Synthesis, stereochemistry confirmation and biological activity evaluation of a constituent from Isodon excisus

Article abstract of DOI:10.1016/j.tet.2003.10.025

A synthesis and stereochemistry confirmation of a constituent recently isolated from the whole plant Isodon excisus is reported. An enantioselective catalytic boron-mediated reduction of an α-bromoketone was utilized in the key synthetic transformation. The methodology described herein was also used for the synthesis of the natural product's enantiomer and several derivatives. In addition, the compounds were evaluated for inhibitory activity in a caspase induction assay. The natural product was found to be devoid of activity, but several derivatives had moderate inhibitory activity (EC ₅₀<1 μM).

Full text of DOI:10.1016/j.tet.2003.10.025

Tetrahedron 59 (2003) 9961–9969
Synthesis, stereochemistry confirmation and biological activity
evaluation of a constituent from Isodon excisus
*
Xuechao Xing, Pei Ho, Geoffroy Bourquin, Li-An Yeh and Gregory D. Cuny
Laboratory for Drug Discovery in Neurodegeneration, Brigham and Women’s Hospital and Harvard Medical School,
65 Landsdowne St., Cambridge, MA 02139, USA
Received 22 September 2003; revised 9 October 2003; accepted 9 October 2003
Abstract—A synthesis and stereochemistry confirmation of a constituent recently isolated from the whole plant Isodon excisus is reported.
An enantioselective catalytic boron-mediated reduction of an a-bromoketone was utilized in the key synthetic transformation. The
methodology described herein was also used for the synthesis of the natural product’s enantiomer and several derivatives. In addition, the
compounds were evaluated for inhibitory activity in a caspase induction assay. The natural product was found to be devoid of activity, but
several derivatives had moderate inhibitory activity (EC50,1 mM).
q 2003 Elsevier Ltd. All rights reserved.
1. Introduction
Recently, two structurally related natural products, calebin
A (1),10a isolated from turmeric (Curcuma longa L.,
Zingiberaceae) and 2a,11 isolated from the whole plant
Isodon excisus, have been independently reported. Both
compounds were described as apoptosis inhibitors. Com-
pound 1 protected PC12 rat pheochromocytoma and IMR-
32 human neuroblastoma cells from b-amyloid insult,10a
while 2a has been reported to inhibit etoposide-induced
apoptosis in U937 cells by impeding the induction of
caspase-3/7.11 A synthesis of calebin A (1) and several
analogs has recently been published.10b
Neurodegenerative diseases are a group of maladies that
afflict a significant portion of the human population. The
medical and socio-economic impacts of these diseases are
significant. Although the etiology of each neurodegenera-
tive disease is likely different, one common feature that
many share is progressive and irreversible neuronal cell
death in specific regions of the central nervous system.1
Compelling evidence is emerging that one of the primary
mechanisms responsible for the observed neuron cell death
in Parkinson’s disease (PD),2 Huntington’s disease (HD),3
amyotrophic lateral sclerosis (ALS)4 and human immuno-
deficiency virus associated dementia (HAD)5 is apoptosis.
Studies have also suggested that this mode of cell death may
also be involved in Alzheimer’s disease (AD).6 Albeit,
neurons present in AD may be chronically dysfunctional
without necessarily undergoing active cell death.7 Apopto-
sis is a genetically regulated process involving an elaborate
sequence of biochemical events culminating in cell death
and disassembly.8 Many key cellular components in this
cascade have been identified and some serve as potential
targets for therapeutic intervention including caspases,
which are a family of cysteine proteases.6,9 Identifying
and preparing low molecular weight molecules that interact
directly with or alter the expression of these cellular
constituents can assist in the understanding of the patho-
physiology of disease and can provide lead compounds for
therapeutic development.
In the present study, we report the synthesis of 2a, its
enantiomer 2b and several derivatives. The synthesis
described also allowed for a confirmation of the absolute
stereochemistry of the natural product reported by Kho
et al.11 They inferred the stereochemistry based on the
work reported by Zwanenburg, et al.12a and Huang
et al.12b In addition, the compounds were evaluated for
their ability to inhibit etoposide-induced caspase induc-
tion in U937 cells.
Keywords: apoptosis; caspase-induction; inhibitor; boron; Isodon excisus.
*
Corresponding author. Tel.: þ1-617-768-8640; fax: þ1-617-768-8606;
e-mail: gcuny@rics.bwh.harvard.edu
0040–4020/$ - see front matter q 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2003.10.025

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X. Xing et al. / Tetrahedron 59 (2003) 9961–9969
the alcohol in a manner similar to Zaponakis and
Katerinopoulos (Scheme 1).13c
The synthesis began by preparing the tert-butyldimethylsi-
lyl (TBDMS) ether 6a from 4-hydroxyacetophenone (5,
Scheme 2).14 Next, the a-bromoketone 4a was generated
through a two step process. First, 6a was allowed to react
with trimethylsilyl triflate (TMSOTf) in the presence of
diisopropylethylamine (i-Pr2NEt) to generate the corre-
sponding silyl enol ether in situ. Then bromination was
effected with N-bromosuccinimide (NBS) to yield 4a in
97% yield.15 The a-bromoketone 4a was reduced enantio-
selectively with borane–methyl sulfide complex and a
catalytic amount of (S)-2-methyl-CBS-oxazaborolidine
utilizing methodology developed by Corey et al. to give
the bromohydrin 7a in 89% yield.16–18 Displacement of the
bromine (0.27 M in water) with sodium azide employing
phase-transfer catalysis with tetrabutylammonium bromide
(TBABr) provided 8a in moderate (60%) yield.19 Next,
alcohol 8a was converted to ether 9a in 50–90% yield via
deprotonation with sodium hydride followed by alkylation
with methyl iodide.20a Alternatively, alcohol 8a was
alkylated with methyl iodide in the presence of silver
oxide to give ether 9a in 72% yield.20b Reduction of the
azide was readily accomplished by hydrogenation (H2,
1 atm) in the presence of 5% Pd/C to give 3a.21 The
unpurified amin0 e 3a was coupled utilizing O-benzotriazol-
2. Results and discussion
A retrosynthetic analysis of 2a began with a disconnection
of the amide bond to give 3a. Lawrence and Bushell
reported an enantioselective synthesis of a structurally
similar amine utilizing an asymmetric dihydroxylation of a
styrene derivative in the synthesis of chelonin B,13a while
Hergenrother recently described the regio- and chemose-
lective synthesis of 1-aryl-2-amino ethanol utilizing asym-
metric aminohydroxylation of styrenes.13b However, we
envisioned the assembly of the aminoether 3a via an
enantioselective catalytic boron-mediated reduction of a-
bromoketone 4a as the key synthetic transformation
followed by introduction of the amine and alkylation of
1-yl-N,N,N0,N -tetramethyluronium
(HBTU)22 in dichloromethane to the cinnamic acid
tetrafluoroborate
Scheme 1.
Scheme 2. (i) t-BuMe2SiCl, imidazole, DMF, 08C to room temperature, 98%; (ii) TMSOTf, i-Pr2EtN, DCM, 08C, 30 min; (iii) NBS, 08C, 4 h, 97% for two
steps; (iv) BH3·Me2S, (S)-2-methyl-CBS-oxazaborolidine, 08C to room temperature, 89%; (v) NaN3, TBABr, H2O, 408C, 60–80%; (vi) NaH in THF then MeI,
50–90% or Ag2O, MeI, 708C, 72%; (vii) H2 (1 atm), 5% Pd/C, MeOH; (viii) 3a or 3b, HBTU, i-Pr2EtN, DCM, 86% for two steps; (ix) TBAF, THF, 97%.

X. Xing et al. / Tetrahedron 59 (2003) 9961–9969
9963
Scheme 3.
derivative 10, which was prepared from the corresponding
commercially available ethyl ester23 to yield 11a (86% for
two steps). HBTU was not very soluble in dichloromethane.
However, addition of small amounts of dimethylformamide
to increase its solubility was detrimental, leading to
formation of undesired side products. Finally, deprotection
of the silyl ether was accomplished using tetrabutylammo-
nium fluoride (TBAF) to give the natural product 2a. The 1H
NMR spectra of the synthetic product was identical to that
reported for the natural product.
cinnamic acid derivative 10 utilizing HBTU provided
amides 16 and 17, respectively.
Compounds 2a and 2b were evaluated for their ability to
inhibit etoposide-induced caspase induction in U937 cells.
Etoposide (40 mM) was added to the cells (4£104 cells/
well) in the presence or absence of various concentrations of
2a, 2b or ammonium pyrrolidinedithiocarbamate (PDTC).
Following incubation (4.5 h), caspase-3/7 protease activity
was determined using an Apo-ONE Homogeneous Caspase-
3/7 Assay kit that employs z-DEVD-R110 as substrate. This
modified peptide substrate produces a fluorescent rhoda-
mine product upon enzymatic cleavage. The incubation
time of 4.5 h was selected because it provided robust
induction of caspase-3/7 with cells undergoing apoptotic
cell death as observed microscopically in agreement with
literature reports.25 The positive control, PDTC, was able to
prevent caspase induction with an EC50¼0.51 mM
(Graph 1). However, neither the natural product 2a nor its
enantiomer 2b were active even at high concentration
(.10 mM). Therefore, we have been unable to confirm the
caspase induction inhibitory activity previously attributed to
this natural product.11,26 Similar results were obtained with
various concentrations of etoposide (10 and 20 mM) and
with different cell numbers (1£104 and 2£104 cells/well).
The enantiomer 2b was also prepared employing the same
synthetic protocols described for 2a, except that (R)-2-
methyl-CBS-oxazaborolidine was utilized in the enantiose-
lective boron-mediated reduction of a-bromoketone 4a.
Furthermore, starting with 4-tert-butoxyacetophenone (6b),
the tert-butoxy derivative 11b was also prepared utilizing
the same methodology. The synthesis proceeded in a similar
manner, except for the conversion of bromide 7b to azide
8b, which occurred in higher yield (88%).
The enantiomeric purity of 3a was assessed by analysis of
the corresponding L-alanine derivative 13a (Scheme 3).
This material was prepared by allowing 3a to react with N-
Boc-L-alanine (12), in the presence of HBTU and i-Pr2NEt
in dichloromethane/DMF (10:1). Likewise, diastereomer
13b was prepared from 3b in a similar manner.24 Analysis
of the 1H NMR spectra of 13a and 13b clearly demonstrated
very high diastereomeric purities for each, indicative of high
enantiomeric purities (.98% ee) for 3a and 3b, respect-
ively. Racemization of the chiral center during the
remaining reactions in the synthesis of 2a and 2b are quite
improbable.
The derivatives 11b, 16 and 17 were similarly evaluated
(Graph 1). The tert-butoxy derivative 11b was found to have
activity (0.1–10 mM) with a maximum inhibition of 50%.
Derivative 16 was quite potent (EC50¼100 nM) at inhibit-
ing caspase induction, but only provided a maximum
inhibition of ,60%. However, derivative 17, which lacks
the aliphatic methoxy group, was moderately potent
(EC50¼570 nM) and provided a maximum inhibition of
,65% at the highest dose tested (10 mM). Control
experiments demonstrated that 11b, 16, and 17 did not
quench the fluorescent by-product formed by enzymatic
cleavage of z-DEVD-R110. In addition, these three
compounds failed to inhibit in vitro caspase-3/7 protease
activity indicating their mechanism of action was on
impeding enzyme induction.
Two additional compounds were prepared for the structure–
activity relationship (SAR) study (Scheme 4). Azide 14a
was prepared from 8a utilizing sodium hydride, methyl
iodide and prolonged reaction time. Presumably under these
conditions the tert-butyldimethylsilyl group in 8a was
cleaved to give a phenol that was subsequently alkylated
along with the aliphatic alcohol upon addition of methyl
iodide. The product was then hydrogenated to give amine
14b. Coupling of amines 14b (unpurified) and 15 with the
These results highlight several interesting points. First, a
Scheme 4. (i) H2 (1 atm), 5% Pd/C, MeOH; (ii) 10, HBTU, i-Pr2EtN, DCM, 85% for 16 over two steps, 81% for 17.

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X. Xing et al. / Tetrahedron 59 (2003) 9961–9969
Graph 1. Inhibition of etoposide-induced caspase-3/7 induction in U937 cells by PDTC (P), 11b (O), 16 (B) and 17 (X). Cells (4£104 cells/well) were
incubated in the presence of etoposide (40 mM) and in the presence or absence of various concentrations of test compounds for 4.5 h. Caspase-3/7 protease
activity was determined using an Apo-ONE Homogeneous Caspase-3/7 Assay kit that employs z-DEVD-R110 as the substrate. Concentration-percent
inhibition curves for each test compound were generated using a sigmoidal dose-response with variable slope nonlinear regression analysis (R 2.0.93). From
these curves, EC50 values were determined using GraphPad Prismw.
hydroxy group on the phenethyl portion of the molecules
was detrimental to activity. This SAR diverges from that
reported for 1, where derivatives with hydroxy groups on
the cinnamate portion of the molecules were most
protective.10b However, conversion of the hydroxy group
on the phenethyl portion of 2a to an ether (e.g. methyl or
tert-butyl) resulted in active compounds. Furthermore, the
aliphatic methoxy group is not necessary for caspase
induction inhibitory activity. The limited maximum inhi-
bition (at 10 mM) observed for 11b (50%), 16 (58%) and 17
(65%), as compared with PDTC (.95%), might be the
result of cellular toxicity caused by the test compounds via
non-caspase-3/7 dependent mechanisms resulting in fewer
cells undergoing etoposide-induced apoptosis via caspase-3/
7 induction.27 Additional studies will be necessary to
determine the precise reasons for these observed effects.
Likewise, additional SAR may reveal compounds that are
able to more effectively inhibit caspase induction than those
reported herein.
the natural product. The synthesis featured the utilization of an
enantioselective catalytic boron-mediated reduction of an a-
bromoketone in the key transformation. In addition, 2a was
inactive at inhibiting etoposide-induced caspase induction in
U937 cells contrary to a previous report. But, several
derivatives (11b, 16 and 17) were found to have activity.
The preliminary SAR revealed that the hydroxy group on the
phenethyl portion of the molecules is detrimental to activity,
replacement of this substituent with an ether resulted in
activity and the aliphatic methoxy group is not necessary for
caspase induction inhibitory activity.
4. Experimental
4.1. General experimental procedures
Unless otherwise noted, all reagents and solvents were
purchased from commercial sources and used without
further purification. The NMR spectra were obtained using
a Bruker 400 MHz, Varian 400 MHz or Varian 500 MHz
1
3. Conclusions
spectrometer. All H NMR spectra are reported in d units
ppm and are reference to tetramethylsilane (TMS) if
conducted in CDCl3 or to the central line of the quintet at
3.30 ppm for samples in CD3OD. All 13C NMR spectra are
reported in d units ppm and are reference to the central line
of the triplet at 77.23 ppm if conducted in CDCl3 or to the
The methodology described in this communication provided a
convenient means for assembling the natural product 2a, its
enantiomer 2b and several derivatives. It also provides
confirmation of the stereochemistry originally assigned to

X. Xing et al. / Tetrahedron 59 (2003) 9961–9969
9965
central line of the septet at 49.0 ppm for samples in CD3OD.
Column chromatography was performed on silica gel
(Merck, grade 60, 230–400 mesh) or utilizing a Combi-
Flash Sg 100c separation system (ISCO) with RediSep
disposable silica gel columns (ISCO). Optical rotations
were measured using an AUTOPOL IV digital polarimeter
(Rudolph Research Analytical, NJ). High-resolution mass
spectra were obtained by using a SX-102A mass spec-
trometer (JEOL USA, Inc., Peabody, MA) or a LCT mass
spectrometer (Micromass Inc., Beverly, MA). All com-
pounds, unless otherwise noted, are $95% pure as
determined by 1H NMR.
by column chromatography on silica gel using hexane/ethyl
acetate (95:5) as eluant to give 4a (1.28 g, 97% yield) as a
1
colorless oil. H NMR (400 MHz, CDCl3): d 0.27 (s, 6H),
1.00 (s, 9H), 4.42 (s, 2H), 6.91 (d, 2H, J¼8.8 Hz), 7.93 (d,
2H, J¼8.8 Hz).
4.1.4. 20-Bromo-4-tert-butoxyacetophenone (4b). Pre-
1
pared using the procedure described in Section 4.1.3. H
NMR (500 MHz, CDCl3): d 1.45 (s, 9H), 4.42 (s, 2H), 7.05
(d, 2H, J¼9.0 Hz), 7.93 (d, 2H, J¼9.0 Hz); 13C NMR
(100.5 MHz, CDCl3): d 29.13 (3£CH3), 30.96 (CH2), 80.18
(C),122.24 (2£CH), 128.35 (C), 130.82 (2£CH), 161.53
(C), 190.40 (C); FT-IR (film, nmax, cm21): 2978s, 2939w,
1676s, 1597s, 1504m, 1283s, 1257s, 1160s, 1051s, 900m,
848m; HREIMS [M]þ: 270.0251 (calcd for [C12H15BrO2]þ,
270.0255).
4.1.1. 4-tert-Butyldimethylsiloxyacetophenone (6a). A
round-bottom flask was charged with 4-hydroxyaceto-
phenone (5, 2.5 g, 18.36 mmol), imidazole (3.09 g,
45.35 mmol), and DMF (20 mL). After cooling the solution
to 08C, tert-butyldimethylsilyl chloride (3.32 g,
22.03 mmol) was added in one portion. After stirring at
08C for 15 min, the solution was maintained at room
temperature for 1 h. The solution was diluted with ethyl
acetate (100 mL) and then washed sequentially with 1N
sodium hydroxide (50 mL), water (3£50 mL) and brine
(40 mL). The organic layer was dried over anhydrous
magnesium sulfate, filtered, and concentrated to give a
colorless oil. The oil was purified by column chromato-
graphy on silica gel using hexane/ethyl acetate (90:10) as
4.1.5. (S)-2-Bromo-1-(4-tert-butyldimethylsiloxy)pheny-
lethanol (7a). To a stirred solution of (S)-2 methyl-CBS-
oxazaborolidine (1.0 M in toluene, 0.1 mL, 0.1 mmol) and
borane–methyl sulfide (2.0 M in THF, 0.05 mL, 0.1 mmol)
in anhydrous THF (2 mL) were added simultaneously a
solution of the a-bromoketone 4a in THF (1 mL) and
borane–methyl sulfide (2.0 M in THF, 0.33 mL,
0.66 mmol) at 08C under argon over a period of 20 min.
After addition, the reaction mixture was allowed to warm to
room temperature and stirred for 1 h. The reaction solution
was cooled to 08C before 1 mL of methanol was added
carefully (CAUTION: gas evolution!!). The reaction
mixture was then concentrated in vacuo (Me2S was trapped
and oxidized with household bleach) and the residue
dissolved in 20 mL of ethyl acetate. The solution was
washed with 1N HCl (3£5 mL) and water (2£5 mL), dried
over MgSO4, filtered, and concentrated. The product was
purified by column chromatography on silica gel using
hexane/ethyl acetate (90:10) as eluant to give 293 mg of 7a
as a colorless oil (89%). [a]2D4.4¼þ26.48 (c 0.5, CHCl3); 1H
NMR (400 MHz, CDCl3): d 0.20 (s, 6H), 0.99 (s, 9H), 2.59
(s, 1H), 3.54 (q, 1H, J¼10.0 Hz), 3.61 (dd, 1H, J¼3.2,
10.0 Hz), 4.88 (dd, 1H, J¼3.2, 10.0 Hz), 6.84 (d, 2H,
J¼8.4 Hz), 7.24 (d, 2H, J¼8.4 Hz); 13C NMR (100.5 MHz,
CDCl3): d 24.21 (2£CH3–Si), 22.20 (C), 29.66 (3£CH3),
44.36 (CH2), 77.61 (CH), 124.28 (2£CH), 131.17 (2£CH),
136.96 (C), 159.89 (C); FT-IR (film, nmax, cm21): 3391m
(br), 2957s, 2930s, 2858s, 1608s, 1510s, 1264s, 916s, 841s,
781s; HREIMS [M]þ: 330.0654 (calcd for [C14H23BrO2-
Si]þ, 330.0651).
1
eluant to give 6a (4.51 g, 98% yield) as a colorless oil. H
NMR (400 MHz, CDCl3): d 0.24 (s, 6H), 0.99 (s, 9H), 2.55
(s, 3H), 6.87 (d, 2H, J¼8.8 Hz), 7.88 (d, 2H, J¼8.8 Hz).
4.1.2. 4-tert-Butoxyacetophenone (6b). The isobutylene
gas (ca. 14 g, 250 mmol) was condensed and added
dropwise to a mixture of 4-hydroxyacetophenone (6.8 g,
50 mmol) in dichloromethane (100 mL) containing a few
drops of concentrated sulfuric acid. The reaction mixture
was stirred overnight at room temperature in a two-necked
flask with a condenser filled with dry ice. After dilution with
50 mL of dichloromethane, the mixture was washed with
water (2£60 mL). The organic layer was dried over
anhydrous sodium sulfate, filtered, and concentrated. The
crude product was purified by chromatography on silica gel
using hexane/ethyl acetate (80:20) as eluant to give 6b
1
(6.1 g, 63% yield) as a colorless oil. H NMR (500 MHz,
CDCl3): d 1.42 (s, 9H), 2.57 (s, 3H), 7.03 (d, 2H, J¼9.0 Hz),
7.90 (d, 2H, J¼9.0 Hz); 13C NMR (100.5 MHz, CDCl3): d
26.61 (CH3), 29.13 (3£CH3), 79.84 (C), 122.54 (2£CH),
130.02 (2£CH), 132.02 (C), 160.62 (C), 197.35 (C); FT-IR
(film, nmax, cm21): 2979s, 2935w, 1679s, 1598s, 1504m,
1365s, 1255s, 1162s, 1051s, 957w, 897m, 840w; HREIMS
[M]þ: 193.1233 (calcd for [C12H16O2]þ, 193.1228).
4.1.6. (S)-2-Bromo-1-(4-tert-butoxy)phenylethanol (7b).
Prepared using the procedure described in Section 4.1.5.
[a]D23.9¼þ24.08 (c 0.5, CH3OH); 1H NMR (500 MHz,
CDCl3): d 1.34 (s, 9H), 2.62 (d, 1H, J¼3.0 Hz), 3.54 (dd,
1H, J¼9.0, 10.5 Hz), 3.62 (dd, 1H, J¼3.0, 10.0 Hz), 4.89
(dt, 1H, J¼3.0, 9.0 Hz), 6.99 (d, 2H, J¼8.5 Hz), 7.28 (d, 2H,
J¼8.5 Hz); 13C NMR (100.5 MHz, CDCl3): d 29.04
(3£CH3), 40.50 (CH2), 73.80 (CH), 78.94 (C), 124.40
(2£CH), 126.79 (2£CH), 135.17 (C), 155.82 (C); FT-IR
(film, nmax, cm21): 3421s (br), 2977s, 2933w, 1606m,
1506s, 1367m,1238s, 1160s, 1070m, 895s, 854m; HREIMS
[M]þ: 272.0413 (calcd for [C12H17BrO2]þ, 272.0412).
4.1.3. 20-Bromo-4-tert-butyldimethylsiloxyacetophenone
(4a). A Schlenk flask, under argon, was charged with 6a
(1.0 g, 4 mmol) and dichloromethane (8 mL). The solution
was cooled to 08C and then diisopropylethylamine
(0.88 mL, 5.06 mmol) was added followed by trimethylsilyl
trifluoromethanesulfonate (TMSOTf, 0.88 mL, 4.86 mmol).
The resulting solution was maintained at 08C for 30 min.
Next, N-bromosuccinimide (0.854 g, 4.8 mmol) was added
in one portion. The resulting mixture was stirred at room
temperature for 4 h. The reaction mixture was concentrated
to give a pale yellow oily solid. This material was purified
4.1.7. (S)-2-Azido-1-(4-tert-butyldimethylsiloxy)phenyl-
ethanol (8a). A mixture of the bromohydrin 7a (132 mg,

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X. Xing et al. / Tetrahedron 59 (2003) 9961–9969
0.4 mmol), sodium azide (52 mg, 0.8 mmol), tetrabutylam-
monium bromide (80 mg, 0.24 mmol) and water (1.5 mL)
was stirred at 408C for 2 days and then extracted with ethyl
acetate (30 mL). The organic layer was washed with water
(2£8 mL), dried over MgSO4, filtered, and concentrated.
The residue was purified by column chromatography on
silica gel using hexane/ethyl acetate (90:10) as eluant to
afford 70 mg of 8a as a colorless oil (60%). [a]2D5.9¼þ46.08
(c 1.0, CHCl3); 1H NMR (400 MHz, CDCl3): d 0.20 (s, 6H),
0.99 (s, 9H), 2.26 (s, 1H), 3.45 (m, 2H), 4.83 (m, 1H), 6.64
(d, 2H, J¼8.4 Hz), 7.23 (d, 2H, J¼8.4 Hz); 13C NMR
(100.5 MHz, CDCl3): d 24.22 (2£CH3–Si), 18.41 (C),
25.86 (3£CH3), 58.30 (CH2), 73.36 (CH), 120.50 (2£CH),
cm21): 2956s, 2930s, 2859s, 2102s, 1608m, 1510s,
1256s, 1110s, 915s, 840s, 782m; HREIMS [M]þ:
307.1719 (calcd for [C15H25N3O2Si]þ, 307.1716).
4.1.10. (S)-1-(2-Azido-1-methoxy)ethyl-4-tert-butoxy-
benzene (9b).28 Prepared using the procedure described in
Section 4.1.9, Method B. [a]2D4.0¼þ120.08 (c 0.5, CH3OH);
1H NMR (500 MHz, CDCl3): d 1.35 (s, 9H), 3.20 (dd, 1H,
J¼4.0, 13.0 Hz), 2.30 (s, 3H), 3.48 (dd, 1H, J¼8.5,
13.0 Hz), 4.32 (dd, 1H, J¼4.0, 8.5 Hz), 6.99 (d, 2H,
J¼9.0 Hz), 7.20 (d, 2H, J¼9.0 Hz); 13C NMR
(100.5 MHz, CDCl3): d 29.06 (3£CH3), 56.79 (CH3),
57.02 (CH2), 78.83 (C), 82.95 (CH), 124.37 (2£CH),
127.45 (2£CH), 133.37 (C), 155.83 (C); FT-IR (film,
127.34 (2£CH), 133.44 (C), 156.01 (C); FT-IR (film, nmax
,
cm21): 3404m (br), 2957m, 2930m, 2859m, 2105s, 1608m,
1511s, 1263s, 915s, 840s, 781s; HREIMS [M]þ: 293.1564
(calcd for [C14H23N3O2Si]þ, 293.1560).
n
max, cm21): 2979s, 2933w, 2102s, 1608m, 1506s,
1366m,1238s, 1162s, 1110s, 896s, 859m.
4.1.11. (S)-1-(2-Amino-1-methoxy)ethyl-4-tert-butyldi-
methylsiloxybenzene (3a). A mixture of azido methyl
ether 9a (31 mg, 0.1 mmol), 5% Pd/C (30 mg) and methanol
(1.5 mL) was stirred for 1.5 h at room temperature under
hydrogen (1 atm), then diluted with methanol (20 mL) and
1 mL of 2.0 M ammonia in ethanol. The catalyst was
removed by filtration and the filtrate concentrated. The
residue was dissolved in ethyl acetate (20 mL) and dried
over anhydrous Na2SO4, filtered and evaporated to afford
30 mg of crude product 3a, which was used without further
purification.
4.1.8. (S)-2-Azido-1-(4-tert-butoxy)phenylethanol (8b).
Prepared using the procedure described in Section 4.1.7.
[a]D24.0¼þ71.68 (c 0.5, CH3OH); 1H NMR (500 MHz,
CDCl3): d 1.34 (s, 9H), 2.34 (s, 1H), 3.42 (dd, 1H, J¼3.5,
12.5 Hz), 3.48 (dd, 1H, J¼8.0, 12.5 Hz), 4.89 (dt, 1H,
J¼3.5, 8.0 Hz), 6.99 (d, 2H, J¼8.5 Hz), 7.26 (d, 2H,
J¼8.5 Hz); 13C NMR (100.5 MHz, CDCl3): d 29.03
(3£CH3), 58.29 (CH2), 73.34 (CH), 78.94 (C), 124.46
(2£CH), 126.75 (2£CH), 135.51 (C), 155.71 (C); FT-IR
(film, nmax, cm21): 3427s (br), 2978s, 2933w, 2104s,
1608m, 1507s, 1367m, 1238s, 1161s, 1070m, 894s, 856m;
HREIMS [M]þ: 235.1313 (calcd for [C12H17N3O2]þ,
235.1321).
4.1.12. (S)-1-(2-Amino-1-methoxy)ethyl-4-tert-butoxy-
benzene (3b). A mixture of azido methyl ether 9b
(125 mg, 0.5 mmol), 5% Pd/C (50 mg) and methanol
(6 mL) was stirred for 2 h at room temperature under
hydrogen (1 atm), then diluted with methanol (20 mL) and
1 mL of 2.0 M ammonia in ethanol. The catalyst was
removed by filtration and the filtrate concentrated. The
residue was dissolved in ethyl acetate (20 mL) and dried
over anhydrous Na2SO4, filtered and evaporated to afford
111 mg of crude product 3b, which was used without further
purification.
4.1.9. (S)-1-(2-Azido-1-methoxy)ethyl-4-tert-butyldi-
methylsiloxybenzene (9a). Method A. To a stirred solution
of the azido alcohol 8a (30 mg, 0.1 mmol) in THF (1.5 mL)
was added sodium hydride (95%, 5 mg, 0.2 mmol) at room
temperature under argon. The mixture was stirred for
15 min and then iodomethane (0.02 mL, 0.3 mmol) was
added. Stirring was continued for another 1 h before the
reaction mixture was diluted with 20 mL of ethyl acetate.
The mixture was washed with 1N HCl (5 mL), saturated
NaHCO3 (5 mL), and brine (5 mL). The organic layer was
dried over anhydrous MgSO4, filtered and concentrated. The
residue was purified by column chromatography on silica
gel using hexane/ethyl acetate (95:5) as eluant to give 15–
27 mg of 9a as a colorless oil (50–90%).
4.1.13. 4-Hydroxy-3-methoxycinnamic acid (10). A
round-bottom flask was charged with ethyl 4-hydroxy-3-
methoxycinnamate (50 mg, 0.225 mmol), THF (2 mL), and
2N sodium hydroxide (5 mL). The reaction mixture was
stirred at room temperature for 8 h and then made acidic
with concentrated hydrochloric acid. The mixture was
extracted with ethyl acetate (3£10 mL). The extracts were
combined, washed with brine (10 mL), dried over anhy-
drous sodium sulfate, filtered, and concentrated to give a
pale yellow solid. The solid was purified by column
chromatography on silica gel using chloroform/ethyl
acetate/acetic acid (10:1:0.1) as eluant to give 10 (43 mg,
98% yield) as a white solid. 1H NMR (400 MHz, CD3OD): d
3.90 (s, 3H), 4.99 (bs, 2H), 6.32 (d, 1H, J¼16 Hz), 6.82 (d,
1H, J¼8.2 Hz), 7.07 (dd, 1H, J¼1.6, 8.2 Hz), 7.18 (d, 1H,
J¼1.6 Hz), 7.61 (d, 1H, J¼16 Hz).
Method B. A mixture of the azido alcohol 8a (29 mg,
0.1 mmol), silver (I) oxide (24 mg, 0.11 mmol), iodo-
methane (1 mL) and acetonitrile (2 mL) was stirred at
688C for 2 days. The solid was then filtered and washed
with ethyl acetate (2£3 mL). After removal of the
solvents, the residue was purified by column chromatog-
raphy on silica gel using hexane/ethyl acetate (90:10) as
eluant to give 22 mg of 9a as a colorless oil (72%).
[a]D24.6¼þ95.48 (c 1.0, CHCl3); 1H NMR (400 MHz,
CDCl3): d 0.21 (s, 6H), 0.99 (s, 9H), 3.19 (dd, 1H, J¼3.2,
12.4 Hz), 3.29 (s, 3H), 3.47 (dd, 1H, J¼8.4, 12.4 Hz),
4.30 (dd, 1H, J¼3.2, 8.4 Hz), 6.84 (d, 2H, J¼8.4 Hz),
7.18 (d, 2H, J¼8.4 Hz); 13C NMR (100.5 MHz, CDCl3):
d 24.22 (2£CH3–Si), 18.38 (C), 25.85 (3£CH3), 56.80
(CH2), 51.90 (CH3), 82.92 (CH), 120.43 (2£CH), 128.07
4.1.14. (S)-3-(4-Hydroxy-3-methoxyphenyl)-N-[2-(4-tert-
butyldimethylsiloxyphenyl)-2-methoxyethyl]acrylamide
(11a). A mixture of the cinnamic acid 10 (20 mg,
0.1 mmol), HBTU (34 mg, 0.09 mmol), i-Pr2NEt
(0.04 mL, 0.2 mmol) and the amino methyl ether 3a
(2£CH), 131.34 (C), 156.03 (C); FT-IR (film, nmax
,

X. Xing et al. / Tetrahedron 59 (2003) 9961–9969
9967
(10 mg, 0.03 mmol) in 1 mL of CH2Cl2 was stirred at room
temperature for 1 h. The reaction mixture was diluted with
20 mL of ethyl acetate before being washed sequentially
with 1N HCl (2£5 mL), saturated NaHCO3 (2£5 mL) and
brine (8 mL). The organic layer was dried over Na2SO4,
filtered and concentrated. The product was purified by
column chromatography on silica gel using hexane/ethyl
acetate (60:40) as eluant to give 14 mg of 11a as a pale
yellow oil (86% for two steps). [a]2D4.5¼21.68 (c 1.0,
J¼15.6 Hz); 13C NMR (100.5 MHz, CDCl3): d 46.09
(CH2), 56.15 (CH3), 56.80 (CH3), 82.16 (CH), 109.74
(CH), 114.90 (CH), 115.74 (2£CH), 118.27 (CH), 122.49
(CH), 127.99 (C), 128.35 (2£CH), 131.13 (C), 141.60 (CH),
146.88 (C), 147.61 (C), 155.96 (C), 166.37 (C); HRESMS
[MþH]þ: 344.1491 (calcd for [C19H21NO5þH]þ,
344.1498).
4.1.17. (R)-3-(4-Hydroxy-3-methoxyphenyl)-N-[2-(4-
hydroxyphenyl)-2-methoxyethyl]acrylamide (2b). Pre-
pared using the same procedure as described in Section
4.1.16. [a]2D6.0¼þ6.18 (c 1.0, CH3OH); 1H NMR (400 MHz,
CD3OD): d 3.21 (s, 3H), 3.41 (dd, 1H, J¼8.4, 14.0 Hz), 3.50
(dd, 1H, J¼4.8, 14.0 Hz), 3.88 (s, 3H), 4.25 (dd, 1H, J¼4.4,
8.0 Hz), 6.47 (d, 1H, J¼15.6 Hz), 6.79 (d, 1H, J¼8.8 Hz),
6.80 (d, 1H, J¼8.8 Hz), 7.03 (dd, 1H, J¼2.4, 8.4 Hz), 7.13
(d, 1H, J¼2.4 Hz), 7.17 (d, 2H, J¼8.8 Hz), 7.44 (d, 1H,
J¼15.6 Hz); HRESMS [MþH]þ: 344.1494 (calcd for
[C19H21NO5þH]þ, 344.1498).
1
CHCl3); H NMR (400 MHz, CDCl3): d 0.21 (s, 6H), 0.99
(s, 9H), 3.24 (s, 3H), 3.30 (m, 1H), 3.84 (m, 1H), 3.92 (s,
3H), 4.26 (dd, 1H, J¼4.0, 8.8 Hz), 5.93 (s, 1H), 6.03 (m,
1H), 6.26 (d, 1H, J¼15.6 Hz), 6.84 (d, 2H, J¼8.8 Hz), 6.91
(d, 1H, J¼7.6 Hz), 7.00 (s, 1H), 7.07 (d, 1H, J¼7.6 Hz),
7.19 (d, 2H, J¼8.8 Hz), 7.56 (d, 1H, J¼15.6 Hz); 13C NMR
(100.5 MHz, CDCl3): d 24.19 (2£CH3–Si), 18.40 (C),
25.87 (3£CH3), 45.96 (CH2), 56.15 (CH3), 56.80 (CH3),
82.19 (CH), 109.84 (CH), 114.91 (CH), 118.45 (CH),
120.47 (2£CH), 122.46 (CH), 127.62 (C), 128.12 (2£CH),
131.81 (C), 141.35 (CH), 146.90 (C), 147.57 (C), 155.85
(C), 166.23 (C); FT-IR (KBr, nmax, cm21): 3412m (br),
2955m, 2933m, 2857m, 1729m, 1659m, 1606m, 1510s,
1261s, 1121s, 915m, 841m, 782m; HRESMS [MþH]þ:
458.2369 (calcd for [C25H35NO5SiþH]þ, 458.2363).
4.1.18. N-[2-(4-tert-Butyldimethylsiloxyphenyl)-2(S)-
methoxyethyl]-N0-BOC-L-alaninamide (13a). A solution
of N-Boc-L-alanine (12, 19 mg, 0.1 mmol), HBTU (34 mg,
0.09 mmol), i-Pr2NEt (0.06 mL, 0.3 mmol) in 1.1 mL of
CH2Cl2/DMF (10:1) was added to the amino methyl ether
3a (10 mg, 0.03 mmol). The reaction mixture was stirred at
room temperature for 1 h. The reaction mixture was diluted
with 20 mL of ethyl acetate before being washed sequen-
tially with 1N HCl (2£5 mL), saturated NaHCO3 (2£5 mL)
and brine (5 mL). The organic layer was dried over Na2SO4,
filtered and concentrated. The product was purified by
column chromatography on silica gel using hexane/ethyl
acetate (50:50) as eluant to give 12 mg of 13a as a colorless
oil (88%). 1H NMR (500 MHz, CDCl3): d 0.20 (s, 6H), 0.98
(s, 9H), 1.34 (d, 3H, J¼7.0 Hz), 1.46 (s, 9H), 3.22 (s, 3H),
3.22 (m, 1H), 3.64 (m, 1H), 4.17 (m, 2H), 5.06 (bs, 1H), 6.40
(m, 1H), 6.82 (d, 2H, J¼8.5 Hz), 7.15 (d, 2H, J¼8.5 Hz);
13C NMR (100.5 MHz, CDCl3): d 24.21 (2£CH3–Si),
18.39 (C), 18.96 (CH3), 25.85 (3£CH3), 28.55 (3£CH3),
45.75 (CH2), 50.29 (CH), 56.82 (CH3), 80.17 (C), 81.94
(CH), 120.34 (2£CH), 128.04 (2£CH), 131.66 (C), 155.59
(C), 155.83 (C), 172.66 (C); FT-IR (film, nmax, cm21):
3319s (br), 2957s, 2930s, 2858s, 1716s, 1660s, 1608m,
1510s, 1366s, 1253s, 1168s, 916s, 841s, 781m; HRESMS
[MþNa]þ: 475.2601 (calcd for [C23H40N2O5SiþNa]þ,
475.2604).
4.1.15. (S)-3-(4-Hydroxy-3-methoxyphenyl)-N-[2-(4-tert-
butyoxyphenyl)-2-methoxyethyl]acrylamide (11b). Pre-
pared using the procedure described in Section 4.1.14.
[a]D23.9¼227.28 (c 0.5, MeOH); 1H NMR (500 MHz,
CDCl3): d 1.35 (s, 9H), 3.26 (s, 3H), 3.30 (m, 1H), 3.84
(m, 1H), 3.93 (s, 3H), 4.28 (dd, 1H, J¼4.0, 9.0 Hz), 5.83 (s,
1H), 6.00 (m, 1H), 6.26 (d, 1H, J¼16.0 Hz), 6.91 (d, 1H,
J¼8.5 Hz), 6.98 (d, 2H, J¼9.0 Hz), 7.00 (d, 1H, J¼2.0 Hz),
7.07 (dd, 1H, J¼2.0, 8.5 Hz), 7.22 (d, 2H, J¼9.0 Hz), 7.55
(d, 1H, J¼16.0 Hz); 13C NMR (100.5 MHz, CDCl3): d
29.09 (3£CH3), 45.97 (CH2), 56.16 (CH3), 56.93 (CH3),
78.80 (C), 82.24 (CH), 109.76 (CH), 114.93 (CH), 118.43
(CH), 122.46 (CH), 124.35 (2£CH), 127.49 (2£CH), 127.62
(C), 133.87 (C), 141.38 (CH), 146.93 (C), 147.61 (C),
155.63 (C), 166.26 (C); FT-IR (film, nmax, cm21): 3512–
3057s (br), 2979m, 2935m, 2827w, 1658s, 1605s, 1514s,
1268m,1205s, 1162s, 1110s, 895w, 847m, 737s; HRESMS
[MþH]þ: 400.2113 (calcd for [C23H29NO5þH]þ,
400.2124).
4.1.16. (S)-3-(4-Hydroxy-3-methoxyphenyl)-N-[2-(4-
hydroxyphenyl)-2-methoxyethyl]acrylamide (2a). A sol-
ution of 1.0 M tetrabutylammonium fluoride (1 mL) in THF
was added to 11a (14 mg, 0.03 mmol) at room temperature.
The mixture was stirred for 5 min before removal of the
solvent. The residue was dissolved in 20 mL of ethyl
acetate, washed with 1N HCl (2£5 mL) and brine (10 mL),
dried over anhydrous MgSO4, filtered and concentrated. The
product was purified by column chromatography on silica
gel using hexane/ethyl acetate (40:60) as eluant to give
10 mg of 2a as a pale yellow oil (97%). [a]2D6.1¼25.68 (c
1.0, CH3OH), literature reference [a]2D5¼228 (c 1.0,
CH3OH);11 1H NMR (400 MHz, CD3OD): d 3.21 (s, 3H),
3.41 (dd, 1H, J¼8.4, 14.0 Hz), 3.50 (dd, 1H, J¼4.8,
14.0 Hz), 3.88 (s, 3H), 4.25 (dd, 1H, J¼4.4, 8.0 Hz), 6.47
(d, 1H, J¼15.6 Hz), 6.79 (d, 1H, J¼8.8 Hz), 6.80 (d, 1H,
J¼8.8 Hz), 7.03 (dd, 1H, J¼2.4, 8.4 Hz), 7.13 (d, 1H,
J¼2.4 Hz), 7.17 (d, 2H, J¼8.8 Hz), 7.44 (d, 1H,
4.1.19. 2-(4-tert-Butyldimethylsiloxyphenyl)-2(R)-meth-
oxyethyl]-N0-BOC-L-alaninamide (13b). A solution of N-
Boc-L-alanine (12, 23 mg, 0.12 mmol), HBTU (38 mg,
0.10 mmol), i-Pr2NEt (0.08 mL, 0.3 mmol) in 1.1 mL of
CH2Cl2/DMF (10:1) was added to the amino methyl ether
3b (12 mg, 0.04 mmol). The reaction mixture was stirred at
room temperature for 1 h. The reaction mixture was diluted
with 20 mL of ethyl acetate before being washed sequen-
tially with 1N HCl (2£5 mL), saturated NaHCO3 (2£5 mL)
and brine (5 mL). The organic layer was dried over Na2SO4,
filtered and concentrated. The product was purified by
column chromatography on silica gel using hexane/ethyl
acetate (50:50) as eluant to give 16 mg of 13b as a colorless
oil (84%). 1H NMR (500 MHz, CDCl3): d 0.20 (s, 6H), 0.98
(s, 9H), 1.36 (d, 3H, J¼7.0 Hz), 1.46 (s, 9H), 3.18 (m, 1H),
3.22 (s, 3H), 3.68 (m, 1H), 4.17 (m, 2H), 4.96 (bs, 1H), 6.46

9968
X. Xing et al. / Tetrahedron 59 (2003) 9961–9969
(m, 1H), 6.82 (d, 2H, J¼8.5 Hz), 7.15 (d, 2H, J¼8.5 Hz);
13C NMR (100.5 MHz, CDCl3): d 24.21 (2£CH3–Si),
18.38 (C), 18.86 (CH3), 25.86 (3£CH3), 28.52 (3£CH3),
45.73 (CH2), 50.50 (CH), 56.83 (CH3), 80.24 (C), 82.03
(CH), 120.32 (2£CH), 128.04 (2£CH), 131.65 (C), 155.54
(C), 155.81 (C), 172.64 (C); FT-IR (film, nmax, cm21):
3320s (br), 2957s, 2930s, 2858s, 1716s, 1660s, 1608m,
1509s, 1366s, 1253s, 1168s, 916s, 841s, 781m; HRESMS
[MþNa]þ: 475.2581 (calcd for [C23H40N2O5SiþNa]þ,
475.2604).
895w, 844m; HRESMS [MþH]þ: 358.1649 (calcd for
[C20H23NO5þH]þ, 358.1654).
4.1.23. 3-(4-Hydroxy-3-methoxyphenyl)-N-[2-(4-meth-
oxyphenyl)ethyl]acrylamide (17). Prepared using the
procedure described in Section 4.1.14. 1H NMR
[500 MHz, CDCl3(CD3OD)]: d 2.82 (t, 2H, J¼7.0 Hz),
3.57 (t, 2H, J¼7.0 Hz), 3.81 (s, 3H), 3.91 (s, 3H), 6.23 (d,
1H, J¼15.5 Hz), 6.85 (d, 1H, J¼8.0 Hz), 6.87 (d, 2H,
J¼9.0 Hz), 7.01 (d, 1H, J¼2.0 Hz), 7.03 (dd, 1H, J¼2.0,
8.0 Hz), 7.26 (d, 2H, J¼9.0 Hz), 7.49 (d, 1H, J¼15.5 Hz);
13C NMR [100.5 MHz, CDCl3(acetone-d6)]: d 34.77 (CH2),
41.02 (CH2), 55.11 (CH3), 55.81 (CH3), 109.88 (CH),
113.91 (2£CH), 114.92 (CH), 118.40 (CH), 121.98 (CH),
127.34 (C), 129.68 (2£CH), 131.09 (C), 140.54 (CH),
147.12 (C), 147.66 (C), 158.20 (C), 166.38 (C); FT-IR (film,
4.1.20. (S)-1-(2-Azido-1-methoxy)ethyl(4-methoxy)ben-
zene (14a). To a stirred solution of the azido alcohol 8a
(110 mg, 0.37 mmol) in THF (5 mL) was added sodium
hydride (95%, 40 mg, 1.6 mmol) at room temperature under
argon. The mixture was stirred for 15 min and then
iodomethane (0.08 mL, 1.2 mmol) was added. Stirring was
continued for an additional 2 h and then the reaction mixture
was diluted with 40 mL of ethyl acetate. The mixture was
washed with 1N HCl (10 mL), saturated NaHCO3 (10 mL)
and brine (10 mL). The organic layer was dried over
anhydrous MgSO4, filtered and concentrated. The residue
was purified by column chromatography on silica gel using
hexane/ethyl acetate (90:10) as eluant to give 14a (50 mg,
43% yield) as a colorless oil. [a]2D2.9¼þ86.08 (c 0.5,
CHCl3); 1H NMR (500 MHz, CDCl3): d 3.18 (dd, 1H,
J¼4.0, 13.0 Hz), 3.28 (s, 3H), 3.48 (dd, 1H, J¼8.5,
13.0 Hz), 3.81 (s, CH3), 4.31 (dd, 1H, J¼4.0, 8.5 Hz),
6.90 (d, 2H, J¼9.0 Hz), 7.24 (d, 2H, J¼9.0 Hz); 13C NMR
(100.5 MHz, CDCl3): d 55.51 (CH3), 56.83 (CH3), 56.90
(CH2), 82.86 (CH), 114.32 (2£CH), 128.16 (2£CH), 130.77
(C), 159.94 (C); FT-IR (film, nmax, cm21): 2991w, 2934m,
2835w, 2102s, 1612m, 1513s, 1249s, 1107s, 1033s, 832m;
n
1615s, 1514s, 1460m, 1275s,1209s, 1123m, 1030s, 846s;
max, cm21): 3736–3100s (br), 2930m, 2850m, 1659s,
HRESMS
[MþH]þ:
328.1545
(calcd
for
[C19H21NO4þH]þ, 328.1549).
4.2. Etoposide-induced Caspase-3/7 induction assay
A 20 mL aliquot of U937 cells (4£104 cells/well; obtained
from American Tissue Culture Co.) in growth media
(DMEM supplemented with 10% fetal bovine serum,
100 units/mL penicillin–streptomycin, 2 mM glutamine,
1 mM sodium pyruvate, 25 mM HEPES, 1.5 g/L sodium
bicarbonate) was added to a 384-well plate. Etoposide
(40 mM) was added to the cells in the presence or absence of
various concentrations of test compounds (10–0.001 mM)
to give a final volume of 50 mL. Stock solutions of
compounds were prepared in DMSO at a concentration of
10 mM. The vehicle control contained 0.16% DMSO. Cells
were incubated for 4.5 h at 378C in an incubator containing
5% CO2–95% air atmosphere. After observing apoptotic
cells by microscopy, the caspase-3/7 protease activity was
estimated by addition of 50 mL of substrate Z-DEVD-R110
(Rhodamine 110 labeled bis-(N-CBZ-L-aspartyl-L-gluta-
myl-L-valyl-aspartic acid amide; Apo-ONE Homogeneous
Caspase-3/7 Assay kit from Promega). Incubation was
carried out at room temperature for 1 h and the fluorescent
intensity (reported as RFU) was monitored by excitation at
485 nm and emission at 530 nm in a fluorescence plate
reader (Molecular Devices). Each sample represents the
average of four replicates. Concentration-percent inhibition
curves for each test compound were generated using a
sigmoidal dose-response with variable slope nonlinear
regression analysis (R 2.0.93). From these curves, EC50
values were determined using GraphPad Prismw.
Ammonium pyrrolidine dithiocarbamate (PDTC; Sigma-
Aldrich, St. Louis, MO) was used as a standard caspase
induction inhibitor. The activity of PDTC in the assay
described herein was lower (EC50¼0.51 mM) than reported
by Kho et al. (EC50¼50 mM).11
HRCIMS
[MþNH4]þ:
225.1360
(calcd
for
[C10H13N3OþNH4]þ, 225.1351).
4.1.21. (S)-1-(2-Amino-1-methoxy)ethyl(4-methoxy)ben-
zene (14b). A mixture of azido methyl ether 14a (30 mg,
0.15 mmol), 5% Pd/C (30 mg) and methanol (2 mL) was
stirred for 3 h at room temperature under hydrogen (1 atm),
then diluted with methanol (20 mL) and 1 mL of 2.0 M
ammonia in ethanol. The catalyst was removed by filtration
and the filtrate concentrated. The residue was dissolved in
ethyl acetate (20 mL) and dried over anhydrous Na2SO4,
filtered and evaporated to afford 24 mg of crude product
14b, which was used without further purification.
4.1.22. (S)-N-[2-(4-tert-Butoxyphenyl)-2-methoxyethyl]-
3-(4-hydroxy-3-methoxyphenyl)acrylamide (16). Pre-
pared using the procedure described in Section 4.1.14.
[a]D22.2¼224.08 (c 0.5, MeOH); 1H NMR (500 MHz,
CDCl3): d 3.26 (s, 3H), 3.30 (m, 1H), 3.82 (s, 3H), 3.83
(m, 1H), 3.93 (s, 3H), 4.27 (dd, 1H, J¼4.0, 9.0 Hz), 5.81 (s,
1H), 5.99 (m, 1H), 6.26 (d, 1H, J¼15.0 Hz), 6.91 (d, 1H,
J¼8.0 Hz), 6.92 (d, 2H, J¼8.5 Hz), 7.01 (d, 1H, J¼2.0 Hz),
7.07 (dd, 1H, J¼2.0, 8.0 Hz), 7.26 (d, 2H, J¼8.5 Hz), 7.56
(d, 1H, J¼15.0 Hz); 13C NMR (100.5 MHz, CDCl3): d
45.97 (CH2), 55.49 (CH3), 56.15 (CH3), 56.79 (CH3), 82.13
(CH), 109.79 (CH), 114.24 (2£CH), 114.93 (CH), 118.41
(CH), 122.44 (CH), 127.59 (C), 128.18 (2£CH), 131.23 (C),
141.38 (CH), 146.93 (C), 147.61 (C), 159.76 (C), 166.26
(C); FT-IR (film, nmax, cm21): 3600–3100s (br), 2979m,
2932m, 2858m, 1647s, 1516s, 1272s,1205s, 1162m, 1115s,
Acknowledgements
We thank the Harvard Center for Neurodegeneration and
Repair (HCNR) for financial support and the Department of
Chemistry at Tufts University for use of their digital
polarimeter. In addition, we would like to thank Drs C. Lee

X. Xing et al. / Tetrahedron 59 (2003) 9961–9969
9969
16. (a) Corey, E. J.; Bakshi, R. K.; Shibata, S. J. Am. Chem. Soc.
1987, 109, 5551–5553. (b) (S)-2-Methyl-CBS-oxazaboroli-
dine is also known as (S)-tetrahydro-1-methyl-3,3-diphenyl-
1H,3H-pyrrolo[1,2-c][1,3,2]oxazaborole.
and Y. Kho (Korea Research Institute of Bioscience and
1
Biotechnology) for H and 13C NMR spectra of 2a and
Professor Paul Hergenrother (University of Illinois) for
sharing the results of his evaluation of 2a, which was
independently prepared.
17. Corey, E. J.; Bakshi, R. K.; Shibata, S.; Chen, C.-P.; Singh,
V. K. J. Am. Chem. Soc. 1987, 109, 7925–7926.
18. Corey, E. J.; Shibata, S.; Bakshi, R. K. J. Org. Chem. 1988, 53,
2861–2863.
References
¨
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