Design, synthesis, and evaluation of an α-tocopherol analogue as a mitochondrial antioxidant

Article abstract of DOI:10.1016/j.bmc.2010.08.030

An efficient synthesis has provided access to a novel α-tocopherol analogue (2), as well as its trifluoroacetate salt and acetate ester. An annulation reaction was used to establish the pyridinol core structure and a Stille coupling reaction was employed

Full text of DOI:10.1016/j.bmc.2010.08.030

Bioorganic & Medicinal Chemistry 18 (2010) 7628–7638
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis, and evaluation of an
as a mitochondrial antioxidant
a
-tocopherol analogue
⇑
Jun Lu, Omar M. Khdour, Jeffrey S. Armstrong, Sidney M. Hecht
Center for BioEnergetics, The Biodesign Institute, Arizona State University, Tempe, AZ 85287, USA
Department of Chemistry, Arizona State University, Tempe, AZ 85287, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
An efficient synthesis has provided access to a novel a-tocopherol analogue (2), as well as its trifluoro-
Received 12 July 2010
Revised 11 August 2010
Accepted 16 August 2010
Available online 26 August 2010
acetate salt and acetate ester. An annulation reaction was used to establish the pyridinol core structure
and a Stille coupling reaction was employed for conjugation with the tocopherol side chain. This analogue
was shown to suppress the levels of reactive oxygen species in cultured cells, and to quench peroxidation
of mitochondrial membranes.
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
a-Tocopherol analogue
Mitochondrial dysfunction
Mitochondrial antioxidant
Lipid peroxidation
Reactive oxygen species
1. Introduction
scavenging antioxidants with better properties than a-TOH has
been a goal of a number of studies. Strategies for the targeting of
antioxidants to mitochondria have also been considered.26–28
Recently Porter, Pratt and co-workers have described a new
Mitochondria are organelles found in eukaryotic cells.1 They
produce much of the ATP employed by eukaryotic cells. Mitochon-
dria are also a major source of intracellular reactive oxygen species
(ROS); as such, they are potentially vulnerable to oxidative stress.
When ROS production exceeds the capacity for detoxification and
repair, oxidative damage to proteins, DNA, and phospholipids can
occur, disrupting mitochondrial oxidative phosphorylation and
potentially leading to impairment of cell function and death. In
addition to this pathological role, ROS can also act as redox signal-
ing molecules.2,3 Accordingly, mitochondrial ROS production and
oxidative damage are attractive targets for pharmacological inter-
vention.4–8
antioxidant (4) reported to be ꢀ90-fold more potent than
a-
tocopherol as an antioxidant, as judged by suppression of the
autoxidation of methyl linoleate in benzene solution (Figure 1).23–
25
We reasoned that conjugation of the pyridinol antioxidant core
to a lipophilic side chain might facilitate delivery of the antioxidant
to membranes, including mitochondrial membranes, and thereby
confer cytoprotective properties at the level of mitochondrial func-
tion. The phytyl side chain of a-tocopherol seemed to be a logical
choice for the requisite lipophilic side chain. Herein, we report the
synthesis of a novel tocopherol analogue (2) incorporating the
pyridinol core of compound 4. Also reported is the evaluation of
the ability of 2 to protect mitochondrial membranes from AAPH
(2,20-azobis(2-amidinopropane) dihydrochloride)-induced lipid
peroxidation, and to diminish reactive oxygen species (ROS) in cul-
tured CEM leukemia cells pretreated with diethyl maleate.
Increasing evidence suggests that oxidative stress and mito-
chondrial dysfunction can play crucial roles in neurodegenerative
diseases.9–19 It seems logical to anticipate that reducing mitochon-
drial oxidative stress may blunt the progression of these neurode-
generative disorders.
a-Tocopherol (a-TOH, 1) is among the most
potent natural lipophilic antioxidants studied, exhibiting excellent
properties in quenching lipid peroxidation.20–22 Suppression of li-
pid peroxidation results from the ability of a-tocopherol to transfer
2. Results and discussion
its phenolic H atom to an incipient lipid carbon-centered radical,
and thereby quench the free radical chain reaction that occurs in
the presence of O2.23–25 The development of synthetic radical-
The retrosynthetic analysis of compound 2 is outlined in Figure 2.
Preparation of the desired compound was envisioned by the cou-
pling of pyridinol 17 and a side chain such as alkyne 9 or its tributyl-
tin derivative 10. Alkyne 9 should be accessible from commercially
available (R)-phytol.29,30 Pyridinol derivative 17 could be assembled
from13 through functional group transformations. Compound 13, in
⇑
Corresponding author. Tel.: +1 480 965 6625; fax: +1 480 965 0038.
E-mail address: sidney.hecht@asu.edu (S.M. Hecht).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.08.030

J. Lu et al. / Bioorg. Med. Chem. 18 (2010) 7628–7638
7629
OH
HO
O
N
RO
HO
N
α
-tocopherol (1)
2 R = H
4
3
R = Ac
N
N
Figure 1. Structures of a-tocopherol (1), novel antioxidants 2 and 3, as well as the pyridinol core antioxidant 4.
OH
coupling
OTf
HO
OBn
R
OHCO
+
N
N
N
N
9
10
R = H
R = SnBu3
2
17
annulation
OH
MeOOC
N
N
HO
13
phytol
MeOOC
COOMe
MeO
+
MeO
N
11
NH2
12
Figure 2. Retrosynthetic analysis of compound 2.
turn, could be obtained by the annulation reaction of 1131 and 12.32
Compounds 11 and 12 themselves were easily prepared from N-
methylpyrrolidine and dimethyl malonate, respectively.
C-16 chain can be obtained from the natural product phytol, which
has the same configuration as the -tocopherol side chain. The
third chiral center was established by Sharpless epoxidation.33
Thus, by the use of (
mer of 5 was prepared in 96% yield. The primary alcohol could
be converted to the chloride by heating in carbon tetrachloride in
the presence of triphenylphosphine.29,30 Although the primary
alcohol 5 could be converted directly to chloride 7, a large amount
a
D
)-(ꢁ)-diethyl tartrate, the desired (R,R)-iso-
2.1. Synthesis of side chain derivatives 9 and 10
The synthesis of the side chain derivatives is outlined in
Scheme 1. The configuration of the stereogenic centers in the
D-(-)-DET, Ti(Oi-Pr) 4,
t-BuOOH, -20 oC, 3 h
HO
HO
2
O
2
96%
5
7
phytol
LiCl, DMF
60 oC, 1 h
TsCl, NEt3, DMAP
Cl
CH2Cl2, 25 oC, 14 h
TsO
O
2
2
O
91%
80%
6
NaH, BnBr, DMF
oC to 25 oC, 1 h
OH
OBn
n-BuLi, THF
-20 oC, 15 min
0
2
2
80%
100%
9
8
OBn
BuLi, n-Bu3SnCl
THF, -78 oC to 25 oC, 5 h
100%
SnBu3
Scheme 1. Synthesis of side chain derivative 10.
10

7630
J. Lu et al. / Bioorg. Med. Chem. 18 (2010) 7628–7638
Table 1
of triphenylphosphine by-product made 7 difficult to purify. This
problem could be resolved by tosylation of the primary alcohol
followed by conversion to the chloride by treatment with
lithium chloride. This two-step process was more efficient and
cleaner than the direct route, and is better suited to larger scale
preparation. Chloride 7 was then treated with n-butyllithium and
converted quantitatively into alkyne alcohol 8.29,30,34,35 Finally,
the alcohol functionality was protected with a benzyl group in
80% yield to form 9. Although alkyne 9 itself could be used in the
coupling reaction, its tin derivative 10 was found to be more effi-
cient in the coupling reaction with formate ester 17. The tin deriv-
ative 10 was prepared by treating alkyne 9 with butyllithium
followed by exchange with tributyltin chloride in quantitative
yield.
Conditions studied for oxidation of alcohol 11 to aldehyde 12
OTf
OTf
OTf
N
OHC
HO
HO
oxidation conditions
or
N
N
N
N
N
22
15
16
Experiment
Oxidation conditions
Producta (yield)
1
2
3
4
5
6
7
Pyridinium chlorochromate (PCC)
Swern oxidation
16 (20%)
16 (<10%)
16 (<10%)
22 (52%)
16 (18%)
16 (95%)
16 (<10%)
Corey–Kim oxidation
MnO2
Dess–Martin periodinane
o-Iodoxybenzoic acid (IBX)
NMO/TPAPb
a
Isolated yield.
2.2. Synthesis of pyrrolo[2,3-b]pyridine core structure 17
b
Tetra-n-propylammonium perruthenate/4-methylmorpholine N-oxide.
The core structure methyl 1,6-dimethyl-4-hydroxy-2,3-dihy-
dro-1H-pyrrolo[2,3-b]pyridine-5-carboxylate (13) could be pre-
pared readily by an annulation reaction of 11 and 12 (Scheme 2).
The conversion of 13 to its triflate was accomplished easily using
PhNTf2 in the presence of potassium carbonate in dry DMF; triflate
14 was obtained in 94% yield. Direct reduction of the methyl ester
to the corresponding aldehyde by using 1 equiv of DIBAL-H was at-
tempted but found to be unsuccessful. Thus the methyl ester in 14
was reduced to the corresponding alcohol by the use of DIBAL-H at
low temperature in dichloromethane, providing alcohol 15 in 95%
yield.
The oxidation of alcohol 15 to aldehyde 16 required some opti-
mization. Several oxidization methods were studied for this pur-
pose (Table 1), including Swern oxidization36 and Corey–Kim
oxidization.37 While these methods are widely used for the prepa-
ration of aldehydes from primary alcohols, 16 was formed only in
low yield from alcohol 15. Dess–Martin periodinane is a mild and
effective reagent, but in this case provided only 18% of aldehyde
16 with >50% of an unidentified by-product. Pyridinium chloro-
chromate (PCC)38 provided ꢀ20% of desired product and the
TPAP/NMO (tetra-n-propylammonium perruthenate (VII)/4-meth-
ylmorpholine N-oxide) system35 gave multiple products. MnO2
oxidized the pyrrolidine moiety selectively to form 7-azaindole
derivative 22 in 52% yield. o-Iodoxybenzoic acid (IBX)39 was finally
found to be an excellent oxidant for the conversion of 15 ? 16 in
high yield.
The oxidation of aldehyde 16 to the corresponding formate es-
ter 17 was also challenging (Table 2). Peracids such as m-chloro-
perbenzoic acid or AcOOH did not effect the desired conversion.
Fortunately, the modified selenium catalyzed Baeyer–Villiger reac-
tion (Table 2, experiment 4) worked reasonably well.40
2.3. Preparation of acetate ester 3 and tocopherol analogue 2
With pyridinol ester 17 and side chains 9 and 10 in hand, the
coupling reaction of the side chains 9/10 and 17 was investigated
(Scheme 3). Direct coupling of side chain 9 with ester 17 in the
presence of Pd(PPh3)4, CuI, and i-Pr2NEt in DMF was successful,
but proceeded in low yield either at room temperature or at
80 °C (Table 3). Changing the ligand to tri-cyclohexylphosphine
or tri-t-butylphosphine resulted in little or no product. Likewise,
activating the side chain as a zinc derivative resulted in only trace
amounts of the desired product. In comparison, when the side
chain derivative 10 was employed in this coupling reaction using
Pd(PPh3)4 as catalyst and N-methylpyrrolidin-2-one as solvent,
the reaction proceeded smoothly at 105 °C for 2 h to give the de-
sired product 18 in 88% yield (Table 3). DIBAL-H reduction of the
formate ester and O-acetylation then provided 19 in 78% yield.
Treatment of 19 with Pearlman’s catalyst in methanol overnight
then gave compound 3 in 71% yield. Removal of the acetyl group
from pyridinol 3 was carried out at low temperature (ꢁ78 °C) to
(1) toluene, reflux, 2h
(2) NaOCH3,
90 oC, 16 h
OH
PhNTf2, K2CO3
DMF, 1.5 h
MeOOC
+
COOMe
NH2
MeO
MeO
MeOOC
N
94%
N
90%
N
13
12
11
OTf
OTf
IBX, DMSO/acetone (1:1)
25 oC, 16 h
DIBAL-H, CH2Cl2
-78 oC, 2 h
MeOOC
HO
95%
95%
N
N
N
N
14
15
OTf
OTf
SeO2, H2O2/CH2Cl2
25 oC, 24 h
O
OHC
O
N
N
N
N
57%
16
17
Scheme 2. Synthesis of pyrrolo[2,3-b]pyridine core structure 17.

J. Lu et al. / Bioorg. Med. Chem. 18 (2010) 7628–7638
7631
Table 2
2.4. Biochemical and biological evaluation of the tocopherol
analogue
Conditions studied for the Baeyer–Villiger reaction
OTf
OTf
N
OHC
OHCO
To investigate the ability of tocopherol analogue 2 to inhibit lipid
peroxidation, bovine mitochondria were incubated with cis-parina-
ric acid (Figure 3). This fatty acid is incorporated into the mitochon-
drial membrane and fluoresces within this lipid environment. The
conjugated double bond of the fluorophore is susceptible to
oxidation by oxygen radicals; consequently, the disappearance of
fluorescence provides a measure of lipid peroxidation. As shown
in Figure 3, compound 2 was found to be slightly more effective in
blocking the oxidation of cis-parinaric acid by peroxyl radicals than
Baeyer-Villiger
N
N
N
16
17
Experiment
Oxidation conditions
Yield of 17a
b
1
2
3
4
5
m-CPBA
—
AcOOH/AcOH
Na2CO3ꢂ1.5H2O2/TFAA
SeO2/H2O2
—
<10%
57%
20%
PhSeSePh/H2O2
was a-tocopherol (naturally occurring isomer).
a
Cellular ROS production was determined in CEM leukemia cells
by monitoring the fluorescence derived from the oxidant-sensitive
probe DCFH-DA. DCFH-DA is a non-polar compound that readily
diffuses into cells, where it is hydrolyzed by esterases to the
non-fluorescent polar derivative DCFH and thereby trapped within
the cell. In the presence of an appropriate oxidant, DCFH is oxi-
dized to the highly fluorescent DCF (dichlorofluorescein). In the
present case, cellular oxidative stress was induced by pharmaco-
logical depletion of glutathione (GSH) using the chemical diethyl
maleate (DEM).
Increased DCF fluorescence, a measure of intracellular ROS pro-
duction, was determined by a shift in DCF fluorescence (FL1-H
channel) to the right on the x-axis of the FACS histogram stained
with DCF (Figure 4, panel A). DEM treatment caused the DCF fluo-
rescence to shift right on the x-axis of the FACS histogram, indicat-
ing increased ROS production as a result of glutathione depletion.
Pretreatment of CEM cells with compound 2 was able to afford sig-
Isolated yield.
No desired product.
b
avoid decomposition. DIBAL-H reduction afforded 2 in quantitative
yield. The final product was found to be unstable, decomposing
completely in one day. This problem was resolved by forming the
trifluoroacetate salt of 2, which proved to be reasonably stable.
Compounds 2 and 4 were purified by reversed-phase HPLC prior
to bioassay, and were tested as their trifluoroacetate salts.
Another possible route for the preparation of 2 was also tried
(Scheme 4). In this route, the coupling reaction was carried out be-
fore the Baeyer–Villiger reaction. The coupling reaction was quite
successful using alkyne 9; the yield was almost quantitative. How-
ever, in the subsequent Baeyer–Villiger reaction, aldehyde 20 re-
acted with m-CPBA in dichloromethane to give only acid 21 as a
product in 49% yield (Scheme 4). Several different substrates were
also examined in the coupling reaction with alkyne 9 (Table 4).
When there was an electron withdrawing group such as methyl
carboxylate or aldehyde in the o-position, the coupling reaction
could be carried out in high yield. However, weaker electron with-
drawing groups hindered the reaction; using a formyloxy substitu-
ent, only a 22% yield of the product was achieved and no desired
product was obtained using an acetoxy substrate.
nificantly better protection than
a-tocopherol or compound 4
against the DEM-induced oxidative stress. Again, the fluorescence
microscopy analysis (Figure 4, panel B) shows that compound 2
provided the best protection against ROS in the glutathione-de-
pleted CEM cells. Also shown in panel C is the bright image (differ-
ential interference contrast, (DIC)) for the treated cells. Thus both
the pyridinol core and phytyl side chain of 2 are important to its
demonstrated ability to suppress cellular ROS levels.
OBn
1) DIBAL-H, THF, -78 oC, 2 h
OTf
N
Pd(PPh3)4, 10, NMP
105 oC, 2 h
2) Ac2O, NEt3, DMAP, 25 oC, 24 h
O
O
OHC
OHC
88%
78%
N
N
N
17
18
OBn
OH
H2, Pd(OH)2/C 20%
MeOH, 25 oC, 18 h
O
O
Ac
Ac
N
N
N
71%
N
3
19
OH
1) DIBAL-H, THF, -78 oC, 2 h
HO
2) TFA/Et2O, -78 o
C
100%
CF3COOH
N
N
2
Scheme 3. Preparation of acetate ester 3 and tocopherol analogue 2.

7632
J. Lu et al. / Bioorg. Med. Chem. 18 (2010) 7628–7638
Table 3
Conditions studied for coupling reaction with formate ester 17
OBn
OTf
side chain
Pd (cat.)
O
OHC
O
OHC
N
N
N
N
17
18
Experiment
Side chain
Catalyst and conditions
Yield of 18a
R
R
R
R
R
R
H
H
H
H
ZnCl
SnBu3
1
2
3
4
5
6
Pd(PPh3)4, CuI, i-Pr2NEt, DMF, 25 °C, 20 h
Pd(PPh3)4, CuI, i-Pr2NEt, DMF, 80 °C, 2 h
Pd2(OAc)2, PCy3,i-Pr2NEt, DMF, 25 °C, 20 h
Pd2(CH3CN)2Cl2, Pt-Bu3, CuI, i-Pr2NH, 25 °C, 24 h
Pd(PPh3)4, THF, 25 °C, 20 h
31%
22%
2%
b
—
Trace
88%
Pd(PPh3)4, NMP, 105 °C, 2 h
Me
Me
Me
R =
Me
OBn
a
Isolated yield.
No desired product.
b
OBn
OTf
9
Pd(PPh3)4, CuI, NEt3,
DMF, 80 oC, 2 h
OHC
m-CPBA, CH2Cl2, 0 oC, 3 h
49%
OHC
96%
N
N
N
N
16
20
OBn
HOOC
N
N
21
Scheme 4. Alternative route studied for the preparation of 2.
Tocopherol analogue 2 afforded significant protection of mito-
chondrial membranes against lipid peroxidation and suppressed
ROS levels in oxidatively stressed cultured CEM cells. More de-
tailed biochemical experiments comparing compounds 2 and 4
have reinforced the importance of the phytyl side chain of 4 in con-
ferring these protective effects.41 These findings parallel in a gen-
eral way those in earlier reports that utilized lipophilic
pyridinol42 and naphthyridinol analogues.43
Table 4
Sonogashira coupling using different substrates
OBn
OTf
N
Pd(PPh3)4,CuI, NEt3
9
R
, DMF, 80 oC, 3 h
R
N
N
N
3. Conclusions
Experiment
R
Producta (yield)
A novel antioxidant analogue of a-tocopherol (2) has been pre-
1
2
3
4
–COOMe
–CHO
–OCHO
–OAc
23 (95%)
20 (96%)
18 (22%)
24 (—b)
pared. The pyridinol core of this analogue was obtained via an
annulation reaction. Functional group transformation of a primary
alcohol to the aldehyde was carried out efficiently using IBX. The
Baeyer–Villiger reaction was found to be workable using H2O2 cat-
alyzed by SeO2. Stille coupling was the most efficient method for
a
Isolated yield.
No desired product.
b

J. Lu et al. / Bioorg. Med. Chem. 18 (2010) 7628–7638
7633
Figure 3. Time course of AAPH-induced oxidation of cis-parinaric acid incorporated into bovine heart mitochondrial membranes (1 mg/mL) detected by fluorescence decay
(cis-parinaric acid oxidation) in presence and absence of compound 2 or -tocopherol (1).
a
Figure 4. Effect of treatment with compound 2 on intracellular ROS accumulation induced by glutathione depletion with 5 mM diethyl maleate (DEM) in cultured CEM
leukemia cells. Following pretreatment of CEM cells with 5 M compound 2, -tocopherol or compound 4 for 1 h, the medium was changed, followed by treatment with
5 mM DEM for 30 min to deplete glutathione. Intracellular ROS accumulation was determined by measuring the DCF-derived fluorescence after incubation of the stressed
cells with 5 M DCFH-DA for an additional 20 min. Left panel (A) represents the flow cytometric histograms of ROS production in CEM cells. The green fluorescence (DCF) was
l
a
l
measured by flow cytometry using the FL1-H channel. The histogram panel shows a representative example of three independent experiments. In each analysis, 10,000
events were recorded. Increased DCF fluorescence, a measure of intracellular oxidation and ROS production, was determined by a shift in DCF fluorescence to the right on the
x-axis of the FACS histogram. Center and right panel images are the imaging conditions (green emission (DCF fluorescence) (B) and bright image (differential interference
contrast (DIC)) (C) with an exposure time of 250 ms showing fluorescence microscopy analysis of CEM cells upon treatment with DEM (5 mM) (highly fluorescent) and in the
presence or absence of 5 lM compound 2, a-tocopherol (1) or compound 4.
conjugation of the phytyl side chain. The final product was found
to be unstable, but could be stabilized as an acetate ester or as
its TFA salt. Tocopherol analogue 2 was found to quench the
AAPH-mediated peroxidation of mitochondrial membranes more
4. Experimental section
4.1. Chemistry
effectively than
in cultured cells depleted of glutathione.
a-tocopherol, and to strongly diminish ROS levels
All chemicals and solvent were of reagent grade and were used
without further purification, except as noted below. Anhydrous

7634
J. Lu et al. / Bioorg. Med. Chem. 18 (2010) 7628–7638
tetrahydrofuran (THF) was distilled from sodium/benzophenone
under dry argon. Dichloromethane (CH2Cl2) was distilled from cal-
cium hydride under argon. Thin layer chromatography (TLC) was
performed on Merck pre-scored Silica Gel 60 F-254 plates; visual-
ization employed 254 nm UV light, or staining with iodine, ceric
ammonium molybdate, or p-anisaldehyde. Silica gel (200–400
mesh, Silicycle) was used for flash chromatography. 1H NMR spec-
tra were recorded in chloroform-d, CD3OD, acetone-d6, DMSO-d6 or
D2O on Varian 400 or 500 MHz spectrometers. Chemical shifts
were reported in parts per million (ppm, d) referenced to the resid-
ual 1H resonance of the solvent (CDCl3, 7.26 ppm). 13C spectra were
referenced to the residual 13C resonance of the solvent (CDCl3,
77 ppm). Splitting patterns were designated as follows: s, singlet;
br, broad; d, doublet; dd, doublet of doublets; t, triplet; q, quartet;
m, multiplet. The low resolution mass spectrum was obtained on a
Voyager DE-STR, MALDI-TOF instrument at Arizona State Univer-
sity. The sample was prepared by mixing droplets of the sample
solution in chloroform with 2,5-dihydroxybenzoic acid and 4-
hydroxybenzylidenemalononitrile solution in acetonitrile, where
the latter served as the matrix. High resolution mass spectra were
obtained at the Ohio State University Mass Spectrometry Facility.
4.1.3. (2R,3S)-3-Methyl-3-((4R,8R)-4,8,12-trimethyltridecyl)-
oxiran-2-yl)methyl chloride (7)30
To a solution of 1.12 g (2.40 mmol) of tosylate 6 in 20 mL of
DMF was added 0.12 g (2.6 mmol) of lithium chloride. The reaction
mixture was stirred at 60 °C for 1 h. The cooled reaction mixture
was poured into 40 mL of water and extracted with three 30-mL
portions of EtOAc. The combined organic layer was washed with
satd aq NaHCO3 and brine, then dried (Na2SO4) and concentrated
under diminished pressure. The residue was purified by flash chro-
matography on a silica gel column (25 ꢃ 3.2 cm). Elution with
100:1 hexanes/ether gave 7 as a colorless oil: yield 0.72 g (91%);
½
a 2D5
ꢄ
ꢁ7.69 (c 0.77, CHCl3), lit.30
½
a 2D7
ꢄ
ꢁ9.47 (c 1.07, CHCl3); silica
gel TLC Rf 0.66 (5:1 hexanes/ether); 1H NMR (CDCl3) d 0.83–0.88
(m, 12H), 1.00–1.70 (m, 21H), 1.31 (s, 3H), 3.03 (t, 1H, J = 6.6 Hz),
3.45 (dd, 1H, J = 11.4, 7.2 Hz), and 3.70 (dd, 1H, J = 11.7, 6.0 Hz).
4.1.4. (3R,7R,11R)-3,7,11,15-Tetramethylhexadec-1-yn-3-ol (8)30
To a solution of 720 mg (2.20 mmol) of epoxy chloride 7 in
10 mL of dry THF was added 4.4 mL (11.0 mmol, 2.5 M in hexane)
of n-butyllithium at ꢁ20 °C under argon. After 20 min the reaction
was quenched by adding 10 mL of satd aq ammonium chloride and
allowed to warm to room temperature. The reaction mixture was
extracted with three 10-mL portions of ethyl acetate. The com-
bined organic layer was washed with brine and dried over MgSO4.
The solvent was removed under diminished pressure and the res-
idue was purified by flash chromatography on a silica gel column
(20 ꢃ 3.2 cm). Elution with 5:1 hexanes/ether gave 8 as a colorless
4.1.1. ((2R,3R)-3-Methyl-3-((4R,8R)-4,8,12-trimethyltridecyl)-
oxiran-2-yl)methanol (5)29,30
To a solution of 4.70 g (22.0 mmol) of (ꢁ)-diethyl
D-tartrate and
3.50 g of 4 Å molecular sieves in 300 mL of dichloromethane was
added 13.7 g (20.0 mmol) of Ti(i-PrO)4 and 5.5 mL (30 mmol) of
t-BuOOH (5.5 M in nonane), at ꢁ20 °C under argon. After 20 min
a solution of 5.93 g (20.0 mmol) of phytol in 80 mL of dichloro-
methane was added. The reaction mixture was stirred at ꢁ20 °C
for 3 h. The absence of starting material was confirmed by silica
gel TLC. The reaction was quenched by adding 250 mL of a 1 N
NaOH–brine mixture, then the mixture was allowed to warm to
room temperature. The mixture was filtered through Celite then
the mother liquor was separated into layers and the solid was
washed with two 400-mL portions of ethyl acetate. The combined
organic layer was dried (Na2SO4) and concentrated under dimin-
ished pressure. The residue was purified by flash chromatography
on a silica gel column (25 ꢃ 3.2 cm). Elution with 10:1 hexanes/
ethyl ether gave the product (R)-5 as colorless oil: yield 5.98 g
oil: yield 640 mg (100%); ½a D25
ꢄ
+1.88 (c 0.87 CHCl3), lit.30
½
a 2D7
+1.37
ꢄ
(c 0.73 CHCl3); silica gel TLC Rf 0.16 (1:1 hexanes/ether); 1H NMR
(CDCl3) d 0.82–0.87 (m, 12H), 0.99–1.72 (m, 21H), 1.49 (s, 3H),
2.12 (br s, 1H), and 2.42 (s, 1H).
4.1.5. 3-Benzoxy-(3R,7R,11R)-3,7,11,15-tetramethylhexadec-1-
yne (9)
To a solution of 71.8 mg (0.24 mmol) of 8 in 2 mL of dry DMF
was added 16.0 mg (0.40 mmol) of sodium hydride (60% mixture
in mineral oil). The reaction mixture was stirred at 0 °C for
20 min, then 68.0 mg (0.40 mmol) of benzyl bromide was added.
The reaction mixture was allowed to warm to room temperature
and stirred for 1 h. Two milliliters of satd aq ammonium chloride
solution and 2 mL of water were added to quench the reaction.
The reaction mixture was extracted with three 5-mL portions of
ethyl acetate. The combined organic layer was washed with brine
and dried (MgSO4) then the solvent was concentrated under
diminished pressure. The residue was purified by flash chromatog-
raphy on a silica gel column (25 ꢃ 3.2 cm). Elution with 100:1 hex-
(96%); ½a 2D5
ꢄ
+4.50 (c 0.20, EtOH), lit.30
½
a 2D6
+4.88 (c 2.80, EtOH),
ꢄ
lit.44
½
a 2D0
+4.39 (c 2.78, EtOH); silica gel TLC Rf 0.26 (1:1 ether/hex-
ꢄ
anes); 1H NMR (CDCl3) d 0.83–0.87 (m, 12H), 1.00–1.65 (m, 21H),
1.21 (s, 3H), 2.27 (br s, 1H), 2.97 (dd, 1H, J = 4.8, 3 Hz), 3.68 (dd,
1H, J = 8.1, 5.4 Hz) and 3.83 (br d, 1H, J = 8.7 Hz); 13C NMR (CDCl3)
d 17.0, 19.9, 20.0, 22.8, 22.9, 23.0, 24.7, 25.1, 26.1, 28.2, 37.2, 37.5,
37.6, 37.7, 39.1, 39.6, 61.65, 61.7, 63.3, and 80.9.
anes/ether gave 9 as a colorless oil: yield 75 mg (80%); ½a D25
ꢄ
+3.08 (c
1.32, CHCl3); silica gel TLC Rf 0.95 (20:1 hexanes/ether); 1H NMR
(CDCl3) d 0.83–0.88 (m, 12H), 1.02–1.81 (m, 19H), 1.50 (s, 3H),
4.1.2. (2R,3R)-3-Methyl-3-((4R,8R)-4,8,12-trimethyltridecyl)-
oxiran-2-ylmethyl p-toluenesulfonate (6)30
2.48 (s, 3H), 4.63 (ABq, 2H,
DmAB = 25.2 Hz, JAB = 11.2 Hz), 7.24–
To a solution of 0.93 g (3.00 mmol) of epoxy alcohol 5 in 20 mL
of dichloromethane was added 0.75 g (7.43 mmol) of triethylamine
and a catalytic amount of N,N-dimethylaminopyridine. Then 0.68 g
(3.58 mmol) of p-toluenesulfonyl chloride was added portionwise
to the reaction mixture. The reaction mixture was stirred at room
temperature overnight, then quenched by the addition of 30 mL of
water. The organic layer was washed with brine and dried over
Na2SO4. The solvent was concentrated under diminished pressure
and the residue was purified by flash chromatography on a silica
gel column (25 ꢃ 3.2 cm). Elution with 100:1 ? 10:1 hexanes/
7.27 (m, 1H) and 7.30–7.37 (m, 4H); 13C NMR (CDCl3) d 19.7,
19.8, 21.7, 22.65, 22.7, 24.5, 24.8, 26.3, 26.4, 28.0, 32.7, 32.8,
37.1, 37.3, 37.4, 39.4, 41.8, 66.2, 73.7, 85.5, 127.3, 127.6, 128.3,
and 139.1; mass spectrum (ESI), m/z 385.3471 (M+H)+ (C27H45O re-
quires m/z 385.3470).
4.1.6. 3-Benzoxy-((3R,7R,11R)-3,7,11,15-tetramethylhexadec-1-
ynyl)tri-n-butylstannane (10)
To a solution of 424 mg (1.40 mmol) of alkyne 9 in 6 mL of dry
THF was added 1.12 mL (2.8 mmol, 2.5 M in hexane) of n-butyllith-
ium at ꢁ78 °C. The reaction mixture was allowed to warm to 0 °C
and stirred for 20 min, then 470 mg (1.40 mmol) of n-Bu3SnCl was
added. The reaction mixture was allowed to warm to room tem-
perature and stirred for 4 h. The reaction mixture was poured into
10 mL of satd aq NH4Cl, then extracted with three 10-mL portions
of ethyl ether. The combined organic layer was washed with brine,
ethyl ether gave 6 as colorless oil: yield 1.12 g (80%); ½a D25
ꢄ
+22.6
(c 0.97, CHCl3), lit.30
½
a 2D7
+15.92 (c 1.06, CHCl3); silica gel TLC Rf
ꢄ
0.20 (5:1 hexanes/ether); 1H NMR (CDCl3) d 0.82–0.86 (m, 12H),
0.99–1.92 (m, 21H), 1.19 (s, 3H), 2.44 (s, 3H), 2.96 (t, 1H,
J = 6.0 Hz), 4.09 (dd, 1H, J = 11.0, 6.0 Hz), 4.15 (dd, 1H, J = 11.0,
5.5 Hz), 7.34 (d, 2H, J = 8.0 Hz), and 7.80 (d, 2H, J = 6.8 Hz).

J. Lu et al. / Bioorg. Med. Chem. 18 (2010) 7628–7638
7635
dried (Na2SO4) and concentrated under diminished pressure. The
residue was purified by flash chromatography on a silica gel col-
umn (25 ꢃ 3.2 cm). Elution with hexanes gave 10 as a colorless
101.1, 102.2, 162.2, 163.8, 165.6 and 172.4; mass spectrum (ESI),
m/z 223.1082 (M+H)+ (C11H15N2O3 requires m/z 223.1083).
oil: yield 768 mg (100%); ½a D25
ꢄ
+2.25 (c 0.80, CHCl3); silica gel TLC
4.1.10. Methyl 1,6-dimethyl-4-(trifluoromethylsulfoxy)-2,3-di-
hydro-1H-pyrrolo[2,3-b]pyridine-5-carboxylate (14)
Rf 0.96 (20:1 hexanes/ether); 1H NMR (CDCl3) d 0.78–0.92 (m,
39H), 0.96–1.84 (24H), 4.64 (ABq, 2H,
D
m
AB = 25.2 Hz, JAB = 12.0 Hz)
To a solution of 74 mg (0.33 mmol) of methyl 1,6-dimethyl-4-
hydroxy-2,3-dihydro-1H-pyrrolo[2,3-b]pyridine-5-carboxylate
(13) and 150 mg of potassium carbonate in 2.5 mL of dry DMF was
added 180 mg (0.50 mmol) of N-phenyl-bis-trifluoromethanesulf-
onimide at room temperature. The reaction mixture was stirred
at room temperature for 1.5 h then quenched by adding 5 mL of
satd aq ammonium chloride. The reaction mixture was extracted
with three 5-mL portions of ethyl acetate. The combined organic
layer was washed with brine and dried (MgSO4). The solvent was
concentrated under diminished pressure and the residue was puri-
fied by flash chromatography on a silica gel column (20 ꢃ 2 cm).
Elution with 40:1 hexane/ethyl acetate gave 14 as a colorless solid:
yield 110 mg (94%); mp 56–58 °C; silica gel TLC Rf 0.30 (1:1 hex-
anes/ethyl acetate); 1H NMR (CDCl3) d 2.54 (s, 3H), 3.01 (s, 3H),
3.11 (t, 2H, J = 8.5 Hz), 3.62 (t, 2H, J = 8.0 Hz), and 3.85 (s, 3H);
13C NMR (CDCl3) d 23.5, 24.4, 31.8, 51.3, 52.0, 108.5, 110.0, 111.9,
149.3, 161.8, 164.9, and 165.6; mass spectrum (ESI), m/z
355.0566 (M+H)+ (C12H14N2O5SF3 requires m/z 355.0576).
and 7.24–7.36 (m, 5H); 13C NMR (CDCl3) d 8.8, 11.2, 13.75, 13.8,
19.8, 22.2, 22.7, 22.8, 24.7, 24.9, 27.0, 27.5, 28.1, 29.0, 29.4, 29.9,
32.9, 37.6, 39.5, 66.2, 73.3, 74.6, 87.4, 127.1, 127.7, 128.2, 128.4,
and 139.8; mass spectrum (ESI), m/z 675.4536 (M+H)+ (C39H71OSn
requires m/z 675.4527).
4.1.7. 2,2-Dimethoxy-1-methylpyrrolidine (11)31
A mixture of 52.0 mL (546 mmol) of N-methyl-2-pyrrolidinone
and 52.0 mL (548 mmol) of dimethyl sulfate was stirred and
heated at 90 °C for 1.5 h, then allowed to cool to room temperature.
A solution containing 130 mL of 25% methanolic sodium methox-
ide and 370 mL of methanol was added at ꢁ10 °C under Ar over
a period of 1 h. The precipitated solid was filtered and the solvent
was concentrated under diminished pressure. The residue was dis-
solved in 500 mL of ether and stirred for 1 h, then the precipitated
solid was filtered. The solid was washed with two 50-mL portions
of ether. After the ether was concentrated, the residue was distilled
in vacuo to give 11 as a yellow liquid: yield 31.0 g (40%); 1H NMR
(CDCl3) d 1.62–1.74 (m, 2H), 1.83 (t, 2H, J = 7.8 Hz), 2.28 (s, 3H),
2.78 (t, 2H, J = 6.6 Hz), and 3.15 (s, 6H).
4.1.11. 1,6-Dimethyl-5-(hydroxymethyl)-2,3-dihydro-1H-pyr-
rolo[2,3-b]pyridin-4-yl trifluoromethanesulfonate (15)
To a solution of 170 mg (0.48 mmol) of methyl ester 14 in 2 mL
of dichloromethane was added dropwise 1.43 mL (1.43 mmol, 1 M
in toluene) of DIBAL-H at ꢁ78 °C under Ar. The reaction mixture
was stirred at ꢁ78 °C for 2 h then 5 mL of satd aq sodium potas-
sium tartrate was added by syringe. The reaction mixture was al-
lowed to warm to room temperature and stirred for an
additional 2 h. After separation, the aqueous layer was extracted
with three 5-mL portions of dichloromethane. The combined or-
ganic layer was washed with brine and dried over MgSO4. The sol-
vent was concentrated under diminished pressure and the residue
was purified by flash chromatography on a silica gel column
(20 ꢃ 2 cm). Elution with 2:1 hexanes/ethyl acetate gave 15 as a
colorless solid: yield 148 mg (95%); mp 67–70 °C (decomp.); silica
gel TLC Rf 0.20 (1:1 hexanes/ethyl acetate); 1H NMR (CDCl3) d 1.72
(br s, 1H), 2.52 (s, 3H), 2.96 (s, 3H), 3.08 (t, 2H, J = 8.4 Hz), 3.54 (t,
2H, J = 8.4 Hz), and 4.58 (s, 2H); 13C NMR (CDCl3) d 22.0, 24.0, 32.3,
51.8, 76.7, 111.5, 114.1, 120.0, 149.8, 159.9, and 164.7; mass spec-
trum (ESI), m/z 327.0622 (M+H)+ (C11H14N2O4SF3 requires m/z
327.0626).
4.1.8. 2-(1-Aminoethylidene)malonic acid dimethyl ester (12)32
To a solution of 1.32 g (10.0 mmol) of dimethyl malonate in
20 mL of 1,2-dichloromethane was added 0.41 g (10.0 mmol) of
acetonitrile and 20.0 mL (20.0 mmol, 1 M in dichloromethane) of
tin chloride under Ar at room temperature. The reaction mixture
was heated at reflux and stirred for 3 h. The solvent was concen-
trated under diminished pressure. The residue was dissolved in
20 mL of acetone and treated slowly with satd aq sodium carbon-
ate. After filtration, the solid was washed with ethyl acetate and
the mother liquor was extracted with three 20-mL portions of
ethyl acetate. The combined organic layer was washed with brine
and dried (MgSO4). The solvent was concentrated under dimin-
ished pressure, and the residue was purified by flash chromatogra-
phy on a silica gel column (20 ꢃ 2 cm). Elution with 10:1 hexanes/
ethyl acetate gave 12 as a colorless solid: yield 0.66 g (38%); mp
82–83 °C, lit.32 mp 83–84 °C; silica gel TLC Rf 0.30 (1:1 hexanes/
ethyl acetate); 1H NMR (CDCl3) d 2.15 (s, 3H), 3.71 (s, 3H), 3.75
(s, 3H), 5.14 (br s, 1H), and 9.01 (br s, 1H); 13C NMR (CDCl3) d
22.4, 51.3, 51.9, 92.8, 164.1, 169.0, and 169.2.
4.1.12. 1,6-Dimethyl-5-formyl-2,3-dihydro-1H-pyrrolo[2,3-b]-
pyridin-4-yl trifluoromethanesulfonate (16)
4.1.9. Methyl 1,6-dimethyl-4-hydroxy-2,3-dihydro-1H-pyrrolo-
[2,3-b]pyridine-5-carboxylate (13)
To a solution of 134 mg (0.41 mmol) of alcohol 15 in 4 mL of
DMSO and 4 mL of acetone was added 116 mg (0.41 mmol) of o-
iodoxybenzoic acid (IBX). The reaction mixture was stirred at room
temperature for 16 h, then cooled to 0 °C. Five milliliters of a 1:1
mixture of 5% aq NaHCO3 and 5% aq Na2S2O3 was added slowly
by syringe. After stirring for an additional 20 min, the mixture
was extracted with three 5-mL portions of ethyl acetate. The com-
bined organic layer was washed with brine and dried over MgSO4.
The solvent was concentrated under diminished pressure, and the
residue was purified by flash chromatography on a silica gel col-
umn (20 ꢃ 2 cm). Elution with 10:1 hexanes/ethyl acetate gave
16 as colorless needles: yield 126 mg (95%); mp 96–98 °C (de-
comp.); silica gel TLC Rf 0.20 (4:1 hexanes/ethyl acetate); 1H
NMR (CDCl3) d 2.70 (s, 3H), 3.09 (s, 3H), 3.18 (t, 2H, J = 8.5 Hz),
3.72 (t, 2H, J = 8.5 Hz) and 10.09 (s, 1H); 13C NMR (CDCl3) d 23.2,
24.1, 31.5, 51.2, 112.3, 112.4, 120.0, 151.2, 165.5, 166.5, and
185.2; 19F NMR (CDCl3) d ꢁ73.5 (s, 3F); mass spectrum (ESI), m/z
325.0465 (M+H)+ (C11H12N2O4SF3 requires m/z 325.0470).
A solution containing 100 mg (0.57 mmol) of 2-(1-aminoethy-
lidene)malonic acid dimethyl ester (12) and 168 mg (1.20 mmol)
of 2,2-dimethoxy-1-methylpyrrolidine (11) in 2 mL of toluene
was heated at reflux for 3 h, then cooled to 90 °C and treated with
160 mg (1.67 mmol) of sodium methoxide in one portion. The
resulting reaction mixture was heated at 90 °C and stirred over-
night. The cooled solution was treated with 5 mL of satd aq ammo-
nium chloride and was then extracted with three 10-mL portions
of ethyl acetate. The combined organic layer was washed with
brine and dried (MgSO4). The solvent was concentrated under
diminished pressure and the residue was purified by flash chroma-
tography on
a
silica gel column (20 ꢃ 2 cm). Elution with
20:1 ? 10:1 hexane/ethyl acetate gave 13 as a colorless solid: yield
115 mg (90%); mp 131–132 °C; silica gel TLC Rf 0.46 (4:1 hexanes/
ethyl acetate); 1H NMR (CDCl3) d 2.62 (s, 3H), 2.93 (t, 2H,
J = 8.4 Hz), 2.98 (s, 3H), 3.55 (t, 2H, J = 9.0 Hz), 3.90 (s, 3H) and
11.79 (s, 1H); 13C NMR (CDCl3) d 22.5, 27.8, 31.9, 52.0, 52.4,

7636
J. Lu et al. / Bioorg. Med. Chem. 18 (2010) 7628–7638
4.1.13. 1,6-Dimethyl-4-(trifluoromethylsulfoxy)-2,3-dihydro-
1H-pyrrolo[2,3-b]pyridin-5-yl formate (17)
20.5, 22.0, 22.65, 22.7, 24.5, 24.8, 25.5, 26.5, 28.0, 32.79, 32.83,
32.9, 37.2, 37.3, 37.5, 39.4, 42.0, 52.5, 66.5, 74.4, 78.0, 100.1,
120.9, 124.3, 127.4, 127.6, 128.3, 136.1, 139.0, 146.4, and 169.2;
mass spectrum (ESI), m/z 589.4383 (M+H)+ (C38H57N2O3 requires
m/z 589.4369).
To a solution of 80 mg (0.25 mmol) of aldehyde 16 in 3 mL of
dichloromethane was added 1 mg of SeO2 and 1.5 mL of aq 30%
H2O2. The reaction mixture was stirred at room temperature for
16 h then cooled to 0 °C. Twelve milliliters of 5% aq NaHCO3 was
added slowly. The mixture was stirred for an additional 20 min
then extracted with three 15-mL portions of ethyl acetate. The
combined organic layer was washed with brine and dried over
Na2SO4. The solvent was concentrated under diminished pressure,
and the residue was purified by flash chromatography on a silica
gel column (20 ꢃ 2 cm). Elution with 10:1 hexanes/ethyl acetate
afforded 17 as a light yellow solid: yield 47.6 mg (57%); mp 59–
61 °C; silica gel TLC Rf 0.28 (4:1 hexanes/ethyl acetate); 1H NMR
(CDCl3) d 2.25 (s, 3H), 2.94 (s, 3H), 3.08 (t, 2H, J = 8.4 Hz), 3.57 (t,
2H, J = 8.4 Hz), and 8.23 (s, 1H); 13C NMR (CDCl3) d 19.2, 23.7,
32.4, 52.1, 113.4, 116.8, 120.0, 126.7, 150.2, 158.2, and 162.8; 19F
NMR (CDCl3) d ꢁ73.8 (s, 3F); mass spectrum (ESI), m/z 340.0413
(M)+ (C11H11N2O5SF3 requires m/z 340.0419).
4.1.16. 1,6-Dimethyl-4-((3R,7R,11R)-3-hydroxy-3,7,11,15-tetra-
methylhexadecyl)-2,3-dihydro-1H-pyrrolo[2,3-b]pyridin-5-yl
acetate (3)
To a solution of 25 mg (0.04 mmol) of acetate ester 19 in 5 mL of
methanol was added 13 mg of Pearlman’s catalyst. Hydrogen was
bubbled through the stirred reaction mixture for 2 h, then the reac-
tion mixture was maintained under a hydrogen atmosphere over-
night. The reaction mixture was filtered through a Celite pad and
the pad was washed with methanol. The solvent was concentrated
under diminished pressure and the residue was purified by flash
chromatography on a silica gel column (20 ꢃ 1.7 cm). Elution with
5:1 hexanes/ethyl acetate gave 3 as a colorless oil: yield 15.7 mg
(71%); ½a 2D2
ꢄ
+17.5 (c 0.80, CH3OH); silica gel TLC Rf 0.10 (4:1 hex-
ane/ethyl acetate); 1H NMR (CDCl3) d 0.83–0.87 (m, 12H), 1.00–
1.56 (m, 23H), 1.20 (s, 3H), 2.20 (s, 3H), 2.30 (s, 3H), 2.38 (t, 2H,
J = 7.2 Hz), 2.87 (t, 2H, J = 8.4 Hz), 2.89 (s, 3H) and 3.44 (t, 2H,
J = 8.0 Hz); 13C NMR (CDCl3) d 19.64, 19.67, 20.5, 21.4, 22.3, 22.6,
22.7, 24.3, 24.4, 24.7, 26.57, 26.60, 27.9, 29.6, 32.7, 33.2, 37.2,
37.36, 37.39, 37.5, 39.3, 40.1, 42.1, 52.8, 72.4, 118.9, 128.5, 136.3,
146.3, and 169.9; mass spectrum (ESI), m/z 503.4213 (M+H)+
(C31H55N2O3 requires m/z 503.4207).
4.1.14. 4-((3R,7R,11R)-3-Benzoxy-3,7,11,15-tetramethylhexadec-
1-ynyl)-1,6-dimethyl-2,3-dihydro-1H-pyrrolo[2,3-b]pyridin-5-yl
formate (18)
To a solution of 28.0 mg (0.08 mmol) of 17 in 2 mL of N-meth-
ylpyrrolidin-2-one (NMP) was added 9.1 mg (0.008 mmol) of
Pd(PPh3)4 and 66.0 mg (0.10 mmol) of 10. The reaction mixture
was heated at 105 °C and stirred for 2 h then cooled to 0 °C. Five
milliliters of 5% aq NaHCO3 was added slowly. The mixture was
stirred at 0 °C for additional 20 min, and then extracted with three
8-mL portions of ethyl acetate. The combined organic layer was
washed with brine and dried over Na2SO4. The solvent was concen-
trated under diminished pressure and the residue was purified by
flash chromatography on a silica gel column (20 ꢃ 1.7 cm). Elution
with 10:1 hexanes/ethyl acetate gave 18 as a colorless oil: yield
4.1.17. ((3R,7R,11R)-3-Hydroxy-3,7,11,15-tetramethylhex-
adecyl)-1,6-dimethyl-2,3-dihydro-1H-pyrrolo[2,3-b]pyridin-5-
ol (2) and its trifluoroacetic acid salt
To a solution of 10.3 mg (20.5
lmol) of acetyl ester 3 in 2 mL of
CH2Cl2 was added 84.0 L (84.0
l
lmol, 1 M in toluene) of DIBAL-H
at ꢁ78 °C. The reaction mixture was stirred at same temperature
for 2 h then 2 mL of aq satd sodium potassium tartrate was added
slowly. The reaction mixture was allowed to slowly warm to room
temperature and stirred for 3 h, then the layers were separated.
The aqueous layer was extracted with two 5-mL portions of ethyl
acetate. The combined organic layer was washed with brine, dried
(Na2SO4) and concentrated under diminished pressure to give 2 as
an orange oil: yield 9.44 mg (100%). This product was analyzed by
C8 reversed-phase HPLC column (150 mm ꢃ 4.6 mm) using a gradi-
ent of methanol and water. Linear gradients were employed using
42 mg (88%); ½a 2D5
ꢄ
+21.1 (c 0.38, CH3OH); silica gel TLC Rf 0.38
(4:1 hexanes/ethyl acetate); 1H NMR (CDCl3) d 0.84–0.89 (m,
12H), 1.05–1.83 (m, 21H), 1.56 (s, 3H), 2.47 (s, 3H), 2.92 (s, 3H),
2.96 (t, 2H, J = 6.4 Hz), 3.50 (t, 2H, J = 6.4 Hz), 4.65 (ABq, 2H,
DmAB = 24.3 Hz, JAB = 11.5 Hz), 7.27–7.39 (m, 5H), and 8.16 (s,
1H); 13C NMR (CDCl3) d 19.1, 19.7, 19.7, 21.9, 22.6, 22.7, 24.5,
24.8, 25.5, 26.3, 28.0, 32.75, 32.77, 32.8, 37.1, 37.3, 37.5, 39.4,
41.9, 52.3, 66.5, 74.5, 101.0, 105.0, 106.4, 124.4, 127.4, 127.5,
128.3, 130.2, 135.2, 139.0, 146.4, and 159.2; mass spectrum (ESI),
m/z 575.4244 (M+H)+ (C37H55N2O3 requires m/z 575.4213).
1:4 methanol/water ? 4:1 methanol/water over
a period of
20 min, and then 4:1 methanol/water ? methanol over a period
of 40 min, both at a flow rate of 1.5 mL/min (monitoring at
260 nm); compound 2 eluted at 21.8 min, and was judged to be
>90% pure; mass spectrum (MALDI-TOF), m/z 461.4 (M+H)+ (theo-
retical m/z 461.4). This compound decomposed after storage for
one day at 25 °C.
4.1.15. 4-((3R,7R,11R)-3-Benzoxy-3,7,11,15-tetramethylhexa-
dec-l-ynyl)-2,3-dihydro-1,6-dimethyl-1H-pyrrolo[2,3-b]pyridin-
5-yl acetate (19)
To a solution of 25.0 mg (0.04 mmol) of formate ester 18 in 2 mL
of CH2Cl2 was added 160
H at ꢁ78 °C. The reaction mixture was stirred at ꢁ78 °C for 1.5 h,
then 300 L of triethylamine, 150 L of acetic anhydride and a cat-
lL (0.16 mmol, 1 M in toluene) of DIBAL-
Freshly prepared compound 2 was dissolved in 1 mL of ether
and cooled to ꢁ78 °C. To this solution was added 0.3 mL of triflu-
oroacetic acid (0.1 M in ether solution). The mixture was stirred
at ꢁ78 °C for 1 h, then the solvent was concentrated under dimin-
ished pressure. The residue was transferred in portions to small
vials as a CH3CN solution, cooled to ꢁ78 °C and lyophilized to
l
l
alytic amount of N,N-dimethylaminopyridine were added. The
reaction mixture was allowed to warm to room temperature and
stirred for 24 h. The reaction was quenched with 2 mL of satd aq
NH4Cl and extracted with three 5-mL portions of ethyl acetate.
The combined organic layer was washed with brine, dried (Na2SO4)
and concentrated under diminished pressure. The residue was
give 2 as a trifluoroacetic acid salt: yield 11.5 mg (97%); ½a D25
ꢄ
ꢁ0.86 (c 0.14, CH3CN); 1H NMR (CD3CN) d 0.89–0.92 (m, 12H),
1.03–1.53 (m, 19H), 1.20 (s, 3H), 1.53–1.61 (m, 1H), 1.67 (t, 2H,
J = 8.0 Hz), 2.37 (s, 3H), 2.64 (t, 2H, J = 7.5 Hz), 3.09 (t, 2H,
J = 8.5 Hz), 3.09 (s, 3H), 3.79 (t, 2H, J = 8.0 Hz) and 12.35 (br s,
1H); 13C NMR (CD3CN) d 19.1, 21.2, 21.9, 21.9, 22.0, 24.1, 24.2,
24.5, 25.9, 27.8, 32.4, 32.5, 32.5, 37.0, 37.0, 37.1, 37.1, 37.3,
38.5, 39.1, 42.0, 53.2, 72.3, 125.8, 127.7, 140.4, 145.9, 152.1,
162.5, and 165.0; mass spectrum (ESI), m/z 461.4106 (M+H)+
(C29H53N2O2 requires m/z 461.4107).
purified by flash chromatography on
a silica gel column
(20 ꢃ 1.7 cm). Elution with 50:3 hexanes/ethyl acetate gave 19 as
a yellowish oil: yield 20 mg (78%); ½a D25
ꢄ
+11.2 (c 0.20, CH3OH); sil-
ica gel TLC Rf 0.48 (4:1 hexane/ethyl acetate); 1H NMR (CDCl3) d
0.83–0.88 (m, 12H), 1.02–1.99 (m, 21H), 1.55 (s, 3H), 2.21 (s, 3H),
2.22 (s, 3H), 2.89 (s, 3H, J = 7.6 Hz), 2.94 (t, 2H, J = 8.0 Hz), 3.46 (t,
2H, J = 8.0 Hz), 4.76 (ABq, 2H,
DmAB = 21.2 Hz, JAB = 10.4 Hz) and
7.24–7.36 (m, 5H); 13C NMR (CDCl3) d 19.0, 19.70, 19.74, 19.8,

J. Lu et al. / Bioorg. Med. Chem. 18 (2010) 7628–7638
7637
4.1.18. 4-((3R,7R,11R)-3-Benzoxy-3,7,11,15-tetramethylhexa-
dec-1-ynyl)-1,6-dimethyl-2,3-dihydro-1H-pyrrolo[2,3-
b]pyridin-5-carbaldehyde (20)
4.2.2. Reactive oxygen species (ROS) assay
Intracellular ROS production was measured using the oxidant-
sensitive fluorescent probe 20,70-dichlorodihydrofluorescein diace-
tate (DCFH-DA) (Molecular Probes, Eugene, OR). One milliliter
(1 ꢃ 106 cells/mL) of CEM leukemia cells were plated in 12-well
plates (RPMI with 10% fetal bovine serum (FBS), 2 mM glutamine
and 1% penicillin–streptomycin mix antibiotics supplements), trea-
To a solution of 74 mg (0.23 mmol) of aldehyde 16 in 2 mL of
DMF was added 26 mg (0.023 mmol) of Pd(PPh3)4, 4.3 mg (0.23
mmol) of CuI, 0.6 mL of i-Pr2NEt and 136 mg (0.35 mmol) of 9.
The reaction mixture was stirred at 80 °C overnight. The cooled
reaction mixture was treated with 5 mL of 1 N NaOH in satd aq
NaCl. The mixture was extracted with three 8-mL portions of ether.
The combined organic layer was washed with brine and dried over
MgSO4. The solvent was concentrated under diminished pressure
and the residue was purified by flash chromatography on a silica
gel column (20 ꢃ 2 cm). Elution with 20:1 hexanes/ethyl acetate
ted with the tested compounds (final concentration 5 lM), and
incubated at 37 °C for 1 h in a humidified atmosphere of 5% CO2
in air. Cells were treated with 5 mM diethyl maleate for 30 min
and collected by centrifugation at 300g for 3 min, then washed
twice with phosphate-buffered saline (PBS). The cells were resus-
pended in Hanks’ Balanced Salt Solution buffer and incubated at
afforded 20 as a reddish oil: yield 122 mg (96%); ½a D22
ꢄ
+28.0 (c
37 °C in the dark for 20 min with 5 lM DCFH-DA. The cells were
0.05, CH3OH); silica gel TLC Rf 0.55 (4:1 hexanes/ethyl acetate);
1H NMR (CDCl3) d 0.83–0.88 (m, 12H), 1.04–1.57 (m, 19H), 1.60
(s, 3H), 1.74–1.87 (m, 2H), 2.70 (s, 3H), 3.01 (t, 2H, J = 8.4 Hz),
3.04 (s, 3H), 3.62 (t, 2H, J = 8.4 Hz), 4.70 (q, 2H, J = 11.2 Hz), 7.25–
7.38 (m, 5H), and 10.34 (s, 1H); 13C NMR (CDCl3) d 19.66, 19.69,
19.7, 22.6, 22.7, 24.5, 24.79, 24.87, 24.93, 26.4, 28.0, 31.4, 32.7,
32.8, 37.1, 37.3, 37.4, 37.5, 39.4, 41.9, 51.2, 66.6, 74.5, 79.1,
103.0, 117.7, 123.8, 127.4, 128.3, 128.4, 139.0, 163.04, 163.07,
and 189.8; mass spectrum (ESI), m/z 559.4259 (M+H)+
(C37H55N2O2 requires m/z 559.4264).
collected by centrifugation at 300g for 3 min and then washed
twice with phosphate-buffered saline. The samples were analyzed
immediately by flow cytometry (FACS Caliber instrument, Becton–
Dickinson; Cell Quest software, BD Biosciences) using 488 nm exci-
tation laser and FL1-H channel 538 nm emission filter. In each
analysis, 10,000 events were recorded. The results obtained were
verified by repeating the experiments in triplicate. For fluorescence
microscopy analysis, the cells were treated as described above and
transferred to culture dishes (glass bottom SensoPlate™, 24 well,
Greiner Bio-One) and analyzed with a Zeiss Axiovert 200 M in-
verted microscope, equipped with a 40ꢃ oil immersion objective
and AxioCam MR. The generation of reactive oxygen species was
detected as a result of the oxidation of DCFH (kex 488 nm; kem
515–540 nm).
4.1.19. 4-((3R,7R,11R)-3-Benzoxy-3,7,11,15-tetramethylhexa-
dec-1-ynyl)-1,6-dimethyl-2,3-dihydro-1H-pyrrolo[2,3-
b]pyridin-5-carboxylic acid (21)
To a solution of 20 mg (0.036 mmol) of aldehyde 20 in 2 mL of
dichloromethane was added 8 mg (77% purity; 0.036 mmol) of m-
chloroperbenzoic acid and a catalytic amount of p-TsOHꢂH2O. The
reaction mixture was stirred at room temperature overnight. The
cooled (0 °C) reaction mixture was treated slowly with 5 mL of a
1:1 mixture of 5% aq NaHCO3 and 5% aq Na2S2O3. After stirring for
an additional 20 min, the mixture was extracted with three 5-mL
portions of dichloromethane. The combined organic layer was
washed with brine and dried over Na2SO4. The solvent was concen-
trated under diminished pressure to afford a residue, which was
purified by preparative silica gel TLC. Development with 4:1 hex-
anes/ethyl acetate gave 21 as a colorless oil: yield 10 mg (49%);
Acknowledgments
We thank Dr. Damien Duveau, ASU, for assistance with the
interpretation of several NMR spectra. This work was supported
in part by a grant from the Friedreich’s Ataxia Research Alliance
(FARA).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.08.030. These data
include MOL files and InChiKeys of the most important compounds
described in this article.
½
a 2D5
ꢄ
ꢁ30.0 (c 0.04, CHCl3); silica gel TLC Rf 0.47 (4:1 hexanes/ethyl
acetate); 1H NMR (CDCl3) d 0.83–0.88 (m, 12H), 1.03–1.61 (m,
19H), 1.64 (s, 3H), 1.79–1.98 (m, 2H), 2.73 (s, 3H), 2.80–3.20 (m,
4H), 3.00 (s, 3H), 4.70 (q, 2H, J = 6.8 Hz), 7.25–7.35 (m, 5H), and
10.55 (s, 1H); 13C NMR (CDCl3) d 19.62, 19.65, 19.7, 21.9, 22.6, 22.7,
24.1, 24.8, 25.2, 26.3, 28.0, 32.7, 32.8, 37.3, 37.5, 39.4, 41.9, 46.9,
54.1, 66.7, 74.6, 78.0, 108.1, 114.8, 124.0, 127.4, 127.5, 128.4,
137.4, 138.6, 158.7, 163.5, 190.90, and 190.94; mass spectrum
(ESI), m/z 575.4210 (M+H)+ (C37H55N2O3 requires m/z 575.4213).
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