Design and synthesis of α-ketoamides as cathepsin s inhibitors with potential applications against tumor invasion and angiogenesis

Article abstract of DOI:10.1021/jm100089e

A series of small molecules bearing an α-ketoamide warhead were synthesized and evaluated for their ability to inhibit cathepsin S, a key proteolytic enzyme upregulated in many cancers during tumor progression and metastasis. Most of the synthetic compounds were noncytotoxic, but several robustly inhibited cathepsin S (IC₅₀ < 10 nM) and potently suppressed cell migration, invasion, and capillary tube formation. These results highlight the potential of α-ketoamide therapy for preventing or delaying cancer spread.

Full text of DOI:10.1021/jm100089e

J. Med. Chem. 2010, 53, 4545–4549 4545
DOI: 10.1021/jm100089e
Design and Synthesis of r-Ketoamides as Cathepsin S Inhibitors with Potential Applications
against Tumor Invasion and Angiogenesis
Jo-Chun Chen,† Biing-Jiun Uang,† Ping-Chiang Lyu,‡ Jang-Yang Chang,§ Ko-Jiunn Liu,§ Ching-Chuan Kuo,§
Hsing-Pang Hsieh, Hsin-Chieh Wang,‡ Chao-Sheng Cheng,‡ Yi-Hsun Chang,§ Margaret Dah-Tsyr Chang,*,‡
Wun-Shaing Wayne Chang,*,§ and Chun-Cheng Lin*,†
†Department of Chemistry, ‡Department of Life Science, National Tsing Hua University, 101, Section 2, Kuang-Fu Road, Hsinchu 30013, Taiwan,
§National Institute of Cancer Research, and Division of Biotechnology and Pharmaceutical Research, National Health Research Institutes, 35,
Keyan Road, Zhunan 35053, Taiwan
Received January 21, 2010
A series of small molecules bearing an R-ketoamide warhead were synthesized and evaluated for their
ability to inhibit cathepsin S, a key proteolytic enzyme upregulated in many cancers during tumor
progression and metastasis. Most of the synthetic compounds were noncytotoxic, but several robustly
inhibited cathepsin S (IC50 < 10 nM) and potently suppressed cell migration, invasion, and capillary
tube formation. These results highlight the potential of R-ketoamide therapy for preventing or delaying
cancer spread.
Introduction
His164 and Asn184.11 The active form of Cat S cleaves target
substrates through nucleophilic attack by the thiolate anion of
Cys25 (forming an ion pair with His164) to the carbonyl
carbon of the peptide bond followed by hydrolysis of the
resulting thioester.11 On the basis of this proteolytic mecha-
nism, a variety of peptide analogue inhibitors have been
designed to irreversibly12,13 or reversibly14 inactivate Cat S.
Although most irreversible inhibitors, such as low-selective
LHVS (N-morpholinurea-leucine-homophenyl-alanine-vinyl-
sulfone-phenyl)12 or E-64 (trans-epoxy succinyl-L-leucylamido-
(4-guanidino)butane),13 form covalent complexes with Cat S to
inactivate the enzyme, they may trigger undesired immune
responses and side effects. In contrast, some reversible inhibi-
tors show less toxicity, but their inhibitory activities against Cat
S are not as substantial. Here, we report the design and
synthesis of a new class of noncytotoxic R-ketoamides as potent
Cat S inhibitors. These compounds not only robustly inhibit
Cat S but also exert profound anti-invasive effects on CL1-5
cells of lung adenocarcinoma (the most common form of
lung cancer) and antiangiogenic activities against HUVEC cells
(the representative model of endothelial cells undergoing
angiogenesis).
For most cancer patients, tumor invasion and distant
metastasis are the major limitations of successful treatment.1
It is therefore important to develop effective strategies to
delay, if not prevent, malignant cells from spreading and
forming secondary metastatic colonies. Cathepsin S, also
known as Cat S or CTSS,a is a cellular protease required
for invasion and angiogenesis of cancers.2,3 This proteolytic
enzyme is localized primarily in lysosomes and participates in
the generation of class II major histocompatibility complex
(MHC)-antigenic peptide complexes.4 It has been suggested
that Cat S can also be translocated to the cell surface or
secreted into the extracellular milieu to help degrade extra-
cellular matrix (ECM) components.5 Unlike other members
of the cathepsin family, Cat S is unique because it is highly
activeand stableat neutral pH,6 properties compatible with its
distinct role in mediating matrix degradation and cell inva-
sion. Consistent with these characteristics, an increase in Cat S
expression is often associated with tumor progression and
adverse outcome.7,8 Recently, a Cat S specific antibody
termed Fsn0503 has been generated and shown not only to
inhibit tumor cell invasion but also to block endothelial cell
capillary tube formation.9 These studies indicate that Cat S
may be a prognostic indicator and a molecular target for
anticancer drug development.2,3,9,10
Results and Discussion
Chemistry. As shown in Figure 1, a known cyano com-
pound, 1, was previously demonstrated to inhibit Cat S with
an IC50 of 6 nM.14 Recently, several ketone-based inhibitors
have been identified as promising electrophilic warhead
candidates with oral bioavailability.15 Prompted by these
observations, we designed and optimized a series of Cat S
inhibitors derived from compound 1; the P1 and P2 substit-
uents were retained and the cyano warhead was replaced
with an R-ketoamide (Figure 1). To increase the affinity
between the synthetic inhibitor and Cat S, various P3
substituents were incorporated by coupling with different
acids. Initially, the serine-based aldehyde 2 was synthesized
from Boc-Ser-OH (Scheme 1). This compound then underwent
As a cysteine protease, Cat S contains a catalytic triad
formed by the unpaired Cys25 residue along with residues
*To whom correspondence should be addressed. For L.C.C.: phone,
þ886-3-5753147; fax, þ886-3-5711082; E-mail, cclin66@mx.nthu.edu.tw.
For W.S.W.C.: E-mail, wayne@nhri.org.tw. For M.D.T.C.: E-mail,
lscmdt@life.nthu.edu.tw.
a Abbreviations: Cat S or CTSS, cathepsin S; Cat L, cathepsin L; Cat
B, cathepsin B; MHC, major histocompatibility complex; HUVEC,
human umbilical vein endothelial cells; ECM, extracellular matrix;
DMSO, dimethyl sulfoxide; EDC, 1-ethyl-3-(3-dimethylaminopropyl)-
carbodiimide; TEMPO, 2,2,6,6-tetramethylpiperidine-1-oxyl; MTT,
3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide; AMC,
7-amino-4-methyl coumarin.
r
2010 American Chemical Society
Published on Web 05/18/2010
pubs.acs.org/jmc

4546 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 11
Chen et al.
Table 1. Inhibition of Cat S Enzymatic Activity and Cellular Migration
by R-Ketoamide-Based Inhibitors
Figure 1. Proposed structural optimization of compound 1, a
cyano-based inhibitor of Cat S. The dashed box (right) represents
the ketone-based warhead.
Scheme 1. Synthesis of R-Ketoamide Inhibitorsa
a Reagents and conditions: (a) BnBr, NaH, DMF, 0 °C; (b) N-methyl-
morpholine, isobutyl chloroformate, CH2Cl2, -20 °C, then NaBH4,
H2O, 0 °C (80% yield for two steps); (c) Dess-Martin periodinane,
CH2Cl2, rt (96%); (d) Boc-Cha-OH, ethyl isocyanoacetate, CH2Cl2,
rt (75%); (e) TFA, CH2Cl2, rt, then Et3N, rt (62%); (f) 4-morpholine-
carbonyl chloride, Et3N, DMF, rt (82%); (g) R1COOH (789 different
acids were used), HBTU, Et3N, DMF. The Dess-Martin oxidation of
hydroxyl amide intermediates gave R-ketoamide inhibitors as a mixture
of diastereomers at the R-center of the P1 site,19,20 with diastereomeric
ratios being 2:1 to 1:1 (S,S:R,S).
a DR = Diastereomeric ratio of the compounds was determined by
1H NMR spectrometry in CDCl3. b The DMSO control group represents
cells (2 ꢀ 105) treated with a mixture of DMSO (10 μL) and medium
(2 mL) per assay well. c HUVEC cells are very sensitive to 6g in low
serum condition, thus the antimigration effect of 6g was not evaluated.
d The P2 of 6r is L-leucine. e N.D.: not determined.
the key multicomponent Passerini reaction16 with N-Boc-
protected cyclohexyl alanine and ethyl isocyanoacetate to
yield compound 3. Acidic deprotection of the N-Boc moieties
on 3 was followed by O- to N-acyl group migration to
generate the R-hydroxyl dipeptide 4 in high yield by adjust-
ing the solution pH to 9.0 with triethylamine.17 The
R-ketoamide precursor 4 was then coupled with 789 different
acids to generate a library of R-hydroxyl compounds (5).
Some of the selected library compounds were subsequently
oxidized using Dess-Martin reagent to yield corresponding
R-ketoamides 6a-6t (Scheme 1).
Previous studies have suggested that R-hydroxyl amides
may be protease inhibitors18 even though they are less
effective inhibitors than the corresponding R-ketoamides.
Thus, to facilitate the identification and development of
potent Cat S inhibitors, the crude R-hydroxyl amides were
subjected to 96-well ELISA-based assays (Supporting
Information (SI) Figure S1) to quantify their effects on the
activity of Cat S. Only those compounds that inhibited Cat S
activity by more than 30% at 100 μM were selected as target
compounds. Of a total 789 crude R-hydroxyl amides
screened using this method, 30 compounds were found to
exhibit significant inhibitory activity against Cat S. These
molecules were then resynthesized and purified as pure
R-ketoamides and the IC50 values were determined in vitro
(Table 1 and SI Table S1).
During the synthesis of derivatives with a hydroxyl or
amino group at the P3 position, we observed that the yields
of desired products were low in the last oxidation step using
Dess-Martin reagent. Alternative attempts using mild

Brief Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 11 4547
Scheme 2. Synthesis of R-Ketoamide Inhibitors with Amino
Group at P3 Sitea
Figure 2. Comparison of inhibition of cell invasion and tube for-
mation by R-ketoamides 6i, 6p, and 6r. (A) Human high-invasive
CL1-5 lung cancer cells were applied for the in vitro invasion assay
using the FluoroBlok invasion system (www.bdbiosciences.com) to
examine the anti-invasive effects of the synthetic molecules. The
results show that, in comparison with the invasiveness of control
cells in dimethyl sulfoxide (DMSO) set as 100%, the addition of
compounds 6i, 6p, and 6r (schematically represented on top)
resulted in a significant decrease of cell invasiveness by 82%,
67%, and 68%, respectively (lower panel). Bar graphs reflect the
mean percent CL1-5 cell traversal and invasion relative to the
control DMSO group ( standard deviation (SD) from hexaplicate
experiments. (B) In vitro HUVEC capillary tube formation assays
were performed using 96-well plates coated with BD Matrigel. The
cells were treated with R-ketoamides in the presence of VEGF
(20 ng/mL). Cultures were photographed after 16 h of incubation
and capillary tube formation was determined by counting under
microscope (original magnification 100ꢀ). While VEGF alone was
shown to stimulate the branch point formation (top panel), the
addition of R-ketoamides 6i, 6p, and 6r resulted in a substantial
reduction of VEGF-induced capillary-like tube formation by 36%,
57%, and 32%, respectively (lower panel). Data reflect mean tube
formation relative to the control (DMSO) group ( standard devia-
tion (SD) from three separate experiments.
a Reagents and conditions: (a) allyl bromide, Cs2CO3, DMF, rt (95%);
(b) (Boc)2O, MeOH, rt (25%); (c) Pd(PPh3)4, MeNHPh. THF, rt (90%);
(d) 4, HBTU, Et3N, DMF, rt (83%); (e) Dess-Martin periodinane,
CH2Cl2, rt (78%); (f) TFA, CH2Cl2, rt (86%).
oxidation reagents such as 1-ethyl-3-(3-dimethylaminopropyl)
carbodiimide (EDC)/dichloroacetic acid, EDC/pyridinium
trifluoroacetate, or 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO)
also resulted in low yields (i.e., less than 5%, 27%, and 15%,
respectively). This problem was finally circumvented by pro-
tection of the amino (or hydroxyl) group at P3 before oxida-
tion (Scheme 2). For instance, in the synthesis of compounds
6p and 6q, the carboxylic acid moiety of aminobenzoic acid
was protected as an allyl ester followed by amine protection
using di-tert-butyldicarbonate (Boc2O). However, the low
reaction rate between the poorly nucleophilic aromatic amine
and Boc2O resulted in a low yield. The yield of the protected
product was improved by adding protic solvents such as
methanol21 to give 7a and 7b. Removal of the allyl group
was followed by coupling with core structure 4 to generate
compounds 8a and 8b, which were oxidized and then depro-
tected under acidic conditions to produce R-ketoamides 6p and 6q.
Notably, compounds 6s and 6t, which do not possess inhibitory
activity against Cat S, were synthesized with intent to serve as the
negative control compounds in the subsequent bioassays.
Bioassays. The inhibition assays of human recombinant
Cat S, which was expressed in Escherichia coli (SI Figure S2),
were performed mainly as described previously.22 A total of
22 R-ketoamides were identified to inhibit Cat S with IC50
values less than 10 nM (Table 1 and SI Table S1), indicating a
successful screening strategy. Unlike cyano inhibitor 1,
which inhibits primarily against Cat S, some of the synthetic
R-ketoamides (i.e., 6a, 6b, 6f, 6n, and 6p) were found to exert
strong cross-inhibitory activity (at 40 nM) against cathepsin
L (SI Figure S3). Compound 6p was further revealed to
partially inactivate cathepsin B but required a higher con-
centration (100 nM) for inhibition (SI Figure S3). These
additional inhibitory activities may provide extra benefit for the
developed R-ketoamides as anticancer agents due to the in-
volvement of cathepsins L and B in malignant progression.4
Notably, the presence of amino group at the P3 site shows the
enhancement of activity (6p and 6q vs 6n and 6o). On the basis
of the IC50 values listed in Table 1, those efficient compounds
were subjected to cell-based assays to evaluate anti-invasive and
antiangiogenic activities. Human umbilical vein endothelial
cells (HUVEC) and high-invasive CL1-5 lung cancer cells, both
of which express high levels of Cat S, were used for the cell-
based bioassays. MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-di-
phenyl-tetrazolium bromide) proliferation assay was carried
out to examine the overall cytotoxicity of these peptidyl
R-ketoamides. Most compounds had no obvious cytotoxicity
at concentrations as high as 50 μM (SI Table S2). As sum-
marized in Table 1, some of the R-ketoamides inhibited cell
migration to varying degrees. It is of interest to note that few
compounds with the lowest IC50 values (i.e., 6e, 6l, and 6m) do
not correlate with their potency in inhibiting cell migration.
This may be best elucidated by their rapid decomposition
during the cell-based assays (SI Figure S4a-d). Compounds
6i, 6p, and 6r had an IC50 < 10 nM and exhibited the greatest
inhibition of both CL1-5 and HUVEC cell migrations (∼50%).
The promising bioactivities of these inhibitors may be explained
by the small molecule inhibitors that do not enter cells but have
a profound effect by targeting cell-surface or secreted cysteine
cathepsins. Because of leaving the intracellular cysteine cathe-
psins inside cells untouched, the toxicity of inhibitor was
minimized. Therefore, these three compounds were further
analyzed for their ability to inhibit CL1-5 invasiveness and
HUVEC angiogenic tube formation. As shown in Figure 2, all
three compounds were capable of significantly suppressing the
invasive and angiogenic properties of the test cells, with com-
pound 6p being the most suppressive. In addition, results from
ECM degradation assays revealed that these molecules can
repress the cleavage of fibronectin protein by Cat S in a dose-
dependent manner, thus suggesting a direct interaction of the
R-ketoamides with Cat S that, subsequently, leads to decreased
extracellular fibronectin degradation and tumor cell invasion
(unpublished observation).

4548 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 11
Chen et al.
Figure 3. Models of Cat S-inhibitor complexes generated using the LigandFit docking program. The interaction between 6p and Cat S is
illustrated at left and the interaction between 6t with Cat S is illustrated at right. Red dashed lines indicate hydrogen bonds.
To evaluate the Cat S binding characteristics of the
R-ketoamide inhibitors generated in this study, the LigandFit
docking program23 was used to dock various inhibitors with
Cat S (SI Figure S5). Two complex models, one with
compound 6p, a robust Cat S inhibitor, and another with
compound 6t, a weak Cat S inhibitor, were constructed and
are represented in Figure 3 (see also SI Figures S6 and S7).
Through charge-charge interaction, 6p was predicted to
utilize the electron-rich methyl ester group (CH3OOC) at
the P3 site to interact with the ammonium group of Lys64 in
Cat S, whereas no such negatively charged group is available
at the P3 site of 6t. Compounds with other extended nega-
tively charged substituents (NO2, OCH3, Cl, Br) at the P3
position also had better inhibitory activity than their counter-
parts lacking a charged substituent at P3. For example, the
IC50 values of 6n and 6o, which contain an N-Boc moiety at
the P3 site, are approximately 8-fold higher than those of
6p and 6q, respectively, which contain a NH2 moiety at P3.
Furthermore, formation of a unique hydrogen bond was
predicted between the P3 amino group of 6p and a carbonyl
oxygen of Gly62 in Cat S, whereas 6t has a rather large
hydrophobic pyrene group at the P3 site which may limit its
flexibility and its ability to inhibit Cat S. It should also be
noted that the target compounds listed in Table 1 have a
negative charge extending from the warhead (carbonyl
group of the ethyl ester) available for interaction with the
S10 site (Arg141) of Cat S that subsequently stabilize the
active site region. The docked models also suggest that an
oxygen atom from the R-ketoamide of the inhibitor is near
the sulfur atom of Cys25 of Cat S, which may allow forma-
tion of a reversible thiohemiketal bond.11,24 This is sup-
ported by the observation that the IC50 values of the R-keto-
amides are approximately 100- to 1900-fold lower than
those of the corresponding R-hydroxyl-based structures.
For example, the IC50 value of 6a is 4.4 nM, whereas the corres-
ponding R-hydroxyl compound 5 is greater than 10 μM.
The double-reciprocal plot shown in SI Figure S8, in which
Z-VVR-AMC (AMC, 7-amino-4-methyl coumarin) is used
as a substrate, confirms that 6a is a good competitive inhi-
bitor of Cat S.
activity measurement was developed at the present study. The
screening results revealed that some 30 compounds from 789
R-hydroxyl amides exert potent Cat S inhibition with IC50
lower than 10 nM. In particular, compounds 6i, 6p, and 6r
were found to not only decrease cell migration and invasive-
ness but also inhibit endothelial tube formation. Such promis-
ing in vitro characteristics suggest that the newly synthesized
R-ketoamide Cat S inhibitors may give rise to alternative drug
therapies to prevent or delay the spread of malignant tumor
cells.
Experimental Section
Glycine, N-[(1,1-Dimethylethoxy)carbonyl]-cyclohexyl-L-alanyl-
(2S)-3,4,5-trideoxy-3-[[(1,1-dimethylethoxy)carbonyl]amino]-4-
benzyloxy-L-glycero-butanoyl, Ethyl Ester (3). Aldehyde 2 (1.0 g,
3.6 mmol), Boc-Cha-OH (0.981 g, 3.9 mmol), and ethyl iso-
cyanoacetate (432 μL, 3.9 mmol) were dissolved in dry CH2Cl2
(20 mL), and the resulting solution was stirred for 48 h at room
temperature (rt). The mixture was then concentrated and puri-
fied by silica gel chromatography (hexane/EtOAc = 2/1, Rf =
0.5) to give isomers 3 as a yellow syrup (1.8 g, 75%). FAB-
HRMS (m/z) [M þ H]þ 624.3489. Calcd for [C31H50N3O10]þ:
624.3496.
Glycine, N-[(3S)-4-Benzyloxy-3-[[[1-[(4-morpholinylcarbonyl)-
amino]cyclohexyl-L-alanyl]carbonyl]amino]-2-hydroxybutanoyl],
Ethyl Ester (5a). Compound 3 (1.8 g, 2.7 mmol) was dissolved in
50% TFA (5 mL) in CH2Cl2 (10 mL), and the resulting solution
was stirred for 30 min and then concentrated. The residue was
redissolved in CH2Cl2 and neutralized with triethylamine. The
solution was stirred for 10 min and then concentrated. The
resulting mixture was dried under vacuum to give isomers 4,
which were used for next step without further purification. To a
solution of compound 4 (0.4 g, 0.9 mmol), 4-morpholinecarbonyl
chloride (0.1 mL, 1.0 mmol) in DMF (9 mL) was added Et3N
(0.4 mL, 2.7 mmol) and the resulting solution was stirred for
overnight. The solvent was removed under reduced pressure,
and the resulting mixture was diluted with EtOAc. The organic
layer was washed with water, dried over MgSO4, filtered, and
concentrated. The mixture was purified by silica gel chromato-
graphy (EtOAc, Rf = 0.2) to give 5a as a white syrup (415 mg,
82%). ESI-HRMS (m/z) [M þ H]þ 577.3228. Calcd for [C29H45-
N4O8]þ: 577.3237.
Glycine, N-[(3S)-4-Benzyloxy-3-[[[1-[(4-morpholinylcarbonyl)-
amino]cyclohexyl-L-alanyl]carbon-yl]amino]-1,2-dioxobutyl], Ethyl
Ester (6a). To a solution of compound 5a (86 mg, 0.15 mmol) in
CH2Cl2 (2 mL) was added Dess-Martin periodinane (0.19 g, 0.45
mmol), and the resulting solution was stirred for 2 h at rt. The
mixture was quenched with Na2S2O3 in saturated bicarbonate
solution (2 mL). The residue was extracted with CH2Cl2. The
organic layer was washed with H2O, dried over MgSO4, filtered,
Conclusion
This report describes the design and synthesis of a series of
noncytotoxic R-ketoamide compounds. These small mole-
cules target and suppress the proteolytic activity of cathepsin
S, a pivotal enzyme involved in the progression of cancer. A
96-well ELISA-based method for rapid synthesis and effective

Brief Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 11 4549
and concentrated. The residue was purified by silica gel chromato-
graphy (EtOAc, Rf =0.4) to give 6a as a white syrup (76 mg,
89%; purity >99%). 1H NMR (CDCl3, 400 MHz, mixture of two
diastereomers) δ 7.51-7.46 (m, 1H), 7.33-7.17 (m, 5H), 7.09 (d,
J = 5.8 Hz, 1H), 5.46-5.42 (m, 1H), 5.12 (s, 1H), 4.52-4.45 (m,
1H), 4.42 and 4.44, 4.38 (s and d, J = 13.8 and 12.0 Hz, 2H), 4.20
and 4.19 (q, J = 7.2 and 7.2 Hz, 2H), 4.14 and 4.13 (dd, J = 9.8,
3.6 and 10.0, 3.8 Hz, 1H), 4.00-3.97 (m, 2H), 3.76 and 3.74 (t, J =
3.3 and 3.3 Hz, 1H), 3.63-3.60 (m, 4H), 3.35-3.28 (m, 4H), 1.74
(d, J = 12.6 Hz, 1H), 1.66-1.57 (m, 5H), 1.51-1.44 (m, 1H),
1.36-1.29 (m, 1H), 1.26 and 1.25 (t, J = 7.1 Hz, 3H), 1.22-1.04
(m, 3H), 0.96-0.78 (m, 2H). 13C NMR (CDCl3, 100 MHz,
mixture of two diastereomers) δ 193.0, 192.9, 173.5, 168.6,
159.2, 157.5, 157.3, 137.2, 128.3, 127.8, 127.6, 127.6, 73.3, 69.4,
69.3, 66.4, 61.8, 55.5, 55.4, 52.1, 51.9, 44.0, 43.9, 41.0, 40.3, 40.0,
34.1, 34.0, 33.5, 32.8, 32.7, 26.3, 26.1, 26.0, 14.1. ESI-HRMS (m/z)
[M þ H]þ 575.3090. Calcd for [C29H43N4O8]þ: 575.3081.
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benzoyl)amino]cyclohexyl-L-alanyl]carbonyl]amino]-1,2-dioxo-
butyl], Ethyl Ester (6q). To a solution of compound 6o in
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solution was stirred for 30 min and then concentrated. The
residue was purified by silica gel chromatography (hexane/
EtOAc=1/1, Rf = 0.3) to give 6p as a white syrup (0.022 g,
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1
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(12) Bromme, D.; Klaus, J. L.; Okamoto, K.; Rasrick, D.; Palmer, J. T.
78%; purity >99%). H NMR (CDCl3, 400 MHz, mixture of
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two diastereomers) δ 7.36 (t, J=5.1 Hz, 1H), 7.28-7.21 (m, 5H),
7.19-7.17 (m, 2H), 7.10 and 6.95 (d, J = 7.2 and 7.6 Hz, 1H),
6.99 (dd, J = 8.2, 2.0 Hz, 1H), 6.64 (d, J = 8.1 Hz, 1H),
5.47-5.44 (m, 1H), 4.77-4.70 (m, 1H), 4.43 and 4.43, 4.39 (s and
d, J = 11.8 and 12.0 Hz, 2H), 4.22 (q, J = 7.2 Hz, 2H), 4.16 (dd,
J = 9.8, 3.5 Hz, 1H), 4.01 and 3.98 (dd and d, J = 5.4, 3.2, and
5.6 Hz, 2H), 3.78 and 3.76 (dd, J = 9.9, 3.3 Hz, 1H), 1.81-1.56
(m, 7H), 1.35 (s, 1H), 1.28 (t, J = 7.2 Hz, 3H), 1.25-1.14 (m,
3H), 1.01-0.86 (m, 2H). FAB-HRMS (m/z) [M þ H]þ 615.2592.
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Acknowledgment. This article was supported by National
Science Council (98-2323-B-007-001, 97-2752-B-007-001-PAE,
97-2752-B-007-002-PAE, 97-2752-B-007-003-PAE, and 97-2752-
B-400-001-PAE), National Tsing Hua University (95N2517E1),
National Health Research Institutes (CA-096-PP-27), and
Department of Health, Taiwan (DOH97-TD-G-111-014
and DOH99-TD-C-111-004). We thank Dr. Tian-Ren Lee,
Yi-Mei Hong, Shu-Chuan Lin, Pei-Yu Lee, Yi-Ting Shih,
Hsin-Tzu Chang, Hsin-Ru Wu, Chun-Wei Chen, Kuo-Chang
Cheng, Li-Hsiung Lin, Wen-Yang Lai, Sheng-Chieh Lin,
Tain-Sheng Tzeng, Meng-Chi Sun, and Ghien-Hung Chiang
for technical assistance.
Supporting Information Available: Experimental details for
chemistry, purity, retention time, and characterization of target
compounds; enzyme inhibition and cell-based bioassays. This
material is available free of charge via the Internet at http://
pubs.acs.org.
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