–1.19 (m, 6 H, CH-Si), 1.09 [d, J =
6.0 Hz, 36 H, (CH3)2CTIPS] ppm. 13C NMR (125 MHz, CDCl3): δ
= 159.5, 157.7, 105.0, 101.9, 70.2, 22.3, 18.2, 12.9 ppm. HRMS
(ESI-TOF): calcd. for C27H53O3Si2 [M + H]+ 481.3527; found
481.3537.
5-Methoxybenzene-1,3-diol (9a): A freshly prepared saturated solu-
tion of dry HCl (gas) in MeOH (17 n; 4 mL) was added to a sus-
pension of phloroglucinol (0.40 g, 3.17 mmol) in dioxane (1 mL).
The mixture was stirred at room temperature for 3 h. An additional
amount of the saturated HCl solution (1 mL) was added, and the
mixture was kept at 70 °C for 1 h. The solvents were evaporated
under vacuum, and the residue was purified by chromatography on
silica gel (CH2Cl2/MeOH, 98:2) to give 9a (0.28 mg, 62%) as a
white solid. Dimethylated compound 14a[43] was also isolated
(25 %). Data for 9a: Rf (CH2Cl2/MeOH, 95:5): 0.47. 1H NMR
(500 MHz, [D4]methanol): δ = 5.88 (s, 3 H, CHaro), 3.69 (s, 3 H,
CH3O) ppm. 13C NMR (125 MHz, CDCl3): δ = 161.7, 158.1, 95.7,
94.0, 55.2 ppm. HRMS (ESI-TOF): calcd. for C7H7O3 [M – H]–
139.0395; found 139.0397.
3-Methoxy-5-(triisopropylsilyloxy)phenol (11a): Diprotected
phloroglucinol 10a (92 mg, 0.19 mmol) was dissolved in dry THF
(6.50 mL). Et3N·3HF (33 μL, 0.19 mmol) was added dropwise. The
reaction was followed by TLC, and it was stopped so as to limit as
much as possible the proportion of the fully deprotected derivative.
The mixture was stirred for 7 h at room temperature, then EtOAc
(15 mL) was added, and the organic layer was washed with water
(10 mL) and brine (10 mL). The organic phase was dried (MgSO4),
and concentrated under vacuum. The residue was purified by
chromatography on silica gel (hexane/EtOAc, 95:5 to 70:30) to give
the monoprotected phloroglucinol 11a (37 mg, 61%) as a white so-
lid. The fully deprotected derivative was also isolated (3.20 mg,
11 %). Data for 11a: Rf (hexane/EtOAc, 70:30): 0.6. 1H NMR
(500 MHz, CDCl3): δ = 6.05 (s, 1 H, CHaro), 6.02–6.00 (m, 2 H,
CHaro), 4.86 (br. s, 1 H, OH), 3.73 (s, 3 H, CH3O), 1.29–1.20 (m,
3 H, CH-Si), 1.09 [d, J = 7.5 Hz, 18 H, (CH3)2C] ppm. 13C NMR
(125 MHz, CDCl3): δ = 161.3, 157.9, 157.2, 100.1, 98.8, 94.6, 55.2,
5-Isopropoxybenzene-1,3-diol (9b): A freshly prepared saturated
solution of dry HCl (gas) in iPrOH (32 n; 4 mL) was added to a
suspension of phloroglucinol (0.40 g, 3.17 mmol) in dioxane
(1 mL). The mixture was stirred at room temperature for 1 h. An
additional amount of the saturated HCl solution (1 mL) was
added, and the mixture was kept at 70 °C for 7 h. The solvents
were evaporated under vacuum, and the residue was purified by
chromatography on silica gel (CH2Cl2/MeOH, 98:2) to give 9b
Eur. J. Org. Chem. 2014, 4548–4561
© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
4557

J. Vercauteren et al.
FULL PAPER
17.9, 12.6 ppm. HRMS (ESI-TOF): calcd. for C16H27O3Si
[M – H]– 295.1729; found 295.1730.
colourless oil after purification by silica gel chromatography (hex-
ane/EtOAc, 90:10). Rf (hexane/EtOAc, 80:20): 0.29. 1H NMR
(500 MHz, CDCl3): δ = 6.25 (s, 1 H, CHaro), 6.22 (s, 1 H, CHaro),
6.18 (s, 1 H, CHaro), 5.49–5.28 (m, 12 H, CH=CH), 5.21 (br., 1 H,
OH), 3.75 (s, 3 H, CH3O), 2.88–2.80 (m, 10 H, CH2 bis-allylic),
2.60 (t, J = 7.3 Hz, 2 H, CH2-C=O), 2.52–2.46 (m, 2 H, CH2 all-
ylic), 2.07 (quint, J = 7.3 Hz, 2 H, CH2 allylic), 0.97 (t, J = 7.5 Hz,
3 H, CH3) ppm. 13C NMR (125 MHz, CDCl3): δ = 171.8, 161.2,
157.2, 152.0, 132.0, 129.7, 128.5, 128.3, 128.2, 128.2, 128.0, 128.0,
127.9, 127.8, 127.4, 127.0, 101.9, 100.0, 99.4, 34.3, 25.6, 25.6, 25.5,
22.7, 20.5, 14.2 ppm. HRMS (ESI-TOF): calcd. for C29H39O4 [M
+ H]+ 451.2848; found 451.2851.
3-Isopropoxy-5-(triisopropylsilyloxy)phenol (11b): Diprotected
phloroglucinol 10b (100 mg, 0.21 mmol) was dissolved in dry THF
(6 mL), and Et3N·3HF (68 μL, 0.42 mmol) was added. The reac-
tion was followed by TLC, and it was stopped so as to limit as
much as possible the formation on the fully deprotected derivative.
The mixture was stirred for 5 h at room temperature, then EtOAc
(15 mL) was added, and the organic layer was washed with water
(10 mL) and brine (10 mL). The organic phase was dried (MgSO4),
and concentrated under vacuum. The residue was purified by
chromatography on silica gel (hexane/EtOAc, 95:5) to give mono-
protected phloroglucinol 11b (42 mg, 62%) as colourless oil. The
fully deprotected derivative was also isolated (5 mg, 14%). Data for
(4,7,10,13,16,19Z)-3-Hydroxy-5-isopropoxyphenyl Docosa-4,7,10,
13,16,19-hexaenoate (13b): Deprotection of protected DHA–
phloroglucinol 12b (120 mg, 0.19 mmol) was carried out according
to the general procedure to give 13b (82 mg, 90%) as a colourless
oil after purification by silica gel chromatography (hexane/EtOAc,
90:10). Rf (hexane/EtOAc, 90:10): 0.30. 1H NMR (500 MHz,
CDCl3): δ = 6.25 (t, J = 2.0 Hz, 1 H, CHaro), 6.21 (t, J = 2.0 Hz,
1 H, CHaro), 6.17 (t, J = 2.0 Hz,1 H, CHaro), 5.48–5.29 (m, 12 H,
CH=CH), 4.95 (br., 1 H, OH), 4.47 (quint, J = 6.0 Hz, 1 H, CHip),
2.89–2.81 (m, 10 H, CH2 bis-allylic), 2.61–2.58 (m, 2 H, CH2-
C=O), 2.54–2.48 (m, 2 H, CH2 allylic), 2.08 (quint, J = 7.5 Hz, 2
H, CH2 allylic), 1.32 [d, J = 6.0 Hz, 6 H, (CH3)2Cip], 0.98 (t, J =
7.5 Hz, 3 H, CH3) ppm. 13C NMR (125 MHz, CDCl3): δ = 171.3,
159.6, 156.9, 152.1, 132.0, 129.7, 128.5, 128.3, 128.3, 128.2, 128.1,
128.0, 128.0, 127.8, 127.5, 127.0, 101.9, 101.5, 100.8, 70.2, 34.3,
25.6, 25.6, 25.6, 25.5, 22.7, 21.9, 20.5, 14.2 ppm. HRMS (ESI-
TOF): calcd. for C31H41O4 [M – H]– 477.3005; found 477.3007.
1
11b: Rf (hexane/EtOAc, 70:30): 0.7. H NMR (500 MHz, CDCl3):
δ = 6.04 (t, J = 2.5 Hz, 1 H, CHaro), 6.01 (t, J = 2.5 Hz, 1 H,
CHaro), 5.99 (t, J = 2.5 Hz, 1 H, CHaro), 4.87 (br., 1 H, OH), 4.44
(quint, J = 6.0 Hz, 1 H, CHip), 1.31 [d, J = 6.0 Hz, 6 H,
(CH3)2Cip], 1.28–1.20 (m, 3 H, CH-Si), 1.10 [d, J = 7.5 Hz, 18 H,
(CH3)2CTIPS] ppm. 13C NMR (125 MHz, CDCl3): δ = 159.6, 157.9,
157.0, 100.7, 100.0, 96.6, 70.0, 22.0, 17.9, 12.6 ppm. HRMS (ESI-
TOF): calcd. for C18H31O3Si [M – H]– 323.2042; found 323.2045.
(4,7,10,13,16,19Z)-3-Methoxy-5-(triisopropylsilyloxy)phenyl Do-
cosa-4,7,10,13,16,19-hexaenoate (12a): Coupling of protected
phloroglucinol-OMe 11a (96 mg, 0.32 mmol) and DHA (106 mg,
0.32 mmol) following the general procedure gave 12a (120 mg,
60%) as a colourless oil after purification by silica gel chromatog-
1
raphy (hexane/EtOAc, 99:1). Rf (hexane/EtOAc, 99:1): 0.28. H
NMR (500 MHz, CDCl3): δ = 6.30 (s, 1 H, CHaro), 6.25–6.23 (m,
2 H, CHaro), 5.48–5.28 (m, 12 H, CH=CH), 3.74 (s, 3 H, CH3O),
2.87–2.79 (m, 10 H, CH2 bis-allylic), 2.60–2.57 (m, 2 H, CH2-
C=O), 2.52–2.48 (m, 2 H, CH2 allylic), 2.07 (quint, J = 7.5 Hz, 2
H, CH2 allylic), 1.29–1.20 (m, 3 H, CH-Si), 1.09 [d, J = 7.4 Hz, 18
H, (CH3)2C], 0.97 (t, J = 7.5 Hz, 3 H, CH3) ppm. 13C NMR
(125 MHz, CDCl3): δ = 171.2, 160.8, 157.4, 152.0, 132.0, 129.6,
128.5, 128.3, 128.2, 128.2, 128.1, 128.1, 128.0, 127.8, 127.5, 127.0,
106.3, 103.8, 100.4, 55.4, 34.3, 25.6, 25.6, 25.5, 22.7, 20.5, 17.8,
14.2, 12.6 ppm. HRMS (ESI-TOF): calcd. for C38H59O4Si
[M + H]+ 607.4183; found 607.4185.
(4,7,10,13,16,19Z)-3,5-Dimethoxyphenyl docosa-4,7,10,13,16,19-
hexaenoate (15a): Coupling of commercially available di-OMe-
phloroglucinol 14a (47 mg, 0.30 mmol) and DHA (100 mg,
0.30 mmol) was carried out according to the general procedure to
give 15a (110 mg, 77%) as a colourless oil after purification by
silica gel chromatography (hexane/EtOAc, 97:3). Rf (hexane/
1
EtOAc, 95:5): 0.36. H NMR (500 MHz, CDCl3): δ = 6.33 (t, J =
2.0 Hz, 1 H, CHaro), 6.25 (d, J = 2.0 Hz, 2 H, CHaro), 5.49–5.28
(m, 12 H, CH=CH), 3.76 (s, 6 H, CH3O), 2.88–2.79 (m, 10 H, CH2
bis-allylic), 2.60 (t, J = 7.3 Hz, 2 H, CH2-C=O), 2.54–2.49 (m, 2
H, CH2 allylic), 2.07 (quint, J = 7.3 Hz, 2 H, CH2 allylic), 0.97 (t,
J = 7.5 Hz, 3 H, CH3) ppm. 13C NMR (125 MHz, CDCl3): δ =
171.4, 161.0, 152.1, 131.9, 129.8, 128.5, 128.3, 128.2, 128.2, 128.0,
128.0, 127.9, 127.8, 127.4, 126.9, 100.1, 98.1, 55.4, 34.3, 25.6, 25.5,
25.4, 22.7, 20.4, 14.2 ppm. HRMS (ESI-TOF): calcd. for C30H41O4
[M + H]+ 465.3005; found 465.3011.
(4,7,10,13,16,19Z)-3-Isopropoxy-5-(triisopropylsilyloxy)phenyl Do-
cosa-4,7,10,13,16,19-hexaenoate (12b): Coupling of mono-TIPS-
mono-isopropyl-phloroglucinol 11b (100 mg, 0.31 mmol) and
DHA (101 mg, 0.31 mmol) was carried out according to the general
procedure to give 12b (132 mg, 67%) as a colourless oil after purifi-
cation by silica gel chromatography (hexane/EtOAc, 99:1). Rf (hex-
(4,7,10,13,16,19Z)-3,5-Diisopropoxyphenyl Docosa-4,7,10,13,16,19-
hexaenoate (15b): Coupling of di-OiPr-phloroglucinol 14b[38]
(96 mg, 0.45 mmol) and DHA (150 mg, 0.45 mmol) was carried out
according to the general procedure to give 15b (168 mg, 70%) as a
colourless oil after purification by silica gel chromatography (hex-
ane/EtOAc, 97:3). Rf (hexane/EtOAc, 95:5): 0.57. 1H NMR
(500 MHz, CDCl3): δ = 6.28 (s, 1 H, CHaro), 6.19 (s, 2 H, CHaro),
5.48–5.28 (m, 12 H, CH=CH), 4.46 (quint, J = 5.9 Hz, 2 H, CH),
2.89–2.78 (m, 10 H, CH2 bis-allylic), 2.59 (t, J = 7.4 Hz, 2 H, CH2-
C=O), 2.53–2.48 (m, 2 H, CH2 allylic), 2.07 (quint, J = 7.3 Hz, 2
H, CH2 allylic), 1.31 [d, J = 6.0 Hz, 12 H, (CH3)2C], 0.97 (t, J =
7.5 Hz, 3 H, CH3) ppm. 13C NMR (125 MHz, CDCl3): δ = 171.2,
159.3, 152.1, 131.9, 129.6, 128.5, 128.3, 128.2, 128.2, 128.0, 128.0,
127.9, 127.8, 127.5, 126.9, 101.4, 101.2, 70.0, 34.2, 25.6, 25.5, 25.4,
22.7, 21.9, 20.5, 14.2 ppm. HRMS (ESI-TOF): calcd. for C34H49O4
[M + H]+ 521.3631; found 521.3636.
1
ane/EtOAc, 99:1): 0.30. H NMR (500 MHz, CDCl3): δ = 6.29 (t,
J = 2.0 Hz,1 H, CHaro), 6.24 (t, J = 2.0 Hz,1 H, CHaro), 6.21 (t, J
= 2.0 Hz,1 H, CHaro), 5.49–5.29 (m, 12 H, CH=CH), 4.45 (quint,
J = 6.0 Hz, 1 H, CHip), 2.88–2.81 (m, 10 H, CH2 bis-allylic), 2.60–
2.57 (m, 2 H, CH2-C=O), 2.52–2.48 (m, 2 H, CH2 allylic), 2.08
(quint, J = 7.5 Hz, 2 H, CH2 allylic), 1.32 [d, J = 6.0 Hz, 6 H,
(CH3)2Cip], 1.28–1.22 (m, 3 H, CH-Si), 1.10 [d, J = 7.5 Hz, 18 H,
(CH3)2CTIPS], 0.98 (t, J = 7.5 Hz, 3 H, CH3) ppm. 13C NMR
(125 MHz, CDCl3): δ = 171.1, 159.1, 157.3, 151.9, 132.0, 129.6,
128.5, 128.3, 128.2, 128.2, 128.1, 128.0, 128.0, 127.8, 127.6, 127.0,
106.0, 105.3, 102.3, 70.1, 34.3, 25.6, 25.6, 25.5, 22.7, 21.9, 20.5,
17.9, 14.2, 12.6 ppm. HRMS (ESI-TOF): calcd. for C40H63O4Si [M
+ H]+ 635.4496; found 635.4502.
(4,7,10,13,16,19Z)-3-Hydroxy-5-methoxyphenyl Docosa-4,7,10,
13,16,19-hexaenoate (13a): Deprotection of protected DHA–
phloroglucinol-OMe 12a (75 mg, 0.12 mmol) was carried out ac-
cording to the general procedure to give 13a (49 mg, 88%) as a
(E)-4-(3,5-Dihydroxystyryl)phenyl Acetate (16): Resveratrol
(200 mg, 0.88 mmol) was dissolved in 2-methylbutan-2-ol (20 mL)
4558
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© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2014, 4548–4561

Polyunsaturated Fatty Acid–Phenol Conjugates
and vinyl acetate (5 mL) in the presence of supported Candida ant-
arctica lipase (Novozyme 435, CALB; 1 g). The mixture was stirred
using a rotary evaporator at 40 °C for 4 d. The lipase was removed
by filtration, and the filter residue was washed with EtOAc (2ϫ)
and diethyl ether. The filtrate was concentrated under reduced pres-
sure, and the resulting residue was purified by chromatography on
silica gel (CH2Cl2/MeOH, 99:1 to 90:10) to give 4Ј-O-acetyl resver-
chromatography (hexane/EtOAc, 99:1). Rf (hexane/EtOAc, 95:5):
0.73. H NMR (500 MHz, CDCl3): δ = 7.50 (d, J = 8.5 Hz, 2 H,
1
2Ј-H and 6Ј-H), 7.07 (d, J = 8.4 Hz, 2 H, 3Ј-H and 5Ј-H), 6.98 (d,
J = 16.5 Hz, 1 H, 8-H), 6.92 (d, J = 16.5 Hz, 1 H, 7-H), 6.64 (d, J
= 2.3 Hz, 2 H, 2-H, 6-H), 6.36 (t, J = 2.3 Hz, 1 H, 4-H), 5.50–5.29
(m, 12 H, CH=CH), 2.90–2.80 (m, 10 H, CH2 bis-allylic), 2.64 (t,
J = 7.0 Hz, 2 H, CH2-C=O), 2.55–2.51 (m, 2 H, CH2 allylic), 2.08
atrol 16 (135 mg, 57 %) as white solid. Starting material (5 mg, (quint, J = 7.0 Hz, 2 H, CH2 allylic), 1.29–1.22 (m, 6 H, CH-Si),
27 %) was also recovered after purification. Data for 16: Rf 1.12 [d, J = 7.5 Hz, 36 H, (CH3)2C], 0.98 (t, J = 7.5 Hz, 3 H, CH3)
1
(CH2Cl2/MeOH, 95:5): 0.30. H NMR (500 MHz, [D4]methanol):
ppm. 13C NMR (125 MHz, CDCl3): δ = 171.8, 157.4, 150.4, 139.1,
δ = 7.54 (d, J = 7.6 Hz, 2 H, 2Ј-H and 6Ј-H), 7.07 (d, J = 7.6 Hz, 135.3, 132.3, 130.0, 129.3, 128.9, 128.7, 128.6, 128.6, 128.4, 128.4,
2 H, 3Ј-H and 5Ј-H), 7.04 (d, J = 16.2 Hz, 1 H, 8-H), 6.97 (d, J = 128.3, 128.2, 127.9, 127.8, 127.7, 127.3, 122.0, 111.7, 111.6, 34.6,
16.2 Hz, 1 H, 7-H), 6.49 (s, 2 H, 2-H, 6-H), 6.21–6.19 (m, 1 H, 4- 25.9, 25.9, 25.8, 23.1, 20.9, 18.2, 14.6, 13.0 ppm. HRMS (ESI-
H), 2.27 [s, 3 H, CH3(OAc)] ppm. 13C NMR (125 MHz, [D4]meth-
anol): δ = 171.1, 159.7, 151.5, 140.6, 136.58, 130.24, 128.3, 128.3,
122.9, 106.1, 103.2, 20.9 ppm. HRMS (ESI-TOF): calcd. for
C16H15O4 [M + H]+ 271.0964; found 271.0972.
TOF): calcd. for C54H83O4Si2 [M + H]+ 851.5824; found 851.5825.
(4,7,10,13,16,19Z)-4-[(E)-3,5-Dihydroxyphenylstyryl]phenyl Do-
cosa-4,7,10,13,16,19-hexaenoate (20): Deprotection of protected
DHA–resveratrol 19 (142 mg, 0.17 mmol) was carried out accord-
ing to the general procedure to give 20 (55 mg, 61%) as a white
solid after reaction for 7 h and purification by silica gel chromatog-
raphy (hexane/EtOAc, 95:5 to 70:30). Rf (hexane/EtOAc, 70:30):
(E)-4-[3,5-Bis(triisopropylsilyloxy)styryl]phenyl Acetate (17):
Resveratrol-4Ј-OAc 16 (100 mg, 0.37 mmol) was dissolved in dry
THF (6 mL). Triethylamine (109 μL, 0.78 mmol) and TIPS-OTf
(208 μL, 0.78 mmol) were added dropwise to the solution, and the
reaction mixture was stirred at room temperature for 2 h. Further
Et3N (109 μL, 0.78 mmol) and TIPS-OTf (208 μL, 0.78 mmol) were
added to drive the reaction to completion. After a further 3 h, the
solvent was evaporated under reduced pressure. The residue was
dissolved in EtOAc (10 mL), and this solution was washed with
water (10 mL) and brine (10 mL). The organic phase was dried
(MgSO4), and concentrated under vacuum. The residue was puri-
fied by chromatography on silica gel (pentane/EtOAc, 80:20) to
give protected resveratrol 17 (152 mg, 71%) as a colourless oil. Rf
(pentane/EtOAc, 95:5): 0.5. 1H NMR (500 MHz, CDCl3): δ = 7.51
(d, J = 8.0 Hz, 2 H, 2Ј-H and 6Ј-H), 7.09 (d, J = 8.0 Hz, 2 H, 3Ј-
H and 5Ј-H), 6.98 (d, J = 16.3 Hz, 1 H, 8-H), 6.92 (d, J = 16.3 Hz,
1 H, 7-H), 6.65 (s, 2 H, 2-H, 6-H), 6.37–6.36 (m, 1 H, 4-H), 2.31
[s, 3 H, CH3(OAc)], 1.26 (m, 6 H, CH-Si), 1.12 [d, J = 7.6 Hz, 36 H,
(CH3)2C] ppm. 13C NMR (125 MHz, CDCl3): δ = 169.7, 157.3,
150.2, 139.0, 135.3, 129.3, 127.8, 127.7, 122.0, 111.6, 111.5, 21.4,
18.2, 12.9 ppm. HRMS (ESI-TOF): calcd. for C34H55O4Si2 [M +
H]+ 583.3633; found 583.3640.
1
0.22. H NMR (500 MHz, CDCl3): δ = 7.45 (d, J = 8.6 Hz, 2 H,
2Ј-H and 6Ј-H), 7.07 (d, J = 8.5 Hz, 2 H, 3Ј-H and 5Ј-H), 6.95 (d,
J = 16.2 Hz, 1 H, 8-H), 6.85 (d, J = 16.2 Hz, 1 H, 7-H), 6.51 (d, J
= 2.1 Hz, 2 H, 2-H, 6-H), 6.26 (t, J = 2.1 Hz, 1 H, 4-H), 5.52–5.29
(m, 12 H, CH2 bis-allylic), 5.13 (br., 2 H, OH), 2.90–2.80 (m, 10
H, CH2 allylic), 2.66 (t, J = 7.4 Hz, 2 H, CH2-C=O), 2.52–2.56 (m,
2 H, CH2 allylic), 2.08 (quint, J = 7.8 Hz, 2 H, CH2 allylic), 0.98
(t, J = 7.3 Hz, 3 H, CH3) ppm. 13C NMR (125 MHz, CDCl3): δ =
172.3, 157.3, 150.4, 140.0, 135.2, 132.4, 130.1, 128.9, 128.7, 128.6,
128.6, 128.6, 128.5, 128.4, 128.4, 128.3, 128.2, 127.8, 127.7, 127.3,
122.1, 106.4, 102.7, 34.6, 25.9, 25.8, 23.1, 20.9, 14.6 ppm. HRMS
(ESI-TOF): calcd. for C36H43O4 [M + H]+ 539.3155; found
539.3160.
1-Lyso-2-docosahexaenoyl-sn-glycero-3-phosphatidylcholine (21):
Commercially available PC-16:0-DHA (Coger, France; 25 mg,
0.03 mmol) was dissolved in ethanol (96%; 250 μL) of in the pres-
ence of the supported lipozyme (immobilized, from Mucor miehei;
25 mg). The mixture was stirred at room temperature for 9 h, and
then an additional amount of the supported enzyme (25 mg) was
added to the mixture. After an overall reaction time of 29 h, the
supported enzyme was removed by filtration, and the filter residue
was washed with absolute EtOH (3ϫ 3 mL) and chloroform (3ϫ
3 mL). The filtrate was concentrated under vacuum, and the re-
sulting residue was purified on a Sep-Pak SiOH cartridge (CHCl3/
MeOH, 100:0 to 60:40) to give sn-1-Lyso-PC-DHA 21 (15 mg,
85%) as a colourless oil. Starting material (4 mg, 15%) was reco-
vered after the purification. Rf (CHCl3/MeOH/H2O, 65:25:4): 0.19.
1H NMR (500 MHz, CDCl3): δ = 5.41–5.29 (m, 12 H, CH=CH),
4.96–4.91 (m, 1 H, CH-O), 4.34–4.28 (m, 2 H, CH2-O), 4.06–4.00
[m, 1 H, CH2(a)-O], 3.98–3.92 [m, 1 H, CH2(b)-O], 3.80–3.75 (m, 2
H, CH2-N), 3.69–3.65 (m, 2 H, CH2-O), 3.32 [s, 9 H, (CH3)3-N+],
2.85–2.79 (m, 10 H, CH2 bis-allylic), 2.37–2.34 (m, 4 H, CH2-C=O
and CH2 allylic), 2.07 (quint, J = 7.5 Hz, 2 H, CH2 allylic), 0.97
(t, J = 7.5 Hz, 3 H, CH3) ppm. 13C NMR (125 MHz, CDCl3): δ =
172.9, 132.2, 129.5, 128.8, 128.6, 128.5, 128.5, 128.3, 128.3, 128.2,
128.1, 128.1, 127.2, 73.7, 66.4, 63.3, 60.0, 59.6, 54.5, 34.3, 25.8,
25.8, 25.8, 25.8, 25.8, 22.8, 20.8, 14.5 ppm. HRMS (ESI-TOF):
calcd. for C30H51NO7P [M + H]+ 568.3403; found 568.3409. HPLC
[Atlantis C18 5 μm (4.6 ϫ 250 mm), A: MeOH/H2O/MeCN,
90:35:2.5, B: MeOH/H2O/MeCN, 100:4:2.5, t0Ј = 100/0, t15Ј = 100/
0, t30Ј = 0/100, t50Ј = 0/100, detection 205 nm]: tR = 25.80 min.
(E)-4-[3,5-Bis(triisopropylsilyloxy)styryl]phenol (18): Protected
resveratrol 17 (239 mg, 0.41 mmol) was dissolved in dry MeOH
(2 mL) and CH2Cl2 (1 mL). A catalytic amount of sodium meth-
oxide (6.60 mg, 0.12 mmol) was added to the solution, and the re-
action mixture was stirred at room temperature for 2 h. Further
NaOMe was added to drive the reaction to completion. After 5 h,
the solvent was evaporated under reduced pressure. The residue
was purified by chromatography on silica gel (hexane/EtOAc, 95:5)
to give 4Ј-deprotected resveratrol 18 (214 mg, 97%) as a colourless
oil. Rf (hexane/EtOAc, 90:10): 0.41. 1H NMR (500 MHz,
[D4]methanol): δ = 7.38 (d, J = 8.5 Hz, 2 H, 2Ј-H and 6Ј-H), 6.95
(d, J = 16.2 Hz, 1 H, 8-H), 6.84 (d, J = 16.2 Hz, 1 H, 7-H), 6.77
(d, J = 8.5 Hz, 2 H, 3Ј-H and 5Ј-H), 6.64–6.63 (m, 2 H, 2-H, 6-H),
6.30–6.29 (m, 1 H, 4-H), 1.30–1.22 (m, 6 H, CH-Si), 1.14 [d, J =
7.5 Hz, 36 H, (CH3)2C] ppm. 13C NMR (125 MHz, [D4]methanol):
δ = 158.5, 158.3, 141.3, 130.1, 129.9, 129.0, 126.5, 116.5, 112.1,
111.4, 18.4, 13.9 ppm. HRMS (ESI-TOF): calcd. for C32H53O3Si2
[M + H]+ 541.3527; found 541.3536.
(4,7,10,13,16,19Z)-4-[(E)-3,5-Bis(triisopropylsilyloxy)styryl]phenyl
Docosa-4,7,10,13,16,19-hexaenoate (19): Coupling of diprotected
resveratrol 18 (103 mg, 0.18 mmol) and DHA (67 mg, 0.20 mmol)
was carried out according to the general procedure to give 19
(130 mg, 80%) as a colourless oil after purification by silica gel
4-[3,5-Bis(triisopropylsilyloxy)phenoxy]-4-oxobutanoic Acid (22):
Diprotected phloroglucinol 2 (1.30 g, 2.97 mmol) was dissolved in
Eur. J. Org. Chem. 2014, 4548–4561
© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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4559

J. Vercauteren et al.
FULL PAPER
CH2Cl2 (30 mL) in the presence of pyridine (283 μL, 3.56 mmol).
Succinic anhydride (358 mg, 3.57 mmol) and DMAP (36 mg,
0.29 mmol) were added to the solution at room temperature, and
the mixture was heated at 50 °C. The mixture was stirred for 1 d,
and then additional amounts of the reagents [succinic anhydride
(1 equiv.), and DMAP (0.1 equiv.)] were added. The reaction was
stopped after 2 d by the addition of water (20 mL). The organic
phase was washed with water and brine, dried (MgSO4), and con-
centrated under vacuum. The residue was purified by chromatog-
raphy on silica gel (hexane/EtOAc, 95:5 to 70:30) to give dipro-
tected succinate phloroglucinol 22 (670 mg, 42%) as a white solid.
128.3, 128.2, 128.1, 128.0, 127.2, 109.5, 107.0, 70.8, 66.8, 63.4, 63.3,
59.4, 54.9, 34.3, 33.5, 33.2, 25.8, 25.8, 25.7, 22.8, 20.8, 20.1, 18.1,
14.5, 12.8 ppm. HRMS (ESI-TOF): calcd. for C59H101NO12PSi2
[M + H]+ 1102.6594; found 1102.6608.
1-[5-(3,5-Dihydroxyphenoxy)-5-oxopentanoyl]-2-docosahexaenoyl-
sn-glycero-3-phosphatidylcholine (25): Et3N·3HF (10 μL,
0.06 mmol) was added to a solution of 24 (17 mg, 0.01 mmol) in
dry THF (0.50 mL). The mixture was stirred at room temperature
for 4 h. The solvent was evaporated under reduced pressure, and
the residue was purified on a Sep-Pak SiOH cartridge (CHCl3/
MeOH, 100:0 to 80:20) to give deprotected polyphenolic-PC-DHA
25 (10.20 mg, 84 %) as a colourless oil. Rf (CHCl3/MeOH/H2O,
65:25:4): 0.30. 1H NMR (500 MHz, CDCl3): δ = 6.43 (s, 1 H,
CHaro), 6.10 (s, 2 H, CHaro), 5.42–5.28 (m, 12 H, CH=CH), 5.18–
5.14 (m, 1 H, CH-O), 4.42–4.37 [m, 1 H, CH2(a)-O], 4.21–4.13 [m,
3 H, CH2(b)-O and CH2-O], 4.04–3.98 (m, 2 H, CH2-O), 3.52–3.46
(m, 2 H, CH2-N), 3.03 [s, 9 H, (CH3)3-N+], 2.84–2.81 (m, 10 H,
CH2 bis-allylic), 2.56–2.51 [m, 2 H, CH2-C=O(glut)], 2.46–2.41 [m,
2 H, CH2-C=O(glut)], 2.37–2.33 [m, 4 H, CH2-C=O(DHA) and CH2
allylic], 2.07 (quint, J = 7.5 Hz, 2 H, CH2 allylic), 2.01–1.95 [m, 2
H, CH2(glut)], 0.97 (t, J = 7.5 Hz, 3 H, CH3) ppm. 13C NMR
(125 MHz, CDCl3): δ = 172.7, 172.6, 172.1, 159.5, 152.5, 132.2,
129.5, 128.8, 128.5, 128.5, 128.5, 128.3, 128.3, 128.3, 128.1, 128.0,
127.2, 101.4, 101.2, 70.5, 66.1, 63.5, 62.3, 59.7, 54.0, 34.0, 33.1,
33.0, 25.9, 25.8, 25.8, 25.8, 22.8, 20.8, 20.4, 14.5 ppm. HRMS (ESI-
TOF): calcd. for C41H61NO12P [M + H]+ 790.3931; found
790.3940. HPLC [Atlantis C18 5 μm (4.6ϫ250 mm), A: MeOH/
H2O/MeCN, 90:35:2.5, B: MeOH/H2O/MeCN, 100:4:2.5, t0Ј = 100/
1
Rf (hexane/EtOAc, 75:25): 0.24. H NMR (500 MHz, CDCl3): δ =
6.29 (t, J = 2.1 Hz, 1 H, CHaro), 6.26 (d, J = 2.1 Hz, 2 H, CHaro),
2.86–2.27 (m, 4 H, CH2succ), 1.27–1.19 (m, 6 H, CH-Si), 1.09 [d, J
= 7.3 Hz, 36 H, (CH3)2C] ppm. 13C NMR (125 MHz, CDCl3): δ =
178.0, 170.2, 157.1, 151.6, 109.4, 106.7, 29.0, 28.8, 17.8, 12.4 ppm.
H R M S ( E S I - T O F ) : c a l c d . f o r C 2 4 H 4 5 O 3 S i 2 [ M –
COCH2CH2COOH]– 437.2907; found 437.2905.
5-[3,5-Bis(triisopropylsilyloxy)phenoxy]-5-oxopentanoic Acid (23):
Diprotected phloroglucinol 2 (100 mg, 0.23 mmol) was dissolved
in CH2Cl2 (5 mL) in the presence of pyridine (27 μL, 0.23 mmol).
Glutaric anhydride (39 mg, 0.34 mmol) and DMAP (3 mg,
0.02 mmol) were added to the solution at room temperature, and
the mixture was heated at 40 °C. The mixture was stirred for 1 d,
and then additional amounts of the reagents [glutaric anhydride
(1.5 equiv.) and DMAP (0.1 equiv.)] were added. The reaction was
stopped after 4 d by the addition of water (10 mL). The organic
phase was washed with water and brine, dried (MgSO4), and con-
centrated under vacuum. The residue was purified by chromatog-
raphy on silica gel (pentane/EtOAc, 90:10) to give diprotected glut-
arate phloroglucinol 23 (90 mg, 71%) as a colourless oil. Rf (pent-
ane/EtOAc, 60:40): 0.50. 1H NMR (500 MHz, CDCl3): δ = 6.28 (t,
J = 2.2 Hz, 1 H, CHaro), 6.25 (d, J = 2.2 Hz, 2 H, CHaro), 2.61 (t,
J = 7.4 Hz, 2 H, CH2glut), 2.51 (t, J = 7.3 Hz, 2 H, CH2glut), 2.06
(quint, J = 7.3 Hz, 2 H, CH2glut), 1.27–1.20 (m, 6 H, CH-Si), 1.08
[d, J = 7.3 Hz, 36 H, (CH3)2C] ppm. 13C NMR (125 MHz, CDCl3):
δ = 178.2, 170.8, 157.1, 151.6, 109.3, 106.7, 33.2, 32.7, 19.7, 17.8,
12.6 ppm. HRMS (ESI-TOF): calcd. for C29H51O6Si2 [M – H]–
551.3224; found 551.3214.
0, t15Ј = 100/0, t30Ј = 0/100, t50Ј = 0/100, detection 272 nm]: tR
=
24.12 min.
Drug Treatment of ARPE-19 Cells: ARPE-19 cells were obtained
from ATCC, and were grown following the instructions in Dulbec-
co’s Modified Eagle’s Medium (DMEM)/HamЈF12 (GIBCO) con-
taining Foetal Bovine Serum (10% v/v) and antibiotics (1% v/v)
under an air (95%)/CO2 (5%) atmosphere at 37 °C. ARPE-19 cells
were put into 96-well plates (3ϫ104 cells/well) and cultured for
24 h to reach confluence before drug treatment. The cell cultures
were treated with a serum-free medium containing drugs at dif-
ferent concentrations (10–40 μm) for 1 h, and then trans-retinal
(25 μm) was added for 4 h, before rinsing with medium. The viabil-
ity of the cells was determined in triplicate samples 16–20 h later,
using an MTT colourimetric assay. After incubation for 2 h with
MTT (0.5 mg/mL), the insoluble purple formazan produced was
dissolved in DMSO. The absorbance at 570 nm and 655 nm of indi-
vidual wells was measured using a microplate reader (BioRad 550).
The percentage of viable cells was calculated as [(OD570 sample –
OD655 sample)/(OD570 control – OD655 control)]ϫ100%; con-
trol cells were incubated with DMSO (0.2%) and DMF (0.14%).
1-{5-[3,5-Bis(triisopropylsilyloxy)phenoxy]-5-oxopentanoyl}-2-
docosahexaenoyl-sn-glycero-3-phosphatidylcholine (24): LysoPC-
DHA 21 (15 mg, 0.02 mmol) and diprotected glutaric phloroglu-
cinol 23 (16 mg, 0.02 mmol) were dissolved in dry CH2Cl2 (2 mL).
DCC (6 mg, 0.03 mmol) and DMAP (1 mg, 8.30 μmol) were added
to the solution, and the mixture was stirred at room temperature
for 24 h under nitrogen. The solvent was evaporated under reduced
pressure, and the residue was purified on a Sep-Pak SiOH cartridge
(CHCl3/MeOH, 100:0 to 60:40) to give polyphenolic-PC-DHA 24
(19 mg, 65%) as a colourless oil. Starting material (5 mg, 33%) was
recovered during the purification. Data for 24: Rf (CHCl3/MeOH/
Supporting Information (see footnote on the first page of this arti-
cle): 1H NMR spectrum of DHA from cod liver oil (p 2); 1H NMR,
13C NMR, HSQC, HMBC, and HPLC analysis of chromene A (pp
3–10); 1H NMR and JMOD 13C NMR spectra of compounds 1,
2, 3, 4, 5, 6, 7, 8, 9a, 9b, 10a, 10b, 11a, 11b, 12a, 12b, 13a, 13b, 15a,
15b, 16, 17, 18, 19, and 20 (pp 11–60); 1H NMR, JMOD 13C NMR,
HMBC, HSQC, and HPLC analysis of compound 21 (pp 61–67);
1H NMR, and JMOD 13C NMR spectra of compounds 22, 23,
and 24 (p 68–73); 1H NMR, JMOD 13C NMR, and HPLC analysis
of 25 (pp 74–76).
1
H2O, 65:25:4): 0.40. H NMR (500 MHz, CDCl3): δ = 6.28 (t, J =
2.5 Hz, 1 H, CHaro), 6.23 (t, J = 2.5 Hz, 2 H, CHaro), 5.41–5.28
(m, 12 H, CH=CH), 5.25–5.20 (m, 1 H, CH-O), 4.43 [dd, J = 3.0,
J = 12.0 Hz, 1 H, CH2(a)-O], 4.36–4.30 (m, 2 H, CH2-O), 4.17 [dd,
J = 6.5, J = 12.0 Hz, 1 H, CH2(b)-O], 4.02–3.95 (m, 2 H, CH2-O),
3.80–3.77 (m, 2 H, CH2-N), 3.35 [s, 9 H, (CH3)3-N+], 2.84–2.79 (m,
10 H, CH2 bis-allylic), 2.57 [t, J = 7.5 Hz, 2 H, CH2-C=O(glut)],
2.43 [t, J = 7.5 Hz, 2 H, CH2-C=O(glut)], 2.39–2.35 [m, 4 H, CH2-
C=O(DHA) and CH2 allylic], 2.07 (quint, J = 7.5 Hz, 2 H, CH2
allylic), 2.00 [quint, J = 7.5 Hz, 2 H, CH2(glut)], 1.24–1.18 (m, 6 H,
CH-Si), 1.08 [d, J = 7.5 Hz, 36 H, (CH3)2C], 0.97 (t, J = 7.5 Hz, 3
H, CH3) ppm. 13C NMR (125 MHz, CDCl3): δ = 172.8, 172.7,
171.2, 157.4, 151.9, 132.2, 129.5, 128.7, 128.5, 128.5, 128.5, 128.3,
Acknowledgments
The authors thank Dr Eric Spokas of Sebastian, FL, for proofread-
ing the manuscript. The University of Montpellier and the Centre
4560
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Eur. J. Org. Chem. 2014, 4548–4561

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Received: March 19, 2014
Published Online: June 4, 2014
Eur. J. Org. Chem. 2014, 4548–4561
© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
4561