Preparation of fluorescent tocopherols for use in protein binding and localization with the α-tocopherol transfer protein

Article abstract of DOI:10.1016/j.bmc.2006.01.053

Sixteen fluorescent analogues of the lipid-soluble antioxidant vitamin α-tocopherol were prepared incorporating fluorophores at the terminus of ω-functionalized 2-n-alkyl-substituted chromanols (1a-d and 4a-d) that match the methylation pattern of α-tocop

Full text of DOI:10.1016/j.bmc.2006.01.053

Bioorganic & Medicinal Chemistry 14 (2006) 3721–3736
Preparation of fluorescent tocopherols for use in protein binding
and localization with the a-tocopherol transfer protein
Phillip Nava,a Matt Cecchini,a Sara Chirico,a Heather Gordon,a Samantha Morley,b
Danny Manorb and Jeffrey Atkinsona,*
aDepartment of Chemistry and Centre for Biotechnology, Brock University, St. Catharines, Ont., Canada L2S 3A1
bDivision of Nutritional Sciences, Savage Hall, Cornell University, Ithaca, NY 14853, USA
Received 2 December 2005; revised 11 January 2006; accepted 12 January 2006
Available online 14 February 2006
Abstract—Sixteen fluorescent analogues of the lipid-soluble antioxidant vitamin a-tocopherol were prepared incorporating fluoro-
phores at the terminus of x-functionalized 2-n-alkyl-substituted chromanols (1a–d and 4a–d) that match the methylation pattern of
a-tocopherol, the most biologically active form of vitamin E. The fluorophores used include 9-anthroyloxy (AO), 7-nitrobenz-2-oxa-
1,3-diazole (NBD), N-methyl anthranilamide (NMA), and dansyl (DAN). The compounds were designed to function as fluorescent
reporter ligands for protein-binding and lipid transfer assays. The fluorophores were chosen to maximize the fluorescence changes
observed upon moving from an aqueous environment (low fluorescence intensity) to an hydrophobic environment such as a pro-
tein’s binding site (high fluorescence intensity). Compounds 9d (anthroyloxy) and 10d (nitrobenzoxadiazole), having a C9-carbon
chain between the chromanol and the fluorophore, were shown to bind specifically and reversibly to recombinant human tocopherol
transfer protein (a-TTP) with dissociation constants of approximately 280 and 60 nM, respectively, as compared to 25 nM for the
natural ligand 2R,40R,80R-a-tocopherol. Thus, compounds have been prepared that allow the investigation of the rate of a-TTP-
mediated inter-membrane transfer of a-tocopherol and to investigate the mechanism of a-TTP function at membranes of different
composition.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
tocopherol between different intracellular sites. A signif-
icant barrier to studies with this vitamin is that current
techniques for determining tissue or plasma levels re-
quire extraction in organic solvents followed by chroma-
tographic analysis and often mass spectrometry. These
analytical barriers especially complicate determinations
of where the tocopherol resides in a cell and of the
dynamics of a-TTP-mediated tocopherol transfer be-
tween membranes. While tocopherol is weakly fluores-
cent (kabs ꢀ 290 nm, kem ꢀ 330 nm) in EtOH,12,13 its
absorption spectrum overlaps with those of many other
cell components and does not allow for selective moni-
toring techniques such as fluorescence resonance energy
transfer (FRET) or fluorescence microscopy.
a-Tocopherol is now accepted to be the dominant lipid-
soluble antioxidant in biological membranes.1 The
bioavailability and biokinetics of a-tocopherol2,3 and
c-tocopherol4,5 have been well studied in humans and
it is clear that the selective retention of the natural ste-
reoisomer (RRR) of a-tocopherol6 is due to the presence
of the a-tocopherol transfer protein (a-TTP) expressed
predominantly in liver.7–9 Of all the ligands tested so
far, a-TTP binds a-tocopherol with the highest affinity,
approximately 25 nM.10 However, the mechanisms by
which a-TTP delivers a-tocopherol from hepatocytes
to carrier lipoproteins in the plasma are poorly under-
stood. Particularly opaque are the mechanisms by which
very-low density lipoprotein (VLDL) particles are en-
riched with vitamin E prior to or during their secretion
from the liver6,11 and the molecular basis of transport of
Similar difficulties were encountered in the study of
other hydrophobic ligands and their transfer proteins,
and these were overcome by the preparation of novel
fluorescently labeled reporters such as the anthroyloxy
stearic acids14,15 and more recently the BODIPY fatty
acids.16,17 Fluorescently-labeled ligands have been par-
ticularly useful probes for quantitative binding assays
and for the elucidation of the mechanism underlying
protein-mediated inter-membrane transfer of the ligand
Keywords: Tocopherol; Vitamin E; Tocopherol transfer protein;
a-TTP; Fluorescence resonance energy transfer.
Corresponding author. Tel.: +1 905 6885550x3967; fax: +1 905
6829020; e-mail: jatkin@brocku.ca
*
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.01.053

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P. Nava et al. / Bioorg. Med. Chem. 14 (2006) 3721–3736
in the case of fatty acid-binding protein (FABP),18–20
sterol transfer protein,21–24 and cholesterol transfer pro-
tein.25–28 Based on the documented success of fluores-
cent hydrophobic ligands, we designed and prepared
fluorescent analogues of a-tocopherol that enable quan-
titative studies of ligand binding and transfer by a-TTP.
x-anthroyloxy tocopherols 5a–d that are readily depro-
tected (TBAF, THF) to give the free phenols 9a–d. De-
spite the fact that these compounds lack the normal
phytyl side-chain of the tocopherols, for simplicity we call
these ‘anthroyloxy-a-tocopherols’ or AO-a-Tocs.
In order to prepare conjugates of other fluorophores, it
was necessary to transform the alcohol functional group
in compounds 1a–d to the terminal amines in 4a–d. This
was accomplished in a straightforward manner by mesy-
lation, substitution with azide, and hydride reduction.
The amines 4a–d were coupled to fluorophores follow-
ing standard procedures.34 It is wisest to store the silyl
ether-protected compounds rather than the free phenols
as the easily cleavable silyl ethers (TBAF, THF) are less
prone to oxidative decomposition—a problem common
to the tocopherols. In the end, 16 compounds (four
homologous series) were prepared having four chain
lengths and four different fluorophores attached to a
2-alkyl chromanol that mimics natural source a-tocoph-
erol with respect to the chromanol methyl groups at C-5,
C-7 and C-8, and the C-2 R stereochemistry. The final
compounds are shown in Figure 2.
2. Results and discussion
As we have noted previously in the design of photo-
affinity ligands of tocopherol,29 any manipulation of
the tocopherol skeleton must not interfere with the chro-
manol head group as this will lessen or abolish the affin-
ity of such ligands to a-TTP.10,30 As a consequence, the
phytyl tail remains the only place available for modifica-
tion with a fluorescent group. To simplify this task the
side-chain methyl groups can be omitted, as their pres-
ence is not necessary for the biological activity of the
tocopherols.31 Thus, the sole remaining possible varia-
tion in structure is the choice of fluorophore and the
length of the alkyl chain connecting the fluorophore to
the chromanol. The recent X-ray structure determina-
tions of human a-TTP32,33 offer some guidance in this
regard. We chose to prepare structures with varying
chain lengths that ‘bracket’ the approximate side-chain
length of a-tocopherol. The general synthetic approach
follows very closely that reported in our earlier work
with tocopherol photoaffinity labels29 and is outlined
in Figure 1.
We were primarily interested in developing a fluorescent
form of tocopherol that would be useful in FRET assays
to monitor inter-membrane tocopherol transfer. We
focused our attention on the AO-a-Tocs, as they are
analogous to the anthroyloxy fatty acids that have been
well characterized as FRET partners with NBD-labeled
phospholipids and used for monitoring the fatty acid
transfer activity of FABP.18 The NBD absorption spec-
trum (kabs ꢀ 465 nm) overlaps the emission spectrum of
the AO-a-Tocs (kem ꢀ 460 nm). Thus, when a donor
membrane or a-TTP containing AO-Toc is mixed with
an acceptor membrane containing an NBD-phospho-
lipid, one can monitor the decay in fluorescence of the
AO-a-Toc as it arrives in the acceptor vesicle. A similar
transfer assay system can be constructed using an
The terminal alcohols 1a–d are prepared from the com-
mercially available (S)-Troloxꢁ after conversion to the
methyl ester, protection of phenol as the tert-butyldim-
ethylsilyl ether, reduction to the aldehyde, and reaction
with the x-functionalized phosphonium salts of varying
chain lengths.29 Compounds 1a–d can easily be esterified
to 9-anthracenecarboxylic acid (oxalyl chloride, cat.
DMF, CH2Cl2 then Et3N, and 1a–d) giving the protected
Br-
Ph3P
OH
RO
RO
n
OH
O
CHO
O
2.1 eq LiHMDS, THF
n
TBDMS-Trolox® aldehyde
10 % Pd/C, H2, EtOAc
RO
RO
a
OH
O
OMs
O
n
n
b
1a-d
2a-d
4a-d
RO
RO
c
N3
O
n
NH2
O
n
3a-d
Figure 1. R = TBDMS, n = 1–4. Reagents and conditions: (a) MsCl, Et3N, DMAP, CH2Cl2, 0 ꢂC, rt, 80–85%; (b) NaN3, DMF, 55 ꢂC, 85–89% from
1a–d; (c) LiAlH4, THF, 0 ꢂC, 83–90%.

P. Nava et al. / Bioorg. Med. Chem. 14 (2006) 3721–3736
3723
O
HO
HO
O
N
N
O
O
O
N
NO2
n
n
H
ω-Nitrobenzoxadiazole-α-tocopherols
(NBD-α-Toc) 10a-d (n = 1-4)
ω-Anthroyloxy-α-tocopherols
(AO-α-Toc) 9a-d (n = 1-4)
HO
HO
O
O
S
O
N
n
H
O
N
O
n
H
N
HN
CH3
ω-Dansyl-α-tocopherols
(DAN-α-Toc) 11a-d (n = 1-4)
ω-N-Methylanthranylamide-α-tocopherols
(NMA-α-Toc) 12a-d (n = 1-4)
Figure 2. Structures of the 16 x-functionalized fluorescent analogues of a-tocopherol prepared in this work.
NBD-a-Toc as FRET donor and a tetramethylrhod-
amine phospholipid such as TRITC-PE as a FRET
1.25
acceptor (kabs ꢀ 540 nm), or a Marina Blue-labeled
kem ꢀ 459 nm) and the NBD-a-Toc as the acceptor.35
1.00
phospholipid as
a
FRET donor (kabs ꢀ 362 nm,
0.75
0.50
0.25
0.00
Cholesterol
RRR-α-Tocopherol
Transfer of a-tocopherol between membranes in vivo is
understood to be mediated by the a-tocopherol transfer
protein (a-TTP).7,8,36 Thus, if the fluorescent analogues
are to be of any utility, it is required that they bind spe-
cifically and reversibly to this protein so that they may
act as competent probes of the unmodified, naturally
occurring vitamin. Figure 3 shows fluorescent titrations
of recombinant human a-TTP with C9-NBD-a-Toc,
10d. Figures 4 and 5 show that both the C9-NBD-a-
Toc and C9-AO-a-TOH analogues exhibit a dose-
dependent decrease in fluorescence upon the addition
of tocopherol. This suggests that the environmentally
sensitive fluorophores are displaced from the hydropho-
bic-binding pocket of a-TTP by the native ligand.
0
10
20
[Competitor] µM
40
50
30
Figure 4. Competitive displacement of C9-NBD-a-Toc (10d) bound to
a-TTP by addition of increasing amounts of RRR-a-tocopherol or
cholesterol. Fluorescence was monitored at 535 nm. The starting
protein–ligand complex included 0.2 lM a-TTP and 1.0 lM C9-NBD-
a-Toc. Competitions were performed in the same buffer as reported in
Figure 3 plus 100 lM Triton X-100.
fluorophore and the competitor are near their solubility
limits. By way of comparison, the water solubility of ret-
inol38 and cholesterol39 in water is 60 and 37 nM, respec-
tively. The solubility of a-tocopherol has been reported
to be 49 lM,40 but in our experience, the preparation
and use of pure tocopherol solutions in aqueous media
does not approach this value. The provision of organic
co-solvents (generally 1% or less of EtOH, acetonitrile,
or DMSO) is useful for preparing low concentration ‘so-
lutions’ (generally below ꢀ20 lM), but higher concen-
trations of ligand with similar content of organic co-
solvent do not exist in solution, but rather as distinct
populations of ligand aggregates in suspension, and/or
adhered to the surfaces of glass or plasticware. As a re-
sult, attempting a competitive displacement of bound
C9-NBD-a-Toc (10d) with a-tocopherol is not possible
in buffer lacking detergent. This is the result of the
inability to supply sufficient dissolved tocopherol in a
solution lacking detergent to achieve competitive equi-
librium in a reasonable length of time. The ability of
the ligand to dissociate from the binding site is predicat-
ed on having a thermodynamically favorable place to
move to, and generally an aqueous buffer did not
Competitive assays with these hydrophobic ligands
are not straightforward to interpret since both the
200000
Kd=56 15 nM
100000
0.2 µM α-TTP
No Protein Control
0
0.00 0.25 0.50 0.75 1.00 1.25 1.50
[C9-NBD-α-Toc] µM
Figure 3. Titration curves showing the increase in fluorescence
intensity at 535 nm during sequential additions of the C9-NBD-a-
Toc, 10d (black squares), to a 0.2 lM solution of a-TTP in SET buffer
(see Section 4). The curves are fitted to a one-site bimolecular
association model.37 Averages and standard errors of triplicate data
sets are reported. kex = 469 nm.

3724
P. Nava et al. / Bioorg. Med. Chem. 14 (2006) 3721–3736
provide this requirement. This was a more pronounced
problem for the anthroyloxy compounds than for the
NBD analogues. In effect, the ligand prefers to stay in
the binding site than dissociate into solution where its
solubility is very low. Complicating this is the observa-
tion that adding detergent to the assay buffer raises
the background fluorescence as the fluorophore is
removed from the protein. The displaced fluorophore
then exists in a hydrophobic complex with detergent
and generates a higher fluorescence signal than in buffer
alone. The result is what looks like an incomplete com-
petition; down to only about 30% of the original fluores-
cence. It is worth noting that detergent alone does not
remove any of the fluorophores once bound to a-TTP.
This is exemplified by the constant fluorescence signal
of the ligand in Figures 4 and 5. Competitions with cho-
lesterol, which has a very low affinity to TTP,10 showed
an apparent increase in fluorescence for the C9-AO-
a-Toc (Figure 5). This is an artifact due to the very
long times required for equilibration of a-TTP with
fluorophore in detergent-containing buffers. During
fluorescence titrations in buffers lacking detergent, equil-
ibration (i.e., a stable fluorescence intensity) was
achieved in 5–10 min with all ligands, but in detergent-
containing buffers the time to a similar stable fluores-
cence signal required 1 h for NBD-a-Toc and >4 h for
AO-a-Toc. As a result the cholesterol competition in
Figure 5 was not yet at equilibrium (ꢀ95%) and conse-
quently normalization of data at higher concentrations
appeared to give >100% relative fluorescence.
specific controls and thus Kd-values could not be
determined. One advantage of the AO-a-Tocs is their
very weak fluorescence in aqueous solution. Contrarily,
while the C9-NBD-a-Toc 10d had less than 2% of the
fluorescence in buffer than an equimolar amount in eth-
anol, the shorter chain lengths had greater fluorescence
in aqueous buffers: 10a (ꢀ20%), 10b (ꢀ10%) and 10c
(ꢀ6%). Dissociation constants for the NBD- and AO-
a-Tocs are listed in Table 1.
Unfortunately, the dansyl (DAN) and N-methylanthra-
nilamide (NMA) compounds did not behave as expected
in fluorescence titrations. While they may show specific
binding to a-TTP, these analogues retained substantial
fluorescence intensity in aqueous solution thus compli-
cating distinction between free and bound ligands. This
is illustrated for C9-DAN-a-Toc in Figure 7. Titration
of a-TTP with sequential additions of any of the DAN
or NMA fluorophores resulted in a linear, non-saturable
increase in fluorescence that did not differ greatly from
controls lacking a-TTP. The minor increase in fluores-
cence in the protein-containing samples (versus no pro-
tein control) likely reflects a combination of specific and
non-specific binding to the protein. It is possible that the
DAN and NMA fluorophores may be too polar to be
accepted into the hydrophobic a-TTP-binding site.
The binding specificity of C9-AO-a-Toc (9d) was further
supported by the results of modeling calculations. The
goal of these calculations was to determine whether
The dissociation constants Kd, defined as [TTP]/[TTP-
ligand][ligand], have also been assessed by fluorescence
titrations. The anthroyloxy tocopherol analogues dis-
play higher Kd values (lower affinity) for a-TTP than
their similar chain length NBD-a-Tocs. The C9-AO-a-
Toc (9d) is the only compound of 9a–d for which a Kd
for binding with a-TTP could be reliably determined
(Figure 6). Compounds 9a–c, although able to bind to
a-TTP, all showed significant non-specific binding (data
not shown) which could not be corrected for by non-
Table 1. Dissociation constants, Kd, for fluorescent tocopherol ana-
logues 9a–d and 10d binding to recombinant a-TTP
NBD-a-Toc
Kd (nM)
AO-a-Toc
Kd (nM)
10a
10b
10c
10d
299 37
106 21
142 35
56 15
9a
9b
9c
9d
nd
nd
nd
279 124
nd, accurate value could not be determined due to non-specific
binding.
1.5
150000
Kd = 279 124 nM
1.0
Cholesterol
α-Tocopherol
100000
0.5
0.2 µM α-TTP
No Protein Control
50000
0.0
0
10
20
30
40
50
[Competitor]µM
0
0
1
2
3
4
Figure 5. Competitive displacement of C9-AO-a-Toc, 9d, bound to a-
TTP by addition of increasing amounts of RRR-a-tocopherol or
cholesterol. Fluorescence was monitored at 460 nm. Averages and
standard errors of triplicate data sets are reported for tocopherol,
duplicates for cholesterol. The starting protein–ligand complex
included 0.2 lM a-TTP and 1.0 lM C9-AO-a-Toc. Competitions were
performed in the same buffer as reported in Figure 3 plus 100 lM
Triton X-100.
[C9-AO-α-Toc] µM
Figure 6. Titration curves showing the increase in fluorescence
intensity at 460 nm during sequential additions of the C9-AO-a-Toc,
9d (black squares), to a 0.2 lM solution of a-TTP in SET buffer (see
Section 4). The curves are fitted to a one-site bimolecular association
model.37 Averages and standard errors of triplicate data sets are
reported. kex = 363 nm.

P. Nava et al. / Bioorg. Med. Chem. 14 (2006) 3721–3736
3725
600000
500000
400000
300000
200000
100000
0
dynamics (MD) simulations were performed to enable
the C9-AO-a-Toc to locate lower-energy conformations
accessible from the one initially assigned. Thirty confor-
mations of the protein–ligand complex, taken at 0.5 ps
intervals from each of the two resultant MD trajectories,
were subjected to energy minimization, under constraint
conditions reflecting those applied during the simula-
tions. Only four distinct conformations of the C9-AO-
a-Toc were found in this manner from the simulation
where none of the protein atoms were allowed to relax
around the initial conformation of the anthroyloxy
ligand. The thirty C9-AO-a-Toc conformations selected
from the simulation boasting less-constrained condi-
tions for the protein environment were divisible into
three conformational clusters having somewhat different
orientations of the alkyl tail. All of the low-energy
C9-AO-a-Toc conformations generated from this
second, less-constrained protein simulation possessed
considerably lower intramolecular energies than those
obtained when all atoms of the protein were fixed to
the X-ray coordinates of the a-Toc/a-TTP complex.
Allowing the side-chains to be conformationally mobile
facilitated the movement of the anthroyloxy moiety
away from the linking alkyl chain and to assume confor-
mations with less steric crowding. The first method
yielded conformers in which the anthroyloxy moiety
was wedged between immobile side-chains into a bent
position. A comparison of the putative ligand position
produced by these simulations and that of a-tocopherol
in the X-ray crystal structure of a-TTP is shown in
Figure 8. The lowest-energy conformation of C9-AO-
a-Toc in a-TTP obtained here is depicted. We do not
claim that this ligand/protein conformation is optimal,
given that the above investigations do not constitute
comprehensive conformational searches. Nevertheless,
it is possible to state that the known binding site of
0.2 µM TTP
No Protein
0
1
2
3
4
5
[C9-DAN-α-Toc] µM
Figure 7. Titration curve showing the increase in fluorescence intensity
at 510 nm during sequential additions of the fluorophore C9-DAN-
a-Toc, 11d (black squares) to a 0.2 lM solution of a-TTP in SET
buffer (see Section 4). No saturable binding was observed.
the known binding site of a-TTP could accommodate a
low-energy conformation of the fluorescent ligand, with-
out requiring significant distortion to accommodate the
bulky fluorophore. Using the X-ray coordinates of hu-
man a-TTP in complexation with a-Toc,32 the initial
conformation of C9-AO-a-Toc (9d) was manually
docked into the binding site, imposing the same atomic
coordinates for the chromanol head group as those ob-
served for the native ligand. The dihedral angles of the
alkyl tail with its pendant anthroyloxy substituent were
manually adjusted to fit within the confines of the bind-
ing site, without regard to energetic considerations. Sub-
sequently, two investigations were carried out—one with
all atoms of the protein fixed in their crystallographic
positions, and in the second, the protein backbone
atoms were subjected to harmonic constraints, whereas
all atoms of the side-chains were unconstrained. In both
studies, short (15 ps) high-temperature (500 K) molecular
Figure 8. Comparison of the calculated structures of C9-AO-a-Toc, 9d bound to a-TTP (panels 1a and 1b) with the a-Toc/a-TTP complex observed
by X-ray diffraction32 (panels 2a and 2b). Perspectives 1a and 2a are looking down the long axis of the binding site; 1b and 2b from ‘above’. Only
amino acid residues 92–225, comprising the bulk of the ligand-binding site, are shown for clarity.

3726
P. Nava et al. / Bioorg. Med. Chem. 14 (2006) 3721–3736
˚
was carried out on silica gel (200–300 A mesh) with
the indicated solvent systems.
a-TTP could accommodate C9-AO-a-Toc without sig-
nificant rearrangement of the protein backbone.
4.3. Fluorescence titrations
3. Summary
Recombinant human a-TTP was expressed and purified
as described previously10 and was stored in the elution
buffer from metal affinity columns used for purification
(250 mM NaCl, 25 mM phosphate at pH 7.4). Fractions
rich in a-TTP were usually in the first 400 mM imidazole
elutions. The imidazole had only minor effects on back-
ground fluorescence and did not affect binding. Titra-
tions were performed in quartz cuvettes containing a
total volume of 3 mL SET buffer (250 mM sucrose,
1 mM EDTA, 50 mM Tris–HCl, and 100 mM KCl,
pH 7.5) and protein sample. Sufficient stock of recombi-
nant a-TTP (typically less than 50 lL) from metal affin-
ity column fractions was added such that the final
concentration was 0.2 lM a-TTP. The cuvette was then
mixed by repeated inversion, equilibrated at room
temperature (19–20 ꢂC), and a baseline fluorescence
measurement was recorded at the wavelength of the
fluorophore being used. To this solution were added
2 lL aliquots of stock solutions of fluorophores
(ꢀ0.2 mM in absolute ethanol). After each addition,
samples were mixed on a Rototorque rotating mixer,
and the fluorescence was monitored until a maximum
signal was obtained, which generally required 3–4 min
for the NBD-a-Tocs and 6–8 min for the AO-a-Tocs.
The final concentration of ethanol did not exceed 2%
(v/v) and was usually less than 1% v/v. Fluorescence
data were fitted to a one-site binding equation37 using
Prism 4.0 software.
The synthetic procedures employed in the preparation of
these fluorescent analogues of a-tocopherol were all rel-
atively straightforward and were routinely performed on
a 10–45 mg scale of the final silyl ether-protected com-
pounds. We have since used the C9-NBD-a-Toc (10d)
in a study of the intracellular location and transport of
tocopherol in hepatocytes41 and have begun work on
the nature of the mechanism of a-TTP transfer of
tocopherol from one lipid environment (liposome and
vesicles) to another. This will explore the effect of vesicle
phospholipid composition (i.e., acyl chain unsaturation,
head group charge) and curvature as well as the effect of
mutant forms of a-TTP on rates by which tocopherol is
transferred between vesicles.
4. Experimental
4.1. Materials
4.1.1. Reagents. Reagent-grade chemicals were from
Aldrich Chemical Co., Oakville, Ontario. Solvents were
from Caledon Labs, Ontario. Activated forms of the
fluorophores (N-methylisatoic anhydride, 4-chloro-7-
nitrobenz-2-oxa-1,3-diazole, and 5-dimethylaminonaph-
thalene-1-sulfonylchlroide) were from Molecular
Probes, Eugene, Oregon.
4.2. Analytical methods
The AO-a-tocopherols 9a–d displayed a degree of non-
specific binding to a-TTP (likely due to their greater
hydrophobicity as compared to the other fluorophores)
that often led to failure of the non-linear regression
analysis. In order to assess the extent of non-specific
binding of the AO-a-tocopherols, the raw fluorescence
data were corrected by fluorescence recorded during a
titration of a-TTP with 9a–d in the presence of 40 lM
a-tocopherol in SET buffer.
4.2.1. Spectroscopy. Optical rotations were registered
on a Rudolph Autopol III polarimeter. Infrared (IR)
spectra were recorded on a Mattson Research Series
FT-IR spectrophotometer and the resonance frequen-
cies are reported in cmꢁ1 1H NMR and 13C NMR
.
spectra were measured on a Bruker Advance DPX-
300 Digital FT-NMR spectrometer (300 and
75 MHz, respectively) in CDCl3 with residual chloro-
form
as
internal
reference
(1H = 7.26 ppm,
Competitive assays were performed under similar condi-
tions. To a 0.2 lM solution of a-TTP in 3 mL SET buff-
er plus 100 lM Triton X-100 was added 2 lL of a stock
solution of fluorophore in absolute EtOH such that the
final concentration of fluorophore was 1 lM. The solu-
tion was then mixed until the fluorescence signal had
reached about 95% of its final value. This took about
15 min for 10d, 11d, and 12d, but four hours for 9d
(AO-a-Tocs). To this solution were added 2 lL aliquots
of RRR-a-tocopherol in absolute ethanol to yield final
concentrations of tocopherol ranging from 1 to 40 lM.
After each addition of tocopherol, the samples were
mixed for 15 min to attain equilibrium, and the final
fluorescence recorded.
13C = 77.0 ppm) unless otherwise noted. Chemical
shifts are reported d values (ppm), and coupling con-
stants (J) are reported in Hertz (Hz). The following
NMR abbreviations are used: s (singlet), d (doublet),
t (triplet), q (quartet), m (multiplet), ar (aryl), and br
(broad). Mass spectra (MS) were recorded on a Carlo
Erba/Kratos GC/MS Concept 1S double focusing mass
spectrometer interfaced to a Kratos DART acquisition
system and a SUN SPARC workstation. Fluorescence
experiments were performed on a Photon Technologies
International (London, Ontario) QuantaMaster Model
QM-2001 L-format, equipped with double-grating
monochromators, a 150 W xenon lamp, running Felix
32 software.
4.4. Computational methods
4.2.2. Chromatography. Analytical thin-layer chroma-
tography (TLC) was performed on 0.25 mm pre-coated
˚
silica gel 60 A F-254 plates. Column chromatography
The structural model is based on the crystal structure
of bound a-tocopherol transfer protein (TTP) with

P. Nava et al. / Bioorg. Med. Chem. 14 (2006) 3721–3736
3727
a-tocopherol in the binding site (pdb code: 1OIP).42,32
The conformation of residues 9–24 was taken from the
crystal structure of unbound TTP (pdb code:
1OIZ).42,32 Using Quanta 4.1,43 a-tocopherol was mod-
ified in isolation from a-TTP to yield a preliminary
structure for C9-AO-a-tocopherol. Since the chromanol
head group of C9-AO-a-tocopherol was superimposed
on a-tocopherol, C9-AO-a-tocopherol was positioned
similarly to a-tocopherol in the binding site of a-TTP.
To yield the initial conformation of the tail of C9-AO-
a-tocopherol, dihedral angles were modified manually
to assume as favorable positions as possible. Partial
charges were assigned to atoms using charge templates
of Quanta and the charge was smoothed over all atoms.
The hydrogen atoms on the protein were generated
using the H-build algorithm of CHARMm 27.1.44
conformation of TTP with bound C9-AO-a-tocopherol
from the previous experiment was used as the starting
conformation, and the system was relaxed by ABNR
energy minimization for 300 steps without any con-
straints. Subsequently, harmonic constraints were
applied to all the atoms of the protein backbone about
their current positions with a force constant of
2
˚
500.0 kcal/mol/A used in conjunction with mass
weighting. The system was heated to 500 K and equili-
brated in the same manner as described formerly. After
dynamics, the harmonic constraints were released and
the protein backbone was fixed in position. Every tenth
conformer in the 300-conformer trajectory was energy-
minimized using the ABNR algorithm for 50,000 steps
or until the root-mean-square gradient of 0.001 kcal/
˚
mol/A was satisfied. This resulted in 30 low-energy con-
formations; however, this set was not as easily catego-
rized into distinct conformers as the above set. The
lowest-energy conformation we found is shown in
Fig. 8. The same procedure was repeated for C6-, C7-,
and C8-AO-a-tocopherol with similar results.
CHARMm was also used for all computer simulations.
All non-bonded interactions were smoothly turned off
by the atom shift function with a start distance of
˚
˚
8.0 A and a cut-off radius of 12.0 A. The radius of the
˚
non-bonded neighbor list was set to 14.0 A, and the list
was updated using heuristic methods.45,46 Covalent
bonds to hydrogen atoms were constrained using the
SHAKE algorithm.47 During molecular dynamics simu-
lations, a time-step of 1 fs was used.
4.5. Synthesis
4.5.1. General procedure for the synthesis of compounds
2a–d.
As the goal of these experiments was to find a low-en-
ergy conformation of the ligand in the binding site of
a-TTP, all the protein atoms were held fixed to their
original positions. The initial conformation of C9-
AO-a-tocopherol in the binding site was manually
implemented, thus it is unlikely that the potential ener-
gy of the system was at a minimum. Post-fixing the
atoms of the protein, possible strain was released via
Adopted Basis Newton-Raphson (ABNR) energy min-
imization for 50,000 steps; however, the minimization
converged in 825 steps with a root-mean-square gradi-
TBDMSO
O
OMs
n
2a-d, n=1-4
The synthesis of compounds 2a–d was conducted under
identical conditions and gave similar product yields
(80–85%).
˚
ent of 0.001 kcal/mol/A satisfied. During the initial 5 ps
of computer simulation, the system was heated to
500 K by rescaling the velocities of each atom and
increasing the temperature by 5 K every 0.05 ps. In pre-
liminary experiments, the system was heated to 300 K,
but this temperature did not provide sufficient energy
to move the ligand out of unfavorable conformations
in which the anthroyloxy moiety was deformed from
its desired planar conformation. Subsequently, the sys-
tem was allowed to equilibrate for 15 ps, during which
time period the velocity of each atom was rescaled only
if the average temperature fell outside the window of
(500 10) K. Every tenth conformer in the 300-con-
former trajectory was energy-minimized using the
ABNR algorithm for 50,000 steps. In all cases, the
4.5.1.1. Methanesulfonic acid 6-[(R)-6-(tert-butyl-di-
methyl-silanyloxy)-2,5,7,8-tetramethyl-chroman-2-yl]-hex-
yl ester (2a, n = 1). To a stirred solution of the alcohol
1a (50.5 mg, 0.120 mmol) in dry CH2Cl2 (5 ml) were
added dry triethylamine (25 ll, 0.180 mmol), metha-
nesulfonyl chloride (14 ll, 0.180 mmol), and a catalytic
amount of DMAP (1.9 mg, 0.015 mmol) at 0 ꢂC under
argon. After 15 min, the ice bath was removed and the
reaction mixture was allowed to stir at room tempera-
ture for an additional 1/2 h. The mixture was then dilut-
ed with ether, washed with saturated aqueous NaHCO3
(15 ml) and water (3· 10 ml). The organic layer was
dried with Na2SO4, decanted and concentrated to give
the crude mesylate as a cloudy yellow oil. This interme-
diate was used in the next reaction without further puri-
fication. (50.3 mg, 84%), clear colorless oil, Rf = 0.30
minimization converged with
a root-mean-square
˚
gradient of 0.001 kcal/mol/A satisfied. This resulted in
30 low-energy conformations; however, there were
approximately 4 distinct conformers. The energy terms
used to evaluate the conformations represent intra-li-
gand interactions and ligand–protein interaction; they
do not represent intra-protein interactions.
20
1
(CH2Cl2/hexane 2:1), ½aꢂ +1.9ꢂ (c 0.041, CHCl3); H
D
NMR (CDCl3): d 4.22 (t, 2H, J = 7 Hz), 3.02 (s, 3H),
2.54 (t, 2H, J = 7 Hz), 2.09 (s, 3H), 2.06 (s, 3H), 2.05
(s, 3H), 1.76 (m, 4H), 1.53 (m, 2H), 1.44 (m, 6H), 1.21
(s, 3H), 1.04 (s, 9H), 0.11 (s, 6H); 13C NMR (CDCl3):
d 145.7, 144.0, 125.8, 123.4, 122.5, 117.3, 74.2, 70.1,
39.3, 37.2, 31.5, 29.4, 29.0, 26.0, 25.3, 23.7, 23.3, 20.8,
18.5, 14.2, 13.3, 11.9, ꢁ3.4; MS (EI) m/z 498 (M+,
In order to allow the ligand more flexibility, another
set of simulations was performed. The minimized

3728
P. Nava et al. / Bioorg. Med. Chem. 14 (2006) 3721–3736
100%), 402 (16.2%), 279 (33.3%), 153 (32.5%), 73
(99.2%). HRMS (EI): calcd for C26H46O5SSi:
498.28352. Found: 498.28360.
4.5.2.1.
[(R)-2-(6-Azido-hexyl)-2,5,7,8-tetramethyl-
chromen-6-yloxy]-(tert-butyl)dimethylsilane (3a, n = 1).
To a solution of mesylate 2a (0.268 mmol) in dry
DMF (3 ml) at room temperature was added sodium
azide (86.9 mg, 1.34 mmol). The mixture was then
warmed to and stirred at 55 ꢂC for 1 h. The reaction
was then diluted with ether (15 ml), washed with water
(3· 25 ml), dried with Na2SO4, decanted, and concen-
trated. The resulting oily residue was purified by column
chromatography (CH2Cl2/hexane 2:1) to afford the de-
sired product 3a.
4.5.1.2.
tyl(dimethyl)silanyloxy-2,5,7,8-tetramethyl-chroman-2-yl]-
Methansulfonic
acid
7-(R)-6-[tert-bu-
heptyl ester (2b, n = 2). (61.2 mg, 85%), clear colorless
20
D
oil, Rf = 0.30 (CH2Cl2/hexane 2:1), ½aꢂ +1.8ꢂ (c 0.084,
1
CHCl3); H NMR (CDCl3): d 4.22 (t, 2H, J = 6 Hz),
2.98 (s, 3H), 2.53 (t, 2H, J = 6 Hz), 2.10 (s, 3H), 2.07
(s, 3H), 2.06 (s, 3H), 1.77 (m, 4H), 1.54 (m, 2H), 1.42
(m, 8H), 1.22 (s, 3H), 1.05 (s, 9H), 0.12 (s, 6H); 13C
NMR (CDCl3): d 145.7, 144.0, 125.7, 123.4, 122.5,
117.3, 74.2, 70.0, 39.3, 37.1, 31.5, 29.8, 29.0, 28.9, 26.0,
25.2, 23.6, 23.3, 20.7, 18.4, 14.2, 13.3, 11.8, ꢁ3.4; MS
(EI) m/z 512 (M+, 99.3%), 416 (23.1%), 279 (30.3%),
153 (39.1%), 73 (100%). HRMS (EI): calcd for
C27H48O5SSi: 512.29917. Found: 512.29835.
(105 mg, 88% over two steps), clear colorless oil,
20
D
1
Rf = 0.41 (CH2Cl2/hexane 1:1), ½aꢂ +2.5ꢂ (c 0.013,
CHCl3), IR (neat): 2098 (–N3); H NMR (CDCl3): d
3.24 (t, 2H, J = 7 Hz), 2.55 (t, 2H, J = 7 Hz), 2.09 (s,
3H), 2.07 (s, 3H), 2.05 (s, 3H), 1.76 (m, 2H), 1.59 (m,
4H), 1.33 (m, 6H), 1.22 (s, 3H), 1.04 (s, 9H), 0.11 (s,
6H); 13C NMR (CDCl3): d 145.8, 144.0, 125.8, 123.5,
122.6, 117.4, 74.3, 51.4, 39.4, 31.5, 29.6, 28.7, 26.7,
26.0, 23.7, 23.4, 20.8, 18.5, 14.3, 13.4, 11.9, ꢁ3.3; MS
(EI) m/z 445 (M+, 15.9%), 415 (59.8%), 358 (20.7%),
279 (27.0%), 73 (100%). HRMS (EI): calcd for
C25H43N3O2Si: 445.31245. Found: 445.31246.
4.5.1.3.
tyl(dimethyl)silanyloxy-2,5,7,8-tetramethyl-chroman-2-yl]-
Methansulfonic
acid
8-(R)-6-[tert-bu-
octyl ester (2c, n = 3). (38.4 mg, 80%), clear colorless oil,
20
D
Rf = 0.32 (CH2Cl2/hexane 2:1), ½aꢂ +1.7ꢂ (c 0.070,
1
CHCl3); H NMR (CDCl3): d 4.22 (t, 2H, J = 7 Hz),
2.99 (s, 3H), 2.56 (t, 2H, J = 7 Hz), 2.11 (s, 3H), 2.09 (s,
3H), 2.07 (s, 3H), 1.75 (m, 4H), 1.54 (m, 2H), 1.40 (m,
4H), 1.31 (m, 6H), 1.23 (s, 3H), 1.05 (s, 9H), 0.12 (s,
6H); 13C NMR (CDCl3): d 145.8, 144.0, 125.8, 123.5,
122.6, 117.4, 74.3, 70.2, 39.5, 37.2, 31.5, 30.0, 29.4, 29.1,
29.0, 26.1, 25.4, 23.8, 23.5, 20.8, 18.5, 14.3, 13.4, 11.9,
ꢁ3.3; MS (EI) m/z 526 (M+, 9.8%), 430 (91.7%), 307
(41.0%), 149 (100%), 57 (63.2%). HRMS (EI): calcd for
C28H50O5SSi: 526.31482. Found: 526.31549.
4.5.2.2. [(R)-2-(7-Azido-heptyl)-2,5,7,8-tetramethyl-
chromen-6-yloxy]-(tert-butyl)dimethylsilane (3b, n = 2).
(182 mg, 88% over two steps), clear colorless oil,
20
D
1
Rf = 0.42 (CH2Cl2/hexane 1:1), ½aꢂ +2.4ꢂ (c 0.040,
CHCl3), IR (neat): 2059 (–N3); H NMR (CDCl3): d
3.25 (t, 2H, J = 7 Hz), 2.56 (t, 2H, J = 7 Hz), 2.10 (s,
3H), 2.08 (s, 3H), 2.06 (s, 3H), 1.77 (m, 2H), 1.59 (m,
4H), 1.33 (m, 8H), 1.22 (s, 3H), 1.05 (s, 9H), 0.12 (s,
6H); 13C NMR (CDCl3): d 145.8, 144.0, 125.8, 123.5,
122.6, 117.4, 74.3, 51.4, 39.4, 31.5, 29.9, 29.1, 28.8,
26.6, 26.0, 23.7, 23.4, 20.8, 18.5, 14.3, 13.4, 11.9, ꢁ3.3;
MS (EI) m/z 459 (M+, 18.6%), 429 (54.3%), 372
(18.2%), 279 (26.4%), 73 (100%). HRMS (EI): calcd
for C26H45N3O2Si: 459.32810. Found: 459.32887.
4.5.1.4. Methansulfonic acid 9-(R)-6-[tert-butyl(dimeth-
yl)silanyloxy-2,5,7,8-tetramethyl-chroman-2-yl]-nonyl ester
(2d, n = 4). (41.1 mg, 81%), clear colorless oil, Rf = 0.32
20
1
(CH2Cl2/hexane 2:1), ½aꢂ +1.6ꢂ (c 0.063, CHCl3); H
D
NMR (CDCl3): d 4.21 (t, 2H, J = 7 Hz), 2.98 (s, 3H),
2.54 (t, 2H, J = 7 Hz), 2.09 (s, 3H), 2.07 (s, 3H), 2.05 (s,
3H), 1.76 (m, 4H), 1.54 (m, 2H), 1.39 (m, 4H), 1.28 (m,
8H), 1.21 (s, 3H), 1.04 (s, 9H), 0.11 (s, 6H); 13C NMR
(CDCl3): d 145.8, 143.9, 125.7, 123.6, 122.5, 117.4, 74.3,
70.1, 39.4, 37.2, 31.4, 30.0, 29.4, 29.2, 29.0, 28.9, 26.0,
25.3, 23.7, 23.5, 20.8, 18.5, 14.2, 13.3, 11.8, ꢁ3.4; MS
(EI) m/z 540 (M+, 42.6%), 428 (80.1%), 279 (17.1%),
153 (56.8%), 73 (100%). HRMS (EI): calcd for
C29H52O5SSi: 540.33047. Found: 540.32960.
4.5.2.3. [(R)-2-(8-Azido-octyl)-2,5,7,8-tetramethyl-
chromen-6-yloxy]-(tert-butyl)dimethylsilane (3c, n = 3).
(180 mg, 88.5% over two steps), clear colorless oil,
20
D
1
Rf = 0.43 (CH2Cl2/hexane 1:1), ½aꢂ +2.5ꢂ (c 0.035,
CHCl3), IR (neat): 2098 (–N3); H NMR (CDCl3): d
3.25 (t, 2H, J = 7 Hz), 2.55 (t, 2H, J = 7 Hz), 2.10 (s,
3H), 2.08 (s, 3H), 2.06 (s, 3H), 1.81 (m, 2H), 1.57 (m,
4H), 1.31 (m, 10H), 1.22 (s, 3H), 1.05 (s, 9H), 0.12 (s,
6H); 13C NMR (CDCl3): d 145.8, 143.9, 125.8, 123.4,
122.6, 117.4, 74.3, 51.4, 39.4, 31.4, 29.9, 29.4, 29.0,
28.7, 26.6, 26.0, 23.7, 23.5, 20.8, 18.5, 14.2, 13.3, 11.9,
ꢁ3.3; MS (EI) m/z 473 (M+, 39.7%), 445 (93.4%), 279
(45.2%), 221 (32.4%), 73 (100%). HRMS (EI): calcd
for C27H47N3O2Si: 473.34375. Found: 473.34235.
4.5.2. General procedure for the preparation of com-
pounds 3a–d.
TBDMSO
4.5.2.4. [(R)-2-(9-Azido-nonyl)-2,5,7,8-tetramethyl-
chromen-6-yloxy]-(tert-butyl)dimethylsilane (3d, n = 4).
O
N3
n
3a-d, n = 1-4
(154 mg, 85% over two steps), clear colorless oil,
20
D
1
Rf = 0.45 (CH2Cl2/hexane 1:1), ½aꢂ +1.9ꢂ (c 0.065,
CHCl3), IR (neat): 2098 (–N3); H NMR (CDCl3): d
3.25 (t, 2H, J = 7 Hz), 2.55 (t, 2H, J = 7 Hz), 2.10 (s,
3H), 2.08 (s, 3H), 2.06 (s, 3H), 1.77 (m, 2H), 1.57 (m,
4H), 1.29 (m, 12H), 1.22 (s, 3H), 1.05 (s, 9H), 0.12 (s,
The synthesis of compounds 3a–d were conducted
under identical conditions and gave similar product
yields (85–88%).

P. Nava et al. / Bioorg. Med. Chem. 14 (2006) 3721–3736
3729
6H); 13C NMR (CDCl3): d 145.8, 143.9, 125.8, 123.4,
122.6, 117.4, 74.3, 51.4, 39.5, 31.4, 30.0, 29.4, 29.3,
29.1, 28.8, 26.6, 26.0, 23.7, 23.5, 20.8, 18.5, 14.2, 13.3,
11.9, ꢁ3.3; MS (EI) m/z 487 (M+, 22.0%), 457 (75.8%),
279 (27.8%), 221 (19.3%), 73 (100%). HRMS (EI): calcd
for C28H49N3O2Si: 487.35940. Found: 487.35939.
m/z 433 (M+, 100%), 372 (19.2%), 279 (35.1%), 221
(24.1%), 73 (76.1%). HRMS (EI): calcd for C26H47
NO2Si: 433.33760. Found: 433.33801.
4.5.3.3. 8-((2R)-6-{[tert-Butyl(dimethyl)silyl]oxy}-
2,5,7,8-tetramethyl-3,4,-dihydro-2H-chromene-2-yl)-octyl-
amine (4c, n = 3). (72.6 mg, 85%), clear colorless oil,
20
D
4.5.3. General procedure for the preparation of com-
pounds 4a–d.
Rf = 0.31 (EtOAc/MeOH+1% HCl 7:2), ½aꢂ +1.9ꢂ (c
0.043, CHCl3), IR (neat): 3368 (N–H), 3291 (N–H);
1H NMR (CDCl3): d 2.67 (t, 2H, J = 7 Hz), 2.52 (t,
2H, J = 7 Hz), 2.32 (br, 2H), 2.07 (s, 3H), 2.05 (s, 3H),
2.03 (s, 3H), 1.77 (m, 2H), 1.48 (m, 6H), 1.27 (br, 8H),
1.19 (s, 3H), 1.02 (s, 9H), 0.09 (s, 6H); 13C NMR
(CDCl3): d 145.6, 143.8, 125.6, 123.3, 122.4, 117.2,
74.2, 41.7, 39.3, 33.0, 31.3, 29.8, 29.3, 29.2, 26.6, 25.8,
23.6, 23.3, 20.6, 18.3, 14.1, 13.1, 11.7, ꢁ3.5; MS (EI)
m/z 447 (M+, 77.7%), 386 (24.8%), 279 (42.2%), 221
(28.7%), 73 (100%). HRMS (EI): calcd for C27H49NO2-
Si: 447.35325. Found: 447.35557.
TBDMSO
O
NH2
n
4a-d, n = 1-4
The synthesis of compounds 4a–d was conducted under
identical conditions to give similar product yields
(83–90%).
4.5.3.4. 9-((2R)-6-{[tert-Butyl(dimethyl)silyl]oxy}-2,5,
7,8-tetramethyl-3,4,-dihydro-2H-chromene-2-yl)-nonylamine
(4d, n = 4). (161.2 mg, 90%), clear colorless oil, Rf = 0.31
4.5.3.1.
6-((2R)-6-{[tert-Butyl(dimethyl)silyl]oxy}-
20
2,5,7,8-tetramethyl-3,4,-dihydro-2H-chromene-2-yl)-hex-
ylamine. To a stirred solution of azide 3a (167.0 mg,
0.37 mmol) in dry THF (3 ml) cooled to 0 ꢂC was added
LiAlH4 (0.48 ml, 0.48 mmol, 1 M in THF) dropwise.
This was then stirred for an additional 5 min at 0 ꢂC
and subsequently for 10 min at room temperature. The
reaction was then acidified using acetic acid (2 M), neu-
tralized with saturated NaHCO3, and extracted with
ether (3· 25 ml). The organic layers were combined,
dried with Na2SO4, and concentrated under reduced
pressure. The resulting crude product was then further
purified by column chromatography by application to
4 ml silica and passing through 100% ethyl acetate
(25 ml) followed by EtAOc/MeOH (8:2) to afford the de-
(EtOAc/MeOH+1% HCl 7:2), ½aꢂ +2.5ꢂ (c 0.032,
D
CHCl3), IR (neat): 3384 (N–H), 3308 (N–H); 1H
NMR (CDCl3): d 2.53 (t, 2H, J = 7 Hz), 2.45 (br, 2H),
2.08 (s, 3H), 2.05 (s, 3H), 2.03 (s, 3H), 1.75 (m, 2H),
1.48 (m, 6H), 1.26 (br, 12H), 1.20 (s, 3H), 1.03 (s, 9H),
0.09 (s, 6H); 13C NMR (CDCl3): d 145.8, 144.0, 125.8,
123.5, 122.6, 117.4, 74.4, 39.6, 33.2, 31.5, 30.9, 30.1,
29.6, 29.4, 26.8, 26.1, 23.8, 23.6, 20.9, 18.6, 14.3, 13.4,
11.9, ꢁ3.3; MS (EI) m/z 461 (M+, 46.9%), 457 (100%),
400 (23.6%), 279 (32.8%), 221 (23.0%), 73 (63.1%).
HRMS (EI): calcd for C28H51NO2Si: 461.36890. Found:
461.36459.
4.5.4. General procedure for the preparation of com-
pounds 5a–d.
sired product. (131.5 mg, 83%), clear colorless oil,
20
D
Rf = 0.31 (EtOAc/MeOH + 1% HCl 7:2), ½aꢂ +1.9ꢂ (c
0.020, CHCl3), IR (neat): 3368 (N–H), 3300 (N–H);
1H NMR (CDCl3): d 2.88 (br, 2H), 2.68 (br, 2H), 2.52
(t, 2H, J = 6 Hz), 2.07 (s, 3H), 2.05 (s, 3H), 2.03 (s,
3H), 1.74 (m, 2H), 1.48 (m, 6H), 1.28 (m, 4H), 1.19 (s,
3H), 1.02 (s, 9H), 0.09 (s, 6H); 13C NMR (CDCl3): d
145.8, 144.0, 125.8, 123.4, 122.6, 117.4, 74.3, 41.6,
39.5, 32.8, 31.5, 29.9, 26.8, 26.0, 23.7, 23.5, 20.8, 18.5,
14.3, 13.3, 11.9, ꢁ3.4; MS (EI) m/z 419 (M+, 100%),
358 (15.1%), 279 (33.3%), 221 (24.6%), 73 (71.1%).
HRMS (EI): calcd for C25H45NO2Si: 419.32195. Found:
419.32160.
TBDMSO
O
O
O
n
5a-d, n = 1-4
The synthesis of compounds 5a–d was conducted under
identical conditions and gave product yields ranging
from 60% to 89%.
4.5.3.2. 7-((2R)-6-{[tert-Butyl(dimethyl)silyl]oxy}-2,5,
7,8-tetramethyl-3,4,-dihydro-2H-chromene-2-yl)-heptyl-
amine (4b, n = 2). (219.4 mg, 87%), clear colorless oil,
4.5.4.1. Anthracene-9-carboxylic acid 6-[(R)-6-(tert-
butyl-dimethyl-silanyloxy)-2,5,7,8-tetramethyl-chroman-
2-yl-hexyl ester (5a, n = 1). To a stirred solution of 9-an-
thracene carboxylic acid (71.4 mg, 0.321 mmol) in dry
DCM (3 ml) under argon were added oxalyl chloride
(41 ll, 0.321 mmol) and a catalytic amount of DMF
(1 ll, 0.016 mmol). It was then allowed to stir for 2 h,
thereby generating the acid chloride in situ. Reaction pro-
gress could be monitored by the poor initial solubility of
9-anthracene carboxylic acid as a cloudy yellow mixture
in DCM to a resulting clear transparent yellow solution
and by the evolution of CO2 and CO bubbles. To this
20
Rf = 0.31 (EtOAc/MeOH + 1% HCl 7:2), ½aꢂ +1.9ꢂ (c
D
0.034, CHCl3), IR (neat): 3368 (N–H), 3275 (N–H);
1H NMR (CDCl3): d 2.64 (t, 2H, J = 7 Hz), 2.52 (t,
2H, J = 7 Hz), 2.16 (br, 2H), 2.07 (s, 3H), 2.05 (s, 3H),
2.03 (s, 3H), 1.76 (m, 2H), 1.50 (m, 2H), 1.48 (br, 4H),
1.26 (br, 6H), 1.19 (s, 3H), 1.02 (s, 9H), 0.09 (s, 6H);
13C NMR (CDCl3): d 145.7, 143.8, 125.6, 123.3, 122.5,
117.3, 74.5, 41.8, 39.4, 33.2, 31.3, 29.9, 29.3, 26.7, 25.9,
23.6, 23.4, 20.7, 18.4, 14.0, 13.2, 11.8, ꢁ3.5; MS (EI)

3730
P. Nava et al. / Bioorg. Med. Chem. 14 (2006) 3721–3736
was added dropwise via syringe a solution of appropriate
alcohol 1a (104.1 mg, 0.247 mmol) in dry DCM (2 ml)
that was stirred for 15 min in the presence of TEA
(38 ll, 0.272 mmol). The combined reaction components
were then mixed for a further 3 h, at which point alcohol
1a could no longer be detected by TLC. The reaction was
quenched with dH2O (15 ml) and extracted with DCM
(3· 25 ml). The combined organic layers were washed
with saturated aqueous NaHCO3 (15 ml) and then
dH2O (15 ml) prior to drying with anhydrous Na2SO4,
decanting, and concentration under reduced pressure.
Purification was achieved by column chromatography
4.5.4.4. Anthracene-9-carboxylic acid 9-[(R)-6-(tert-
butyl-dimethyl-silanyloxy)-2,5,7,8-tetramethyl-chroman-
2-yl-nonyl ester (5d, n = 4). (159.2 mg, 89%), clear
20
light yellow oil, Rf = 0.55 (CH2Cl2/hexane 10:1), ½aꢂ
D
+1.2ꢂ (c 0.008, CHCl3); 1H NMR (CDCl3): d 8.50
(s, 1H), 8.01 (m, 4H), 7.49 (m, 4H), 4.59 (t, 2H,
J = 6 Hz), 2.51 (t, 2H, J = 6 Hz), 2.06 (s, 3H), 2.04
(s, 3H), 2.02 (s, 3 H), 1.83 (m, 2H), 1.73 (m, 2H),
1.47 (m, 6H), 1.37 (m, 8H), 1.19 (s, 3H), 1.02 (s,
9H), 0.09 (s, 6H); 13C NMR (CDCl3): d 169.7
(C@O), 145.8, 144.0, 130.9, 129.1, 128.5, 128.3,
128.1, 126.8, 126.0, 125.4, 125.0, 123.5, 122.6, 117.4,
74.4, 65.9, 39.5, 31.9, 30.1, 29.6, 29.5, 29.4, 29.2,
28.7, 26.0 (3 CH3), 23.7, 23.5, 20.8, 18.5, 14.3, 13.3,
11.9, ꢁ3.3 (2 CH3); MS (EI) m/z 666 (M+, 18.5%),
472 (8.7%), 205 (55.1%), 73 (45.0%), 43 (100%).
HRMS (EI): calcd for C43H58O4Si: 666.41043.
Found: 666.41082.
(Et2O/hexane 1:2). (104.9 mg, 68%), clear light yellow
20
oil, Rf = 0.53 (CH2Cl2/hexane 10:1), ½aꢂ +2.4ꢂ (c 0.010,
D
CHCl3); 1H NMR (CDCl3): d 8.55 (s, 1H), 8.02 (m, 4H),
7.5 (m, 4H), 4.59 (t, 2H, J = 6 Hz), 2.49 (t, 2H,
J = 6 Hz), 2.07 (s, 3H), 2.04 (s, 3H), 2.02 (s, 3H), 1.86
(m, 2H), 1.76 (m, 2H), 1.46 (m, 4H), 1.40 (m, 4H), 1.19
(s, 3H), 1.02 (s, 9H), 0.09 (s, 6H); 13C NMR (CDCl3): d
169.7 (C@O), 145.8, 144.0, 130.9, 129.1, 128.5, 128.3,
128.1, 126.8, 125.8, 125.4, 125.0, 123.5, 122.6, 117.4,
74.3, 65.9, 39.5, 31.5, 29.7, 29.7, 28.7, 26.0 (3 CH3), 23.7,
23.5, 20.8, 18.5, 14.3, 13.4, 11.9, ꢁ3.3 (2 CH3); MS (EI)
m/z 624 (M+, 1.4%), 208 (100%), 180 (66.6%), 152
(46.0%), 76 (35.2%). HRMS (EI): calcd for C40H52O4Si:
624.36348. Found: 624.37719.
4.5.5. General procedure for the preparation of com-
pounds 6a–d.
O
TBDMSO
N
N
O
N
NO2
n
H
6a-d, n = 1-4
4.5.4.2. Anthracene-9-carboxylic acid 7-[(R)-6-(tert-
butyl-dimethyl-silanyloxy)-2,5,7,8-tetramethyl-chroman-
2-yl-heptyl ester (5b, n = 2). (165.2 mg, 60%), clear light
The synthesis of compounds 6a–d was conducted under
identical conditions and gave similar product yields
(73–76%).
20
D
yellow oil, Rf = 0.54 (CH2Cl2/hexane 10:1), ½aꢂ +1.7ꢂ (c
1
0.006, CHCl3); H NMR (CDCl3): d 8.52 (s, 1H), 8.01
(m, 4H), 7.51 (m, 4H), 4.58 (t, 2H, J = 6 Hz), 2.48 (t,
2H, J = 6 Hz), 2.07 (s, 3H), 2.05 (s, 3H), 2.03 (s, 3H),
1.86 (m, 2H), 1.75 (m, 2H), 1.45 (m, 6H), 1.39 (m,
4H), 1.19 (s, 3H), 1.02 (s, 9H), 0.08 (s, 6H); 13C NMR
(CDCl3): d 169.8 (C@O), 145.9, 144.1, 130.7, 129.2,
128.6, 128.4, 128.2, 126.7, 125.7, 125.3, 125.0, 123.5,
122.6, 117.2, 74.2, 65.8, 39.5, 31.5, 30.1, 29.6, 29.5,
28.6, 26.0 (3 CH3), 23.6, 23.5, 20.7, 18.6, 14.4, 13.4,
11.8, ꢁ3.4 (2 CH3); MS (EI) m/z 638 (M+, 21.4%), 279
(19.4%), 205 (48.3%), 73 (36.5%), 43 (100%). HRMS
(EI): calcd for C41H54O4Si: 638.37913. Found:
638.37231.
4.5.5.1.
{9-[(R)-6-(tert-Butyl-dimethyl-silanyloxy)-
2,5,7,8-tetramethyl-chroman-2-yl]-hexyl}-(7-nitro-benzo
[1,2,5]oxadiazol-4-yl)-amine (6a, n = 1). The amine 4a
(44.8 mg, 0.106 mmol) was dissolved in dry THF
(1 ml) and then dry TEA (30 ll, 0.212 mmol) was added
under argon. This was stirred at room temperature for
15 min prior to dropwise addition via cannula of a solu-
tion of NBD-Cl (23.2 mg, 0.116 mmol) in dry THF
(1 ml). After stirring for a further 1 h, the reaction mix-
ture was diluted with dH2O (10 ml), extracted with
EtOAc (3· 30 ml), dried over Na2SO4, and concentrated
under reduced pressure to give the crude product, which
was purified by column chromatography (Et2O/hexane
1:2) to afford the pure compound 6a. (47.3 mg, 76%),
4.5.4.3. Anthracene-9-carboxylic acid 8-[(R)-6-(tert-
butyl-dimethyl-silanyloxy)-2,5,7,8-tetramethyl-chroman-
dark browny orange oil, Rf = 0.28 (Et2O/hexane
2-yl-octyl ester (5c, n = 3). (89.5 mg, 87%), clear light
20
D
20
D
1:1), ½aꢂ
+2.1ꢂ (c 0.005, CHCl3); 1H NMR
yellow oil, Rf = 0.54 (CH2Cl2/hexane 10:1), ½aꢂ +1.5ꢂ
1
(CDCl3): d 8.44 (d, 1H, J = 9 Hz), 6.24 (br, 1H),
6.12 (d, 1H, J = 9 Hz), 3.45 (q, 2H, J = 7 Hz),
2.53 (t, 2H, J = 7 Hz), 2.06 (s, 3H), 2.04 (s, 3H),
2.02 (s, 3H), 1.77 (m, 4H), 1.41 (m, 8H), 1.20
(s, 3H), 1.01 (s, 9H), 0.08 (s, 6H); 13C NMR
(c 0.007 CHCl3); H NMR (CDCl3): d 8.50 (s, 1H),
8.01 (m, 4H), 7.48 (m, 4H), 4.59 (t, 2H, J = 6 Hz),
2.51 (t, 2H, J = 6 Hz), 2.06 (s, 3H), 2.04 (s, 3H), 2.02
(s, 3H), 1.83 (m, 2H), 1.73 (m, 2H), 1.47 (m, 6H),
1.37 (m, 6H), 1.18 (s, 3H), 1.02 (s, 9H), 0.08 (s, 6H);
13C NMR (CDCl3): d 169.8 (C@O), 145.9, 144.1,
131.0, 129.2, 128.6, 128.4, 128.2, 126.9, 125.9, 125.5,
125.1, 123.6, 122.7, 117.5, 74.5, 66.0, 39.6, 31.5, 30.1,
29.7, 29.6, 29.2, 28.8, 26.1 (3 CH3), 23.8, 23.6, 20.9,
18.6, 14.4, 13.4, 12.0, ꢁ3.2 (2 CH3); MS (EI) m/z 652
(M+, 100%), 446 (8.8%), 279 (25.8%), 205 (52.0%), 73
(38.1%). HRMS (EI): calcd for C42H56O4Si:
652.39478. Found: 652.39563.
(CDCl3):
d
145.7, 144.2, 144.0, 143.8, 143.8,
136.5, 125.8, 123.8, 123.5, 122.5, 117.4, 98.5, 74.2,
43.9, 39.3, 31.5, 29.6, 28.4, 26.8, 26.0 (3 CH3),
23.7, 23.3, 20.8, 18.5, 14.3, 13.3, 11.9, ꢁ3.4 (2
CH3); MS (EI) m/z 582 (M+, 50.3%), 548
(31.6%), 418 (21.5%), 279 (24.5%), 73 (100%).
HRMS (EI): calcd for C31H46N4O5Si: 582.32374.
Found: 582.32418.

P. Nava et al. / Bioorg. Med. Chem. 14 (2006) 3721–3736
3731
4.5.5.2. {9-[(R)-6-(tert-Butyl-dimethyl-silanyloxy)-2,5,
7,8-tetramethyl-chroman-2-yl]-heptyl}-(7-nitro-benzo[1,2,
5]oxadiazol-4-yl)-amine (6b, n = 2). (52.5 mg, 76%),
The synthesis of compounds 7a–d was conducted
under identical conditions and gave similar product
yields (81–82%).
dark browny orange oil, Rf = 0.30 (Et2O/hexane 1:1),
20
D
½aꢂ +2.0ꢂ (c 0.007, CHCl3); 1H NMR (CDCl3): d
4.5.6.1. 5-Dimethylamino-naphthalene-1-sulfonic acid
{9-[(R)-6-(tert-butyl-dimethyl-silanyloxy)-2,5,7,8-tetrameth-
yl-chroman-2-yl]hexyl}-amide (7a, n = 1). A solution of
amine 4a (47.8 mg, 0.113 mmol) and dry TEA (47 ll,
0.339 mmol) in dry DCM (2 ml) was stirred at 0 ꢂC for
15 min prior to dropwise addition of a solution of
DAN-Cl (33.5 mg, 0.124 mmol) in dry DCM (1 ml).
The reaction was maintained at this temperature for
another five minutes at which point it was allowed to
proceed at room temperature for a further one hour.
The reaction was then quenched with dH2O (15 ml)
and extracted with CH2Cl2 (3· 20 ml), dried over
Na2SO4, decanted, and concentrated in vacuo to give
the crude product. Further purification by column chro-
matography (Et2O/hexane 2:1) afforded the desired
8.45 (d, 1H, J = 9 Hz), 6.20 (br, 1H), 6.13 (d, 1H,
J = 9 Hz), 3.44 (q, 2H, J = 7 Hz), 2.52 (t, 2H,
J = 7 Hz), 2.06 (s, 3H), 2.04 (s, 3H), 2.02 (s, 3H),
1.76 (m, 4H), 1.41 (m, 10H), 1.19 (s, 3H), 1.01 (s,
9H), 0.08 (s, 6H); 13C NMR (CDCl3): d 145.7, 144.2,
144.0, 143.8, 143.8, 136.5, 125.8, 123.9, 123.5, 122.5,
117.4, 98.5, 74.3, 43.9, 39.8, 31.5, 29.9, 29.1, 28.5,
26.8, 26.0 (3 CH3), 23.7, 23.4, 20.8, 18.5, 14.3, 13.4,
11.9, ꢁ3.3 (2 CH3); MS (EI) m/z 596 (M+, 32.4%),
562 (17.9%), 432 (69.2%), 279 (28.3%), 73 (100%).
HRMS (EI): calcd for C32H48N4O5 Si: 596.33939.
Found: 596.34291.
4.5.5.3. {9-[(R)-6-(tert-Butyl-dimethyl-silanyloxy)-2,5,
7,8-tetramethyl-chroman-2-yl]-octyl}-(7-nitro-benzo[1,2,
5]oxadiazol-4-yl)-amine (6c, n = 3). (39.2 mg, 74%), dark
browny orange oil, Rf = 0.33 (Et2O/hexane 1:1), ½aꢂ
product. (60.1 mg, 81%), lime green solid, mp 46–
20
D
47 ꢂC, Rf= 0.42 (Et2O/hexane 2:1), ½aꢂ +2.7ꢂ (c 0.006,
1
20
D
CHCl3); H NMR (CDCl3): d 8.51 (d, 1H, J = 8 Hz),
8.26 (d, 1H, J = 8 Hz), 8.22 (d, 1H, J = 7 Hz), 7.51 (q,
2H, J = 8 Hz), 7.14 (d, 1H, J = 7 Hz), 4.62 (t, 1H,
J = 6 Hz), 2.84 (s, 8H), 2.50 (t, 2H, J = 6 Hz), 2.07 (s,
3H), 2.07 (s, 6H), 1.70 (m, 2H), 1.39 (m, 2H), 1.35 (m,
4H), 1.31 (m, 4H), 1.15 (s, 3H), 1.02 (s, 9H), 0.09 (s,
6H); 13C NMR (CDCl3): d 151.9, 145.7, 144.0, 134.7,
130.3, 129.8, 129.6, 129.6, 128.3, 125.8, 123.5, 123.2,
122.6, 118.7, 117.4, 115.1, 74.2, 45.4 (2 CH3), 43.2,
39.3, 31.4, 29.4 (2 CH2), 26.3, 26.0 (3 CH3), 23.7, 23.3,
20.8, 18.5, 14.3, 13.3, 11.9, ꢁ3.3 (2 CH3); MS (EI) m/z
652 (M+, 90.5%), 543 (15.6%), 445 (51.6%), 279
(28.8%), 73 (100%). HRMS (EI): calcd for
C37H56N2O4SSi: 652.37300. Found: 652.36645.
1
+2.4ꢂ (c 0.009, CHCl3); H NMR (CDCl3): d 8.47 (d,
1H, J = 9 Hz), 6.21 (br, 1H), 6.15 (d, 1H, J = 9 Hz),
3.45 (q, 2H, J = 7 Hz), 2.53 (t, 2H, J = 7 Hz), 2.07 (s,
3H), 2.04 (s, 3H), 2.03 (s, 3H), 1.77 (m, 4H), 1.41 (m,
4H), 1.30 (m, 8H), 1.19 (s, 3H), 1.01 (s, 9H), 0.08 (s,
6H); 13C NMR (CDCl3): d 145.8, 144.2, 144.0, 143.8,
143.8, 136.5, 125.8, 123.9, 123.5, 122.6, 117.4, 98.5,
74.3, 43.9, 39.4, 31.5, 29.9, 29.3, 29.1, 28.5, 26.9, 26.0
(3 CH3), 23.8, 23.4, 20.8, 18.5, 14.3, 13.4, 11.9, ꢁ3.3 (2
CH3); MS (EI) m/z 610 (M+, 20.2%), 576 (21.0%), 446
(48.2%), 279 (55.2%), 73 (100%). HRMS (EI): calcd
for C33H50N4O5 Si: 610.35504. Found: 610.35132.
4.5.5.4. {9-[(R)-6-(tert-Butyl-dimethyl-silanyloxy)-2,5,
7,8-tetramethyl-chroman-2-yl]-nonyl}-(7-nitro-benzo[1,2,
5]oxadiazol-4-yl)-amine (6d, n = 4). (29.5 mg, 73%),
4.5.6.2. 5-Dimethylamino-naphthalene-1-sulfonic acid
{9-[(R)-6-(tert-butyl-dimethyl-silanyloxy)-2,5,7,8-tetrameth-
yl-chroman-2-yl]heptyl}-amide (7b, n = 2). (48.3 mg,
dark browny orange oil, Rf = 0.36 (Et2O/hexane 1:1),
82%), lime green solid, mp 59–61 ꢂC, Rf = 0.42 (Et2O/
20
D
½aꢂ +1.7ꢂ (c 0.007, CHCl3); 1H NMR (CDCl3): d
20
hexane 2:1), ½aꢂ +2.9ꢂ (c 0.007, CHCl3); 1H NMR
D
8.46 (d, 1H, J = 9 Hz), 6.18 (br, 1H), 6.14 (d, 1H,
J = 9 Hz), 3.45 (q, 2H, J = 6 Hz), 2.53 (t, 2H,
J = 7 Hz), 2.07 (s, 3H), 2.05 (s, 3H), 2.03 (s, 3H), 1.77
(m, 4H), 1.39 (m, 6H), 1.28 (br, 8H), 1.20 (s, 3H),
1.02 (s, 9H), 0.09 (s, 6H); 13C NMR (CDCl3): d
145.8, 144.2, 144.0, 143.8, 143.8, 136.5, 125.8, 124.0,
123.5, 122.6, 117.4, 98.5, 74.4, 43.9, 39.5, 31.5, 30.0,
29.4, 29.3, 29.1, 28.5, 26.9, 26.0 (3 CH3), 23.8, 23.5,
20.8, 18.5, 14.3, 13.4, 11.9, ꢁ3.3 (2 CH3); MS (EI) m/
z 624 (M+, 30.3%), 590 (20.3%), 460 (45.6%), 279
(54.2%), 73 (100%). HRMS (EI): calcd for
C34H52N4O5Si: 624.37069. Found: 624.36959.
(CDCl3): d 8.51 (d, 1H, J = 9 Hz), 8.29 (d, 1H,
J = 9 Hz), 8.23 (d, 1H, J = 7 Hz), 7.54 (t, 1H, J = 8 Hz),
7.50 (t, 1H, J = 8 Hz), 7.15 (d, 1H, J = 7 Hz), 4.77 (t,
1H, J = 6 Hz), 2.87 (s, 8H), 2.52 (t, 2H, J = 6 Hz), 2.08
(s, 3H), 2.04 (s, 6H), 1.73 (m, 2H), 1.41 (m, 2H), 1.32
(m, 4H), 1.18 (s, 3H), 1.07 (s, 6H), 1.03 (s, 9H), 0.10 (s,
6H); 13C NMR (CDCl3): d 151.9, 145.7, 143.9, 134.7,
130.3, 129.8, 129.5 (2 Ar-C), 128.3, 125.7, 123.4, 123.1,
122.5, 118.7, 117.4, 115.1, 74.3, 45.3 (2 CH3), 43.2, 39.4,
31.4, 29.6, 29.4, 28.8, 26.3, 26.0 (3 CH3), 23.7, 23.4,
20.8, 18.5, 14.3, 13.3, 11.9, ꢁ3.4 (2 CH3); MS (EI) m/z
666 (M+, 22.7%), 307 (26.1%), 279 (6.7%), 171 (15.8%),
149 (100%). HRMS (EI): calcd for C38H58N2O4SSi:
666.38865. Found: 666.38290.
4.5.6. General procedure for the preparation of com-
pounds 7a–d.
4.5.6.3. 5-Dimethylamino-naphthalene-1-sulfonic acid
{9-[(R)-6-(tert-butyl-dimethyl-silanyloxy)-2,5,7,8-tetrameth-
yl-chroman-2-yl]octyl}-amide (7c, n = 3). (28.4 mg, 82%),
lime green sticky residue, Rf = 0.44 (Et2O/hexane 2:1),
TBDMSO
O
O
N
H
S
n
20
1
O
½aꢂ +2.6ꢂ (c 0.008, CHCl3); H NMR (CDCl3): d 8.51
D
(d, 1H, J = 8 Hz), 8.26 (d, 1H, J = 9 Hz), 8.22 (d, 1H,
N
7a-d, n = 1-4

3732
P. Nava et al. / Bioorg. Med. Chem. 14 (2006) 3721–3736
J = 7 Hz), 7.54 (t, 1H, J = 8 Hz), 7.50 (t, 1H, J = 8 Hz),
7.15 (d, 1H, J = 7 Hz), 4.57 (t, 1H, J = 6 Hz), 2.87 (s,
8H), 2.51 (t, 2H, J = 7 Hz), 2.07 (s, 3H), 2.03 (s, 3H),
2.03 (s, 3H), 1.73 (m, 2H), 1.48 (m, 2H), 1.30 (m, 4H),
1.18 (s, 3H), 1.07 (s, 8H), 1.02 (s, 9H), 0.09 (s, 6H);
13C NMR (CDCl3): d 151.8, 145.8, 144.0, 134.7, 130.3,
129.7, 129.6, 128.3, 125.8, 123.4, 123.5, 123.2, 122.6,
118.8, 117.4, 115.1, 74.3, 45.4 (2 CH3), 43.3, 39.5, 31.4,
29.9, 29.6, 29.4, 29.3, 26.3, 26.0 (3 CH3), 23.7, 23.5,
20.8, 18.5, 14.3, 13.4, 11.9, ꢁ3.3 (2 CH3); MS (EI) m/z
680 (M+, 6.4%), 473 (10.3%), 279 (6.5%), 73 (43.9%),
43 (100%). HRMS (EI): calcd for C39H60N2O4SSi:
680.40430. Found: 680.40426.
(EtOAc/hexane 2:1) to yield the desired product in excel-
lent yield. (51.9 mg, 92%), clear light yellow sticky resi-
20
D
due, Rf = 0.62 (EtOAc/hexane 2:1), ½aꢂ 2.8ꢂ (c 0.003,
1
CHCl3); H NMR (CDCl3): d 7.56 (br, 1H), 7.30 (m,
2H), 6.65 (d, 1H, J = 9 Hz), 6.57 (t, 1H, J = 7 Hz),
6.06 (br, 1H), 3.35 (q, 2H, J = 6 Hz), 2.83 (s, 3H), 2.51
(t, 2H, J = 6 Hz), 2.08 (s, 3H), 2.06 (s, 3H), 2.04 (s,
3H), 1.75 (m, 2H), 1.54 (m, 4H), 1.34 (m, 6H), 1.20 (s,
3H), 1.03 (s, 9H), 0.10 (s, 6H); 13C NMR (CDCl3): d
169.7 (C@O), 150.2, 145.7, 143.9, 132.6, 126.9, 125.8,
123.4, 122.6, 117.4, 115.5, 114.6, 111.2, 74.3, 39.6,
39.4, 31.4, 29.8, 29.7, 29.6, 26.9, 26.0 (3 CH3), 23.7,
23.5, 20.8, 18.5, 14.2, 13.3, 11.9, ꢁ3.4 (2 CH3); MS
(EI) m/z 552 (M+, 76.0%), 445 (5.5%), 279 (11.8%),
134 (89.6%), 73 (100%). HRMS (EI): calcd for
C33H52N2O3Si: 552.37472. Found: 552.37384.
4.5.6.4. 5-Dimethylamino-naphthalene-1-sulfonic acid
{9-[(R)-6-(tert-butyl-dimethyl-silanyloxy)-2,5,7,8-tetrameth-
yl-chroman-2-yl]nonyl}-amide (7d, n = 4). (34.3 mg,
20
D
81%), lime green oil, Rf = 0.44 (Et2O/hexane 2:1), ½aꢂ
4.5.7.2. N-{9-[(R)-6-(tert-Butyl-dimethyl-silanyloxy)-
2,5,7,8-tetramethyl-chroma-2-yl]-heptyl}-2-methylamino-
1
+2.2ꢂ (c 0.009, CHCl3); H NMR (CDCl3): d 8.52 (d,
1H, J = 9 Hz), 8.28 (d, 1H, J = 9 Hz), 8.22 (d, 1H,
J = 7 Hz), 7.54 (t, 1H, J = 8 Hz), 7.51 (t, 1H,
J = 8 Hz), 7.16 (d, 1H, J = 7 Hz), 4.67 (t, 1H,
J = 6 Hz), 2.85 (s, 8H), 2.52 (t, 2H, J = 7 Hz), 2.07 (s,
3H), 2.05 (s, 3H), 2.03 (s, 3H), 1.74 (m, 2H), 1.48 (m,
2H), 1.30 (m, 4H), 1.19 (s, 3H), 1.15 (s, 4H), 1.06 (s,
6H), 1.03 (s, 9H), 0.09 (s, 6H); 13C NMR (CDCl3): d
151.8, 145.8, 143.9, 134.7, 130.2, 129.7, 129.6, 129.5,
128.3, 125.8, 123.4, 123.2, 122.6, 118.8, 117.4, 115.1,
74.3, 45.4 (2 CH3), 43.2, 39.5, 31.4, 30.0, 29.4, 29.3,
29.2, 28.9, 26.3, 26.0 (3 CH3), 23.7, 23.5, 20.8, 18.5,
14.3, 13.3, 11.9, ꢁ3.3 (2 CH3); MS (EI) m/z 694 (M+,
9.1%), 460 (12.2%), 279 (12.3%), 149 (41.2%), 69
(100%). HRMS (EI): calcd for C40H62N2O4SSi:
694.41995. Found: 694.42075.
benzamide (8b, n = 2). (86.3 mg, 91%), clear light yellow
20
D
sticky residue, Rf = 0.64 (EtOAc/hexane 2:1), ½aꢂ +2.2ꢂ
1
(c 0.004, CHCl3); H NMR (CDCl3): d 7.57 (br, 1H),
7.30 (m, 2H), 6.63 (d, 1H, J = 9 Hz), 6.56 (t, 1H,
J = 7 Hz), 6.08 (br, 1H), 3.34 (q, 2H, J = 7 Hz), 2.83
(s, 3H), 2.53 (t, 2H, J = 7 Hz), 2.08 (s, 3H), 2.06 (s,
3H), 2.04 (s, 3H), 1.75 (m, 2H), 1.54 (m, 4H), 1.31 (m,
8H), 1.20 (s, 3H), 1.03 (s, 9H), 0.10 (s, 6H); 13C NMR
(CDCl3): d 169.7 (C@O), 150.2, 145.7, 143.9, 132.6,
126.9, 125.7, 123.4, 122.5, 117.4, 115.5, 114.5, 111.0,
74.3, 39.6, 39.4, 31.4, 29.9, 29.7, 29.6, 29.2, 26.9, 26.0
(3 CH3), 23.7, 23.5, 20.8, 18.5, 14.2, 13.3, 11.7, ꢁ3.4 (2
CH3); MS (EI) m/z 566 (M+, 100%), 458 (12.9%), 279
(10.1%), 221 (12.2%), 134 (65.6%). HRMS (EI): calcd
for C34H54N2O3Si: 566.39037. Found: 566.39190.
4.5.7. General procedure for the preparation of com-
pounds 8a–d.
4.5.7.3. N-{9-[(R)-6-(tert-Butyl-dimethyl-silanyloxy)-
2,5,7,8-tetramethyl-chroma-2-yl]-octyl}-2-methylamino-benz-
amide (8c, n = 3). (42.3 mg, 93%), clear faint yellow oil,
20
D
Rf = 0.67 (EtOAc/hexane 2:1), ½aꢂ +2.1ꢂ (c 0.004,
1
CHCl3); H NMR (CDCl3): d 7.56 (br, 1H), 7.30 (m,
2H), 6.64 (d, 1H, J = 9 Hz), 6.57 (t, 1H, J = 7 Hz),
6.05 (br, 1H), 3.35 (q, 2H, J = 7 Hz), 2.83 (s, 3H), 2.53
(t, 2H, J = 7 Hz), 2.08 (s, 3H), 2.05 (s, 3H), 2.03 (s,
3H), 1.75 (m, 2H), 1.55 (m, 4H), 1.28 (m, 10H), 1.20
(s, 3H), 1.03 (s, 9H), 0.09 (s, 6H); 13C NMR (CDCl3):
d 169.7 (C@O), 150.2, 145.8, 143.9, 132.6, 126.9, 125.8,
123.4, 122.6, 117.4, 115.5, 114.6, 111.2, 74.4, 39.6,
39.5, 31.4, 30.0, 29.7, 29.6, 29.4, 29.2, 26.9, 26.0 (3
CH3), 23.7, 23.5, 20.8, 18.5, 14.2, 13.3, 11.9, ꢁ3.4 (2
CH3); (EI) m/z 580 (M+, 100%), 446 (32.7%), 279
(24.5%), 134 (70.4%), 73 (95.0%). HRMS (EI): calcd
for C35H56N2O3Si: 580.40602. Found: 580.41120.
TBDMSO
O
O
N
H
n
HN
8a-d, n = 1-4
The synthesis of compounds 8a–d was conducted under
identical conditions and gave similar product yields
(90–93%).
4.5.7.1. N-{9-[(R)-6-(tert-Butyl-dimethyl-silanyloxy)-
2,5,7,8-tetramethyl-chroma-2-yl]-hexyl}-2-methylamino-
benzamide (8a, n = 1). To solid N-methylisatoic anhy-
dride34 (20.0 mg, 0.112 mmol) and 4-dimethylamino-
pyridine (1.2 mg, 0.010 mmol) under argon is added
dry DMF (1 ml) via syringe. After stirring for 1 min, a
solution of amine 4a (43.0 mg, 0.102 mmol) in dry
DMF (1 ml) was added via cannula. The reaction mix-
ture was stirred for an additional 30 min., after which
is diluted with dH2O and extracted with ethyl acetate
(3· 30 ml). The combined organic layers are then dried
with Na2SO4, decanted, and evaporated to give the crude
product which is purified by column chromatography
4.5.7.4. N-{9-[(R)-6-(tert-Butyl-dimethyl-silanyloxy)-
2,5,7,8-tetramethyl-chroma-2-yl]-nonyl}-2-methylamino-
benzamide (8d, n = 4). (60.2 mg, 90%), clear faint yellow
20
D
oil, Rf = 0.68 (EtOAc/hexane 2:1), ½aꢂ +1.6ꢂ (c 0.007,
1
CHCl3); H NMR (CDCl3): d 7.55 (br, 1H), 7.30 (m,
2H), 6.64 (d, 1H, J = 8 Hz), 6.57 (t, 1H, J = 6 Hz),
6.06 (br, 1H), 3.35 (q, 2H, J = 7 Hz), 2.83 (s, 3H), 2.53
(t, 2H, J = 7 Hz), 2.08 (s, 3H), 2.06 (s, 3H), 2.03 (s,
3H), 1.75 (m, 2H), 1.51 (m, 4H), 1.27 (m, 12H), 1.20
(s, 3H), 1.03 (s, 9H), 0.10 (s, 6H); 13C NMR (CDCl3):

P. Nava et al. / Bioorg. Med. Chem. 14 (2006) 3721–3736
3733
d 169.7 (C@O), 150.2, 145.8, 143.9, 132.6, 126.9, 125.7,
123.4, 122.6, 117.4, 115.5, 114.6, 111.2, 74.4, 39.6,
39.5, 31.4, 30.0, 29.7, 29.6, 29.5, 29.4, 29.2, 26.9, 26.0
(3 CH3), 23.7, 23.5, 20.8, 18.5, 14.8, 13.3, 11.9, ꢁ3.4 (2
CH3); MS (EI) m/z 594 (M+, 100%), 322 (23.7%), 279
(16.7%), 134 (78.2%), 73 (61.2%). HRMS (EI): calcd
for C36H58N2O3Si: 594.42167. Found: 594.42157.
4.5.8.3. Anthracene-9-carboxylic acid 8-(R)-6-
hydroxy-2,5,7,8-tetramethyl-chroman-2-yl-octyl ester
(9c, n = 3). (20.8 mg, 84%), light yellow oil, Rf = 0.58
20
(Et2O/hexane 4:1), ½aꢂ +0.6ꢂ (c 0.007, EtOH); UV
D
(EtOH) kabs 362 nm (e = 6835); fluorescence kem
1
470 nm; H NMR (CDCl3): d 8.51 (s, 1H), 7.99 (t, 4H,
J = 8 Hz), 7.50 (m, 4H), 4.60 (t, 2H, J = 6 Hz), 4.18
(br, 1H), 2.57 (t, 2H, J = 7 Hz), 2.14 (s, 3H), 2.09 (s,
6H), 1.86 (m, 2H), 1.76 (m, 2H), 1.38 (m, 12H), 1.19
(s, 3H); 13C NMR (CDCl3): d 169.7, 145.5, 144.5,
130.9, 129.1, 128.5, 128.3, 128.1, 126.8, 125.4, 125.0,
122.5, 121.0, 118.4, 117.3, 74.4, 65.9, 39.4, 31.5, 30.0,
29.4, 29.1, 28.7, 26.0, 23.7, 23.5, 20.7, 12.2, 11.7, 11.2;
MS (EI) m/z 538 (M+, 3.0%), 376 (19.3%), 262
(49.5%), 165 (62.2%), 43 (100%). HRMS (EI): calcd
for C36H42O4: 538.30831. Found: 538.30037.
4.5.8. General procedure for the preparation of com-
pounds 9a–d, 10a–d, 11a–d, and 12a–d. Syntheses provid-
ing the deprotected fluorescent tocopherols 9a–d, 10a–d,
11a–d, and 12a–d were conducted under identical condi-
tions, except where noted, to give similar product yields
(83–89%).
4.5.8.1. x-Anthroyloxy-a-tocopherols (AO-a-Toc)
anthracene-9-carboxylic acid 6-(R)-6-hydroxy-2,5,7,8-
tetramethyl-chroman-2-yl-hexyl ester (9a, n = 1). A solu-
tion of tetrabutylammonium fluoride (TBAF) (1 M in
THF, 61 ll, 0.212 mmol) was added dropwise via
syringe to a stirred solution of TBSO-C6-AO-a-Toc
5a (26.5 mg, 0.042 mmol) in dry THF (1 ml). The
mixture was stirred at room temperature for 30 min
until no starting material 5a was detected by TLC.
The reaction was then quenched with 1 ml of 1 N
HCl and diluted with 15 ml ether, to which an addi-
tional 10 ml dH2O was added. The water phase was
extracted again with ether (2· 15 ml), dried over
anhydrous Na2SO4, filtered, and concentrated under
reduced pressure. The crude product was then chro-
matographed on silica gel (Et2O/hexane 1:2) to give
4.5.8.4. Anthracene-9-carboxylic acid 9-(R)-6-hy-
droxy-2,5,7,8-tetramethyl-chroman-2-yl-nonyl ester (9d,
n = 4). (29.5 mg, 85%), light yellow oil, Rf = 0.61 (Et2O/
hexane 4:1), ½aꢂ20 +0.4ꢂ (c 0.008, EtOH); UV (EtOH) kabs
D
1
363 nm (e = 6933); fluorescence kem 470 nm; H NMR
(CDCl3): d 8.50 (s, 1H), 8.01 (t, 4H, J = 8 Hz), 7.49
(m, 4H), 4.60 (t, 2H J = 7 Hz), 4.24 (br, 1H), 2.57 (t,
2H, J = 7 Hz), 2.13 (s, 3H), 2.09 (s, 6H), 1.84 (m, 2H),
1.74 (m, 2H), 1.48 (m, 4H), 1.36 (m, 2H), 1.28 (m,
8H), 1.20 (s, 3H); 13C NMR (CDCl3): d 169.7, 145.4,
144.4, 130.9, 129.1, 128.5, 128.3, 128.1, 126.8, 125.4,
124.9, 122.5, 121.0, 118.4, 117.3, 74.4, 65.9, 39.4, 31.4,
30.0, 29.5, 29.4, 29.2, 28.7, 26.0, 23.8, 23.5, 20.7, 12.2,
11.7, 11.2; MS (EI) m/z 552 (M+, 16.4%), 222 (14.4%),
205 (100%), 165 (30.2), 83 (31.1%). HRMS (EI): calcd
for C37H44O4: 552.32395. Found: 552.32126.
the pure product 9a. (19.7 mg, 84%), light yellow
20
D
oil, Rf = 0.54 (Et2O/hexane 4:1), ½aꢂ
+0.8ꢂ (c
0.003, EtOH); UV (EtOH) kabs 362 nm (e = 6731);
fluorescence kem 470 nm; 1H NMR (CDCl3): d 8.50
(s, 1H), 8.01 (t, 4H, J = 7 Hz), 7.49 (m, 4H), 4.59
(t, 2H, J = 7 Hz), 2.57 (t, 2H, J = 7 Hz), 2.13 (s,
3H), 2.08 (s, 6H), 1.84 (m, 2H), 1.74 (m, 2H), 1.50
(m, 8H), 1.21 (s, 3H); 13C NMR (CDCl3): d 169.8,
145.5, 144.6, 131.0, 129.2, 128.6, 128.4, 128.2,
126.9, 125.4, 125.0, 122.6, 121.0, 118.5, 117.3, 74.4,
65.9, 39.5, 31.5, 29.7, 28.7, 26.1, 23.7, 23.5, 20.7,
12.2, 11.8, 11.3; MS (EI) m/z 510 (M+, 4.9%), 205
(16.1%), 165 (18.8%), 138 (54.0%), 43 (100%).
HRMS (EI): calcd for C34H38O4: 510.27701. Found:
510.27581.
4.5.8.5. x-Nitrobenzoxadiazole-a-tocopherols (NBD-
a-Toc). The following change was made to the above
protocol: (i) column chromatography was conducted
using an ethyl acetate/hexane (1:2) solvent system.
4.5.8.6. (R)-2,5,7,8-tetramethyl-chroman-2-[9-(7-ni-
tro-benzo[1,2,5]oxadiazol-4-ylamino)-hexyl]-chroman-6-
ol (10a, n = 1). (7.6 mg, 85 %), dark browny orange oil,
20
D
Rf = 0.43 (EtOAc/hexane 1:1), ½aꢂ +4.5ꢂ (c 0.005,
EtOH); UV (EtOH) kabs 468 nm (e = 18,750); fluores-
1
cence kem 527 nm; H NMR (CDCl3): d 8.49 (d, 1H,
J = 9 Hz), 6.17 (br, 1H), 6.16 (d, 1H, J = 9 Hz), 4.20
(s, 1H), 3.46 (q, 2H, J = 6 Hz), 2.60 (t, 2H, J = 6 Hz),
2.15 (s, 3H), 2.11 (s, 3H), 2.10 (s, 3H), 1.77 (m, 4H),
1.48 (m, 8H), 1.22 (s, 3H); 13C NMR (CDCl3): d
145.3, 144.6, 144.2, 143.8, 143.7, 136.4, 124.0, 122.5,
121.0, 118.5, 117.3, 98.5, 74.2, 43.9, 39.2, 31.6, 29.6,
28.4, 26.9, 23.7, 23.3, 20.7, 12.2, 11.8, 11.3; MS (EI)
m/z 468 (M+, 8.3%), 304 (20.2%), 203 (20.9%), 165
(100%), 43 (84.7%). HRMS (EI): calcd for
C25H32N4O5: 468.23727. Found: 468.22557.
4.5.8.2. Anthracene-9-carboxylic acid 7-(R)-6-hy-
droxy-2,5,7,8-tetramethyl-chroman-2-yl-heptyl ester (9b,
n = 2). (33.9 mg, 83%), light yellow oil, Rf = 0.56 (Et2O/
hexane 4:1), ½aꢂ20 +0.4ꢂ (c 0.007, EtOH); UV (EtOH) kabs
D
1
362 nm (e = 6718); fluorescence kem 470 nm; H NMR
(CDCl3): d 8.54 (s, 1H), 8.06 (t, 4H, J = 9 Hz), 7.53
(m, 4H), 4.64 (t, 2H, J = 7 Hz), 4.26 (br, 1H), 2.61 (t,
2H, J = 7 Hz), 2.13 (s, 3H), 2.09 (s, 6H), 1.91 (m, 2H),
1.79 (m, 2H), 1.28 (m, 10H), 1.24 (s, 3H); 13C NMR
(CDCl3): d 169.8, 145.4, 144.5, 130.9, 129.2, 128.6,
128.3, 128.2, 126.8, 125.4, 124.9, 122.5, 121.0, 118.5,
117.3, 74.4, 65.9, 39.4, 31.5, 30.0, 29.1, 28.7, 26.0, 23.7,
23.5, 20.7, 12.2, 11.7, 11.2; MS (EI) m/z 524 (M+,
3.4%), 348 (20.7%), 205 (24.1%), 165 (62.0%), 43
(100%). HRMS (EI): calcd for C35H40O4: 524.29266.
Found: 524.29270.
4.5.8.7. (R)-2,5,7,8-tetramethyl-chroman-2-[9-(7-ni-
tro-benzo[1,2,5]oxadiazol-4-ylamino)-heptyl]-chroman-6-
ol (10b, n = 2). (9.2 mg, 84%), dark browny orange oil,
20
D
Rf = 0.45 (EtOAc/hexane 1:1), ½aꢂ +3.9ꢂ (c 0.005,
EtOH); UV (EtOH) kabs 468 nm (e = 18,415); fluores-
1
cence kem 529 nm; H NMR (CDCl3): d 8.48 (d, 1H,
J = 9 Hz), 6.19 (br, 1H), 6.16 (d, 1H, J = 9 Hz), 4.18

3734
P. Nava et al. / Bioorg. Med. Chem. 14 (2006) 3721–3736
(s, 1H), 3.44 (q, 2H, J = 6 Hz), 2.58 (t, 2H, J = 7 Hz),
2.13 (s, 3H), 2.08 (s, 3H), 2.07 (s, 3H), 1.76 (m, 4H),
1.56 (m, 2H), 1.48 (m, 8H), 1.19 (s, 3H); 13C NMR
(CDCl3): d 145.4, 144.5, 144.2, 143.8, 143.7, 136.4,
123.9, 122.5, 121.0, 118.5, 117.3, 98.4, 74.3, 43.9, 39.3,
31.5, 29.8, 29.1, 28.5, 26.8, 23.7, 23.4, 20.7, 12.2, 11.8,
11.3; MS (EI) m/z 482 (M+, 7.6%), 318 (31.0%), 203
(16.1%), 165 (100%), 43 (92.8%). HRMS (EI): calcd
for C26H34N4O5: 482.25292. Found: 482.25347.
(s, 3H), 1.08 (br, 2H); 13C NMR (CDCl3): d 151.4,
145.4, 144.5, 134.7, 130.2, 129.7, 129.6, 129.5, 128.2,
123.2, 122.5, 121.1, 118.9, 118.6, 117.2, 115.1, 74.2,
45.4 (2 CH3), 43.2, 38.9, 31.5, 29.3, 29.3, 26.2, 23.7,
23.2, 20.6, 12.2, 11.7, 11.2; MS (EI) m/z 538 (M+,
100%), 374 (6.7%), 205 (12.5%), 165 (74.6%), 43
(58.1%). HRMS (EI): calcd for C31H42N2O4S:
538.28653. Found: 538.28642.
4.5.8.12. 5-Dimethylamino-naphthalene-1-sulfonic acid
[9-((R)-6-hydroxyl-2,5,7,8-tetramethyl-chroman-2-yl)-heptyl-
4.5.8.8. (R)-2,5,7,8-tetramethyl-chroman-2-[9-(7-ni-
tro-benzo[1,2,5]oxadiazol-4-ylamino)-octyl]-chroman-6-ol
amine (11b, n = 2). (18.1 mg, 87%), light lime green sticky
solid, mp 80–82 ꢂC,Rf = 0.57 (Et2O/hexane 5:1), ½aꢂ
20
D
(10c, n = 3). (8.2 mg, 84%), dark browny orange oil,
20
D
Rf = 0.46 (EtOAc/hexane 1:1), ½aꢂ +4.1ꢂ (c 0.004,
+1.0ꢂ (c 0.009, EtOH); UV (EtOH) kabs 341 nm
1
EtOH); UV (EtOH) kabs 469 nm (e = 5809); fluorescence
(e = 3865); fluorescence kem 507 nm; H NMR (CDCl3): d
k
em 528 nm; 1H NMR (CDCl3): d 8.47 (d, 1H, J = 9 Hz),
8.51 (d, 1H, J = 8 Hz), 8.26 (d, 1H, J = 9 Hz), 8.21 (d,
1H, J = 8 Hz), 7.54 (t, 1H, J = 8 Hz), 7.50 (t, 1H,
J = 8 Hz), 7.15 (d, 1H, J = 7 Hz), 4.61 (t, 1H, J = 6 Hz),
4.24 (s, 1H), 2.87 (s, 6H), 2.83 (m, 2H), 2.56 (t, 2H,
J = 6 Hz), 2.13 (s, 3H), 2.08 (s, 3H), 2.06 (s, 3H), 1.72
(m, 2H), 1.42 (m, 2H), 1.35 (m, 6H), 1.17 (s, 3H), 1.07
(br, 4H); 13C NMR (CDCl3): d 151.4, 145.4, 144.5, 134.7,
130.2, 129.7, 129.6, 129.6, 128.2, 123.2, 122.5, 121.0,
118.9, 118.5, 117.2, 115.1, 74.3, 45.4 (2 CH3), 43.2, 39.2,
31.4, 29.7, 29.4, 28.8, 26.2, 23.7, 23.3, 20.6, 12.2, 11.7,
11.2; MS (EI) m/z 552 (M+, 76.4%), 388 (5.8%), 203
(15.6%), 165 (60.6%), 69 (100%). HRMS (EI): calcd for
C32H44N2O4S: 552.30218. Found: 552.30216.
6.24 (br, 1H), 6.14 (d, 1H, J = 9 Hz), 4.23 (br, 1H), 3.46
(q, 2H, J = 6 Hz), 2.58 (t, 2H, J = 7 Hz), 2.13 (s, 3H),
2.08 (s, 3H), 2.03 (s, 3H), 1.73 (m, 4H), 1.57 (m, 2H),
1.42 (m, 10H), 1.20 (s, 3H); 13C NMR (CDCl3): d
145.4, 144.5, 144.2, 143.9, 143.8, 136.5, 123.9, 122.5,
121.0, 118.5, 117.3, 98.4, 74.4, 43.9, 39.1, 31.6, 29.8,
29.2, 29.0, 28.5, 26.9, 23.8, 23.4, 20.7, 12.2, 11.8, 11.3;
MS (EI) m/z 496 (M+, 11.8%), 332 (22.2%), 203
(17.1%), 165 (100%), 43 (43.0%). HRMS (EI): calcd
for C27H36N4O5: 496.26857. Found: 496.26402.
4.5.8.9. (R)-2,5,7,8-tetramethyl-chroman-2-[9-(7-ni-
tro-benzo[1,2,5]oxadiazol-4-ylamino)-nonyl]-chroman-
6-ol (10d, n = 4). (14.9 mg, 86%), dark browny orange
4.5.8.13. 5-Dimethylamino-naphthalene-1-sulfonic acid
[9-((R)-6-hydroxyl-2,5,7,8-tetramethyl-chroman-2-yl)-oc-
oil, Rf = 0.49 (EtOAc/hexane 1:1), ½aꢂ20 +4.7ꢂ (c 0.004,
D
EtOH); UV (EtOH) kabs 469 nm (e = 17,602); fluores-
tyl-amine (11c, n = 3). (8.0 mg, 87%), light lime green
20
D
1
cence kem 527 nm; H NMR (CDCl3): d 8.49 (d, 1H,
sticky residue, Rf = 0.58 (Et2O/hexane 5:1), ½aꢂ +1.5ꢂ
J = 9 Hz), 6.26 (br, 1H), 6.17 (d, 1H, J = 9 Hz), 4.24
(s, 1H), 3.47 (q, 2H, J = 6 Hz), 2.58 (t, 2H,
J = 7 Hz), 2.15 (s, 3H), 2.10 (s, 3H), 2.05 (s, 3H),
1.77 (m, 4H), 1.55 (m, 2H), 1.43 (m, 12H), 1.22 (s,
3H); 13C NMR (CDCl3): d 145.5, 144.5, 144.2,
143.9, 143.8, 136.5, 123.9, 122.5, 121.0, 118.5,
117.3, 98.5, 74.4, 43.9, 39.3, 31.5, 29.9, 29.4, 29.3,
29.2, 28.5, 26.9, 23.8, 23.5, 20.7, 12.2, 11.8, 11.3;
MS (EI) m/z 510 (M+, 13.5%), 346 (21.4%), 203
(17.6%), 165 (100%), 43 (18.5%). HRMS (EI): calcd
for C28H38N4O5: 510.28422. Found: 510.28290.
(c 0.004, EtOH); UV (EtOH) kabs 342 nm (e = 3655);
1
fluorescence kem 511 nm; H NMR (CDCl3): d 8.51 (d,
1H, J = 9 Hz), 8.25 (d, 1H, J = 8 Hz), 8.22 (d, 1H,
J = 8 Hz), 7.53 (t, 1H, J = 7 Hz), 7.49 (t, 1H,
J = 7 Hz), 7.15 (d, 1H, J = 8 Hz), 4.60 (t, 1H,
J = 6 Hz), 4.24 (s, 1H), 2.86 (s, 6H), 2.84 (t, 2H,
J = 7 Hz), 2.56 (t, 2H, J = 7 Hz), 2.13 (s, 3H), 2.08 (s,
3H), 2.06 (s, 3H), 1.73 (m, 2H), 1.45 (m, 2H), 1.31 (m,
6H), 1.17 (s, 3H), 1.14 (br, 6H); 13C NMR (CDCl3): d
151.4, 145.4, 144.4, 134.6, 130.2, 129.7, 129.6, 129.5,
128.2, 123.2, 122.5, 121.1, 118.9, 118.6, 117.2, 115.1,
74.3, 45.4 (2 CH3), 43.2, 39.2, 31.4, 29.7, 29.5, 29.4,
28.8, 26.2, 23.7, 23.3, 20.6, 12.1, 11.6, 11.1; MS (EI)
m/z 566 (M+, 24.9%), 205 (20.1%) 165 (37.3%), 57
(62.5%), 43 (100%). HRMS (EI): calcd for
C33H46N2O4S: 566.31783. Found: 566.31688.
4.5.8.10. x-Dansyl-a-tocopherols (DAN-a-Toc). The
following changes were made to the above protocol: (i)
extraction of the crude product was done using dichlo-
romethane, (ii) column chromatography was conducted
using an ether/hexane (2:1) solvent system.
4.5.8.14. 5-Dimethylamino-naphthalene-1-sulfonic acid
[9-((R)-6-hydroxyl-2,5,7,8-tetramethyl-chroman-2-yl)-non-
4.5.8.11. 5-Dimethylamino-naphthalene-1-sulfonic acid
[9-((R)-6-hydroxyl-2,5,7,8-tetramethyl-chroman-2-yl)-hex-
yl-amine (11d, n = 4). (20.8 mg, 88%), lime green sticky
20
D
yl-amine (11a, n = 1). (20.8 mg, 88%), light lime green
20
solid, mp 60–63 ꢂC,Rf = 0.56 (Et2O/hexane 5:1), ½aꢂ
residue, Rf = 0.59 (Et2O/hexane 5:1), ½aꢂ +0.3ꢂ (c
0.010, EtOH); UV (EtOH) kabs 340 nm (e = 4009); fluo-
D
1
+0.8ꢂ (c 0.010, EtOH); UV (EtOH) kabs 340 nm
rescence kem 507 nm; H NMR (CDCl3): d 8.52 (d, 1H,
1
(e = 3797); fluorescence kem 510 nm; H NMR (CDCl3):
J = 9 Hz), 8.26 (d, 1H, J = 8 Hz), 8.22 (d, 1H, J = 8 Hz),
7.54 (t, 1H, J = 7 Hz), 7.50 (t, 1H, J = 7 Hz), 7.16 (d,
1H, J = 8 Hz), 4.62 (t, 1H, J = 6 Hz), 4.25 (s, 1H), 2.87
(s, 6H), 2.84 (t, 2H, J = 7 Hz), 2.57 (t, 2H, J = 7 Hz),
2.13 (s, 3H), 2.08 (s, 6H), 1.74 (m, 2H), 1.47 (m, 2H),
1.34 (m, 6H), 1.19 (s, 3H), 1.15 (br, 8H); 13C NMR
(CDCl3) d 151.5, 145.5, 144.5, 134.7, 130.3, 129.8,
d 8.51 (d, 1H, J = 9 Hz), 8.25 (d, 1H, J = 8 Hz), 8.23 (d,
1H, J = 8 Hz), 7.53 (t, 1H, J = 8 Hz), 7.50 (t, 1H,
J = 8 Hz), 7.14 (d, 1H, J = 7 Hz), 4.57 (t, 1H,
J = 6 Hz), 4.26 (s, 1H), 2.87 (s, 6H), 2.82 (m, 2H), 2.56
(t, 2H, J = 7 Hz), 2.13 (s, 3H), 2.08 (s, 3H), 2.06
(s, 3H), 1.71 (m, 2H), 1.41 (m, 2H), 1.33 (m, 6H), 1.15

P. Nava et al. / Bioorg. Med. Chem. 14 (2006) 3721–3736
3735
129.7, 129.6, 128.3, 123.3, 122.5, 121.0, 119.0, 118.5,
117.3, 115.2, 74.4, 45.4 (2 CH3), 43.3, 39.3, 31.5, 30.0,
29.5, 29.3, 29.2, 28.9, 26.3, 23.8, 23.5, 20.7, 12.2, 11.8,
11.3; MS (EI) m/z 580 (M+, 21.5%), 205 (22.7%), 149
(30.8%), 57 (100%), 43 (97.6%). HRMS (EI): calcd for
C34H48N2O4S: 580.33348. Found: 580.33409.
(M+, 5.6%), 329 (3.6%), 165 (16.4%), 134 (23.4%), 69
(100%). HRMS (EI): calcd for C29H42N2O3:
466.31954. Found: 466.31689.
4.5.8.19. N-[9-((R)-6-hydroxy-2,5,7,8-tetramethyl-
chroma-2-yl)-nonyl]-2-methylamino-benzamide (12d,
n = 4). (19.2 mg, 89%), light yellow oil, Rf = 0.67
20
D
4.5.8.15.
x-N-Methylanthranylamide-a-tocopherols
(EtOAc/hexane 2:1), ½aꢂ +1.3ꢂ (c 0.006, EtOH); UV
(NMA-a-Toc). The following change was made to the
above protocol: (i) column chromatography was con-
ducted using an ethyl acetate/hexane (1:1) solvent
system.
(EtOH) kabs 346 nm (e = 3969); fluorescence kem
424 nm; H NMR (CDCl3): d 7.30 (t, 2H, J = 7 Hz),
1
6.68 (d, 1H, J = 8 Hz), 6.60 (t, 1H, J = 7 Hz), 6.10 (br,
1H), 4.31 (br, 1H), 3.34 (q, 2H, J = 7 Hz), 2.83 (s,
3H), 2.57 (t, 2H, J = 7 Hz), 2.13 (s, 3H), 2.08 (s, 6H),
1.74 (m, 2H), 1.55 (m, 4H), 1.25 (m, 12H), 1.20 (s,
3H); 13C NMR (CDCl3): d 169.7, 149.8, 145.4, 144.5,
132.7, 127.0, 122.5, 121.1, 118.6, 117.3, 115.9, 115.2,
111.7, 74.4, 39.7, 39.3, 31.5, 30.0, 29.6, 29.5, 29.4, 29.3,
27.0, 23.8, 23.5, 20.7, 12.2, 11.8, 11.3; MS (EI) m/z 480
(M+, 6.4%), 163 (39.9%), 149 (32.3%), 105 (100%), 69
(38.5%). HRMS (EI): calcd for C30H44N2O3:
480.33519. Found: 480.33222.
4.5.8.16.
chroma-2-yl)-hexyl]-2-methylamino-benzamide
n = 1). (12.3 mg, 88%), light yellow solid, mp 48–49 ꢂC,
Rf = 0.61 (EtOAc/hexane 2:1), ½aꢂ +3.0ꢂ (c 0.005,
N-[9-((R)-6-hydroxy-2,5,7,8-tetramethyl-
(12a,
20
D
EtOH); UV (EtOH) kabs 340 nm (e = 2729); fluorescence
kem 423 nm; 1H NMR (CDCl3): d 7.27 (q, 2H,
J = 8 Hz), 6.73 (d, 1H, J = 7 Hz), 6.62 (t, 1H, J = 7 Hz),
6.19 (br, 1H), 4.31 (br, 1H), 3.28 (q, 2H, J = 7 Hz), 2.80
(s, 3H), 2.52 (t, 2H, J = 7 Hz), 2.08 (s, 3H), 2.03 (s, 6H),
1.71 (m, 2H), 1.51 (m, 4H), 1.28 (m, 6H), 1.14 (s, 3H);
13C NMR (CDCl3): d 169.4, 150.0, 145.4, 144.5, 132.8,
127.1, 122.5, 121.1, 118.6, 117.3, 116.5, 115.8, 112.4,
74.4, 39.8, 39.3, 31.5, 29.8, 29.5, 27.0, 23.7, 23.5, 20.7,
12.2, 11.8, 11.3; MS (EI) m/z 438 (M+, 23.8%), 305
(6.9%), 165 (22.2%), 134 (100%), 69 (53.2%). HRMS
(EI): calcd for C27H38N2O3: 438.28824. Found:
438.28643.
Acknowledgments
This work was supported by grants from NSERC,
Cottrell College Science Award, Research Corporation,
AZ (for support of S. Chirico), and the Premier’s
Research Excellence Award (JA), and by NIH awards
T32-DK715827 (SM) and R01-DK067494 (DM) from
the National Institutes of Health.
4.5.8.17.
N-[9-((R)-6-hydroxy-2,5,7,8-tetramethyl-
(12b,
n = 2). (15.2 mg, 89%), light yellow solid, mp 53–
chroma-2-yl)-heptyl]-2-methylamino-benzamide
20
55 ꢂC, Rf = 0.63 (EtOAc/hexane 2:1), ½aꢂ +5.3ꢂ (c
D
References and notes
0.007, EtOH); UV (EtOH) kabs 346 nm (e = 3898); fluo-
rescence kem 423 nm; 1H NMR (CDCl3): d 7.29 (m, 2H),
6.66 (d, 1H, J = 8 Hz), 6.58 (t, 1H, J = 7 Hz), 6.07 (br,
1H), 4.33 (br, 1H), 3.32 (q, 2H, J = 7 Hz), 2.83 (s,
3H), 2.57 (t, 2H, J = 7 Hz), 2.13 (s, 3H), 2.08 (s, 6H),
1.74 (m, 2H), 1.55 (m, 4H), 1.30 (m, 8H), 1.19 (s, 3H);
13C NMR (CDCl3): d 169.7, 150.0, 145.4, 144.5, 132.6,
127.0, 122.5, 121.1, 118.6, 117.3, 115.6, 114.8, 111.4,
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20.7, 12.2, 11.7, 11.2; MS (EI) m/z 452 (M+, 11.3%),
319 (4.2%), 165 (16.7%), 134 (53.9%), 69 (100%). HRMS
(EI): calcd for C28H40N2O3: 452.30389. Found:
452.30454.
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