"Clicked" bivalent ligands containing curcumin and cholesterol as multifunctional Aβ oligomerization inhibitors: Design, synthesis, and biological characterization

Article abstract of DOI:10.1021/jm100601q

In our effort to develop multifunctional compounds that cotarget beta-amyloid oligomers (AβOs), cell membrane/lipid rafts (CM/LR), and oxidative stress, a series of bivalent multifunctional Aβ oligomerization inhibitors (BMAOIs) containing cholesterol and curcumin were designed, synthesized, and biologically characterized as potential treatments for Alzheimer's disease (AD). The in vitro assay results established that the length of spacer that links cholesterol and curcumin and the attaching position of the spacer on curcumin are important structural determinants for their biological activities. Among the BMAOIs tested, 14 with a 21-atom-spacer was identified to localize to the CM/LR of human neuroblastoma MC65 cells, to inhibit the formation of AβOs in MC65 cells, to protect cells from AβOs-induced cytotoxicity, and to retain antioxidant properties of curcumin. Furthermore, 14 was confirmed to have the potential to cross the blood-brain barrier (BBB) as demonstrated in a Caco-2 cell model. Collectively, these results strongly encourage further optimization of 14 as a new hit to develop more potent BMAOIs.

Full text of DOI:10.1021/jm100601q

6198 J. Med. Chem. 2010, 53, 6198–6209
DOI: 10.1021/jm100601q
“Clicked” Bivalent Ligands Containing Curcumin and Cholesterol As Multifunctional
Aβ Oligomerization Inhibitors: Design, Synthesis, and Biological Characterization
James A. Lenhart,† Xiao Ling,† Ronak Gandhi,† Tai L. Guo,‡ Phillip M. Gerk,§ Darlene H. Brunzell,‡ and Shijun Zhang*,†
§
†Department of Medicinal Chemistry, ‡Department of Pharmacology and Toxicology, and Department of Pharmaceutics,
Virginia Commonwealth University, Richmond, Virginia 23298
Received May 17, 2010
In our effort to develop multifunctional compounds that cotarget beta-amyloid oligomers (AβOs), cell
membrane/lipid rafts (CM/LR), and oxidative stress, a series of bivalent multifunctional Aβ oligomer-
ization inhibitors (BMAOIs) containing cholesterol and curcumin were designed, synthesized, and
biologically characterized as potential treatments for Alzheimer’s disease (AD). The in vitro assay
results established that the length of spacer that links cholesterol and curcumin and the attaching
position of the spacer on curcumin are important structural determinants for their biological activities.
Among the BMAOIs tested, 14 with a 21-atom-spacer was identified to localize to the CM/LR of human
neuroblastoma MC65 cells, to inhibit the formation of AβOs in MC65 cells, to protect cells from AβOs-
induced cytotoxicity, and to retain antioxidant properties of curcumin. Furthermore, 14 was confirmed
to have the potential to cross the blood-brain barrier (BBB) as demonstrated in a Caco-2 cell model.
Collectively, these results strongly encourage further optimization of 14 as a new hit to develop more
potent BMAOIs.
Introduction
dementia.4,5 Despite the fact that multiple assemblies of AβOs
and a variety of underlying mechanisms have been suggested
in the literature,6-11 one point of consensus remains clear: the
requirement of AβOs. Collectively, these findings provide
compelling support for developing Aβ oligomerization inhi-
bitors as novel therapeutic agents for the treatment of AD.
Increased oxidative damage by reactive oxygen species (ROS)
and reactive nitrogen species is another feature consistently
found in the brains of AD patients.2,12 Many factors have
been demonstrated to cooperatively contribute to the produc-
tion of ROS in the AD brain such as biometals, mitochondria
dysfunction, and Aβ.13 Transgenic mouse studies have also
shown a correlation of increased oxidative stress and Aβ
accumulation.14
Recently, a wealth of data has implicated the roles of
neuronalcellmembrane/lipid rafts (CM/LR) in the oligomeri-
zation and toxicity of Aβ.15,16 Once associated with the mem-
branes, Aβ exhibits an enhanced rate of aggregation that is
dependent on pH, metal ion, and ganglioside interactions.17-19
Recently, evidence has also indicated that lipid rafts, a cell
membrane microdomain enriched in cholesterol and sphingo-
lipids, can accelerate the cell membrane binding of Aβ and
AβOs formation.15,16 On the other hand, destruction of lipid
rafts affects Aβ membrane binding and protects cells from Aβ
toxicity.20 Furthermore, Aβ precursor protein (APP), APP
cleavage enzymes (β- and γ-secretases), Aβ and AβOs have all
been identified in lipid rafts, suggesting that lipid rafts may be
a critical platform for Aβ production and oligomerization.21
In addition, oxidative stress has been shown to upregulate
presenilin-1, the critical component of γ-secretase, in lipid
rafts of neuronal cells to promote Aβ production.22 Alto-
gether, it is apparent that CM/LR are important regulators in
Alzheimer’s disease (ADa) is a devastating neurodegenera-
tive disease and is the most common cause of dementia. The
etiology of AD still remains elusive, and multiple factors have
been suggested to contribute to the development of AD,
among which amyloid-β (Aβ) and oxidative stress have been
well documented.1,2 Recently emerging evidence indicates
that small Aβ oligomers (AβOs), rather than insoluble Aβ
fibrils, are responsible for disruption of neuronal synaptic
plasticity and the resulting early cognitive impairment asso-
ciated with AD.3 Studies of brain samples from AD patients
also confirmed the correlation of AβOs with the severity of
*Correspondence address: Shijun Zhang, Ph.D., Department of
Medicinal Chemistry, School of Pharmacy, Virginia Commonwealth
University, Richmond, Virginia 23298-0540. Tel: 804-6288266. Fax:
804-8287625. E-mail: szhang2@vcu.edu.
a Abbreviations: Aβ, amyloid-β; AβOs, amyloid-β oligomers; AD,
Alzheimer’s disease; APP, Aβ precursor protein; ATCC, American Type
Culture Collection; BBB, blood-brain barrier; BMAOIs, bivalent
multifunctional Aβ oligomerization inhibitors; CHO, Chinese hamster
ovary; CM/LR, cell membrane/lipid rafts; CTX-B, cholera toxin sub-
unit B; DAPI, 40,6-diamino-2-phenylindole; DBU, 1,8-diazabicycloun-
dec-7-ene; DCFH-DA, dichlorofluorescein diacetate; DIC, differential
interference contrast; DMF, dimethylformamide; DMEM, Dulbecco’s
modified eagle’s medium; DMSO, dimethyl sulfoxide; EDC, 1-ethyl-
3-(3-dimethylaminopropyl)-carbodiimide; ELISA, sandwich enzyme-
linked immunoassays; FBS, fetal bovine serum; HBSS, Hank’s balanced
salt solution; HOBt, hydroxybenzotriazole; HPLC, high performance
liquid chromatography; NAC, N-acetylcysteine; NK, natural killer;
PBS, phosphate buffered saline; PMA, phosphomolybdic acid; PVDF,
polyvinylidene fluoride; ROS, reactive oxygen species; SDS-PAGE,
sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SEM, stan-
dard error of mean; TBS, Tris buffered saline; TC, tetracycline; TEM,
transmission electron microscope; TFA, trifluoroacetic acid; THF,
tetrahydrofuran; TLC, thin-layer chromatography; TMS, tetramethyl-
silane.
r
pubs.acs.org/jmc
Published on Web 07/28/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 16 6199
AD development and this relationship can be exploited to
design and develop novel AD therapies.
distinct ligands with the potential to overcome the limits
posted by the traditional single-factor based approach. Con-
ceptually, these BMAOIs contain a multifunctional AβO-
inhibitor pharmacophore that accommodates additional anti-
oxidant properties as well as a CM/LR anchor pharmaco-
phore linked by a spacer (Figure 1). The use of bivalent
strategies to explore protein-protein interactions has been
particularly successful in opioid receptor research field.28 Re-
cently, this concept has been extended to neurodegenerative
diseases in developing acetylcholinesterase inhibitors and me-
tal chelators.25 We envisaged that such BMAOIs would
chaperone the multifunctional AβO-inhibitor moiety in close
proximity to CM/LR in which AβOs and oxidative stress are
produced to increase its accessibility to interfere with these
multiple processes, thus improving its clinical efficacy (Figure 1).
In this report, we rationally designed, synthesized, and bio-
logically characterized a series of BMAOIs, and one com-
pound was identified as a new hit for further investigation.
Numerous chemical ligands have been developed as poten-
tial AD treatments by targeting Aβ and oxidative stress.23,24
However, very few of themmoved toclinical trialsand noneof
them has been approved by FDA, which suggests that target-
ing a single risk factor is not an ideal strategy for developing
treatments for this multifaceted disease. In contrast, new
approaches that cotarget multiple risk factors involved in
AD are emerging as promising strategies for developing effec-
tive treatment agents for AD.25-27 Herein, we hypothesized
that a bivalent multifunctional Aβ oligomerization inhibitors
(BMAOIs) strategy that targets AβOs, oxidative stress, and
CM/LR would be a novel approach to design strategically
Design and Chemistry
The desired BMAOIs must contain an AβO-inhibitor
moiety with intrinsic antioxidant effects, as well as incorpo-
rate a residue able to efficiently interact with CM/LR, span-
ned by a stable linkage. Thus, in our designed BMAOIs,
curcumin (1) was selected as the multifunctional AβO-inhi-
bitor pharmacophore, and on the other end, connected by a
spacer, cholesterol (2) was selected as the anchor pharmaco-
phore to the CM/LR (Figure 2). The selections of 1 and 2 were
based on the following reasons: (1) 1 is an important phyto-
chemical that has long been known for its antioxidant, anti-
inflammatory properties as well as recently discovered anti-
Aβ properties;29-32 (2) it has been demonstrated that 2 and
Figure 1. BMAOIs strategy and design.
Figure 2. Chemical structures of 1, 2, designed BMAOIs and monovalent ligands.

6200 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 16
Scheme 1. Synthesis of intermediates 20 and 23a
Lenhart et al.
a Reagents and conditions: (a) propargyl bromide, K2CO3, DMF; (b) i. B2O3, acetylacetone; ii. (BuO)3B, piperidine; iii. 1 N HCl; (c) i. B2O3, ii. 16,
(BuO)3B, piperdine; iii. 1 N HCl; (d) propargyl bromide, DBU, benzene; (e) i. B2O3, 17, (BuO)3B, n-BuNH2; ii. 1 N HCl.
other sterols linked with another moiety can anchor CM/LR
in mammalian cells and function as a carrier to induce
internalization via endocytosis.33,34 The crucial consideration
in designing BMAOIs is to determine the loci on the two
pharmacophores for attaching the spacer and the nature and
lengthofthe spacer. Given the fact thatalkylation of the 3-OH
of 2/sterol does not affect their binding affinities to CM/
LR,33,34 we selected this position as the spacer attachment
position. On the other end, one of the phenolic oxygens and
the C-4 position (methylene carbon between the two carbonyl
groups) of 1 were selected to design two series of BMAOIs to
investigate the optimal attachment. Since it is not clear whet-
her Aβ oligomerization occurs on the surface or inside of CM/
LR and optimal spacer length range cannot be predicted from
the existing literature, we varied the spacer length as a key
parameter for investigation. Since the cell membrane thick-
ness is frequently cited as 3 nm (although ranging from 2.5 to
10 nm), we have decided to initially vary the spacer length
from 11-21 atoms (Figure 2). Two monovalent ligands (1 at-
tached to spacer but not cholesterol) (15 and 16) were also
designed to evaluate the influence of spacer attachment on 1’s
activity. Recently “click chemistry”35 methodology has been
successfully applied to connect 1 to peptides by Ouberai et al.36
Therefore, to efficiently assemble the two pharmacophores
together, we adopted this “click chemistry” methodology to
include a 1,4-disubstituted triazole ring in the spacer.
The synthesis began with the preparation of alkyne inter-
mediates 20 and 23 through a well established Pabon reaction
(Scheme 1).37 Briefly, alkylation of vanillin 17 with propargyl
bromide provided 18. Aldol reaction of 17 with 2,4-pentane-
dione followed by another Aldol reaction with 18 afforded
intermediate 20. Alkylation of 2,4-pentane-dione with pro-
pargyl bromide in the presence of 1,8-diazabicycloundec-7-
ene (DBU) in benzeneyielded 22 which on Aldol reaction with
17 afforded intermediate 23.
As shown in Scheme 2, carboxylic acid 25 was synthesized
following the reported procedure.34 Then, coupling reactions
of 25 with various azidoamines 26-31 which were synthesized
through coupling reactions of azidoalkylamines 26 and 27
with Boc protected β-alanine followed by Boc deprotection
afforded azido intermediates 32-37.
Scheme 2. Synthesis of Intermediates 32-37a
a Reagents and conditions: (a) tert-butyl-2-bromoacetate, NaH,
THF; (b) formic acid/Et2O; (c) i. Boc protected beta-alanine or Boc
protected beta-alanylalanine, EDC, HOBt, CH2Cl2; ii. TFA/CH2Cl2;
(d) EDC, HOBt, CH2Cl2.
butylamine with succinic anhydride followed by amide cou-
pling with 6-azidohexylamine achieved the synthesis of 15 or
16, respectively.
Results and Discussion
Inhibition of AβOs Production by Designed BMAOIs. The
rational design of BMAOIs targeting CM/LR and AβOs as
well as oxidative stress will require demonstration of antici-
pated effects in a biologically relevant system. The whole cell
assay is a composite of not only Aβ oligomerization inhibi-
tion, but also permeability, stability, and other factors will
validate the accessibility and function of our BMAOIs.
MC65 is a human neuroblastoma cell line that conditionally
expresses C99, the C-terminus fragment of APP using tetra-
cycline (TC) as transgene suppressor.38 Upon removal of
TC, these cells can produce intracellular Aβ aggregates
including small AβOs. Most importantly, the induced cyto-
toxicity in these cells by TC removal has been associated with
the accumulation of AβOs.39 Furthermore, oxidative stress
has been indicated as one potential effector to impart neuro-
toxicity upon the accumulation of intracellular AβOs in
these cells.40 Therefore, MC65 cells were initially employed
Once all the required intermediates were available, the click
reactions of the alkynes 20 or 23 with 32-37 were applied
under sodium ascorbate and CuSO4 in THF/H2O conditions
to obtain the designed BMAOIs 3-8 or 9-14, respectively
(Scheme 3). All the designed BMAOIs are in keto-enol forms
in chloroform judged by 1HNMR and 13CNMR. The synth-
esis of the monovalent compounds 15 or 16 is similar to the
synthesis of BMAOIs. Click reactions of 20 or 23 with azido
intermediate 38 which was synthesized from the reaction of

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 16 6201
Scheme 3. Synthesis of Designed BMAOIs and Monovalent Ligandsa
a Reagents and conditions: (a) CuSo4, sodium ascorbate, THF/H2O (1:1).
Figure 3. Inhibition of AβOs formation by 14 in MC65 cells and ML60 cells. (A) MC65 cells were cultured under þTC or -TC conditions for
varying intervals (0, 2, 18, 27 h), and then cell lysates were analyzed by Western blot using 6E10 antibody. (B) MC65 cells were treated with
indicated compounds (10 μM) for 24 h immediately after the removal of TC. Lysates from cultures were analyzed by Western blotting using
6E10 antibody. The image represents the results from one of three independent experiments. (C) ML60 cells were treated with test compounds
(10 μM) for 24 h and extracellular AβOs in conditioned medium were analyzed by ELISA. Data were expressed as mean percentage of AβOs
(n = 4) with parallel DMSO cultures set at 100%. Error bars represent standard error of mean (SEM).
to validate and test our BMAOIs using Western blot anal-
ysis. All BMAOIs were first evaluated at a single concentra-
tion of 10 μM. Candidate compounds with inhibitory
activities at this concentration were further evaluated in a
dose-dependent manner in the following assays. As shown in
Figure 3A, withdrawal of TC induced the production of
AβOs consistent with reported results.39 1 did not exhibit
inhibition on the formation of AβOs (Figure 3B). Spacer
attachment at both positions (15 and 16) did not change the
activity of 1. BMAOIs 3, 4 and 9, 10 (spacer length ranging
from 11 to 13 atoms) showed no inhibition on the formation
of small AβOs. BMAOIs 5-7 and 11-13 (spacer length
ranging from 15 to 19 atoms) slightly inhibited the formation
of AβOs with specific suppression of the 24-kDa bands.
Notably, among the BMAOIs tested, 14 (with 21 atoms in
the spacer) significantly inhibited AβOs production. This
may indicate that spacer length is an important structural
determinant for their inhibition on AβOs formation in
MC65 cells with a 21-atom-spacer best supporting the design
of BMAOIs tested here. Most importantly, it is notable that
8, with the same spacer length (21 atoms) as 14 but different
spacer attaching position on 1, did not show inhibitory
effects on AβOs formation, which suggests the importance
of attachment position on 1 as well. Next, another cell line,
ML60, was employed to evaluate the inhibition of AβOs
production. ML60 cell line is a line of Chinese hamster ovary
(CHO) cells stably expressing wild type APP and mutant
presenilin 1 (M146L missense mutation) and can specifically
produce high levels of extracellular AβOs.41 As shown in
Figure 3C, only 14 inhibited the production of extracellular
AβOs in ML60 cells, and surprisingly all the other com-
pounds increased the production of AβOs at a tested con-
centration (10 μM). It has been demonstrated that AβOs are
formed intracellularly and then excreted outside the cells.42
The results from ML60 cells may further reflect 14’s ability to
reduce intracellular AβOs, which is consistent with the re-
sults from MC65 cells. Altogether, these results suggest that
spacer length and attachment position on 1 are important

6202 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 16
Lenhart et al.
Figure 5. Binding interactions of 14 with Aβ42 and the CM/LR of
MC65 cells. (A) TEM image of AβOs. (B) Aβ42 (5 μM) was
incubated with or without compounds (20 μM), and then samples
were analyzed by Western blot using 6E10 antibody. Lane 1 - Aβ42
without incubation; Lane 2 - Aβ42 with incubation; Lane 3 - Aβ42
with 1; Lane 4 - Aβ42 with 14. (C) MC65 cells were treated and
imaged as in Figure 4. Left panel - differential interference contrast
(DIC) images of MC65 cells; central panel - fluorescence of 14 and 1;
right panel - overlay of left and central panels plus DAPI staining of
nucleus.
Figure 4. Immunocytochemistry of 1 and 14 in MC65 cells. MC65
cells were treated with the indicated compounds (10 μM) immedi-
ately after the removal of TC. After 24 h, the cells were fixed and
immunofluorescently stained for AβOs (red), CM/LR (green), and
nucleus (blue) and imaged with Leica TCS-SP2 AOBS confocal
laser scanning microscope. White arrows indicate the red puncta of
AβOs. The image represents one of five areas examined.
and inside of MC65 cells as well (bottom panel). 1 was
detected inside of MC65 cells but not on the cell membrane
(middle panel). The results demonstrate that 14 can directly
interact with CM/LR of MC65 cells and anchor the ligand
primarily to the CM/LR. Given the fact that Aβ aggregates
on the cell surface,17-19 the anchorage of 14 to CM/LR may
increase its target accessibility and consequently increase its
potency. Collectively, these results support our design ratio-
nale of using BMAOIs to cotarget AβOs and CM/LR.
Protective Effects of 14 on AβOs-Induced Cytotoxicity in
MC65 Cells and Differentiated Human Neuroblastoma SH-
SY5Y Cells. The production of intracellular AβOs has been
suggested to be the major factor leading to cytotoxicity in
MC65 cells.39 Therefore, to test whether the suppression of
AβOs formation by 14 correlate with functional activities, 14
was further evaluated for its protective effects on MC65 cell
viability upon removal of TC. As shown in Figure 6A, 1 and
14 exhibited no toxic effects at tested concentrations in the
presence of TC. Upon removal of TC, MC65 cell viability
was significantly decreased and 14 protected MC65 cell
survival in a dose-dependent manner with nearly full rescue
at 16 μM. 1 only exhibited minimal protective effects on
MC65 cell viability consistent with reported results.39 8 and
12 exhibited no protective effects under these conditions
(data not shown) which further suggests the importance of
spacer length and attachment position on their activities.
Together with the results from Western blot and immuno-
cytochemistry assays, these data suggest that the localization
of 14 to the CM/LR may increase 14’s target accessibility and
produce a more profound inhibition of the formation of
AβOs and elevate the survival of MC65 cells. To further
verify whether 14 can protect cells from extracellular AβOs-
induced cytotoxicity, all trans-retinoic acid differentiated
human SH-SY5Y cells were employed. As shown in Figure 6B,
freshly prepared AβOs (1 μM) from Aβ42 significantly
decreased SH-SY5Y cell viability (∼40% decrease). Not-
ably, 14 completely restored the cell viability at all of the
tested concentrations. On the other hand, 1 only exhibited
moderateprotective activities at2, 4, and8 μM concentrations
structural determinants for inhibitory activities on the for-
mation of AβOs and BMAOIs with optimal spacer length
can improve their potencies.
In order to further confirm the inhibition of small AβOs by
14 in MC65 cells, an AβO-specific antibody A1143 combined
with Alexa Fluor 568 conjugated secondary antibodies was
employed to detect the expression of AβOs in MC65 cells
using immunocytochemistry techniques. As shown in Figure
4, removal of TC induced rapid intracellular accumulation of
AβOs (red fluorescence puncta). Consistent with Western
blot results, 14 significantly inhibited the formation of AβOs
in MC65 cells upon the removal of TC. Surprisingly, 1 slig-
htly suppressed the formation of AβOs in this assay while it
exhibited no inhibitory effects on the formation of AβOs in
Western blot analysis. This might be due to the different
antibodies used for detection in these two assays with A11
antibody more specific to AβOs. In addition to confirming
Western blot data, these results also indicate that both 14 and
1 can cross the cell membrane of MC65 cells.
Interactions of 14 with AβOs and Cell Membrane of MC65
Cells. In order to confirm 14 can bind to AβOs, the inhibition
of Aβ42 oligomerization was performed and assessed using
Western blot analysis as described in the literature.29 As
shown in Figure 5A, Aβ42 formed oligomers under the
reported protocol as demonstrated by transmission electron
microscopy (TEM) analysis. After incubation in Ham’s F-12
medium for 4 h at 37 °C, higher order species of AβOs were
formed (Figure 5B, lane 2). Notably, both 1 and 14 inhibited
the oligomerization of Aβ42 (Figure 5B, lanes 3 and 4),
which demonstrates their direct binding to Aβ42. This fur-
ther confirms that the addition of spacer in 14 does not affect
its binding interactions with Aβ42. Next, immunocytochem-
istry studies were conducted to confirm the interactions of 14
with the CM/LR taking advantage of the intrinsic fluores-
cence of 14. As shown in Figure 5C, 14 was detected pri-
marily on the cell membrane of MC65 cells (yellow puncta)

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 16 6203
but not at 16 μM. This may be due to its toxic effect on SH-
SY5Y cells at this concentration since 1 has been reported
to have cytotoxicity on SH-SY5Y cells at higher con-
centrations.44 These results suggest that 14 can protect cells
from both intracellular and extracellular AβOs-induced
cytotoxicity, while 1 only exhibits protective activity toward
extracellular AβOs-induced cytotoxicity even though it can
cross the cell membrane under these experimental condi-
tions. This may further indicate that while both 1 and 14 can
bind to AβOs, CM/LR anchorage of 14 can increase its
accessibility to intracellular target AβOs. Since CM/LR are
crucial for many aspects of cell signaling and functions, 14
was further evaluated for its potential cytotoxicity in mouse
spleen and natural killer (NK) cells. 14 showed minimal cyto-
toxic effects in mouse spleen (Figure 6C) and no cytotoxic
effects in NK cells (Figure 6D). This suggests that localiza-
tion of BMAOIs to the CM/LR will not affect the normal
cellular functions. Taken together, it is clear that 14 is more
active than 1 in inhibiting the production of AβOs and in
protecting cells from the in situ AβOs-induced cytotoxicity.
Antioxidant Activity of 14. One of the BMAOIs design
goals is to reduce oxidative stress that potentially contributes
to the development of AD. Furthermore, oxidative stress has
been indicated as one potential effector to impart neurotoxi-
city upon the accumulation of intracellular AβOs in MC65
cells.40 Therefore, we decided to further evaluate the anti-
oxidant activity of 14 in MC65 cells. Despite the availability
of several chemical antioxidation assays, the ability to pre-
dict and correlate these chemical assays with in vivo activity
is questionable. In contrast, a cellular antioxidation assay
may provide a more biologically relevant system that best
addresses the permeability, distribution, and metabolism
issues to evaluate potential antioxidant properties. Recently,
a dichlorofluorescein diacetate (DCFH-DA) based cellular
antioxidant assay has been established and widely used for
this purpose.45 We therefore adopted this DCFH-DA assay
in MC65 cells to evaluate the antioxidant effects of 14 and 1.
As shown in Figure 7A, upon TC removal, intracellular oxi-
dative stress, as measured by fluorescence intensity, is signi-
ficantly increased compared to normal growing MC65 cells
in the presence of TC. Notably, both 14 and 1 suppressed the
intracellular oxidative stress in a dose-depndent manner.
These results may indicate that the curcumin moiety in 14
is responsible for its antioxidant activities. Although 1
exhibited antioxidant activities in this cellular model, it did
not protect MC65 cell survival (Figure 6A). To compare
whether other antioxidants can protect MC65 cells from
AβOs-induced cytotoxicity, N-acetylcysteine (NAC) and
trolox (6-hydroxy-2,5,7,8-tetramethyl chroman-2-carbo-
xylic acid), an analogue of vitamin E, were tested in MC65
cells. As shown in Figure 7B, trolox (32 μM) completely
Figure 6. Protective effects of 14. (A) MC65 cells were treated with
1 or 14 at indicated concentrations under þTC or -TC conditions
for 72 h. Cell viability was assayed by MTS assay. Data were
expressed as mean percentage viability (n = 6) with parallel þTC
cultures set at 100% viability. Error bars represent SEM. (B) All-
trans-retinoic acid differentiated SH-SY5Y cells were treated with
AβOs (1 μM) in the presence or absence of test compounds at
indicated concentrations for 48 h. Cell viability was assayed by MTS
assay. Data were expressed as mean percentage viability (n = 6)
with cultures without AβOs set at 100% viability. Error bars
represent SEM. (C) Effects of 14 (10 μM) on anti-CD3 antibody
mediated splenocyte proliferation. (D) Effects of 14 (10 μM) on IL-2
augmented NK cell activity in vitro. The experiments were per-
formed as described in the Experimental Section. Data were pre-
sented as mean (n = 4) ( SEM. *P < 0.05 indicates significant
differences from control group (without TC in A and without AβOs
in B) analyzed by one-way ANOVA.
Figure 7. Antioxidant effects and Caco-2 permeability of 14. (A) MC65 cells were treated with 1 or 14 at indicated concentrations under þTC
or -TC conditions for 24 h, and then DCFH-DA (25 μM) was loaded and fluorescence intensity was analyzed at 485 nm (excitation) and
530 nm (emission). Data were presented as mean percentage of fluorescence intensity (n = 5) with parallel -TC cultures set at 100%. Error bars
represent SEM. (B) MC65 protection was performed as described in Figure 6A with NAC (8 mM) or trolox (32 μM) (n = 5). (C) Caco-2 cells
were plated on transwell filters. Test compounds (10 μM) were added to either the apical or basolateral side, and then samples were analyzed by
HPLC to determine flux (A-B: apical-to-basolateral; B-A: basolateral-to-apical) at indicated time points. Data were presented as mean (n = 3)
( SEM. *P<0.05 indicates significant differences from control group (-TC) analyzed by one-way ANOVA.

6204 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 16
Lenhart et al.
rescued MC65 cells from AβOs-induced cytotoxicity, while
NAC (8 mM) rescued MC65 cells by 48% consistent with re-
ported results.40 Given the fact that NAC is mainly a hydro-
gen peroxide scavenger while trolox, a chain-breaking anti-
oxidant, is particularly effective against lipid peroxidation
within the cell membrane,46 these results may indicate lipid
peroxidation within the cell membrane as a major contribu-
tor underlying the mechanism of AβOs-induced cytotoxicity
in MC65 cells, which is consistent with the results from
Woltjer et al.47 The discrepancy of 1 and the other two anti-
oxidants in MC65 cell-protection may suggest that 1 either
cannot reach the targets or only partially suppress lipid
peroxidation in MC65 cells. Together with the results from
Western blot analysis, immunocytochemistry, and cell pro-
tection, the results of antioxidation assay further suggest that
14 can retain the antioxidant property of 1 while exhibiting
superior capability to reach intracellular AβOs by interact-
ing with the CM/LR, thus efficiently reducing the formation
of AβOs and ultimately exhibiting better overall protective
activities in these cells when compared to 1. This further
supports the idea that our BMAOIs strategy has the poten-
tial to provide clinically efficient multifunctional agents for
treatment of AD.
Assessment of Permeability and P-Glycoprotein Using
Caco-2 Cell Model. Because of the adverse effects of AD in
the central nervous system, effective drug candidates need to
cross the blood-brain barrier (BBB). To test whether 14 has
the potential to reach the brain, we determined its permeabil-
ity and transport directionality using the Caco-2 model.48
Although the Caco-2 cell monolayer model is derived from
the colon rather than the brain, this model expresses efflux
transporters such as P-glycoprotein which are also expressed
at the BBB. The Caco-2 model does not predict BBB pen-
etration as well as other models, such as PAMPA-BLM,
ECV/C6, or hCMEC/D3;49-51 however, this model can pro-
vide early screening regarding the transcellular diffusional
permeability and directional efflux transport across the
BBB.52 As shown in Figure 7C, the apical-to-basolateral
and basolateral-to-apical permeabilities of 14 were 7.1 (
4.6 ꢀ 10-6 and 4.7 ( 0.5 ꢀ 10-6 cm/s, respectively. Thus, 14
exhibits good bidirectional permeability in Caco-2 cells. In
contrast, we were unable to detect transport of 1, likely due
to its extensive metabolism by glutathione-S-transferase en-
zymes.53 This further indicates that CM/LR anchorage of 14
can improve its metabolic stability compared to 1. The
permeability directional ratio (efflux ratio) for 14 is 0.63,
so it does not appear to be a substrate for BBB efflux trans-
porters such as P-glycoprotein, since the efflux ratio is <2.54
These data further support the potential of 14 as a new lead
to develop effective AD treatment agents. Furthermore, in
vivo studies have demonstrated the ability of 1 to cross the
BBB,29,55,56 so 14 is anticipated to be able to cross the BBB
and the results from Caco-2 assay also supports this notion.
Future in vivo studies will assess the BBB permeability more
directly, and studies are being undertaken in our laboratory
to evaluate 14’s BBB permeability in mice.
minants for their biological activities. Among the designed
BMAOIs, 14 with a 21-atom-spacer was identified to
localize to the CM/LR of MC65 cells, to efficiently inhibit
the production of intracellular AβOs in MC65 cells, and to
protect MC65 cells and differentiated SH-SY5Y cells from
the cytotoxicity of AβOs. Furthermore, 14 exhibited anti-
oxidant properties and demonstrated potential to cross the
BBB using a Caco-2 model. These results strongly encou-
rage further optimization of 14 as a new hit to develop more
potent BMAOIs. These results may also help validate
BMAOIs strategy as a novel design strategy to provide
effective multifunctional ligands as potential AD treat-
ment agents.
Experimental Section
Chemistry. Reagents and solvents were obtained from com-
mercial suppliers and used as received unless otherwise indi-
cated. All reactions were carried out under inert atmosphere
(N2) unless otherwise noted. Reactions were monitored by thin-
layer chromatography (TLC) (precoated silica gel 60 F254 plates,
EMD Chemicals) and visualized with UV light or by treatment
with phosphomolybdic acid (PMA). Flash chromatography was
performed on silica gel (200-300 mesh, Fisher Scientific) using
solvents as indicated. 1HNMR and 13CNMR spectra were
routinely recorded on Bruker ARX 400 spectrometer. The
NMR solvent used was CDCl3 or DMSO-d6 as indicated.
Tetramethylsilane (TMS) was used as the internal standard.
The purity of target BMAOIs was determined by HPLC using a
Varian 100-5 C18 250 ꢀ 4.6 mm column with UV detection (288
nm) (40% acetonitrile/60% methanol/0.1% trifluoroacetic acid
(TFA) and 38% acetonitrile/62% H2O/2% acetic acid, pH 3.0
two solvent systems) to be g95%.
4-Methoxy-3-propargyloxy-benzaldehyde (18). A mixture of
vanillin 17 (0.76 g, 4.90 mmol), K2CO3 (1.37 g, 9.90 mmol), and
propargyl bromide (1.19 g, 6.90 mmol) in DMF (30 mL) was
refluxed at 80 °C for 1 h. Reaction mixture was cooled to 0 °C in
an ice bath and filtered through a short bed of Celite. Ethyl
acetate (50 mL) was added and the mixture was washed with 1 N
HCl (20 mL), extracted with ethyl acetate (100 mL). The organic
phase was combined and washed with brine and dried over
anhydrous Na2SO4. After filtration, solvent was removed under
reduced pressure and the crude residue was purified by flash
chromatography (hexane/ethyl acetate: 8/2) to afford 18 as
1
white solid (0.74 g, 80%). HNMR (400 MHz, CDCl3) δ 2.57
(t, 1H), 3.94 (s, 3H), 4.85-4.86 (d, J = 2.44 Hz, 2H), 7.13-7.15
(d, J = 8.16 Hz, 1H), 7.43-7.47 (m, 2H), 9.87 (s, 1H); 13CNMR
(100 MHz, CDCl3) δ 56.06, 56.65, 109.58, 112.71, 126.21,
131.00, 150.11, 152.17, 190.85.
5-Hydroxy-1-(4-hydroxy-3-methoxy-phenyl)-7-(3-methoxy-4-
propargyloxy-phenyl)-hepta-1,4,6-trien-3-one (20). Compound
20 was prepared by Pabon reaction following the reported pro-
1
cedure57 from 2,4-pentane-dione and 17. HNMR (400 MHz,
CDCl3) δ 2.53-2.54 (t, J = 4.84, 3H), 3.92-3.93 (d, J = 6.2 Hz,
6H), 4.79-4.80 (d, J = 2.32 Hz, 2H), 5.81 (s, 1H), 6.45-6.51 (m,
2H), 6.91-6.93 (d, J = 8.2 Hz, 1H), 7.04 (s, 2H), 7.08-7.12 (m,
3H), 7.57-7.61 (d, J = 15.72 Hz, 2H); 13CNMR (100 MHz,
CDCl3) δ 26.78, 55.97, 56.67, 60.41, 101.28, 109.70-129.31,
140.08-149.83, 182.83, 183.68.
3-Propargyl-pentane-2,4-dione (22). The mixture of propargyl
bromide (0.32 g, 2.70 mmol), K2CO3 (2.22 g, 16.10 mmol), and
2,4-pentane-dione (1.34 g, 13.40 mmol) in acetone (30 mL) was
stirred for 24 h at 60 °C. After filtration and removal of solvent
under reduced pressure, the crude residue was purified by flash
chromatography (hexane) to give 22 as colorless liquid (0.30 g,
69%). 1HNMR (400 MHz, CDCl3) δ 2.03-2.04 (t, J = 5.28 Hz,
1H), 2.22 (s, 3H), 2.25 (s, 3H), 2.68-2.71 (m, 2H), 3.84-3.87 (t,
J = 15.08 Hz, 1H); 13CNMR (100 MHz, CDCl3) δ 14.45, 29.33,
29.41, 68.70, 70.79, 86.13, 202.18, 202.63.
Conclusion
In summary, a series of BMAOIs containing 1 and 2 were
designed and synthesized to cotarget AβOs, oxidative
stress, and CM/LR. Biological characterization from in
vitro assays established that spacer length and the spacer
attachment position on 1 are important structural deter-

Article
1,7-Bis-(4-hydroxy-3-methoxy-phenyl)-4-propargyl-hepta-1,6-
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 16 6205
pressure and the crude product was purified by flash chroma-
tography (MeOH/CH2Cl2: 3/97) to afford 32. 1HNMR (400
MHz, CDCl3) δ 0.61 (s, 3H), 0.78-0.80 (dd, J = 4.96 Hz, 1.6
Hz, 6H), 0.81-0.83 (d, J = 9.44 Hz, 3H), 0.85-2.30 (m, 37H),
3.15-3.26 (m, 3H), 3.89 (s, 2H), 5.28-5.29 (t, J = 5.16 Hz, 1H);
13CNMR (100 MHz, CDCl3) δ 10.84-41.32, 49.12, 50.06,
55.16, 55.73, 66.58, 79.22, 121.31, 139.04, 169.30.
Preparation of 33. Compound 25 (1.00 mmol) was reacted
with 27 (3.00 mmol) following Procedure B to give 33. 1HNMR
(400 MHz, CDCl3) δ 0.61 (s, 3H), 0.78-0.80 (dd, J = 4.88 Hz,
1.72 Hz, 6H), 0.83-0.85 (d, J = 9.44 Hz, 3H), 0.94-2.30 (41H),
3.14-3.23 (m, 3H), 3.89 (s, 2H), 5.28-5.29 (t, J = 5.24 Hz, 1H);
13CNMR (100 MHz, CDCl3) δ 10.85-41.32, 49.13, 50.35,
55.16, 55.73, 66.62, 79.22, 121.30, 139.07, 169.18.
Preparation of 34. Compound 25 (1.00 mmol) was reacted
with 28 (3.00 mmol) following Procedure B to give 34. 1HNMR
(400 MHz, CDCl3) δ 0.60 (s, 3H), 0.85-0.87 (dd, J = 4.92 Hz,
1.68 Hz, 6H), 0.90-0.92 (d, J = 6.48 Hz, 3H), 1.00-2.39 (39H),
3.09-3.18 (m, 1H), 3.20-3.25 (m, 4H), 3.48-3.53 (m, 2H), 3.88
(s, 2H), 5.27-5.29 (t, J = 5.04 Hz, 1H); 13CNMR (100 MHz,
CDCl3) δ 10.84-41.32, 49.14, 50.03, 55.16, 55.74, 66.50, 79.29,
121.23, 139.12, 169.86, 169.99.
Preparation of 35. Compound 25 (1.00 mmol) was reacted
with 29 (3.00 mmol) following Procedure B to give 35. 1HNMR
(400 MHz, CDCl3) δ 0.67 (s, 3H), 0.85-0.87 (dd, J = 4.92 Hz,
1.68 Hz, 6H), 0.90-0.92 (d, J = 6.48 Hz, 3H), 1.00-2.43 (43H),
3.16-3.27 (m, 5H), 3.57-3.60 (m, 2H), 3.95 (s, 2H), 5.34-5.35
(t, J = 5.04 Hz, 1H); 13CNMR (100 MHz, CDCl3) δ 11.84-
42.32, 50.15, 51.33, 56.16, 56.74, 67.53, 80.29, 122.21, 140.12,
170.81, 170.87.
diene-3,5-dione (23). Compound 22 (0.81 g, 5.90 mmol) was
reacted with boric anhydride (0.29 g, 4.10 mmol), 17 (0.18 g,
11.70 mmol), tributylborate (5.39 g, 23.40 mmol), and n-buty-
lamine (0.64 g, 8.80 mmol) following a reported procedure57 to
afford 23 as a yellow solid (0.50 g, 21%). 1HNMR (400 MHz,
CDCl3) δ 2.14-2.16 (t, J = 5.08 Hz, 1H), 2.89-2.92 (m, 2H),
3.91 (s, 3H), 3.95 (s, 3H), 6.68-6.72 (d, J = 15.8 Hz, 1H),
6.90-7.26 (m, 8H), 7.56-7.74 (m, 2H); 13CNMR (100 MHz,
CDCl3) δ 16.27, 56.03, 69.61, 70.57, 82.66, 106.24, 109.83-
127.97, 142.43-148.87, 182.68, 193.34.
Cholesteryl-3-acetic Acid (25). 25 was prepared following the
reported procedure33 from cholesterol as a white solid. 1HNMR
(400 MHz, CDCl3) δ 0.67 (s, 3H), 0.85-0.87 (dd, J = 6.64 Hz,
1.68 Hz, 6H), 0.90-0.92 (d, J = 6.52 Hz, 3H), 1.00-2.40 (31H),
3.26-3.34 (m, 1H), 4.14 (s, 2H), 5.36-5.37 (t, J = 5.2 Hz, 1H);
13CNMR (100 MHz, CDCl3) δ 11.86-42.33, 50.13, 56.17,
56.74, 65.20, 80.45, 122.40, 139.98, 173.89.
Procedure A. Preparation of 3-Amino-N-(4-azido-butyl)-pro-
pionamide (28). To a mixture of Boc-protected β-alanine (1.00
mmol) and hydroxybenzotriazole (HOBt) (1.50 mmol) in
CH2Cl2 (10 mL) was added 1-ethyl-3-(3-dimethylaminopro-
pyl)-carbodiimide (EDC) (1.50 mmol) at 0 °C. The reaction
mixture was stirred for 1 h at room temperature. Then, a
solution of TFA salt of 4-azido-butylamine 26 (2.00 mmol)
and Et3N (3.00 mmol) in CH2Cl2 (5 mL) was added to the
reaction mixture at 0 °C. The reaction mixture was then stirred
overnight at room temperature. The reaction mixture was
washed with H2O, NaHCO3, and brine. The organic phase
was dried over anhydrous Na2SO4 and the solvent was removed
under reduced pressure. The crude residue was purified by flash
chromatography (MeOH/CH2Cl2: 3/97) and deprotected using
TFA/CH2Cl2 (0.5 mL per 1 mmol of Boc-protected azido pro-
Preparation of 36. Compound 25 (1.00 mmol) was reacted
with 30 (3.00 mmol) following Procedure B to give 36. 1HNMR
(400 MHz, CDCl3) δ 0.67 (s, 3H), 0.85-0.87 (dd, J = 4.92 Hz,
1.68 Hz, 6H), 0.90-0.92 (d, J = 6.48 Hz, 3H), 1.00-2.58 (41H),
3.17-3.35 (m, 2H), 3.51-3.59 (m, 3H), 3.70 (s, 2H), 3.96 (s, 2H),
5.35-5.36 (t, J = 3.28 Hz, 1H); 13CNMR (100 MHz, CDCl3) δ
10.84-41.31, 49.12, 50.00, 50.79, 55.15, 55.73, 66.56, 79.29,
121.24, 139.09, 169.45, 171.66.
1
duct) to afford 28 as a colorless viscous liquid. HNMR (400
MHz, DMSO-d6) δ 1.41-1.61 (m, 4H), 2.50-2.51 (m, 2H),
2.89-2.98 (m, 2H), 3.01-3.09 (m, 2H), 3.31-3.34 (t, J = 13.32,
2H); 13CNMR (100 MHz, DMSO-d6) δ 25.74, 26.19, 32.01,
35.22, 37.90, 48.51, 50.33, 169.16.
Preparation of 37. Compound 25 (1.00 mmol) was reacted
with 31 (3.00 mmol) following Procedure B to give 37. 1HNMR
(400 MHz, CDCl3) δ 0.67 (s, 3H), 0.85-0.87 (dd, J = 4.88 Hz,
1.72 Hz, 6H), 0.90-0.92 (d, J = 6.52 Hz, 3H), 1.00-2.58 (45H),
3.16-3.26 (m, 1H), 3.50-3.59 (m, 3H), 3.95-3.96 (d, J = 3.56
Hz, 2H), 5.34-5.36 (t, J = 5.76 Hz, 1H); 13CNMR (100 MHz,
CDCl3) δ 11.87-42.33, 50.15, 51.81, 51.85, 56.17, 56.75, 67.59,
80.29, 122.20, 140.12, 170.47, 170.73, 171.09.
Procedure C. Preparation of BMAOI 3. To the solution of
compounds 32 (1 equiv) and compound 20 (2 equiv) in THF/
H2O (5 mL, 1:1) was added sodium ascorbate (0.04 equiv) and
CuSO4 (0.02 equiv) at room temperature. The reaction mixture
was stirred for 24 h at 65 °C. The solvent was removed under
reduced pressure and CH2Cl2 (5 mL) was added. The organic
layer was washed with H2O and brine, and then dried over anhy-
drous Na2SO4. After filtration and removal of the solvent under
reduced pressure, the crude residue was purified by flash chro-
matography (MeOH/CH2Cl2: 5/95) to give BMAOI 3 as a
yellow solid. 1HNMR (400 MHz, CDCl3) δ 0.67 (s, 3H),
0.85-0.87 (dd, J = 4.92 Hz, 1.64 Hz, 6H), 0.90-0.91 (d, J =
6.48 Hz, 3H), 0.99-2.34 (35H), 3.15-3.21 (m, 1H), 3.30-3.35
(m, 2H), 3.91 (s, 2H), 3.94 (s, 3H), 3.95 (s, 3H), 4.37-4.40 (t, J =
14.08 Hz, 2H), 5.32 (s, 2H), 5.32-5.34 (m, 1H), 6.46-6.50 (d,
2H), 6.92-6.94 (d, 1H), 7.05-7.13 (m, 5H), 7.56 (d, 1H), 7.60 (d,
1H), 7.65 (s, 1H); 13CNMR (100 MHz, CDCl3) δ 11.86-42.32,
49.86-50.11, 55.97-56.72, 63.10, 67.50, 80.21, 101.24, 109.66-
128.85, 140.06-149.67, 170.49, 182.92-183.58.
3-Amino-N-(6-azido-hexyl)-propionamide (29). 6-Azido-hex-
ylamine 27 (2.00 mmol) was reacted with Boc-protected β-
alanine (1.00 mmol) following Procedure A to give 29. 1HNMR
(400 MHz, DMSO-d6) δ 1.20-1.46 (m, 8H), 2.40-2.44 (m, 2H),
2.84-2.87 (m, 2H), 2.93-2.98 (m, 2H), 3.21-3.24 (t, J = 13.64,
2H); 13CNMR (100 MHz, DMSO- d6) δ 25.80, 25.89, 28.12,
28.78, 31.98, 35.25, 38.37, 50.55, 169.07.
3-Amino-N-[2-(4-azido-butylcarbamoyl)-ethyl]-propionamide
(30). Compound 28 (2.00 mmol) was reacted with Boc-protected
β-alanine(1.00 mmol) following ProcedureA togive 30. 1HNMR
(400 MHz, DMSO-d6) δ 1.42-1.54 (m, 4H), 2.24-2.28 (t, J =
14.2 Hz, 2H), 2.45-2.50 (m, 4H), 2.9-3.03 (m, 5H), 3.21-3.32
(m, 4H); 13CNMR (100 MHz, DMSO-d6) δ 25.71, 26.18, 26.25,
31.19, 32.03, 34.53, 35.19, 35.23, 35.39, 37.81, 38.83, 50.33, 51.69,
54.88,169.22, 170.15.
3-Amino-N-[2-(6-azido-hexylcarbamoyl)-ethyl]-propionamide
(31). Compound 29 (2.00 mmol) was reacted with Boc-protected
β-alanine(1.00 mmol) following ProcedureA togive 31. 1HNMR
(400 MHz, DMSO-d6) δ 1.22-1.54 (m, 10H), 2.22-2.25 (t, J =
14.12 Hz, 2H), 2.9-3.1 (m, 6H), 3.23-3.27 (m, 2H), 3.31-3.35
(t, 2H); 13CNMR (100 MHz, DMSO-d6) δ 25.80, 25.88, 28.13,
28.87, 32.03, 35.18, 35.24,35.39, 38.31, 38.84, 50.54, 54.89,169.31,
170.04.
Procedure B. Preparation of 32. The mixture of compound 25
(1.00 mmol), EDC (1.50 mmol), and HOBt (1.50 mmol) in
CH2Cl2 (10 mL) was stirred for 1 h at room temperature. To
this solution was added a solution of 26 (3.00 mmoL) and Et3N
(4.00 mmol) in CH2Cl2 (5 mL) at 0 °C. The reaction mixture was
then stirred overnight at room temperature. After filtration
through a short bed of Celite, the organic phase was washed
with H2O, NaHCO3, and brine, followed by drying over anhy-
drous Na2SO4. Organic solvent was removed under reduced
Preparation of BMAOI 9. Compounds 32 (1 equiv) was
reacted with compound 23 (2 equiv) in THF/H2O (5 mL, 1:1)
following Procedure C to give BMAOI 9. 1HNMR (400 MHz,
CDCl3) δ 0.67 (s, 3H), 0.85-0.87 (dd, J = 4.88 Hz, 1.72 Hz, 6H),
0.90-0.92 (d, J = 6.52 Hz, 3H), 0.99-2.35 (35H), 3.16-3.28

6206 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 16
Lenhart et al.
(m, 3H), 3.39-3.34 (m, 2H), 3.90 (s, 3H), 3.93 (s, 3H), 3.94 (s,
2H), 4.27-4.31 (t, J = 14 Hz, 2H), 5.33-5.35 (t, J = 5.08 Hz,
1H), 6.64-6.68 (d, 2H), 6.88-6.91 (m, 3H), 6.92-7.09 (m, 4H),
7.58-7.62 (d, 1H), 7.68-7.72 (d, 1H); 13CNMR (100 MHz,
CDCl3) δ 11.86-42.33, 49.67-50.12, 56.04-56.73, 63.07, 67.48,
80.22, 108.87, 109.83-128.88, 140.09-149.67, 170.51, 182.12,
194.47.
3H), 3.38-3.40 (d, 1H), 3.53-3.67 (m, 3H), 3.90 (s, 2H), 3.94 (s,
3H), 3.96 (s, 3H), 4.22-4.27 (t, J = 14.88 Hz, 2H), 5.33-5.34 (t,
J = 3.48, 1H), 6.65-6.69 (d, 1H), 6.89-6.91 (d, 2H), 6.98-7.09
(m, 4H), 7.20 (s, 1H), 7.55-7.59 (d, 1H), 7.67-7.71 (d, 1H);
13CNMR (100 MHz, CDCl3) δ11.87-42.34, 50.14-50.19, 56.06-
56.75, 63.06, 67.49, 80.33, 108.87, 109.88-128.88, 140.09-
149.67, 170.01-171.23, 182.12, 194.62.
Preparation of BMAOI 4. Compounds 33 (1 equiv) was
reacted with compound 20 (2 equiv) in THF/H2O (5 mL, 1:1)
following Procedure C to give BMAOI 4. 1HNMR (400 MHz,
CDCl3) δ 0.67 (s, 3H), 0.85-0.87 (dd, J = 4.88 Hz, 1.72 Hz, 6H),
0.90-0.91 (d, J = 6.52 Hz, 3H), 1.00-2.34 (39H), 3.21-3.29 (m,
3H), 3.91 (s, 2H), 3.94 (s, 3H), 3.95 (s, 3H), 4.31-4.35 (t, J =
14.36 Hz, 2H), 5.33 (s, 2H), 5.33 (t, 1H), 6.46 (d, 1H), 6.50 (d,
1H), 6.92-6.94 (d, 1H), 7.05-7.13 (m,5H), 7.56-7.57 (d, 1H),
7.60-7.61 (d,1H), 7.62 (s, 1H); 13CNMR (100 MHz, CDCl3) δ
11.86-42.33, 50.11-50.32, 55.98-56.72, 63.10, 67.59, 80.22,
101.24, 109.66-128.85, 140.06-149.67, 170.26, 182.92-183.56.
Preparation of BMAOI 10. Compounds 33 (1 equiv) was
reacted with compound 23 (2 equiv) in THF/H2O (5 mL, 1:1)
following Procedure C to give BMAOI 10. 1HNMR (400 MHz,
CDCl3) δ 0.60 (s, 3H), 0.85-0.87 (dd, J = 4.88 Hz, 1.72 Hz, 6H),
0.90-0.91 (d, J = 6.52 Hz, 3H), 0.99-2.44 (39H), 3.07-3.18 (m,
3H), 3.31-3.33 (m, 4H), 3.82 (s, 3H), 3.84 (s, 3H), 3.89 (s, 2H),
4.16-4.18 (t, J = 11.92 Hz, 2H), 5.27-5.28 (t, J = 5.16 Hz, 1H),
6.58-6.62 (d, 1H), 6.79-6.83 (m, 2H), 6.92-7.00 (m, 4H), 7.24
(s, 1H), 7.49-7.53 (d, 1H), 7.60-7.64 (d, 1H); 13CNMR (100
MHz, CDCl3) δ 10.84-41.31, 49.11-50.15, 55.01-55.71, 62.90,
66.55, 79.24, 108.87, 109.92-128.88, 140.09-149.67, 170.26,
182.12, 193.57.
Preparation of BMAOI 7. Compounds 36 (1 equiv) was
reacted with compound 20 (2 equiv) in THF/H2O (5 mL, 1:1)
following Procedure C to give BMAOI 7. 1HNMR (400 MHz,
CDCl3) δ 0.67 (s, 3H), 0.85-0.87 (d, J = 6.44 Hz, 6H),
0.90-0.91 (d, J = 6.48 Hz, 3H), 0.98-2.4 (39H), 3.20-3.28
(m, 3H), 3.52-3.56 (m, 4H), 3.91 (s, 2H), 3.94 (s, 3H), 3.96 (s,
3H), 4.36-4.39 (t, J = 13.56 Hz, 2H), 5.31 (s, 2H), 5.34 (t, 1H),
6.46-6.47 (d, 1H), 6.50-6.51 (d, 1H), 6.92-6.94 (d, 1H),
7.05-7.13 (m, 5H), 7.56-7.57 (d, 1H), 7.60-7.61 (d, 1H), 7.67
(s, 1H); 13CNMR (100 MHz, CDCl3) δ 11.86-42.33, 50.12-
50.19, 55.98-56.73, 63.06, 67.57, 80.33, 101.26, 109.71-128.88,
140.09-149.67, 170.01-171.23, 182.85-183.64.
Preparation of BMAOI 13. Compounds 36 (1 equiv) was
reacted with compound 23 (2 equiv) in THF/H2O (5 mL, 1:1)
following Procedure C to give BMAOI 13. 1HNMR (400 MHz,
CDCl3) δ 0.67 (s, 3H), 0.82-0.87 (dd, J = 4.88 Hz, 1.72 6H),
0.90-0.91 (d, J = 6.52 Hz, 3H), 0.93-2.4 (39H), 3.17-3.20 (m,
3H), 3.38-3.55 (m, 6H), 3.90 (s, 2H), 3.92 (s, 3H), 3.95 (s, 3H),
4.24-4.30 (t, J = 13.44 Hz, 2H), 5.33-5.34 (t, 1H), 6.65-6.67
(d, 1H), 6.69-6.71 (d, 1H), 6.89-6.91 (d, 1H), 7.00-7.09 (m,
5H), 7.57-7.61 (d, 1H), 7.67-7.71 (d, 1H), 7.63 (s, 1H);
13CNMR (100 MHz, CDCl3) δ11.86-42.33, 50.12-50.19, 56.07-
56.73, 63.06, 67.57, 80.33, 108.87, 109.71-128.88, 140.09-
149.67, 170.01-171.23, 182.12, 193.57.
Preparation of BMAOI 8. Compounds 37 (1 equiv) was
reacted with compound 20 (2 equiv) in THF/H2O (5 mL, 1:1)
following Procedure C to give BMAOI 8. 1HNMR (400 MHz,
CDCl3) δ 0.67 (s, 3H), 0.85-0.87 (dd, J = 4.92 Hz, 1.68 Hz, 6H),
0.90-0.91 (d, J = 6.52 Hz, 3H), 0.93-2.4 (43H), 3.17-3.22 (m,
3H), 3.48-3.56 (m, 4H), 3.89 (s, 2H), 3.94 (s, 3H), 3.96 (s, 3H),
4.36-4.39 (t, J = 12 Hz, 2H), 5.33 (s, 2H), 5.33 (t, 1H),
6.45-6.47 (d, 1H), 6.49-6.51 (d, 1H), 6.92-6.94 (d, 1H),
7.05-7.12 (m, 5H), 7.55-7.57 (d, 1H), 7.59-7.61 (d, 1H), 7.63
(s, 1H); 13CNMR (100 MHz, CDCl3) δ 11.86-42.32, 50.13-
50.19, 55.98-56.73, 63.06, 67.57, 80.29, 101.26, 109.71-128.88,
140.09-149.67, 170.01-171.23, 182.85-183.64.
Preparation of BMAOI 14. Compounds 37 (1 equiv) was
reacted with compound 23 (2 equiv) in THF/H2O (5 mL, 1:1)
following Procedure C to give BMAOI 14. 1HNMR (400 MHz,
CDCl3) δ 0.67 (s, 3H), 0.85-0.87 (dd, J = 4.88 Hz, 1.56 Hz 6H),
0.90-0.91 (d, J = 6.52 Hz, 3H), 0.93-2.4 (43H), 3.17-3.22 (m,
3H), 3.48-3.56 (m, 6H), 3.89 (s, 2H), 3.94 (s, 3H), 3.96 (s, 3H),
4.32-4.36 (t, J = 13.96 Hz, 2H), 5.33 (t, 1H), 6.45-6.47 (d, 1H),
6.49-6.51 (d, 1H), 6.92-6.94 (d, 1H), 7.05-7.12 (m, 5H),
7.56-7.57 (d, 1H), 7.59-7.61 (d, 1H), 7.63 (s, 1H); 13CNMR
(100 MHz, CDCl3) δ 11.86-42.32, 50.13-50.19, 55.98-56.73,
63.06, 67.57, 80.29, 108.87, 109.71-128.88, 140.09-149.67,
170.01-171.23, 182.12, 193.57.
Preparation of BMAOI 5. Compounds 34 (1 equiv) was
reacted with compound 20 (2 equivalent) in THF/H2O (5 mL,
1:1) following Procedure C to give BMAOI 5. HNMR (400
1
MHz, CDCl3) δ 0.66 (s, 3H), 0.84-0.87 (dd, J = 4.96 Hz, 1.64
Hz, 6H), 0.89-0.91 (d, J = 6.48 Hz, 3H), 0.98-2.43 (37H),
3.15-3.2 (m, 1H), 3.26 (m, 2H), 3.55-3.57 (m, 4H), 3.91 (s, 2H),
3.93 (s, 6H), 4.36 (t, 2H), 5.33 (s, 2H), 5.33 (t, 1H), 6.48 (d, 2H),
6.93-6.19 (m,5H), 7.57-7.6610 (m, 3H); 13CNMR (100 MHz,
CDCl3) δ 11.86-42.33, 49.86-50.14, 55.98-56.73, 63.02, 67.47,
80.30, 101.26, 109.79-128.93, 140.10-149.68, 170.09-171.23,
182.85-183.64.
Preparation of BMAOI 11. Compounds 34 (1 equiv) was
reacted with compound 23 (2 equiv) in THF/H2O (5 mL, 1:1)
following Procedure C to give BMAOI 11. 1HNMR (400 MHz,
CDCl3) δ 0.67 (s, 3H), 0.85-0.87 (dd, J = 4.92 Hz, 1.68 Hz, 6H),
0.90-0.91 (d, J = 6.56 Hz, 3H), 0.99-2.44 (37H), 3.07-3.11 (m,
1H), 3.17-3.19 (m, 4H), 3.53-3.57 (m, 2H), 3.90 (s, 2H), 3.92 (s,
3H), 3.95 (s, 3H), 4.26-4.29 (t, J = 13.72 Hz, 2H), 5.33-5.34 (t,
J=2.88 Hz, 1H), 6.66-6.67 (d, 1H), 6.89-6.92 (m, 3H), 6.99-
7.09 (m, 4H), 7.22 (s, 1H), 7.57-7.61 (d, 1H), 7.67-7.70 (d, 1H);
13CNMR (100 MHz, CDCl3) δ11.87-42.34, 49.64-50.15, 56.07-
56.75, 62.90, 67.49, 80.32, 108.87, 109.92-128.88, 140.09-149.67,
170.01-171.13, 182.12, 194.55.
Preparation of BMAOI 6. Compounds 35 (1 equiv) was
reacted with compound 20 (2 equiv) in THF/H2O (5 mL, 1:1)
following Procedure C to give BMAOI 6. 1HNMR (400 MHz,
CDCl3) δ 0.67 (s, 3H), 0.85-0.87 (dd, J = 5 Hz, 1.6 Hz, 6H),
0.90-0.91 (d, J = 6.52 Hz, 3H), 0.99-2.44 (41H), 3.18-3.21 (m,
3H), 3.57-3.58 (m, 4H), 3.91 (s, 2H), 3.94 (s, 6H), 4.32-4.35 (t,
J = 14.16 Hz, 2H), 5.33 (s, 2H), 5.33 (t, 1H), 6.46-6.47 (d, 1H),
6.50-6.51 (d, 1H), 6.92-6.94 (d,1H), 7.05-7.19 (m, 5H),
7.56-7.57 (d, 1H), 7.60-7.61 (d, 1H), 7.62 (s, 1H); 13CNMR
(100 MHz, CDCl3) δ 11.86-42.33, 50.13-50.23, 55.98-56.74,
63.09, 67.52, 80.29, 101.25, 109.68-128.87, 140.09-149.67,
170.01-171.23, 182.95-183.55.
Preparation of BMAOI 12. Compounds 35 (1 equiv) was
reacted with compound 23 (2 equiv) in THF/H2O (5 mL, 1:1)
following Procedure C to give BMAOI 12. 1HNMR (400 MHz,
CDCl3) δ 0.67 (s, 3H), 0.80-0.87 (dd, J = 4.92 Hz, 1.68, 6H),
0.90-0.91 (d, J = 6.64 Hz, 3H), 0.93-2.35 (41H), 3.03-3.22 (m,
Biological Assays. Aβ42 was obtained from American Pep-
tide, Inc. (Sunnyvale, CA). 6E10 antibody was obtained from
Signet (Dedham, MA). A11 oligomer Rabbit polyclonal anti-
body, Alexa Fluor 568 donkey antirabbit IgG, Alexa 488
conjugated cholera toxin subunit B (CTX-B) were obtained
from Invitrogen (CA, USA). 40,6-Diamidino-2-phenylindole
(DAPI) was obtained from Sigma-Aldrich (St. Louis, MO).
MC65 cells (kindly provided by Dr. George M. Martin at the
University of Washington, Seattle) were cultured in Dulbecco’s
Modified Eagle’s Medium (DMEM) (Life Technologies, Inc.,
Grand Island, NY) supplemented with 10% of heat-inactivated
fetal bovine serum (FBS) (Hyclone, Logan, UT), 1 μg/mL TC,
and 0.2 mg/mL G418 (Invitrogen) and maintained at 37 °C in a
fully humidified atmosphere containing 5% CO2. SH-SY5Y
neuroblastoma cells were obtained from American Type

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 16 6207
Culture Collection (ATCC) and were cultured in DMEM/
Ham’s F-12 (Invitrogen) supplemented with 10% FBS. ML60
cells were maintained in DMEM supplemented with 10% FBS,
0.2 mg/mL G418, and 25 μg/mL puromycin. All experiments
were performed on 70% confluent growing cells unless other-
wise indicated.
10% nonfat milk in PBS for 30 min. The blots were probed with
6E10 (1:2000) overnight at 4 °C, followed by horseradish
peroxidase-conjugated secondary antibody (1:2000. Kirkegaard
& Perry, Gaithersburg, MD). The proteins were visualized by
Western Blot Chemiluminescence Reagent (NEN Life Science
Products, Boston, MA).
Western Blot Assay. MC65 cells were seeded in 6-well plates
(1 ꢀ 106 cells/well). After incubation at 37 °C, 5% CO2 for 24 h,
the medium was replaced with fresh Opti-MEM (Invitrogen)
and compounds in Opti-MEM (with or without TC) were
added. After 24 h incubation, cells were collected on ice and
centrifuged. Pellet was lysed by sonication in 1ꢀ lysis buffer
(62.5 mM Tris base, pH 6.8, 2% SDS, 50 mM DTT, 10%
glycerol, 0.1% bromphenol blue, and 5 mg/mL each chymosta-
tin, leupeptin, aprotinin, pepstatin, and soybean trypsin in-
hibitor) and protein level was determined using Coomassie
Protein Assay Reagent (Pierce, Rockford, IL). Equal amounts
of protein (10 μg) were separated by SDS-PAGE on 10-20%
tris-tricine gel (Bio-Rad) and transferred onto a PVDF mem-
brane (Bio-Rad). The blots were blocked with 5% milk in TBS-
Tween 20 (0.1%) at room temperature for 1 h and probed
with primary 6E10 (1:2000) antibody overnight at 4 °C. The
blots were then incubated with horseradish peroxidase-
conjugated secondary antibody (1:2000. Kirkegaard & Perry,
Gaithersburg, MD). The proteins were visualized by Western
Blot Chemiluminescence Reagent (NEN Life Science Products,
Boston, MA).
Transmission Electron Microscope (TEM). Ten microliters of
Aβ42 in Ham’s F-12 (20 μM) were adsorbed onto 200-mesh
carbon and formavar-coated grids (Electron Microscopy Scien-
ces) for 20 min, washed for 1 min in distilled H2O. The samples
were negatively stained with 2% uranyl acetate (Electron Mi-
croscopy Sciences) for 5 min and washed for 1 min in distilled
H2O. The samples were air-dried overnight and viewed with a
Jeol JEM-1230 TEM equipped with a Gatan UltraScan 4000SP
4K ꢀ 4K CCD camera (100 kV).
Cytotoxicity Assay in MC65 Cells. MC65 cells were seeded in
96-well plates (4 ꢀ 104 cells/well) at 37 °C, 5% CO2 for 24 h. The
medium was removed and washed with PBS twice. Opti-MEM
and test compounds were added under þTC and -TC condi-
tions. The plates were incubated at 37 °C, 5% CO2 for 72 h, then
20 μL CellTiter 96 AQueous One Solution (Promega, Madison,
WI) were added to each well and the plates were incubated at 37
°C, 5% CO2 for 2-4 h. The plates were read at 490 nm using
FlexStation III plate reader (Molecular Devices). The blank
with only test compounds in Opti-MEM was set up as back-
ground control for all of the tested concentrations. Each data
point was averaged from six replicates and the experiments were
independently repeated at least three times.
Immunocytochemistry Assay. MC65 cells were plated onto
Lab-Tec chamber slides (1 ꢀ 104 cells/well). After 24 h incuba-
tion at 37 °C and 5% CO2, Opti-MEM was added (with or
without TC) and followed by test compounds. MC65 cells were
incubated for 24 h. MC65 cells were rinsed 3x with PBS and
incubated with Alexa 488-conjugated CTX-B (10 ug/mL) for 15
min on ice. After rinsing once with ice-cold PBS, cells were fixed
for 30 min with 4% paraformaldehyde. MC65 cells were per-
meabilized for 30 min with 0.1% Triton X 100. Then MC65 cells
were stained with A11 rabbit antibody followed by antirabbit
Alexa 568 (1:500). Finally MC65 cells were treated with DAPI
(5 μg/mL) and mounted with Vectashield Mounting Media. Cell
fluorescence was analyzed by a Leica TCS-SP2 AOBS confocal
laser scanning microscope equipped with blue diode, Argon,
and 3 HeNe (lasers as well as a spectrophotometer based
detection system with variable detector windows) using excita-
tion lines at 355, 488, and 568 nm for DAPI, Alexa 488-
conjugated CTX-B, and Alexa Fluor 568 donkey antirabbit
IgG. Sequential scanning was conducted to ensure that there
was no signal cross-talk between channels. Five different areas
around the center were taken and the red puncta was averaged
per cell.
For BMAOI 14 interaction with CM/LR immunocytochem-
istry assay, MC65 cells were incubated with 1 or 14 for 24 h, then
fixed with 4% paraformaldehyde, and permeabilized with 0.1%
Triton X100. MC65 cells were incubated with DAPI and
mounted with Vectashield Mounting Media for confocal laser
scanning using excitation lines at 355 and 494 nm. A series of
optical sections (1024 ꢀ 1024 pixels) of 1.0 μm in thickness were
taken through the cell depth for examined sample and projected
as a single composite image by superimposition.
Cytotoxicity Assay in Differentiated SH-SY5Y Cells. SH-
SY5Y cells were plated at 10 000 cells/well in type 1 collagen
coated 96-well plates (Invitrogen) and were differentiated in
Opti-MEM supplemented with 2% B-27 (Invitrogen) and 10
μM all-trans-retinoic acid for 7 days. The medium was removed
and replaced with fresh maintenance medium. Freshly prepared
Aβ42 oligomers in Ham’s F-12 medium (1 μM) was added to
cells for 48 h at 37 °C with or without test compounds. After
treatment, 20 μL CellTiter 96 AQueous One Solution (Promega,
Madison, WI) were added to each well and the plates were
incubated at 37 °C, 5% CO2 for 2-4 h. The plates were read at
490 nm in FlexStation III plate reader (Molecular Devices).
Each data point was averaged from six replicates and the
experiments were independently repeated at least three times.
ML60 Cell Assay. Sandwich enzyme-linked immunoassays
(ELISAs) for extracellular AβOs in ML60 cells were performed.
ML60 cells (90% confluent) in 96-well plates were treated with
test compounds (10 μM) at 37 °C, 5% CO2 for 24 h. ML60 cells
were centrifuged for 5 min at 6000g, and supernatant media was
collected for AβOs measurement by ELISA. The capture anti-
body 21F12 (to Aβ residues 33-42) was used for capturing both
monomeric and oligomeric Aβ42 species. The detecting anti-
body was biotinylated 21F12B, and the combination of 21F12
and biotinylated 21F12B allows the detection of only AβOs. The
21F12 mAbs were coated at 10 mg/mL into 96-well immunoas-
say plates (Costar) at room temperature overnight. The plates
were then aspirated and blocked with 0.25% human serum
albumin in PBS buffer for 1 h at room temperature. The plates
were rehydrated with wash buffer (0.05% Tween 20 in TBS)
before use. The samples were added to the plates and incubated
at room temperature for 1 h. The plates were washed 3ꢀ with
wash buffer between each step of the assay. The biotinylated
21F12B diluted to 0.5 mg/mL in casein assay buffer (0.25%
casein, 0.05% Tween 20, pH 7.4, in PBS) was incubated in the
wells for 1 h at room temperature. Avidin-horseradish perox-
idase (Vector Laboratories), diluted 1:4000 in casein assay
buffer, was added to the wells for 1 h at room temperature.
The colorimetric substrate, Slow TMB-ELISA (Pierce), was
added and allowed to react for 15 min, after which the enzymatic
reaction was stopped with addition of 1 M H2SO4. Reaction
product was quantified using plate reader by measuring the
difference in absorbance at 450 and 650 nm.
Aβ42 Oligomerization Inhibition Assay. An aliquot of Aβ42
(0.045 mg) was dissolved in 20 μL of DMSO and diluted in
Ham’s F-12 media without phenol red (Caisson’s Laboratory,
Inc., UT). Aβ42 (5 μM) was incubated with 1 or 14 (20 μM) in
37 °C water bath for 4 h. After incubation, the samples (50 μL)
were spun down at 14000g for 10 min. The supernatant (20 μL)
was mixed with an equal part of Tricine sample buffer without
reducing agents (Bio-Rad). The unaggregated Aβ42 control was
not incubated at 37 °C, and mixed with sample buffer (no
centrifuging) and stored at -80 °C before it was electrophor-
esed. Samples (25 μL) were electrophoresed on a 10-20% Tris-
Tricine gel, transferred to PVDF membrane, and blocked with

6208 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 16
Lenhart et al.
Anti-CD3 Antibody Mediated Splenocyte Proliferation. A
single spleen cell suspension from female B6C3F1 mice (N = 4)
was prepared and resuspended in RPMI medium supplemented
with FBS (10%), sodium bicarbonate (GIBCO), HEPES
(GIBCO), L-glutamine, gentamicin, and 2-mercaptoethanol
(0.00035%). The splenocytes (2 ꢀ 105/well) were cultured in
the microtiter wells coated with anti-CD3 Ab (1 μg/mL; BD
PharMingen) in the presence of 1 or 14 (10 μM) at 37 °C in 5%
Acknowledgment. We thank Dr. George M. Martin at the
University of Washington, Seattle, for kindly providing the
MC65 cells. We acknowledge Dr. Pallabi Mitra and Zhenxian
Zhang for their help with Caco-2 permeability experiments.
We also acknowledge Dr. Ting Yang (Harvard Institute of
Medicine) for the ML60 ELISA assay. The work was sup-
ported in part by Alzheimer’s & Related Diseases Research
Award Fund, Commonwealth of Virginia (S.Z.), and new
faculty start-up funds from Virginia Commonwealth Univer-
sity (S.Z.).
3
CO2. Prior to harvest on day 3, the cells were pulsed with H-
thymidine for 18-24 h. The incorporation of 3H-thymidine into
the proliferating cells was used as the end point of the assay, and
the data were expressed as CPM/2 ꢀ 105 cells.
IL-2 Augmented Natural Killer (NK) Cell Activity. To deter-
mine NK activity, single cell suspensions from female B6C3F1
mice (N = 4) were adjusted to 2.5 ꢀ 106 cells/mL in a 96-well
U-bottom plate (0.1 mL/well) for each animal. Recombinant
IL-2 (Chiron, Emeryville, CA) at a volume of 50 μL was added
to each well so that the final concentration of IL-2 was 5000 IU/
mL. The plates were cultured overnight in the presence of 1 or 14
(10 μM), and then assayed for NK cell activity using 51Cr-
labeled YAC-1 cells as the target cells. The 51Cr-YAC-1 cells
were added to each well of a 96-well plate to obtain E:T ratio of
50:1. The spontaneous release and the maximum release were
determined by adding 0.1 mL of medium and Triton X-100
(0.1%) to each of 12 replicate wells containing the target cells,
respectively. Following 4 h incubation, the plates were centri-
fuged, and 0.1 mL of the supernatant was removed from each
well and the radioactivity counted. The mean percentage of
cytotoxicity was determined.
DCFH-DA Antioxidation Assay. MC65 cells were seeded in
96-well plates (4 ꢀ 104 cells/well). After incubation at 37 °C, 5%
CO2 for 24 h, the medium was removed and washed with PBS.
Opti-MEM and test compounds were added (þTC, -TC, and
blank with only test compounds). MC65 cells were incubated at
37 °C, 5% CO2 for 24 h. Then DCFH-DA in Opti-MEM was
added to each well (final DCFH-DA concentration was 25 uM)
and incubate at 37 °C, 5% CO2 for 30 min. The medium was
removed and replaced with fresh opti-MEM and plates were read
for fluorescence intensity at 485 nm (excitation)/530 nm
(emission) using FlexStation III plate reader (Molecular Devices).
Caco-2 Permeability Assay. Caco-2 cells (ATCC, Manassas,
VA) were cultured in high-glucose DMEM supplemented with
10% FBS and used between passages 30-50. Caco-2 cells were
plated on 12-well polyester transwell inserts (0.4 μM pore size)
(Corning #3460) at a density of 80 000 cells/cm2 and grown to
100% confluence (21 days). Filters were rinsed in Hank’s
balanced salt solution (HBSS) and test compounds (10 μM)
were added to either the apical (0.5 mL) or basolateral (1.5 mL)
side and incubated at 37 °C with shaking (100 rpm). Samples
(200 μL) were removed at 30, 60, and 120 min with replacement
of an equal volume of the appropriate buffer containing or
lacking the compounds. Samples were stored at -20 °C until
analysis. After 120 min, lucifer yellow was added to the donor
chamber, with additional sampling as above at 10, 20, and 30
min. Lucifer yellow was analyzed by fluorescence (excitation
450 nm, emission 528 nm) in a Synergy 2 microplate reader.
Monolayer tight junctions and integrity were confirmed by
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