Asymmetric synthesis and biological activity of nor-α-tocopherol, a new vitamin E analogue

Article abstract of DOI:10.1002/cbic.201000511

The vitamin E analogues (2R,4'R,8'R)-nor-α-tocopherol (94% de) and (2RS,4'R,8'R)-nor-α-tocopherol have been synthesized from (all R)-hexahydrofarnesol and phytol, respectively. According to in vitro experiments with murine macrophages nor-α-tocopherol is

Full text of DOI:10.1002/cbic.201000511

DOI: 10.1002/cbic.201000511
Asymmetric Synthesis and Biological Activity of nor-a-
Tocopherol, a New Vitamin E Analogue
Julien Chapelat,[a] Urs Hengartner,[a] Antoinette Chougnet,[a] Kegang Liu,[a] Patricia Huebbe,[b]
Gerald Rimbach,[b] and Wolf-D. Woggon*[a]
The vitamin E analogues (2R,4’R,8’R)-nor-a-tocopherol (94% de)
and (2RS,4’R,8’R)-nor-a-tocopherol have been synthesized from
(all R)-hexahydrofarnesol and phytol, respectively. According to
in vitro experiments with murine macrophages nor-a-toco-
pherol is an anti-inflammatory compound more potent than a-
tocopherol.
Introduction
a-Tocopherol (1) is the biologically most significant member of
the vitamin E family and is known to act as a very efficient rad-
ical-chain-breaking antioxidant in tissues.[1] Ingold’s investiga-
tions suggested[1] that the antioxidant reactivity of the chroma-
nol system in vitamin E compounds is attributable to stereo-
electronic factors: that is, the lone pair of O1 is favorably
oriented to stabilize the tocopheroxyl radical 2 produced by
homolysis of the phenolic OH group.[2] Hence, in attempts to
improve the antioxidant properties of 1, the racemic vitamin E
analogues 3 and 4 in which the oxygen is replaced by sulfur
and selenium were synthesized.[3–5] It was expected that the
larger heteroatoms should stabilize the adjacent radical better
than oxygen. These compounds, however, were less efficient
antioxidants than 1.[6,7] In contrast, replacement of the six-
membered heterocycle with a five-membered ring (see 5–8)
led to greater radical chain-breaking capacities in a two-phase
lipid peroxidation model system.[7,8]
More recent studies have reported on the replacement of
the chiral side chain in 1 by shorter units with polar functional
groups (see, for example, 9 and 10).[9] These compounds show
remarkably increased antioxidant potencies in relation to 1.
Changing of the phenol group of 1 for an amino group yields
tocopheryl amines such as 11, which shows antiproliferative
effects on the neuroblastoma glioma C6 cancer cell line 100
times more efficient than 1.[10] The fact that tocopherols as
well as tocopherol analogues display various biological activi-
ties[11–12] beyond their well established antioxidant properties
led us to synthesize the unexplored chromanol 12 (Scheme 1).
Like 1, this tocopherol analogue shows the same R chirality at
the stereogenic centers C2/C4’/C8’; however, the methyl group
at C2 is replaced by a hydrogen and 12 is hence named nor-a-
tocopherol.
[a] Dr. J. Chapelat, Dr. U. Hengartner, Prof. A. Chougnet, Dr. K. Liu,
Prof. W.-D. Woggon
Department of Chemistry, University of Basel
St. Johanns Ring 19, 4056 Basel (Switzerland)
Fax: (+41)267-1103
E-mail: wolf-d.woggon@unibas.ch
[b] Dr. P. Huebbe, Prof. G. Rimbach
Institute for Human Nutrition and Food Science
Christian-Albrechts-Universitꢀt zu Kiel
H. Rodewald Strasse 6, 24118 Kiel (Germany)
118
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ChemBioChem 2011, 12, 118 – 124

Synthesis and Biological Activity of nor-a-Tocopherol
Scheme 1. Attempted synthesis of 12.
Results and Discussion
Synthesis of nor-a-tocopherol
Our first approach directed towards enantiomerically enriched
nor-a-tocopherol (12) involved the use of a procedure pub-
lished by Overman et al.[13] in which couplings of Z-configured
allyl trichloroacetimidates with various phenols in the presence
of catalytic amounts of the Pd complex 13 were performed
(Scheme 1). Unfortunately, with the phenol 14 and the activat-
ed olefin 15 this reaction did not work at all and it seems that
the scope of this procedure is limited to monosubstituted phe-
nols and short olefins.
Accordingly we developed a more elaborate, but neverthe-
less efficient, route to 12 (Scheme 2). For the synthesis of the
key intermediate 16 two building blocks were required: 1) the
Grignard reagent 17,[14] available from (3R,7R)-hexahydrofarne-
sol 18,[15] and 2) the allyl epoxide 19, which was accessible
from commercially available (R)-epichlorohydrin.[16] The reac-
tion between 17 and 19, catalyzed by lithium cuprate,[17–18]
gave the alcohol 16 in 80% yield and 97% de (diastereomeric
excess), determined on the corresponding p-nitrobenzoate.
Unlike in cases of tertiary alcohols[14] the Mitsunobu reaction
between the non-activated 16 and the phenol 14 proceeded
readily and gave the phenol ether 20 in 60% yield with com-
plete inversion of configuration. Ozonolysis of the double
bond of 20 was unsatisfactory, so cleavage of the olefin to the
aldehyde 21 was pursued in two steps via the diol 22 in excel-
lent yield. Acid-catalyzed cyclization to the chromane system
and subsequent hydrogenation was accomplished in 91%
yield and the resulting chromanol ether 23 was finally depro-
tected to give nor-a-tocopherol (12) in 94% de.
Scheme 2. Synthesis of nor-a-tocopherol (12).
mic at C2 (Scheme 3). For this purpose phytol (25) was con-
verted into the known acid 26[19] in analogy with a published
procedure.[20] Reduction to the alcohol 27 followed by Dess–
Martin oxidation to the corresponding aldehyde and subse-
quent Wittig reaction furnished the unsaturated ester 28. Re-
For the preliminary biological experiments reported here
larger amounts of nor-a-tocopherol were required, and so we
developed a route to (2RS,4’R,8’R)-nor-a-tocopherol (24), race-
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119

W.-D. Woggon et al.
afford pure 24. In order to prepare stable stock solutions for
biological experiments, 24 was quantitatively acetylated to 32.
Evaluation of the biological activity of (2RS,4’R,8’R)-nor-a-
tocopherol (24)
As mentioned above, the antioxidant activities of tocopherols
and analogues are to a large extent related to stabilization of
the radical center adjacent to O1. In order to compare this
property for tocopherol and nor-tocopherol, the electron distri-
butions in the SOMOs of the chromanyl radicals were calculat-
ed. As shown in Figure 1 for the two units 33 and 34, corre-
Figure 1. SOMOs of the chromanyl radicals 33 and 34.
sponding to a-tocopherol and nor-a-tocopherol, respectively,
the electron distributions are superimposable, and hence no
significant difference in the in vitro antioxidant activities of 12
and 24 versus a-tocopherol (1) would be expected. According-
ly we focused our investigations on tocopherol features[11–12]
unrelated to its antioxidant activity.
An increasing volume of evidence suggests a role for a-toco-
pherol in modulating redox-regulated signal transduction
pathways both in cultured cells and in vivo.[21–24] Although no
common transcription factor for a-tocopherol-sensitive molec-
ular targets has yet been identified, different genes have been
reported to be affected by a-tocopherol, including those in-
volved in the regulation of inflammation and atherogenesis.[25]
Monocytes, when stimulated with the endotoxin lipopolysac-
charide (LPS), differentiate into macrophages and produce
large amounts of pro-inflammatory mediators, including tumor
necrosis factor a (TNFa), interleukin 1b (IL-1b), and interleu-
kin 6 (IL-6). Furthermore, release of pro-inflammatory mole-
cules triggers nitric oxide production by activation of the
macrophage-inducible NO synthase (iNOS). For our cell culture
studies we used LPS-stimulated murine RAW264.7 monocytes
to investigate the potential anti-inflammatory activity of
(2RS,4’R,8’R)-nor-a-tocopherol (24) relative to 12 (94% de) and
to a-tocopherol (1).
Scheme 3. Synthesis of (2R/S,4’R,8’R)-nor-a-tocopherol (24).
duction to the alcohol 29 and subsequent oxidation yielded
the desired aldehyde 30 ready for coupling with the phenol
14. Lewis-acid-catalyzed ring closure gave the chromene 31,
which was conveniently hydrogenated and deprotected to
It is important to note that for initial biological experiments
a stable stock solution of the phenol ester 32 was used. How-
ever, 32 is hydrolyzed to 24 under conditions of incubation
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ChemBioChem 2011, 12, 118 – 124

Synthesis and Biological Activity of nor-a-Tocopherol
with cell cultures. Accordingly the results reported in Fig-
ures 2–4 refer to the biological activity of (2RS,4’R,8’R)-nor-a-to-
copherol (24).
We determined the expression of pro-inflammatory mole-
cules after LPS stimulation (100 ngmLÀ1) in RAW264.7 cells pre-
incubated with 50 mmolLÀ1 nor-a-tocopheryl acetate 32, which
is a noncytotoxic concentration. Relative mRNA levels of TNFa,
IL-1b, IL-6, and iNOS were significantly decreased in cells incu-
bated with the nor-a-tocopherol relative to controls (Figure 2).
Figure 4. Effect of 24 (nor-aT) on iNOS protein level in murine macrophages.
Cells were preincubated with 32 (50 mmolLÀ1) for 24 h and stimulated with
interferon g (IFNg) and LPS. Whole cells were lyzed after 24 h and protein
levels were assessed by Western Blotting. Relative protein intensities were
determined by densitometry.
Figure 2. Effect of 24 (nor-aT) on mRNA levels of pro-inflammatory genes in
murine macrophages. RAW264.7 cells were preincubated with 32
(50 mmolLÀ1) for 24 h and subsequently stimulated with LPS. Total RNA was
isolated after 1 h (for TNFa) and 6 h (for IL-1b, IL-6, and iNOS). mRNA levels
were measured by real-time RT-PCR. Values are meansÆSD (n=6). # indi-
cates significant differences (p<0.5) between nor-aT-treated and untreated
control cells.
proved to be (1.8Æ0.3)-, (3.0Æ0.3)-, and (1.8Æ0.1) times more
potent than a-tocopherol (1).
No significant difference between (2RS,4’R,8’R)-nor-a-toco-
pherol (racemic at C2, 24) and compound 12 [(2R,4’R,8’R),
94% de] with regard to inhibition of inflammatory gene ex-
pression in our RAW264.7 cells was observed.
Similar results for (2RS,4’R,8’R)-nor-a-tocopherol (24) were ob-
tained with regard to proinflammatory cytokine levels in the
cell culture supernatant (Figure 3) and iNOS protein levels in
cell lysates of our RAW264.7 cells (Figure 4). In fact,
(2RS,4’R,8’R)-nor-a-tocopherol (24) significantly decreased LPS-
induced secretion of TNF-a, IL-1b, and IL6 relative to untreated
controls, as well as cellular iNOS protein levels.
Conclusions
Nor-a-tocopherol is a potent anti-inflammatory molecule in
murine macrophages in vitro. These properties are unrelated
to the absolute configuration at C2, because 12 and 24 dis-
played the same biological activities. However, nor-a-tocopher-
ol is a significantly better inflammatory agent than a-tocopher-
ol (1).
Experimental Section
General: Chemicals and solvents were purchased from commercial
suppliers or purified by standard techniques. For TLC, silica gel
plates (Merck AG, 60 F254) were used and compounds were visual-
ized by irradiation with UV light and/or by treatment with a solu-
tion of phosphomolybdic acid, cerium(IV) sulfate, and concd. H2SO4
in water (2:4:40:160 by weight) followed by heating. Flash chroma-
tography was performed with Fluka silica gel 60 (particle size
0.040–0.063 mm). 1H and 13C NMR spectra were recorded with a
Bruker DPX NMR (400 MHz and 500 MHz) spectrometer at ambient
temperature. Chemical shifts are given in d units relative to tetra-
methylsilane (TMS, d=0 ppm), and the coupling constants (J) are
given in Hz. If not otherwise stated, spectra were recorded in
CDCl3 at ambient temperature; for 1H NMR spectra tetramethylsi-
lane was used as internal standard and for 13C NMR spectra CDCl3
was used as internal standard (d=77.2 ppm). Melting points are
uncorrected. IR spectroscopy was performed with a PerkinElmer
Figure 3. Effect of 24 (nor-aT) on secretion of the proiinflammatory cyto-
kines TNF-a, IL-1b, and IL6. Cells were preincubated with 32 (50 mmolLÀ1
)
for 24 h and subsequently stimulated with LPS. Cell culture supernatants
were collected after 24 h and cytokine production was measured by ELISA.
Values are meansÆSD (n=3). # indicates significant differences (p<0.5)
between nor-aT and untreated controls.
As far as the inhibition of LPS-induced TNF-a, IL-1b, and IL-6
gene expression is concerned, (2RS,4’R,8’R)-nor-a-tocopherol
ChemBioChem 2011, 12, 118 – 124
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121

W.-D. Woggon et al.
1600 FTIR apparatus. Electron-spray ionization mass spectra (ESI-
MS) were recorded with a Bruker Esquire 3000 plus spectrometer.
HPLC was carried out with an intelligent pump, detector, and inte-
grator and a Hewlett Packard S1100 or a Shimadzu LC-20AB/SPD-
M20A instrument. Microanalyses were performed with a PerkinElm-
er 240 Analyzer by Werner Kirsch at the Department of Chemistry,
University of Basel. Optical rotations were measured with a Per-
kinElmer Polarimeter 341 at l=589 nm.
4.18 (q, J=6 Hz, 1H), 3.66 (s, 3H), 2.34–2.46 (m, 2H), 2.26 (s, 3H),
2.20 (s, 3H), 2.12 (s, 3H), 1.0–1.65 (m, 21H), 0.84–0.88 ppm (m,
12H); 13C NMR (100 MHz, CDCl3): d=152.43, 150.87, 135.07, 130.98,
128.03, 125.50, 117.49, 113.75, 78.37, 60.52, 39.79, 38.71, 37.86,
37.75, 37.71, 37.47, 34.44, 33.21, 33.12, 28.40, 25.22, 24.88, 23.23,
23.14, 23.05, 20.17, 20.11, 16.76, 13.15, 12.65 ppm; IR (neat): n˜ =
1641, 1479, 1229, 1090 cmÀ1; EI-MS: m/z (%): 444.4 (10) [M], 166.1
(100); elemental analysis calcd (%) for C30H52O2 (444.74): C 81.02, H
11.78; found: C 80.86, H 11.65.
Preparation of 16: N-Bromosuccinimide (4.63 g, 26 mmol) was
added portionwise over 30 min at 4–88C to a solution of (3R,7R)-
hexahydrofarnesol (18, 5.25 g, 23 mmol) and triphenylphosphine
(7.23 g, 27.6 mmol) in dichloromethane (35 mL). The reaction mix-
ture was stirred for 20 min and then concentrated on a rotary
evaporator. The two-phase residue was triturated with hexane and
the solid material was filtered off and washed with hexane. The
concentrated filtrate was purified by chromatography on SiO2
(hexane) to afford the corresponding bromide (6.55 g, 98%) as a
colorless oil. This (874 mg, 3.0 mmol) was dissolved in THF (4 mL)
and added over 15 min to a suspension of magnesium turnings
(85 mg, 3.5 mmol) in THF (1 mL)/dibromoethane (15 mL,
0.17 mmol). The mixture was stirred at RT for 30 min and at 508C
for 4 h.
Preparation of 22: A solution of 4-methylmorpholine N-oxide
monohydrate (270 mg, 2.0 mmol) in water (4 mL) and an aqueous
solution of osmium tetroxide (4%, 480 mL, 75 mmol) were added to
a solution of the olefin 20 (427 mg, 0.96 mmol) in acetone (16 mL).
The reaction mixture was stirred at room temperature for 20 h. It
was partitioned between diethyl ether and water, and the organic
phase was washed with sat. aq. Na2SO3 and brine, dried, and con-
centrated. The residual oil was purified by chromatography on SiO2
(hexane/EtOAc 5:4) to afford 7 (382 mg, 83%) as a colorless, vis-
cous oil. 1H NMR (400 MHz, CDCl3): d=6.60 (s, 1H), 4.39–4.50 (m,
1H), 3.96–4.04 (m, 1H), 3.64–3.72 (m, 1H), 3.66 (s, 1.5H), 3.65 (s,
1.5H), 3.46–3.55 (m, 1H), 3.14 (d, J=2 Hz, 0.5H), 2.59 (d, J=4 Hz,
0.5H), 2.25 (s, 3H), 2.19 (s, 3H), 2.10 (s, 3H), 1.0–2.05 (m, 24H),
0.80–0.88 ppm (m, 12H); IR (neat): n˜ =3387, 1479, 1229, 1090 cmÀ1
;
In a second flask, a solution of (R)-2-allyloxirane (19, 190 mg,
2.26 mmol) in THF (4 mL) was cooled to À708C, the Grignard re-
agent 17 was added by syringe, and the mixture was stirred at
À708C for 5 min. The mixture was stirred at À708C for 5 min. A
catalytic amount of dilithium tetrachlorocuprate(II) (0.1m solution
in THF, 40 mmol) was then added. After stirring at À708C for
15 min the reaction mixture was allowed to warm slowly to room
temperature while additional dilithium tetrachlorocuprate solution
(80 mmol) was added portionwise. The mixture was stirred for 2 h,
quenched with half-saturated aq. NH4Cl, and extracted with tert-
butyl methyl ether (TBME). The organic layer was washed with
brine, dried (MgSO4), and concentrated on a rotary evaporator. The
colorless oil was purified by column chromatography on SiO2
(hexane/EtOAc 10:1) to afford 16 (491 mg, 73%) as a colorless, vis-
EI-MS: m/z (%): 478.4 (4) [M], 166.1 (100); elemental analysis calcd
(%) for C30H54O4 (478.75): C 75.26, H 11.37; found: C 75.01, H 11.23.
Preparation of 21: Sodium periodate (300 mg, 1.3 mmol) was
added to a solution of the diol 20 (123.6 mg, 0.26 mmol) in ace-
tone/water (4:1, 5 mL). The heterogeneous mixture was stirred at
room temperature for 2 h. It was partitioned between TBME and
water, and the organic phase was washed with sat. brine, dried,
and concentrated in vacuo to afford the aldehyde 21 (109.5 mg,
95%) as a colorless oil. [a]2D0 =À11.1 (c=1, in hexane); 1H NMR
(400 MHz, CDCl3): d=9.82 (t, J=2 Hz, 1H), 6.56 (s, 1H), 4.66 (q, J=
6 Hz, 1H), 3.65 (s, 3H), 2.78 (ddd, J=16, 6, 3 Hz, 1H), 2.69 (ddd, J=
16, 6, 3 Hz, 1H), 2.25 (s, 3H), 2.18 (s, 3H), 2.08 (s, 3H), 1.0–1.8 (m,
21H), 0.82–0.88 ppm (m, 12H); 13C NMR (100 MHz, CDCl3): d=
201.60, 151.61, 151.44, 131.29, 128.37, 125.70, 114.02, 74.38, 60.51,
48.44, 39.77, 37.84, 37.73, 37.69, 37.28, 35.18, 33.20, 33.10, 28.39,
25.21, 24.87, 23.14, 23.04, 20.15, 20.03, 16.73, 13.15, 12.65; IR
(neat): n˜ =1723, 1476, 1455, 1227, 1088 cmÀ1; EI-MS: m/z (%): 446.4
(21) [M], 166.1 (100); elemental analysis calcd (%) for C29H50O3
(446.71): C 77.97, H 11.28; found: C 78.17, H 11.21.
1
cous oil. [a]2D0 =À4.58 (c=1.47 in CHCl3); H NMR (400 MHz,CDCl3):
d=5.78–5.88 (m, 1H), 5.11–5.16 (m, 2H), 3.6–3.7 (m, 1H), 2.25–2.35
(m, 1H), 2.10–2.18 (m, 1H), 1.0–1.6 (m, 22H), 0.84–0.88 ppm (m,
12H); 13C NMR (100 MHz, CDCl3): d=135.33, 118.50, 71.10, 42.40,
39.77, 37.84, 37.78, 37.69, 37.56, 37.40, 33.20, 33.16, 28.39, 25.21,
24.88, 23.54, 23.13, 23.04, 20.16, 20.10 ppm; IR (neat): n˜ =3348,
1640 cmÀ1; EI-MS: m/z (%): 255.3 (55) [MÀC3H5]; elemental analysis
calcd (%) for C20H40O (296.54): C 81.01, H 13.60; found: C 80.78, H
13.37. Determination of diastereomeric excess de=97%: HPLC on
Chiralpak AD-H column (heptane/propan-2-ol 99.8:0.2), UV 254 nm;
0.5 mLminÀ1; major diastereoisomer tR =18.7 min, minor diastereo-
isomer tR =23.2 min.
Preparation of 23: A solution of the aldehyde 21 (109.5 mg,
0.26 mmol) in dichloromethane (5 mL) was cooled to 08C. Tri-
fluoroacetic acid (50 mL) was added and the solution was stirred at
08C for 25 min. Palladium on carbon (10%, 40 mg) was then
added, the argon was replaced with hydrogen (1 atm), and the
chromane was hydrogenated at room temperature for 1 h. The fil-
tered reaction mixture was concentrated in vacuo. The residual oil
was purified by chromatography on SiO2 (hexane/EtOAc 80:1) to
Preparation of 20:
1.65 mmol), 4-methoxy-2,3,5-trimethylphenol
A
solution of the alcohol 16 (490 mg,
(14, 399 mg,
2.4 mmol), and triphenylphosphine (629 mg, 2.4 mmol) in toluene
(5 mL) was cooled in an ice bath. Diisopropyl azodicarboxylate
(525 mg, 2.6 mmol) in toluene (2.5 mL) was added dropwise over
15 min and the mixture was stirred at room temperature for 1 h.
The reaction mixture was concentrated and the residual viscous oil
was partitioned between hexane and aqueous methanol (85%,
v/v). The aq. methanol phase was reextracted with hexane and the
combined hexane phases were dried (MgSO4) and concentrated in
vacuo. The residual oil was purified by chromatography on SiO2
(hexane/EtOAc 25:1) to yield 20 (438 mg, 60%) as a colorless, vis-
cous oil. [a]2D0 =+3.9 (c=1.08 in CHCl3); 1H NMR (400 MHz,CDCl3):
d=6.52 (s, 1H), 5.86 (ddt, J=17, 10, 7 Hz, 1H), 5.04–5.14 (m, 2H),
afford 23 (96.1 mg, 91%) as a white solid; m.p. 39–418C; [a]D20
=
+49.0 (c=1, CHCl3); 1H NMR (400 MHz, CDCl3): d=3.86 (m, 1H),
3.64 (s, 3H), 2.63 (m, 2H), 2.20 (s, 3H), 2.14 (s, 3H), 2.12 (s, 3H),
2.00 (m, 1H), 1.0–1.75 (m, 22H), 0.84–0.89 ppm (m, 12H); 13C NMR
(100 MHz, CDCl3): d=150.08, 149.63, 128.13, 126.47, 123.10, 119.00,
75.31, 60.80, 39.79, 37.88, 37.77, 37.72, 37.32, 36.16, 33.22, 33.19,
28.40, 28.31, 25.23, 24.89, 23.66, 23.35, 23.15, 23.05, 20.17, 20.16,
12.92, 12.13, 12.08 ppm; IR (neat): n˜ =1456, 1251, 1091 cmÀ1; EI-
MS: m/z (%): 430.4 (100) [M]; elemental analysis calcd (%) for
C29H50O2 (430.71): C 80.87, H 11.70; found: C 80.77, H 11.59.
122
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Synthesis and Biological Activity of nor-a-Tocopherol
.
Preparation of 12: A solution of the chromane 23 (94.6 mg,
0.22 mmol) in dichloromethane (3 mL) was cooled to 08C. Boron
trifluoride methyl sulfide complex (0.5 mL), anhydrous aluminum
chloride (360 mg), and acetonitrile (2 mL) were added, and the
mixture was stirred at room temperature for 4 h. The mixture was
partitioned between ice-cold aqueous NaHCO3 and TBME, and the
organic phase was washed with sat. brine, dried, and concentrated
on a rotary evaporator. The crude product was purified by chroma-
tography on SiO2 (hexane/EtOAc 30:1 to 12:1) to afford nor-a-toco-
quenched with NaSO4 10H2O and the mixture was stirred at RT for
1 h. The solid was filtered and washed twice with ether. The com-
bined organic solvents were concentrated under vacuum to afford
the allyl alcohol 29 (230 mg, yield 83%) as a colorless oil. [a]D20
À0.8 (c=1.10 in CHCl3); H NMR (CDCl3, 400 MHz): d=5.66 (m, 2H),
4.08 (t, J=5.2 Hz, 2H), 2.03 (m, 2H), 1.50 (m, 1H), 1.02–1.45 (m,
19H), 0.85 ppm (m, 12H); 13C NMR (CDCl3, 100 MHz): d=133.85,
128.99, 64.07, 39.56, 37.62, 37.54, 37.48, 36.76, 32.98, 32.87, 32.74,
28.17, 26.82, 24.98, 24.65, 22.91, 22.81, 19.94, 19.89 ppm; ESI-MS:
m/z (%): 305.3 [M+Na]+; elemental analysis calcd (%) for C20H38O2:
C 80.78, H 13.56; found: C 80.90, H 13.30.
=
1
pherol (12, 73.8 mg, 81%) as a colorless, wax-like solid. [a]D20
=
+42.5 (c=1 in CHCl3); 1H NMR (400 MHz,CDCl3): d=4.19 (s, 1H),
3.82 (m, 1H), 2.65 (m, 2H), 2.16 (s, 3H), 2.13 (s, 3H), 2.10 (s, 3H),
2.00 (m, 1H), 1.0–1.75 (m, 19H), 0.83–0.88 ppm (m, 12H); 13C NMR
(100 MHz, CDCl3): d=147.44, 145.25, 122.75, 121.26, 119.13, 118.73,
75.21, 39.79, 37.88, 37.77, 37.72, 37.32, 36.09, 33.22, 33.19, 28.47,
28.40, 25.23, 24.89, 23.70, 23.39, 23.15, 23.05, 20.18, 20.16, 12.56,
12.15, 11.67 ppm; IR (neat): n˜ =3350, 1460, 1254, 1084 cmÀ1; EI-MS:
m/z (%): 416.4 (100) [M]; ESI-MS (MeOH, neg): m/z (%): 415 [MÀH],
831 [2MÀH]; elemental analysis calcd (%) for C28H48O2 (416.69): C
80.71, H 11.61; found: C 80.77, H 11.53. Determination of diastereo-
meric excess de=94%: HPLC on Chiralpak AD-H column (heptane/
propan-2-ol 99:1), UV 295 nm; 0.8 mLminÀ1; major diastereomer
tR =12.1 min, minor diastereomer tR =12.9 min.
Preparation of 30: Activated MnO2 (869 mg, 10 mmol) was added
at RT to a solution of the allylic alcohol 29 (225 mg, 0.80 mmol) in
dichloromethane (5 mL) and the reaction mixture was stirred for
4 h. The solid was then filtered through a celite pad and washed
three times with dichloromethane. The combined organic layers
were concentrated under vacuum to afford the unsaturated alde-
hyde 30 (220 mg, yield 98%) as a colorless oil. [a]2D0 =À2.2 (c=1.10
in CHCl3); 1H NMR (CDCl3, 400 MHz): d=9.50 (d, J=8.00 Hz, 1H),
6.85 (dt, J=15.60, 6.80 Hz, 1H), 6.11 (ddt, J=15.60, 8.00, 1.40 Hz,
1H), 2.32 (m, 2H), 1.02–1.49 (m, 19H), 0.85 ppm (m, 12H); 13C NMR
(CDCl3, 100 MHz): d=194.32, 159.21, 133.17, 39.54, 37.56, 37.45,
37.44, 36.67, 33.24, 32.96, 32.80, 28.15, 25.58, 24.97, 24.60, 22.89,
22.80, 19.92, 19.78 ppm; ESI-MS: m/z (%): 303.3 [M+Na]+.
Preparation of 27: The alcohol 27 was prepared from phytol
25;[19,20] a different synthesis of racemic 27 has been reported pre-
viously.[26] Characteristic data for 27: [a]D20 =+1.1 (c=1.05 in CHCl3);
1H NMR (CDCl3, 400 MHz): d=3.64 (t, J=6.40 Hz, 2H), 1.05–1.56 (m,
21H), 0.85 ppm (m, 12H); 13C NMR (CDCl3, 100 MHz): d=63.32,
39.56, 37.62, 37.54, 37.47, 37.00, 33.35, 32.98, 32.96, 28.17, 24.98,
24.66, 23.41, 22.91, 22.81, 19.94, 19.86 ppm; ESI-MS: m/z (%): 279.2
[M+Na]+; elemental analysis calcd (%) for C17H36O: C 79.61, H
14.15; found: C 79.57, H 13.98.
Preparation of 24: A freshly prepared solution of Ti(OEt)4 in tolu-
ene (0.9 mL) was added to a solution of the hydroquinone 14
(83.1 mg, 0.5 mmol) in toluene (1 mL). The reaction mixture was
heated at reflux for 30 min. The aldehyde 30 (225 mg, 0.8 mmol)
was then added to the mixture, which was heated at reflux for an-
other 3.5 h. The reaction mixture was quenched with sat. aq. NH4Cl
and extracted with ether. The combined organic layers were
washed with brine and dried over Na2SO4. After concentration
under vacuum the residue was purified by flash chromatography
on SiO2 with hexane/ethyl acetate (49:1, v/v) as a eluent to afford
the chromene 31 (150 mg, yield 72%) as a yellowish oil. The air-
and light-sensitive 31 was immediately hydrogenated and depro-
tected as described for the transformation of 23 to 12. Finally,
(2RS,4’R,8’R)-nor-a-tocopherol (24, 117 mg, 80%) was obtained as a
colorless oil, spectroscopically identical with 12.
Preparation of 28: A solution of Dess–Martin periodinane (2.34 g,
5.4 mmol) in dichloromethane (5 mL) was added under Ar to a
solution of the alcohol 27 (1 g, 3.90 mmol) in dichloromethane
(12 mL). The reaction mixture was stirred at RT for 2 h and
quenched with a solution of Na2S2O3 (7.2 g) in saturated aq. NH4Cl
(32 mL). The mixture was extracted twice with ether. The extract
was washed with brine, dried over Na2SO4, and concentrated
under vacuum to afford the crude aldehyde as colorless oil used
immediately for the next step.
Preparation of 32: (2RS,4’R,8’R)-nor-a-Tocopherol (24, 25.1 mg,
0.06 mmol) was dissolved in pyridine (0.5 mL), and acetic anhydride
(0.1 mL,109 mg, 1.06 mmol) was added by syringe. The mixture
was stirred for 3 h at RT and was then poured into HCl (10%, 5 mL)
and extracted three times with dichloromethane (30 mL). The com-
bined organic phases were washed twice with water (20 mL) and
finally dried over Na2SO4. The solution was concentrated under
vacuum and the pure product 32 was obtained as slightly yellow
oil (26.9 mg, 0.59 mmol, 97%). 1H NMR (CDCl3, 400 MHz): d=3.85
(m, 1H), 2.65–2.62 (m, 2H), 2.33 (s, 3H), 2.11 (s, 3H), 2.02 (s, 3H),
1.96 (s, 3H), 1.8–1.6 (m, 2H), 1.6–1.05 (m, 21H), 0.9–0.8 ppm (m,
12H).
The freshly prepared aldehyde (382 mg, 1.50 mmol) in dimethyl
sulfoxide (1 mL was added) under Ar to a solution of Ph3P=
CHCOOMe (1.0 g, 3 mmol) in dimethyl sulfoxide (4 mL). The reac-
tion mixture was stirred overnight, quenched with methanol/water
(85:15, v/v), and extracted with hexane. The organic phases were
washed with brine and dried over Na2SO4. After concentration, a
residue was purified by flash chromatography on SiO2 with di-
chloromethane/hexane (1:1, v/v) to afford the ester 28 (300 mg,
yield 63%) as a colorless oil. [a]2D0 =À1.9 (c=1.22 in CHCl3);
1H NMR (CDCl3, 400 MHz): d=6.97 (dt, J=15.60, 7.00 Hz, 1H), 5.82
(dt, J=15.60, 1.40 Hz, 1H), 3.72 (s, 3H), 2.19 (m, 2H), 1.04–1.52 (m,
19H), 0.85 ppm (m, 12H); 13C NMR (CDCl3, 100 MHz): d=167.40,
150.04, 120.99, 51.55, 39.55, 37.59, 37.47, 36.69, 32.97, 32.82, 32.74,
28.17, 25.75, 24.98, 24.62, 22.90, 22.81, 19.92, 19.81 ppm; ESI-MS:
m/z (%): 333.3 [M+Na]+, 643.3 [2M+Na]+; elemental analysis calcd
(%) for C20H38O2: C 77.36, H 12.33; found: C 77.57, H 12.09.
Cell culture experiments: RAW264.7 cells were cultured in Dulbec-
co’s modified Eagle’s medium supplemented with fetal bovine
serum (10%), together with penicillin (100 UmLÀ1) and streptomy-
cin (100 mgmLÀ1, all reagents from PAA). Cells were grown in a hu-
midified atmosphere at 378C and 5% CO2. For induction of cyto-
kine production, cells were stimulated with LPS from Salmonella
enteriditis (Sigma, 100 ngmLÀ1). For measurement of iNOS levels,
cells were stimulated with IFNg (Invitrogen, 5 ngmLÀ1) and LPS
(100 ngmLÀ1). Total RNA was isolated by chloroform/water separa-
tion and isopropyl alcohol precipitation. RNA concentration was
Preparation of 29: A solution of DIBAL-H in hexane (1m, 2 mL)
was added at 08C under Ar to a solution of the ester 28 (300 mg,
0.96 mmol) in dry ether (4 mL). The reaction mixture was allowed
to warm to RT and stirred for another 2 h. The reaction was
ChemBioChem 2011, 12, 118 – 124
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www.chembiochem.org
123

W.-D. Woggon et al.
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Dr. Thomas Netscher (DSM) for a generous sample of phytol (25),
and Dr. Christian Bartels for calculations.
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Keywords: inflammation
· nor-tocopherol · tocopherol ·
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Published online on December 15, 2010
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