Synthesis and biological evaluation of analogues of a novel inhibitor of β-amyloid secretion

Article abstract of DOI:10.1021/jm100308g

A drug library of 17200 compounds was screened to select small molecules that inhibit the secretion of amyloid β peptide (Aβ), the major component of Alzheimer disease senile plaques, from a human neuronal cell line. Twenty-nine hits were validated that decreased Aβ secretion by >40% at 10 μM, for a 0.17% hit rate. A lead hit was selected for further study based on its activity and low cytotoxicity, and it was found to inhibit Aβ secretion through activation of the α-secretase pathway. Twenty-four commercially available and 53 synthesized analogues were analyzed for activity. Selected analogues were evaluated for biological stability by incubation with hepatoma cells and for transcellular permeability using Caco-2 cell monolayers. The analogue with the best permeability was evaluated in 2-month old amyloid precursor protein transgenic mice and found to acutely reduce cerebral Aβ levels by 40% after a single iv administration.

Full text of DOI:10.1021/jm100308g

5302 J. Med. Chem. 2010, 53, 5302–5319
DOI: 10.1021/jm100308g
Synthesis and Biological Evaluation of Analogues of a Novel Inhibitor of β-Amyloid Secretion
Enakshi Chakrabarti,† Subrata Ghosh,‡,§ Sushabhan Sadhukhan,‡ Lawrence Sayre,‡, Gregory P. Tochtrop,*,‡ and
Jonathan D. Smith*,†
†Department of Cell Biology, Cleveland Clinic, 9500 Euclid Avenue, Cleveland, Ohio 44195, and ‡Department of Chemistry,
Case Western Reserve University, 2074 Adlebert Road, Cleveland, Ohio 44106. §Current address: Department of Basic Sciences and
Social Sciences, North-Eastern Hill University, Mawlai Umshing, Shillong-793 022, India. Deceased.
Received April 2, 2010
A drug library of 17200 compounds was screened to select small molecules that inhibit the secretion of
amyloid β peptide (Aβ), the major component of Alzheimer disease senile plaques, from a human neuronal
cell line. Twenty-nine hits were validated that decreased Aβ secretion by >40% at 10 μM, for a 0.17% hit
rate. A lead hit was selected for further study based on its activity and low cytotoxicity, and it was found to
inhibit Aβ secretion through activation of the R-secretase pathway. Twenty-four commercially available and
53 synthesized analogues were analyzed for activity. Selected analogues were evaluated for biological
stability by incubation with hepatoma cells and for transcellular permeability using Caco-2 cell monolayers.
The analogue with the best permeability was evaluated in 2-month old amyloid precursor protein transgenic
mice and found to acutely reduce cerebral Aβ levels by 40% after a single iv administration.
Introduction
media conditioned by stably transfected H4βAPP695wt human
CNS derived cells.7 The initial screen was performed in 96-well
plates with drugs distributed by the hanging drop method
yielding estimated drug levels of 10-25 μM. Total Aβ levels in
the 24 h conditioned media were measured by a sandwich
ELISA assay, and cytotoxicity was assessed by methylene blue
staining of the cells in the wells, as previously described.8 After
assay validation,8 we screened each compound in a single well
and identified 80 compounds that both reduced Aβ levels in the
conditioned media by at least 40%, and did not reduce methyl-
ene blue staining by 25% or more. Each of the 80 drugs was
rescreened in duplicate, and 29 positive hits were individually
ordered and validated in dose response studies, as previously
described,8 yielding a confirmed hit rate of 0.17% of the
screened compounds. We selected 1 (Figure 1A, ChemBridge
no. 5538506) as the lead hit for three reasons: (1) It displayed Aβ
lowering activity at 1 μM in H4βAPP695wt cells (Figure 1B),
(2) it did not have significant cytotoxicity, measured by total cell
protein content, at 10 μM (Figure 1B), and (3) it had the lowest
miLogP value of 5.165, an indicator of hydrophobicity asso-
ciated with blood-brain barrier penetration, of the hits with
similar potency. 1 was also effective in reducing secreted Aβ
levelsinculturedprimaryneuronsderivedfromAPPtransgenic
mice (Figure 1C).
Alzheimer disease (ADa) is the most common form of senile
dementia, and it is characterized pathologically by decreased
brain mass, extracellular senile plaques, and intracellular neuro-
fibrillary tangles.1 The major risk factor for AD is age, and it
affects more than a third of all people that reach 85 years of
age.1 Although the pathogenesis of AD is still controversial,
rare human mutations that lead to familial early onset AD are
found in genes that lead to increased expression or processing of
the amyloid precursor protein (APP) into amyloid β peptide
(Aβ), which is the major component of senile plaques.2-4 The
so-called “amyloid hypothesis” for AD pathogenesis is sup-
ported by transgenic mouse models that overexpress mutant
APP and genes involved in APP processing, which lead to the
production of senile plaques and cognitive impairment.5,6
We took an unbiased approach to discover small molecule
inhibitors of Aβ release from cultured human cells. Here we
describe the selection of a lead hit, it is mechanism of action,
and biological tests of commercially available and synthesized
analogues. Ultimately, our lead drug was found to be effective
in reducing cerebral Aβ levels in APP transgenic mice.
Results
Drug Library Screen and Lead Hit Selection. We screened
17200 small drug-like molecules obtained from ChemBridge
(San Diego, CA) for their ability to inhibit Aβ secretion into
Mechanism of Action of Lead Hit. The alternate processing
of holoAPP by either the β-secretase (a precursor to Aβ
production) or R-secretase (which clips in the middle of the
Aβ region) is shown in Figure 2A. 1 induced decrease in total
Aβ secretion may be due to its effect on APP processing or due
to decreased production of its precursor APP. To probe its
mechanism, H4βAPP695wt cells were treated with 10 μM 1 for
24 h. Using Western blot assays, we measured secreted APPR
(sAPPR) and APPβ (sAPPβ) in the conditioned media, and
full-length holo-APP and the 83 residue long R-C-terminal
fragment (R-CTF) in cell lysate. 1 caused an increase in
sAPPR and R-CTF level without altering sAPPβ or cellular
*To whom correspondence should be addressed. For G.P.T.: phone,
216-368-2351; E-mail: tochtrop@case.edu. For J.D.S.: Phone: 216-444-
2248; fax, 216-444-9404; E-mail: smithj4@ccf.org.
aAbbreviations: Aβ, amyloid β peptide; AD, Alzheimer disease; APP,
amyloid precursor protein; sAPPR, secreted form of amyloid precursor
protein after R-secretase cleavage; sAPPβ, secreted form of amyloid
precursor protein after β-secretase cleavage; R-CTF, the amyloid pre-
cursor protein C-terminal fragment after R-secretase cleavage; PKC,
protein kinase C; β-BMEA, β-benzylmercaptoethylamine, ADAM, a
disintegrin and metalloproteinase.
r
pubs.acs.org/jmc
Published on Web 06/22/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 14 5303
Figure 2. Compound 1 activates R-secretase processing of APP.
(A) Scheme of APP processing and Western blot bands from
conditioned media (top) and cell lysate (bottom) after 24 h treat-
ment with 10 μM 1 or vehicle control. APP is shown in turquoise,
with the Aβ region shown in the green plus purple ovals. The
cleavage sites of the R, β, and γ secretases are shown. 1 treatment
leads to increased sAPPR and RCTF without altering holoAPP
levels, indicating increased R-secretase cleavage of APP. (B) Quan-
tification of sAPPR induction by 10 μM 1 treatment (N = 3 each (
SD; ***, p < 0.0001). (C) Dose response of sAPPR induction by 1
showing Western blot band (top) and quantification (bottom).
Figure 1. Compound 1 inhibits Aβ secretion. (A) Structure of 1, with
the labeling of the two phenyl rings. (B,C) Dose response of 1 on Aβ
secretion and total cellular protein after 24 h incubation with
H4βAPP695wt cells (B) or primary neurons from APP/PS1 transgenic
mice (C). Data show mean ( SD for quadruplicate wells; *, p < 0.05 vs
0 dose control by ANOVA with Dunnett’s multiple comparison test.
full length APP (Figure 2A). 1 treatment significantly in-
creased sAPPR levels by over 3-fold, with a dose response
similar to that observed for inhibition of Aβ secretion
(Figure 2B,C). 1 also raised cellular R-CTF levels by over
2-fold (Figure 3A), an effect that was observed in as little as
2 h after addition of 1, and reached maximal levels by 8 h
(Figure 3B). The effect of a 4 h treatment with 1 on the
increase in cellular of R-CTF was not blocked by the protein
synthesis inhibitor cyclohexamide, indicating that new pro-
tein synthesis was not required for this effect (data not
shown). Thus, 1 appears to decrease Aβ secretion by activat-
ing the R-secretase pathway, a favorable pathway as the large
secreted sAPPR protein is reported to have neurotrophic and
neuroprotective activities.9
We measured the activity of R-secretase using a fluoro-
genic peptide substrate in lysates of H4βAPP695wt cells.
Pretreatment of the cells with 10 μM 1 for 24 h led to a
significant 20% increase in R-secretase activity (Figure 4A),
with a dose response similar to that observed for inhibition of
Aβ secretion (Figure 4B). However, addition of 1 directly to
Figure 3. RCTF induction by 1. (A) Quantification of RCTF induc-
tion by 24 h treatment with 10 μM 1 or vehicle control (N = 7 each (
SD; *, p < 0.05). (B) Time course of RCTF induction by 10 μM 1.
the cell lysates, rather than to the live cells, had no effect on
R-secretase activity (data not shown), implying an indirect
cellular activation of this pathway rather than direct activa-
tion of the R-secretase protease by 1.
The tumor promoter phorbal myristate acetate is an
activator of protein kinase C (PKC) that has been reported
to activate R-secretase activity.10,11 Thus, we wanted to
determine if protein kinase C inhibitors could block the
effect of 1 on Aβ secretion. The PKC inhibitors Bisindolyl-
maleimide I and Calphostin C had no effect on basal or 1
inhibited Aβ secretion, while the PKC inhibitor Rottlerin
itself reduced Aβ secretion but did not significantly alter the
effect of 1 (Figure 5).

5304 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 14
Chakrabarti et al.
Figure 6. Compound 1 promotes cellular processing of ADAM9 and
ADAM10. 1 at increasing doses was incubated on H4βAPP695wt cells
for 24 h. Cell lysates were prepared for Western blots. Arrows point to
the pro and active forms of ADAM9, ADAM10, and ADAM17.
N= 3 each control and 1) and a 42% increase in the ratio active/
pro ADAM10 (p < 0.05). Although the direct target for 1 has
not been identified, it seems likely that its mechanism is via the
increased ADAM 9 and ADAM10 processing into active
R-secretase, which cleaves APP in a manner that precludes Aβ
production.
Commercial Analogue Evaluation. We searched for com-
pounds having at least 60% similarity to 1 using the Chem-
Bridge Hit2lead.com and PubChem Web sites. We were able to
obtain 24 compounds (2-25) commercially which were tested
for their ability to decrease Aβ secretion from H4βAPP695wt
cells. Each drug, along with 1, was tested in 1-4 independent
experiments, each in quadruplicate, at the 10 μM dose. Table 1
shows the chemical structure of 1 and the 24 analogues, their
source, product number, molecular weight, and the mean % re-
duction in Aβ secretion compared to the 0.02% DMSO vehicle
control. In these experiments, 10 μM 1 yielded a 75.3 ( 11.6%
decrease in Aβ secretion (N=4 ( SD). None of the 24 ana-
logues were as effective as 1. Visual inspection of these com-
pounds revealed that the two ortho-chlorines on aryl region A
are critical, removing one of these chlorines in 3ledtoalargeloss
in activity.
Figure 4. R-Secretase activity measured in vitro is induced by
treatment of cells with 1. (A) R-Secretase in cell lysates from cells treated
for 24 h with 10 μM 1 or vehicle control (N = 6 each ( SD, **, p =
0.001). (B) Dose-response of R-secretase activity induction by 1.
Design, Synthesis, and Evaluation of Novel Analogues.
Figty-three novel analogues (26-78) were synthesized to ex-
amine their efficacy in reducing Aβ secretion. In examining 1,
the molecule can be parsed into three regions: aryl region A,
linker region, and aryl region B. These regions along with our
synthetic approach to constructing this small library are shown
in Scheme 1. We then took the strategy of iterative modification
of the three regions starting from commercially available benzyl
halides (III). Conversion of III to the corresponding β-benzyl-
mercaptoethylamine (β-BMEA) derivatives (II) gave deriva-
tives that could further be reacted with substituted benzoyl
chlorides, giving the desired amides (I).
Figure 5. Effect of 1 on Aβ secretion is not blocked by PKC
inhibitors. Results show secreted Aβ levels compared to the respec-
tive controls with 1 treated cells shown in open bars (N = 4 each,
þ SD; **, p < 0.01; and ***, p < 0.001 for effect of 1 vs the res-
pective control for each treatment in the filled bar).
In general, various β-BMEA derivatives were synthesized
from the corresponding benzyl halides using our newly deve-
loped procedure.15,16 The commercially unavailable benzyl ha-
lides were synthesized from the corresponding benzyl alcohols
or toluene derivatives (Scheme 2),17,18 whereas the commercially
unavailable 2,6-disubstituted benzoyl chlorides were synthesized
from the corresponding acid derivatives (Scheme 2). In parti-
cular, 2,6-dibromobenzoic acid was synthesized from 2,6-dibro-
motoulene using a KMNO4 mediated oxidation strategy.19
By replacing 2,6-dichlorobezoyl moiety of aryl region A,
we synthesized seven new analogues (36, 54, 56, 63, 67, 74,
and 75), whereas the replacement of 3-chloro moiety of aryl
Several members of the ADAM (a disintegrin and metallo-
proteinase) family, ADAM9, ADAM10, and ADAM17, have
been reported to have R-secretase activity.12 These proteases are
made in inactive pro forms and must be proteolytically cleaved
into their active forms.13,14 Using antibodies specific for these
three ADAM proteases, we assessed the levels of the pro and
active forms in cells cultured with increasing concentrations of 1.
We observed a dose dependent increase in active ADAM9 and
ADAM10 but did not observe much effect on the active form of
ADAM17 (Figure 6). At the 10 μM dose of 1, we observed
a 31% increase in the ratio of active/pro ADAM9 (p < 0.05),

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 14 5305
Table 1. Commercial Analogues Inhibition of Aβ Secretiona
a *All drugs tested at 10 μM, % decrease relative to vehicle treated control cells. **Vendors: C, ChemBridge; M, Maybridge; R, Ryan Scientific.
region B gave us a library of 30 new analogues (26-29, 38-41,
43-45, 47-53, 58-62, 65, 68-70, 73, 76, and77). Modification
at the linker region yielded five new analogues (57, 66, 71, 72,
and 78). Finally, on the basis of initial findings in conjunction
with the above chemistry, several analogues were synthesized
whereby more than one region was diversified, yielding an addi-
tional 11 analogues (30-35, 37, 42, 46, 55, and 64). Although
the majority of the molecules synthesized followed the route
outlined in Scheme 2, several required specific manipulations.
Detailed schemes for these transformations can be found in
Supporting Information Schemes 1-7.
The structure of these compounds and their effects on redu-
cing Aβ secretion and total protein levels (cytotoxicity) are
shown in Table 2. Thirteen compounds (27, 29, 40, 48, 50, 51,
52, 53, 54, 55, 65, 73, and 76) at a 10 μM dose had roughly
similar efficacy as 1 (>50% inhibition of Aβ secretion), but five
of these (40, 50, 51, 54, and 55) displayed cytotoxicity with a
decrease in cell protein >20% at the 30 μM dose. Inspection of
these 13 active analogues reveals some flexibility of substitutions
on aryl region B. Replacing the meta-chorine with meta-methyl
ether (27), hydroxyl (29), nitrate (40), methyl (48), carbon
trifluoride (50), bromine (51), or iodine (53) all yielded good
activity. Other substitutions on aryl region B meta position did
not yield good activity such as cyanate (38), amine (41),
carboxylate (44), fluorine (47), formyl (49), and other more
bulky substitutions (39, 43, 45, 68, 69, 70). Removing the aryl
region B meta-chlorine (65) retained activity, as did substituting
an ortho-chlorine (76); however, substitutions with para-chlor-
ine (77), para-methyl ether (26), orpara-hydroxyl (28) ledtoloss
of activity. While an aryl region B with two substituents could be
tolerated, such as meta-chlorine meta-methyl ether (73), meta-
meta-methyl ether (60), ortho-meta-methyl ether (52), and
ortho-amide meta-chlorine (61), other compounds with two sub-
stitutions such as meta-meta-hydroxyl (59) and ortho-meta-
hydroxyl (58) lost activity. On aryl region A, substitutions for
one or both of the two chlorines in the ortho-positions, or their
removal, were not well tolerated (34, 35, 36, 37, 56, 63, 67, 74,
and 75), with the exception of bromine substitution, which

5306 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 14
Chakrabarti et al.
Scheme 1. Synthetic Analysis for Defining a Structure Activity Relationship for 1
Scheme 2. General Synthetic Scheme for the Preparation of 1 Analoguesa
a Reagents: (a) NBS, BPO, CCl4, reflux; (b) SOCl2, Et3N, DCM, 50 °C; (c) LiOH-H2O-EtOH, HSCH2CH2NH2 HCl, 35 °C, 40 min; (d) KMnO4,
3
t-BuOH-H2O, celite, reflux, conc HCl; (e) SOCl2, reflux; (f) Et3N, DCM, 0 °C to RT; (g) DCC, Et3N, DCM, RT.
maintained activity (54 and 55). No tested substitutions in
the linker region were well tolerated (30, 31, 32, 33, 57, 66, 71,
72, and 78).
To screen for bioavailability of 1 and seven active noncyto-
toxic analogues (27, 48, 52, 53, 65, 73, and 76), we assessed
their transcellular transport through confluent monolayers of
Caco-2 cells grown on transwell inserts. Monolayer integrity
was confirmed by high levels of transcellular electrical resis-
tance (>160 ohms/cm2) and by transcellular impermeability
to the fluorescent dye lucifer yellow. The compounds were
placed in the upper chamber, and 4 h later the lower chamber
media was withdrawn and used to treat H4βAPP695wt cells
for 24 h to assay inhibition of Aβ secretion. The concentration
of each compound added to the upper chamber would lead to
10 μM in the lower chamber if equilibration was complete, and
thus the activity of the lower chamber media was compared to
the activity of 10 μM drug added directly to the H4βAPP695wt
cells. Table 4 shows the result of this study. 1 crossed the
monolayer and the bottom chamber media led to a 27%
To screen for resistance to metabolism, 1 and eight non-
cytotoxic analogues with >50% Aβ reducing activity (27, 29,
48, 52, 53, 65, 73, and 76) were incubated at 20 μM for 24 h
with or without THLE-3 human hepatoma cells. This media
was diluted 1:1 with fresh media (drug concentration diluted
to 10 μM) and used to treat H4βAPP695wt cells for 24 h to
assay inhibition of Aβ secretion. Table 3 shows that 1 and
most of the active analogues maintained ∼50% of their Aβ
inhibiting activity during the 24 h incubation with the hepa-
toma cells, with the exception of 52, which lost virtually all of
itsactivity. The three analogues withthe most net activityafter
incubation with the hepatoma cells were 76, 53, and 48 with
49%, 40%, and 38% inhibition of Aβ secretion, respectively.

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Journal of Medicinal Chemistry, 2010, Vol. 53, No. 14 5307
Table 2. Newly Synthesized Analogues and Inhibition of Aβ Secretiona

5308 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 14
Table 2. Continued
Chakrabarti et al.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 14 5309
Table 2. Continued

5310 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 14
Table 2. Continued
Chakrabarti et al.
a MP, melting point given in °C; liq denotes a viscous liquid at room temperature. 30 and 66 were recrystallized from ethanol.
reduction in Aβ production, yielding 38% of the theoretical
activity. The compound with the most activity in the lower
chamber was 48, which decreased Aβ production by 50%,
with 71% of its theoretical activity. The lower chamber
media from the other two compounds that were highly ac-
tive after incubation with hepatocytes, 76 and 53, decreased
Aβ secretion by 32% and 1%, respectively. On the basis of
the combined activity data from these last two studies, 48 was
selected as the lead drug for in vivo study.
In Vivo Activity of Lead Drug. In APP transgenic mice,
cerebral Aβ turns over rapidly prior to the onset of plaque
formation with a half-life of ∼2 h,20 allowing one to test Aβ

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 14 5311
Table 3. Drug Activity Persistence after Incubation with Hepatoma Cells
hepatoma incubationa
cognition. Although there may be several distinct processes
involved in AD pathogenesis, the “amyloid hypothesis” is
supported by the presence of high levels of Aβ containing
senile plaques, the genetics of early onset familial AD, and
transgenic mouse models.21 Age is the major AD risk factor,
with AD affecting over 3% of those 65-74 years of age, 19%
of those 75-84 years of age, and 47% of those over 85 years of
age.22 Thus, we postulate that the inhibition of Aβ production
in middle age subjects, prior to Aβ plaque deposition, could
prevent or significantly delay the onset of AD. Delaying the
age of AD onset by just two years is predicted to decrease AD
incidence by 25%, while delaying the age of onset by five years
would eliminate half of AD incidence.23
no
total Aβ
(% decrease)
yes
total Aβ
(% decrease)
% remaining
activityb
compd
1
69.4
60.6
56.4
70.5
57.8
84.4
51.6
67.7
75.6
34.6
22.2
23.1
38.3
1.9
50
37
41
54
3
27
29
48
52
53
65
73
76
40.0
27.0
33.5
48.8
47
52
49
65
In the current study, we undertook an unbiased chemical
library screen to find compounds that would decrease Aβ
secretion by neuronal cells. We selected a lead hit, 1, and
characterized its mechanism of action. Aβ is produced by
sequential β- and γ-secretase cleavage of APP, while the
alternative APP cleavage by R-secretase destroys the Aβ
peptide. γ-Secretase inhibitors that inhibit Aβ production
have been developed, but γ-secretase also processes other
cellular proteins such as Notch-1, which may lead to detri-
mental side effects.24 Peptide analogue inhibitors for β-secre-
tase activity, mediated by the BACE1 protein, have also been
developed, but they have been hampered by poor cellular and
blood-brain barrier penetration.25 We found that 1 upregu-
lated sAPPR secretion and the cellular accumulation of CTF-R,
thus we concluded that 1 upregulates R-secretase activity.
Because sAPPR has neurotrophic and neuroprotective acti-
vities, R-secretase activation is thought to be an attractive
candidate for the prevention of AD.26
Phorbol esters that activate PKC have been shown to
inhibit Aβ production through activation of R-secretase acti-
vity, although the full pathway for this activation is not well
understood.11,12,27 However, phorbol esters are tumor pro-
moters and are thus not likely to be used as human therapeu-
tics. Other PKC activators such as bryostatin, which does not
have tumor promoter activity, also activate R-secretase and
have been shown to decrease cerebral Aβ levels after chronic
treatment in mutant APP/PS1 transgenic mice.28,29 Addition-
ally, muscarinic agonists and the neuropeptide PACAP acti-
vate R-secretase via their specific G protein-coupled receptors
that also signal through PKC.12 Thus, we sought to determine
if the effect of 1 was also mediated through PKC. Through the
use of inhibitors of various PKC isoforms, we conclude that 1
did not induce R-secretase via PKC activation.
a Drugs incubated with H4 cells at 10 μM, with or without preincubation
with hepatoma cells for 24 h. b % of activity surviving after hepatoma cell
incubation.
Table 4. Epithelial Transcellular Transport of Drug Activity
transcellular transporta
no
total Aβ
(% decrease)
yes
total Aβ
(% decrease)
% theoretical
activityb
compd
1
69.4
60.6
70.5
57.8
84.4
51.6
67.7
75.6
26.7
17.9
49.9
26.5
1.1
38.4
29.5
70.8
45.9
1.3
27
48
52
53
65
73
76
31.0
2.7
60.1
4.0
31.6
41.8
a Drugs alone or after incubation with Caco-2 cells to yield 10 μM if in
equilibrium. b % of activity passing through Caco-2 monolayer.
Three proteins that belong to a disintegrin and metallo-
proteinase (ADAM) family have been found to have
R-secretase activity, ADAM9, ADAM10, and ADAM17.12
These are zinc dependent membrane bound proteins with
“sheddase” activity, which cleave other membrane proteins to
release soluble ectodomain protein fragments. Geneknockout
mice have not been conclusive about the relative role of these
three ADAM proteins in R-secretase activity in vivo, and
ADAM10 and 17 knockout mice are embryonic and perinatal
lethal, respectively.12 ADAM9 and ADAM10 are expressed
highly in neurons, while cerebral ADAM17 is expressed
primarily in endothelial cells and astrocytes.30,31 ADAM
proteases are expressed as precursors and require proteolytic
removal of the prodomain in order to become active; thus,
proprotein convertases which cleave and activate ADAM
proteases may regulate cellular ADAM activity.13,14 Our
results demonstrate that 1 treatment of cells led to increased
R-secretase activity in cell lysates and was accompanied by
Figure 7. Compound 48 decreases cerebral Aβlevels in mice. APP/PS1
transgenic mice were injected iv with 100 to 125 mg/kg 48 or vehicle con-
trol. Six hours later, the mice were sacrificed and cerebral Aβ levels were
measured by an ELISA assay and normalized to cerebral protein. Com-
pound 48 led to a 39.6% reduction in cerebral Aβ levels (p < 0.0001).
lowering drugs acutely after a single administration. Thus, we
administeredasinglehighdoseofourlead drug 48 (100 mg/kg)
or vehicle iv to female APP transgenic mice and sacrificed the
mice 6 h later for assay of cerebral Aβ levels. 48 led to a
39.6% decrease in cerebral Aβ levels (Figure 7, p < 0.0001).
Discussion
Alzheimer disease is a progressive, debilitating, and ulti-
mately fatal disease of the elderly affecting memory and

5312 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 14
Chakrabarti et al.
increases in the active forms of both ADAM9 and ADAM10.
However, because 1 did not activate R-secretase activity
when added directly to cell lysates, we conclude that 1 may
work through either activation of proprotein convertases
or through effects on cell trafficking so that the ADAMs
and their convertases intersect in the same cellular compart-
ment. Additional studies will need to be done to test these
hypotheses.
A major hurdle for AD drug development is the ability of
test compounds to cross the blood-brain barrier. We used
Caco-2 cells, an epithelial cell monolayer with tight junctions,
as a surrogate to help predict which analogues might best
penetrate the blood-brain barrier.32 On the basis of these
results, we selected 48 for our in vivo screen. These studies
employed a very high dose of 48, but we were able to observe a
significant ∼40% decrease in cerebral Aβ levels in the drug
treated mice vs the control mice after a single iv administra-
tion. Whether this analogue is potent enough to be useful for
human studies still needs to be determined; however, we did
not discover any analogues thus far with efficacy at nM doses.
Nevertheless, drugs that work via R-secretase activation may
provetobe ausefulmethodforsignificantlydelaying the onset
of AD, and thus preventing the majority of AD incidence.
transgenic mice on the C57BL/6 genetic background (Jackson
Laboratories, strain no. 5864) as previously described.33 THLE-
3 human hepatoma cells (from ATCC) were plated in 24-well
plates that were precoated with 10 μg/mL bovine serum albu-
min, 10 μg/mL fibronectin, and 30 μg/mL bovine type I col-
lagen. The cells were grown in BEGM media (Clonetics) until
confluent and treated with drugs at 20 μM for 24 h. This
conditioned media was harvested, diluted 1:1 in serum-free
medium, and incubated with H4βAPP695wt cells for 24 h prior
to analysis of secreted Aβ, which was compared to the effect of
10 μM drug treatments without prior incubation with the
THLE-3 cells. Caco-2 cells (ATCC) were plated in 12-well plates
with Transwell inserts (Costar) at 70000 cells per well and grown
for 21 days. The media volume of the apical and basal chambers
was 0.5 and 1.5 mL, respectively. Transepithelial resistance was
measured in each well and found to be >160 Ω/cm2. Drugs at
40 μM and 20 μg/mL of lucifer yellow were added in serum-free
medium to the apical chamber. Four hours later, the basal
chamber media was harvested and used to measure lucifer
yellow fluorescence to confirm monolayer integrity as well as
for treatment of H4βAPP695wt cells for 24 h prior to analysis of
secreted Aβ, which was compared to the effect of direct addition
of 10 μM of the corresponding drug.
Assays for Secreted Aβ and Processed APP. Conditioned
medium was collected, protease inhibitor cocktail was added,
and cell debris was cleared by centrifugation prior to assays for
secreted total Aβ by ELISA and sAPPR and sAPPβ by Western
blot. The total Aβ ELISA, normalized to total cell lysate protein
by BCA assay, was preformed as previously described8 using the
capture antibody 6E10, which recognizes residues 1-16 of
human Aβ, a region present in sAPPR but absent in sAPPβ,
the long processed forms of secreted APP, and 4G8 for the
detection antibody, which is absent in both sAPPR and sAPPβ.
For Western blot analysis of sAPPR and sAPPβ, the volume of
conditioned media was adjusted by the protein level in the cell
lysate prior to SDS gel electrophoresis on 4-12% Bis-Tris SDS
gels (Invitrogen). Protein was transferred to PVDF membranes
(Invitrogen), blocked with casein blocker (Pierce), and incu-
bated with primary antibodies 6E10, which recognizes sAPPR
but not sAPPβ, or with an antibody specific for sAPPβ (both at
1:1000). After washing and incubation with HRP conjugated
secondary antibodies, the signals were detected using ECL
Western Blotting Detection kit (Pierce).
Conclusions
We performed a drug library screen to identify compounds
that inhibit Aβ secretion by cultured neuronal cells. We
determined that the lead hit lowered Aβ secretion by activa-
tion of the R-secretase processing of its precursor protein,
APP. We made a series of analogues of the lead hit and
screened these for activity along with some commercially
available analogues. We then tested the best analogues in cell
based systems to predict which might be most effective in vivo.
This lead drug was effective in lowering cerebral Aβ levels in a
transgenic mouse model of AD-like pathology. Further stud-
ies are required to determine the target of this drug, the
mechanism by which it activates R-secretase activity and its
ability to delay the onset of amyloid plaque formation in a
mouse model via chronic treatment.
Assays for Cell Lysate Proteins. Cells were washed with cold
phosphate buffered saline (PBS) and lysed on ice by vigorous
mixing over 15 min with RIPA buffer containing 50 mM Tris-
HCl (pH 8.0), 150 mM sodium chloride, 1% NP-40, 0.5%
sodium deoxycholate, and 0.1% SDS, supplemented with
10% protease inhibitor cocktail and 2 mM PMSF . After
centrifugation at 14000g for 30 min to remove nuclei and other
debris at 4 °C, the protein content of the supernatants was
measured using the BCA assay kit (Pierce). Twenty μg of cell
protein was mixed with 4ꢀ Nupage loading buffer and with
reducing agent (Invitrogen), heated at 70 °C for 10 min, and
loaded onto a 4-20% Bis-Tris gel with MES gel running buffer.
Blots were prepared and probed with anti APP C-terminus,
which binds to both holoAPP, detected at ∼110 KDa, and
RCTF detected at ∼10 KDa. An anti GAPDH antibody was
also added to detect GAPDH at 38 KDa, which was used as a
loading control for normalization. Although this gel system did
not routinely detect βCTF, preliminary studies using urea gels
demonstrated that the βCTF fragment could be separated from
RCTF but is a minor band compared to the RCTF band. For
analysis of ADAM9, ADAM10, and ADAM17, the blots were
probed with the appropriate antibodies and processed as de-
scribed above.
Experimental Section
Antibodies and Biological Reagents. Mouse anti-Aβ/APP
monoclonal antibodies 6E10 (SIG-39320) and 4G8-biotin
(SIG-39240), and rabbit anti sAPPβ (SIG-39138) were obtained
from Signet (now part of Covance), rabbit polyclonal anti-APP
C-terminal antibody (A8717) was obtained from Sigma-
Aldrich, rabbit monoclonal anti ADAM-9 (4151) was obtained
from Cell Signaling, rabbit polyclonal anti-ADAM-10 (2051)
was obtained from ProSci, rabbit polyclonal anti mouse
ADAM-17 (AB19026) was obtained from Chemicon, and rab-
bit anti-GAPDH (ab9485) was obtained from Abcam. Other
cell treatment reagents were purchased from Sigma-Aldrich
except for the PKC inhibitors (Calbiochem).
Cell Culture and Drug Treatment. H4βAPP695wt cells, a
human neuroblastoma cell line stably transfected with a
wtβAPP695 expression vector construct under the control of a
CMV promoter (kindly provided by Dr. Chris Eckman from
Mayo Clinic, Florida) were cultured as previously described.7
Twenty-four hours before drug treatment, the media was re-
placed with serum-free media. Cells were then incubated with
drugs (dissolved in DMSO) or vehicle for 24 h for collection of
conditioned media and cell lysates. For the PKC inhibitors
experiment, three inhibitors were treated 30 min prior to drug
challenge. Primary cortical neurons were prepared from em-
bryonic day 18 APP and presenilin 1 (APPswe, PSEN1dE9)
r-Secretase Activity Assay. H4βAPP695wt were treated with
vehicle or 1 for 24 h, washed with ice cold PBS, and lysed in
extraction buffer provided with the R-secretase assay kit (R&D
Systems) for 10 min on ice and centrifuged at 10000g for 1 min to

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 14 5313
remove nuclei. Protein content of the supernatant was de-
termined by the BCA assay, and 50 μg of lysate protein were
added to wells of a 96-well assay plate and the volume was
adjusted to 100 μL using the lysis buffer. 100 μL of reaction
buffer and 10 μL of the fluorogenic substrate were added, and
the plates were incubated at 37 °C in the dark with gentle
agitation for 2 h. Activity was detected in a fluorescence plate
reader using an excitation of 355 nm and an emission of
510 nm after subtraction of fluorescence in the absence of
cell lysate.
and are uncorrected. β-Benzylmercaptoethylamine (β-BMEA)
derivatives were prepared following our own procedure.15,16
1-Chloromethyl-3-iodo-benzene, 2-bromomethyl-6-chloro-
benzonitrile, 2-benzyloxy-ethylamine, and 2,6-dibromobenzoic
acid were prepared by following literature procedures.17-19,35-37
All other chemicals were purchased from commercial sources
(Aldrich, Acros, A. K. Scientific, Matrix Scientific, Mile-
stone Pharm Tech, and Wako) and used without any further
purification.
Typical Procedure for the Preparation of β-BMEA Derivatives
(VII of Supporting Information Scheme 7). LiOH (0.245 g, 10.2
mmol) was dissolved in 5 mL of water, and 15 mL of ethanol was
added. The resulting solution was added to a flask containing
cysteamine hydrochloride (0.568 g, 5 mmol), followed by the
dropwise addition of benzyl halides (5 mmol) with continuous
stirring. The reaction mixture was stirred for 40 min at 35 °C,
and ethanol was removed in vacuo. Subsequently, 20 mL of
water was added, and the mixture was extracted with dichlor-
omethane (3 ꢀ 30 mL), dried over anhydrous Na2SO4, concen-
trated in vacuo, and purified via column chromatography over
silica gel) using a mobile phase consisting of a suitable mixture of
DCM-methanol to afford the chromatographically pure de-
sired β-benzylmercaptoethylamine derivatives.
Typical Procedure for the Coupling of β-BMEA Derivatives
(VII of Scheme 2) and 2,6-Disubstituted Benzoyl Chlorides.
(Synthesis of 2,6-dichloro-N-[2-(3-methoxy-benzylsulfanyl)-
ethyl]-benzamide, 27 as representative example.) To a solution
of 2-(3-methoxy-benzylsulfanyl)-ethylamine (0.394 g, 2 mmol)
in dry DCM (15 mL) and Et3N (0.505 g 5 mmol) was added
2,6-dichloro-benzoyl chloride (0.420 g, 2 mmol) at 0 °C under
nitrogen atmosphere. The reaction mixture was further stir-
red for ∼1 h, during which time it was allowed to warm to
room temperature. Solvent evaporation in vacuo gave the
crude product, which was purified via column chromatogra-
phy over silica gel using a mobile phase consisting of a mixture
of hexane-ethyl acetate (gradient from 15% v/v ethyl acet-
ate/hexane to 35% v/v ethyl acetate/hexane) to afford the
chromatographically pure desired amidated product, 27 in
92% yield (0.68 g), 90% pure by HPLC: mp = 60-62 °C.
1H NMR (CDCl3, 400 MHz): δ 2.65 (t, J = 6.4 Hz, 2H), 3.54
(q, J = 6.4 Hz, 2H), 3.68 (s, 2H), 3.76 (s, 3H), 6.36 (t, J = 5.6
Hz, 1H), 6.72-6.74 (m, 1H), 6.84-6.88 (m, 2H), 7.15-7.25
(m, 4H). 13C NMR (CDCl3, 100 MHz): δ 30.67, 35.82, 38.59,
55.21, 112.78, 114.24, 121.17, 127.98, 129.58, 130.62, 132.13,
In Vivo Drug Administration and Cerebral Aβ Assay. Trans-
genic mice expressing humanized mutant versions of APP and
presenilin 1 (APPswe, PSEN1dE9) on the C57BL/6 genetic
background were obtained from Jackson Laboratories (strain
no. 5864) and bred to wild type C57BL/6 mice in order to obtain
∼50% transgenic that were used in these studies. These mice
develop Aβ senile plaques by 6 months of age.34 To prepare the
drug stock, 17.5 mg of compound 48 was dissolved in 40 μL of
DMSO, which was mixed sequentially with 110 μL of Cremo-
phor EL (Sigma) and 900 μL of sterile saline. Two month old
female transgenic mice weighing 20-25 g were anesthetized with
ketamine/xylazine and injected iv via the retroorbital plexus
with vehicle control or with 2.5 mg of compound 48 in 150 μL
of the drug stock (yielding a dose of ∼100 to 125 mg/kg). Six h
later, the mice were euthanized by CO2 inhalation, the brains
were removed, and the cerebellum discarded. One brain
hemisphere was homogenized for 45 s on ice using a tissue
grinder in 1 mL of solution containing 5 M guanadine HCl,
50 mM Tris (final pH adjusted to 8.3), and 5% protease
inhibitor cocktail. Any debris was removed by centrifugation
for 10 min at 2000g. The supernatant was diluted 1:10 in 20%
casein blocker in PBS with 5% protease inhibitor cocktail.
The AB1-40 standards were diluted to have an equal concen-
tration of homogenization buffer and other reagents. The
protein concentration of the brain homogenate was deter-
mined with the BCA assay, and the total Aβ levels were
normalized to brain protein levels.
Statistical Analyses. Western blot band intensities were quan-
tified by densitometric analyses using a BioRad GelDoc 2000
with Quantity One software. Data are presented as the mean (
SD. Statistical analyses were carried out using a two-tailed
Student’s t test using GraphPad Prism software, with p < 0.05
regarded as statistically significant.
Chemistry. Flash chromatography as performed on silica gel
˚
(60 A, 230-400 mesh) purchased from Dynamics Adsorbents
135.79, 139.57, 159.71, 164.50. HRMS calculated for C17H18
-
Cl2NO2S (M þ H)þ 370.04353, found 370.04491.
(Atlanta, GA). Purity of synthesized compounds was assessed
using an Agilent 1200 HPLC equipped with an Agilent Eclipse
XDB-C18 column (4.6 mm ꢀ 150 mm) monitoring absorbance
at 254 nM. All final products were >95% pure unless otherwise
noted in the experimental details. Purities were confirmed by
NMR. All purchased compounds tested were at least 95% pure
as judged by TLC analysis via densitometry using either anisal-
dehyde, 2,4-dinitrophenylhydrazine, or iodine as a stain. TLC
was done on hard layer, organic binder TLC-plates with a
fluorescent indicator and visualized by UV light (254 nm).
Solvents and reagents were of commercially available analytical
grade quality and used as received without any further purifica-
tion. 1H and 13C NMR spectra were recorded on a Varian Inova
spectrometer (at the Department of Chemistry, CWRU) oper-
Typical Procedure for Demethylation of Methoxy Derivatives.
(Synthesis of 2,6-dichloro-N-[2-(3-hydroxy-benzylsulfanyl)-
ethyl]-benzamide, 29 as representative example.) 2,6-Dichloro-
N-[2-(3-methoxy-benzylsulfanyl)-ethyl]-benzamide, 27 (370 mg,
1 mmol), was dissolved in anhydrous dichloromethane (10 mL)
under a nitrogen atmosphere, and the solution was subsequently
cooled to -78 °C. A solution of BBr3 in hexane (2.5 mL of a 1 M
solution, 2.5 mmol) was added dropwise. The reaction mix-
ture was stirred for 30 min at -78 °C, warmed slowly to room
temperature, and stirred an additional 3 h at room temperature.
The reaction was quenched by dropwise addition of 1 M HCl
(5 mL) at 0 °C followed by the addition of 15 mL of room
temperature water, the layers were separated, and the aqueous
phase was extracted with EtOAc (3 ꢀ 20 mL). The combined
organic layers were washed with water followed by brine, dried
over Na2SO4, and then concentrated in vacuo. The crude product
was purified via column chromatography over silica gel using a
mobile phase consisting of a mixture of hexane/ethyl acetate
(gradient from 20% v/v ethyl acetate/hexane to 45% v/v ethyl
acetate/hexane) as eluent to obtain chromatographically pure
desired 29 as off-white solid (0.3 g, 85%): mp = 108-110 °C. 1H
NMR (CDCl3, 400 MHz): δ 2.63 (t, J = 6.4 Hz, 2H), 3.55 (q, J =
6.4 Hz, 2H), 3.61 (s, 2H), 6.43 (t, J = 6.0 Hz, 1H), 6.63-6.66 (m,
1H), 6.78-6.8(m, 2H), 7.07 (t, J = 8 Hz, 1H), 7.19-7.29 (m, 3H),
1
ating at 400 and 100 MHz for the H and 13C NMR spectra,
respectively. The internal references were TMS (δ 0.00) and
CDCl3 (δ 77.2) for 1H and 13C spectra, respectively. NMR data
are presented in the following order: chemical shift, peak multi-
plicity (s = singlet, d = doublet, t = triplet, q = quartet, m =
multiplet, dd=doublet of doublet, bs=broad singlet), coupling
constant, proton number. Mass spectra were obtained on a Kratos
MS 25 mass spectrometer (at the Department of Chemistry,
Case Western Reserve University) using FAB ionization
method in m-nitrobenzyl alcohol or glycerol matrices. Melting
points were determined with a MEL-TEMP capillary apparatus

5314 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 14
Chakrabarti et al.
7.31 (bs, 1H). 13C NMR(CDCl3, 100MHz): δ 30.25, 35.58, 38.97,
114.45, 116.07, 120.64, 128.17, 129.89, 130.99, 132.24, 135.43,
139.52, 156.36, 165.29. HRMS calculated for C16H16Cl2NO2S
(M þ H)þ 356.02788, found 356.02903.
calculated for C18H21ClNOS (M þ H)þ 334.10324, found
334.10221.
Procedure for DCC-Mediated Coupling of Carboxylic Acids.
(Synthesis of N-[2-(3-chloro-benzylsulfanyl)-ethyl]-2-(2,6-di-
chloro-phenyl)-acetamide, 78 as representative example.) To a
solution of 2,6-dichlorophenylacetic acid (0.41 g, 2 mmol) in dry
DCM (10 mL) was added DCC (0.435 g, 2.1 mmol) and
resultant mixture was stirred under N2 for 30 min. A mixture
of β-(3-chlorobenzyl)mercaptoethylamine (0.404 g, 2 mmol)
and triethylamine (0.252 g, 2.5 mmol) in dry DCM (4 mL) was
added and the resultant mixture was left for stirring at room
temperature under N2 for 1 h. The reaction mixture was then
filtered to remove the precipitated dicyclohexylurea (DHU).
The filtrate was concentrated in vacuo and the crude product
was dissolved with cold EtOAc to effect the precipitation of
more DHU, which was removed by further filtration. The
organic layer was concentrated in vacuo and the crude product
was purified by silica gel column chromatography using a
mixture of hexane/ethyl acetate (gradient from 15% v/v ethyl
acetate/hexane to 35% v/v ethyl acetate/hexane) as eluent to
obtain the desired amidated product, 78 (0.62 g, 80%): mp =
Typical Procedure for the Preparation of Sulfone Derivatives.
(Synthesis of 2,6-dichloro-N-[2-(3-methoxy-phenylmethane-
sulfonyl)-ethyl]-benzamide, 30 as representative example.) To
a solution of 2,6-dichloro-N-[2-(3-methoxy-benzylsulfanyl)-
ethyl]-benzamide, 27 (370 mg, 1 mmol), in glacial acetic acid
(6 mL) was added hydrogen peroxide (2 mL, 30% solution).
After 24 h stirring at room temperature, the acetic acid was
removed under vacuum and the crude sulfone was purified by
recrystallization from ethanol (0.3 g, 75%): mp =172-173 °C.
1H NMR (CDCl3, 400 MHz): δ 3.19-3.22 (m, 2H), 3.83 (s, 3H),
3.91-3.96 (m, 2H), 4.27 (s, 2H), 6.55 (bs, 1H), 6.93-6.99 (m,
3H), 7.23-7.35 (m, 4H). 13C NMR (CDCl3, 100 MHz): δ 32.98,
50.72, 55.59, 60.81, 115.19, 116.35, 123.04, 128.30, 128.93,
130.51, 131.14, 132.39, 135.45, 160.31, 164.95. LRMS calculated
for C17H18Cl2NO4S (M þ H)þ 402.0, found 402.1.
Typical Procedure for the Preparation of Sulfoxide Deriva-
tives. (Synthesis of 2,6-dichloro-N-[2-(3-methoxy-phenylme-
thanesulfinyl)-ethyl]-benzamide, 32 as representative example.)
To a solution of 2,6-dichloro-N-[2-(3-methoxy-benzylsulfanyl)-
ethyl]-benzamide, 27 (370 mg, 1 mmol), in chloroform at -10 °C
was added m-chloroperbenzoic acid (1 mmol) and the resulting
mixture was stirred at -10 °C for 12 h. Subsequently, a saturated
solution of NaHCO3 (15 mL) was added, the reaction was
stirred for 5 min, followed by extracting with chloroform (2 ꢀ
20 mL). The organic layer was washed with water, dried over
Na2SO4, concentrated in vacuo, and the crude product was
purified via column chromatography over silica gel using a
mobile phase consisting of a mixture of hexane/ethyl acetate
(gradient from 20% v/v ethyl acetate/hexane to 50% v/v ethyl
acetate/hexane) as eluent to obtain chromatographically pure
desired product, 32 as white solid (0.247 g, 64%): mp =
1
116-118 °C. H NMR (CDCl3, 400 MHz): δ 2.54 (bs, 2H),
3.38-3.39 (m, 2H), 3.63 (s, 2H), 3.92 (s, 2H), 5.88 (bs, 1H),
7.16-7.36 (m, 7H). 13C NMR (CDCl3, 100 MHz): δ 31.35,
35.49, 38.39, 39.06, 127.24, 127.59, 128.64, 129.09, 129.44,
130.04, 131.78, 134.59, 136.52, 140.36, 168.53. HRMS calcu-
lated for C17H17Cl3NOS (M þ H)þ 388.00964, found 388.00983.
Specific Procedures for Reactions Deviating from Typical
Conditions. 2,6-Dichloro-N-(2-[3-(N-hydroxycarbamimidoyl)-
benzylsulfanyl]-ethyl)-benzamide (39). 2,6-Dichloro-N-[2-(3-
cyano-benzylsulfanyl)-ethyl]-benzamide, 38 (0.365 g, 1 mmol),
hydroxylamine hydrochloride (0.278 g, 4 mmol) and K2CO3
(0.276 g, 2 mmol) were dissolved in a mixture of water (6 mL)
and ethanol (9 mL). The solution was gently stirred for 10 min
and then was heated under reflux for 5 h. The resulting solution
was cooled, concentrated in vacuo, and the residue was parti-
tioned between water and DCM. The organic extracts were
dried over Na2SO4, evaporated in vacuo, and the crude
product was purified via column chromatography over silica
gel using a mobile phase consisting of a mixture of hexane/
ethyl acetate (gradient from 25% v/v ethyl acetate/hexane to
80% v/v ethyl acetate/hexane) as eluent to obtain chromato-
graphically pure desired, 39 (0.165 g, 42%) as a white solid:
mp = 126-127 °C. 1H NMR (DMSO-d6, 400 MHz): δ 2.54 (t,
J = 6.8 Hz, 2H), 3.39 (q, J = 6.4 Hz, 2H), 3.78 (s, 2H), 5.75 (bs,
2H), 7.27-7.52 (m, 6H), 7.63 (s, 1H), 8.82 (t, J = 5.2 Hz, 1H),
9.59 (s, 1H). 13C NMR (DMSO-d6, 100 MHz): δ 30.38, 35.39,
39.36, 124.62, 126.60, 128.74, 128.81, 130.10, 131.65, 131.77,
134.16, 137.07, 139.06, 151.32, 164.24. HRMS calculated for
C17H18Cl2N3O2S (M þ H)þ 398.04968, found 398.04691.
N-[2-(3-Amino-benzylsulfanyl)-ethyl]-2,6-dichloro-benzamide
(41). 2,6-Dichloro-N-[2-(3-nitro-benzylsulfanyl)-ethyl]-benza-
mide, 40 (0.385 g, 1 mmol), and sodium acetate (0.41 g, 5 mmol)
were added to 15 mL of EtOH, and the mixture was heated to 75 °C
1
144-146 °C. H NMR (CDCl3, 400 MHz): δ 2.71-2.76 (m,
1H), 3.03-3.09 (m, 1H), 3.81 (s, 3H), 3.82-3.89 (m, 1H), 3.92 (s,
2H), 3.94-4.01 (m, 1H), 6.77 (t, J = 2.0 Hz, 1H), 6.81 (d, J = 8.0
Hz, 1H), 6.87-6.89 (m, 1H), 7.21-7.30 (m, 4H), 7.35 (t, J = 5.6
Hz, 1H). 13C NMR (CDCl3, 100 MHz): δ 34.56, 49.55, 55.53,
58.46, 114.32, 115.84, 122.51, 128.20, 130.35, 130.49, 130.88,
132.45, 135.96, 160.19, 165.15. HRMS calculated for C17H18-
Cl2NO3S (M þ H)þ 386.03844, found 386.03650.
Typical Procedure for Amidation of 2,6-Disubstituted Benzoic
Acids through Acid Chloride Formation. (Synthesis of N-[2-(3-
chloro-benzylsulfanyl)-ethyl]-2,6-dimethyl-benzamide, 75 as re-
presentative example.).One drop of DMF was added to a
solution of 2,6-dimethylbenzoic acid (0.3 g, 2 mmol) in thionyl
chloride (2 mL), and the mixture was refluxed for 2 h. It was then
cooled to room temperature and the excess thionyl chloride was
removed in vacuo. The solid residue was dissolved in dry DCM
(5 mL) and cooled to 0 °C. A mixture of β-(3-chloroben-
zyl)mercaptoethylamine (0.404 g, 2 mmol) and triethylamine
(1 mL, 7 mmol) in dry DCM (5 mL) was added in dropwise
manner at 0 °C under nitrogen atmosphere, and the mixture was
allowed to stir at room temperature for 1.5 h. After the comple-
tion of reaction (as judged by TLC) the solvent was removed in
vacuo and the solid residue was dissolved in DCM (30 mL). The
organic phase was washed with brine (15 mL) and water
(15 mL), dried over Na2SO4, and concentrated in vacuo. The
crude residue was purified by silica gel column chromatography
using a mixture of hexane/ethyl acetate (gradient from 15% v/v
ethyl acetate/hexane to 35% v/v ethyl acetate/hexane) as eluent
to obtain the desired amidated product, 75 (0.507 g, 76%):
mp = 53-55 °C. 1H NMR (CDCl3, 400 MHz): δ 2.27 (s, 6H),
2.65 (t, J = 6.4 Hz, 2H), 3.57 (q, J = 6.4 Hz, 2H), 3.69 (s, 2H),
6.06 (bs, 1H), 6.99 (d, J = 7.6 Hz, 2H), 7.14 (t, J = 7.6 Hz, 1H),
7.19-7.23 (m, 3H), 7.32 (s, 1H). 13C NMR (CDCl3, 100 MHz):
δ 19.41, 31.42, 35.46, 38.11, 127.27, 127.65, 127.71, 128.96,
129.12, 130.07, 134.39, 134.65, 137.61, 140.32, 170.61. HRMS
until the solid dissolved completely. SnCl2 2H2O (1.12 g, 5 mmol)
3
was added, and the reaction mixture was subsequently stirred at
75 °C for 40 min. After removal of solvent in vacuo, the residue was
dissolved in 40 mL EtOAc and washed with 45% K2CO3 (25 mL),
dried over anhydrous Na2SO4, concentrated in vacuo, and purified
via column chromatography over silica gel using a mobile phase
consisting of a mixture of hexane/ethyl acetate (gradient from 25%
v/v ethyl acetate/hexane to 70% v/v ethyl acetate/hexane) as eluent
to obtain chromatographically pure 41 (0.265 g, 75%): mp =
55-56 °C. 1H NMR (CDCl3, 400 MHz): δ 2.67 (t, J = 6.4 Hz,
2H), 3.57 (q, J = 6.4 Hz, 2H), 3.62 (s, 2H), 3.69 (bs, 2H), 6.29 (bs,
1H), 6.49-6.52 (m, 1H), 6.64-6.68 (m, 2H), 7.03 (t, J = 7.6 Hz,
1H), 7.21-7.31 (m, 3H). 13C NMR (CDCl3, 100 MHz): δ 30.82,
35.99, 38.83, 114.22, 115.67, 119.18, 128.25, 129.74, 130.87, 132.42,
136.09, 139.27, 146.97, 164.73. LRMS calculated for C16H17Cl2-
N2OS (M þ H)þ 355.0, found 355.2.

Article
3-[2-(2,6-Dichloro-benzoylamino)-ethylsulfanylmethyl]-benzoic
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 14 5315
(0.24 g, 1 mmol) in a dropwise manner. The resulting mixture
was allowed to stir for 18 h at room temperature. The reaction
mixture was poured into 35 mL of water and stirred for 10 min.
The aqueous layer was extracted with DCM (2 ꢀ 25 mL), the
combined organic extracts was dried over anhydrous Na2SO4,
concentrated in vacuo, and purified via column chromatography
over silica gel using a mobile phase consisting of a mixture of
hexane/ethyl acetate (gradient from 25% v/v ethyl acetate/hexane
to 80% v/v ethyl acetate/hexane) to yield chromatographically
pure 57 as colorless liquid (0.216 g, 60%). 1H NMR (CDCl3, 400
MHz): δ 1.96 (s, 1H), 2.59 (t, J = 6.4 Hz, 2H), 2.77 (t, J = 6.4 Hz,
2H), 3.60 (s, 2H), 4.08 (s, 2H), 7.12-7.22 (m, 4H), 7.27 (s, 1H),
7.31 (d, J = 8.0 Hz, 2H). 13C NMR (CDCl3, 100 MHz): δ 31.95,
35.63, 47.09, 48.18, 127.19, 127.44, 128.63, 129.12, 129.19, 129.94,
134.50, 135.81, 136.16, 140.63. HRMS calculated for C16H17-
Cl3NS (M þ H)þ 360.01473, found 360.01422.
Acid (44). To a solution of 3-[2-(2,6-dichloro-benzoylamino)-
ethylsulfanylmethyl]-benzoic acid methyl ester, 43 (0.398 g,
1 mmol), in THF (7 mL) was added 5 mL of aqueous LiOH
(0.072 g, 3 mmol) solution and the resulting mixture was stirred
at 60 °C (∼2 h) until the ester starting material was consumed
completely (as judged by TLC). The mixture was then allowed to
cool to room temperature, and solvent was removed in vacuo
followed by dilution of the residue with water. The resulting
aqueous mixture was extracted once with EtOAc. The pH of the
aqueous phase was adjusted to approximately 1 via the addition
of 1 M HCl. The precipitate was filtered off, washed with water,
and dried, yielding 44 (0.29 g, 75%) as a white solid, 90% pure
by HPLC: mp = 165-166 °C. 1H NMR (acetone-d6, 400 MHz):
δ 2.69-2.73 (m, 2H), 3.57-3.62 (m, 2H), 3.92 (s, 2H), 7.37-7.47
(m, 4H), 7.65-7.67 (m, 1H), 7.89-7.92 (m, 2H), 8.05 (t, J = 2.0
Hz, 1H). 13C NMR (acetone-d6, 100 MHz): δ 30.48, 35.09,
39.17, 128.21, 128.37, 128.79, 130.32, 130.95, 131.06, 132.00,
133.79, 136.97, 139.69, 164.05, 166.87. HRMS calculated for
C17H16Cl2NO3S (M þ H)þ 384.02279, found 384.02352.
N-[2-(3-Carbamoyl-benzylsulfanyl)-ethyl]-2,6-dichloro-benza-
mide (45). To a stirred solution of 2,6-dichloro-N-[2-(3-cyano-
benzylsulfanyl)-ethyl]-benzamide, 38 (0.365 g, 1 mmol), in
DMSO (1.5 mL) at 0 °C was added 30% H2O2 (0.12 mL) and
anhydrous K2CO3 (0.03 g). The reaction mixture was allowed to
warm to room temperature and stirred for an additional 10 min.
Then 30 mL of water was added to the reaction mixture followed
by stirring for an additional 10 min. The aqueous phase was
extractedwithEtOAc(3ꢀ 20 mL), dried over anhydrous Na2SO4,
concentrated in vacuo, and the crude product was purified via
column chromatography over silica gel using a mobile phase
consisting of a mixture of hexane/ethyl acetate (gradient from
25% v/v ethyl acetate/hexane to 75% v/v ethyl acetate/hexane) as
eluent to obtain chromatographically pure 45 (0.25 g, 65%) as a
white solid: mp = 148-150 °C. 1H NMR (CD3OD, 400 MHz): δ
2.61-2.65 (m, 2H), 3.50-3.53 (m, 2H), 3.84 (s, 2H), 7.32-7.43
(m, 4H), 7.55 -7.57 (m, 1H), 7.73-7.75 (m, 1H), 7.86 -7.87 (m,
1H). 13C NMR (CD3OD, 100 MHz): δ 29.59, 34.94, 39.06, 126.23,
128.02, 128.12, 128.61, 130.95, 132.01, 132.47, 133.98, 136.08,
139.39, 165.98, 171.03. HRMS calculated for C17H17Cl2N2O2S
(M þ H)þ 383.03878, found 383.03804.
2,6-Dichloro-N-(2-[3-(cyclopropanecarbonyl-amino)-benzylsul-
fanyl]-ethyl)-benzamide (69). To a solution of N-[2-(3-amino-ben-
zylsulfanyl)-ethyl]-2,6-dichloro-benzamide, 41 (0.423 g, 1 mmol),
and Et3N (0.303 g, 3 mmol) in dry CH2Cl2 (10.0 mL) was added
cyclopropanecarbonyl chloride (0.105 g, 1 mmol) at room
temperature. The resulting solution was refluxed for 12 h, cooled
to room temperature, and the solvent was removed in vacuo to
afford a thick oily residue which was purified by column
chromatography over silica gel using a mobile phase consisting
of a mixture of hexane/ethyl acetate (gradient from 25% v/v
ethyl acetate/hexane to 50% v/v ethyl acetate/hexane) as eluent
to obtain chromatographically pure 69 (0.21 g, 50%) as a white
solid: mp = 46-47 °C. 1H NMR (CDCl3, 400 MHz): δ 0.63-
0.65 (m, 2H), 0.74 (bs, 2H), 1.47-1.49 (m, 1H), 2.61 (t, J=6.8
Hz, 2H), 3.44 (q, J=6.8 Hz, 2H), 3.64 (s, 2H), 6.98 (d, J=7.6 Hz,
1H), 7.16-7.27 (m, 6H), 7.57 (s, 1H), 8.57 (s,1H). 13C NMR
(CDCl3, 100 MHz): δ 8.05, 15.46, 30.32, 35.83, 38.97, 118.88,
121.24, 124.42, 128.15, 129.47, 130.76, 132.33, 136.09, 138.49,
138.79, 165.16, 172.95. HRMS calculated for C20H21Cl2N2O2S
(M þ H)þ 423.07008, found 423.07069.
2,6-Dichloro-N-(2-(3-[2-(2-hydroxy-ethoxy)-ethoxy]-benzylsul-
fanyl)-ethyl)-benzamide (70). To a stirred solution of 2,6-dichloro-
N-[2-(3-hydroxy-benzylsulfanyl)-ethyl]-benzamide, 29 (0.356 g,
1 mmol) and K2CO3 (0.414 g, 3 mmol) in 4 mL of dry DMF
was added ethylene glycol mono-2-chloroethyl ether (0.13 g, 1.05
mmol). The resulting mixture was allowed to stir for 12 h
at 100 °C. After cooling to room temperature, water was added
(25 mL) and the reaction mixture was extracted with DCM (2 ꢀ 25
mL). The combined organic extracts were dried over anhydrous
Na2SO4, concentrated in vacuo, and purified via column chroma-
tography over silica gel using a mobile phase consisting of a mixture
of hexane/ethyl acetate (gradient from 25% v/v ethyl acetate/
hexane to 80% v/v ethyl acetate/hexane) to yield chromatographi-
cally pure 70 as colorless liquid (0.230 g, 52%), 86% pure by HPLC.
1H NMR (CDCl3, 400 MHz): δ2.68 (t, J = 6.4 Hz, 2H), 3.54-3.63
(m, 4H), 3.66-3.70 (m, 4H), 3.82-3.84 (m, 2H), 4.11-4.13 (m,
2H), 6.76-6.83 (m, 2H), 6.90-6.92 (m, 2H), 7.17-7.29 (m, 4H).
13C NMR (CDCl3, 100 MHz): δ 30.73, 35.97, 38.90, 61.77, 67.55,
69.81, 72.77, 113.59, 115.26, 121.73, 128.14, 129.81, 130.76, 132.34,
136.17, 139.88, 159.02, 164.79. HRMS calculated for C20H24Cl2-
NO4S (M þ H)þ 444.08031, found 444.07924.
2,6-Dichloro-N-[2-(3-formyl-benzylsulfanyl)-ethyl]-benzamide
(49). To a stirred solution of 2,6-dichloro-N-[2-(3-cyano-
benzylsulfanyl)-ethyl]-benzamide, 38 (0.365 g, 1 mmol), in dry
THF (10 mL) at -78 °C under nitrogen atmosphere was added
1 M solution of DIBAL-H in toluene (1.2 mL, 1.2 mmol) drop-
wise. The reaction mixture was stirred and gradually warmed
from -78 °C to room temperature over 2 h. The reaction
mixture was then quenched by addition of a mixture of saturated
aqueous NH4Cl solution and 6 M aqueous HCl (5:1, v/v) at 0 °C.
The organic solvent was removed in vacuo, and the residue was
partitioned between ethyl acetate and brine. The organic solvent
was dried over anhydrous Na2SO4, concentrated in vacuo, and
the crude product was purified via column chromatography
over silica gel using a mobile phase consisting of a mixture of
hexane/ethyl acetate (gradient from 20% v/v ethyl acetate/
hexane to 45% v/v ethyl acetate/hexane) as eluent to obtain
chromatographically pure 49 as white solid (0.19 g, 52%): mp =
63-65 °C. 1H NMR (CDCl3, 400 MHz): δ 2.71 (t, J = 6.4 Hz,
2H), 3.63 (q, J = 6.4 Hz, 2H), 3.83 (s, 2H), 6.28 (bs, 1H),
7.23-7.31 (m, 3H), 7.49 (t, J = 7.6 Hz, 1H), 7.63 (d, J = 7.6 Hz,
1H), 7.76 (d, J = 7.6 Hz, 1H), 7.85 (s, 1H), 9.99 (s, 1H). 13C
NMR (CDCl3, 100 MHz): δ 31.03, 35.56, 38.76, 128.29, 128.86,
129.61, 130.19, 130.96, 132.39, 135.19, 135.94, 136.92, 139.51,
164.78, 192.38. HRMS calculated for C17H16Cl2NO2S (M þ
H)þ 368.02788, found 368.02880.
2,6-Dichloro-N-[2-(4-methoxy-benzylsulfanyl)-ethyl]-benza-
mide (26). The compound was prepared using 2-(4-methoxy-
benzylsulfanyl)-ethylamine as the amine component and follow-
ing the procedure described for 27: mp = 80-81 °C. 1H NMR
(CDCl3, 400 MHz): δ 2.68 (t, J = 6.4 Hz, 2H), 3.60 (q, J = 6.0
Hz, 2H), 3.69 (s, 3H), 3.78 (s, 3H), 6.13 (bs, 1H), 6.80-6.83 (m,
2H), 7.22-7.31 (m, 5H). 13C NMR (CDCl3, 100 MHz): δ 30.75,
35.27, 38.56, 55.38, 114.13, 128.15, 129.91, 130.05, 130.77,
132.33, 135.96, 158.84, 164.59. HRMS calculated for
C17H18Cl2NO2S (M þ H)þ 370.04353, found 370.04217.
2,6-Dichloro-N-[2-(4-hydroxy-benzylsulfanyl)-ethyl]-benzamide
(28). The compound was prepared from 26 using the proce-
dure described for 29: mp = 138-140 °C. 1H NMR (acetone-d6,
[2-(3-Chloro-benzylsulfanyl)-ethyl]-(2,6-dichloro-benzyl)-amine
(27). To a stirred solution of β-(3-chlorobenzyl)mercapto-
ethylamine (0.202 g, 1 mmol) and K2CO3 (0.276 g, 2 mmol) in
5 mL of dry DMF was added 2,6-dichlorobenzyl bromide

5316 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 14
Chakrabarti et al.
400 MHz): δ 2.66-2.69 (m, 2H), 3.54-3.59 (m, 2H), 3.74 (s,
2H), 6.76-6.79 (m, 2H), 7.19-7.23 (m, 2H), 7.36-7.43 (m, 3H),
7.85 (bs, 1H), 8.32 (s, 1H). 13C NMR (acetone-d6, 100 MHz): δ
30.15, 34.93, 39.26, 115.39, 128.22, 129.43, 130.36, 130.95,
132.01, 137.01, 156.64, 164.03. HRMS calculated for C16H16-
Cl2NO2S (M þ H)þ 356.02788, found 356.02820.
2,6-Dichloro-N-[2-(3-cyano-benzylsulfanyl)-ethyl]-benzamide
(38). The compound was prepared using 3-(2-amino-ethyl-
sulfanylmethyl)-benzonitrile as the amine component and follow-
ing the procedure described for 27: mp=123-125 °C. 1H NMR
(CDCl3, 400 MHz): δ 2.69 (t, J = 6.4 Hz, 2H), 3.63 (q, J = 6.4 Hz,
2H), 3.78 (s, 2H), 6.26 (bs, 1H), 7.24-7.32 (m, 3H), 7.43 (t, J = 7.6
Hz, 1H), 7.54 (d, J = 7.6 Hz, 1H), 7.60 (d, J = 7.6 Hz, 1H), 7.65 (s,
1H). 13C NMR (CDCl3, 100 MHz): δ 31.05, 35.33, 38.74, 112.94,
118.81, 128.31, 129.67, 131.00, 131.15, 132.39, 132.55, 133.67,
135.89, 139.91, 164.82. HRMS calculated for C17H15Cl2N2OS
(M þ H)þ 365.02821, found 365.02794.
2,6-Dichloro-N-[2-(3-hydroxy-phenylmethanesulfonyl)-ethyl]-
benzamide (31). The compound was prepared from 30 using the
procedure described for 29: mp = 139-141 °C. 1H NMR (ace-
tone-d6, 400 MHz): δ 3.34-3.38 (m, 2H), 3.82-3.87 (m, 2H),
4.43 (s, 2H), 6.85-6.88 (m, 1H), 6.99 (d, J = 7.6 Hz, 2H), 7.22 (t,
J = 7.6 Hz, 1H), 7.41-7.43 (m, 3H), 7.98 (bs, 1H), 8.56 (s, 1H).
13C NMR (acetone-d6, 100 MHz): δ 33.38, 50.63, 59.29, 115.87,
118.21, 122.40, 128.28, 129.88, 130.21, 131.25, 131.98, 136.45,
157.76, 164.46. LRMS calculated for C16H16Cl2NO4S (M þ H)þ
388.0, found 388.2.
2,6-Dichloro-N-[2-(3-nitro-benzylsulfanyl)-ethyl]-benzamide
(40). The compound was prepared using 2-(3-nitro-benzyl-
sulfanyl)-ethylamine as the amine component and following
the procedure described for 27: mp = 76-78 °C. 1H NMR
(CDCl3, 400 MHz): δ 2.72 (t, J = 6.4 Hz, 2H), 3.64 (q, J = 6.4
Hz, 2H), 3.86 (s, 2H), 6.27 (bs, 1H), 7.23-7.32 (m, 3H), 7.50 (t,
J = 7.6 Hz, 1H), 7.71 (d, J = 7.6 Hz, 1H), 8.11 (d, J = 8.0 Hz,
1H), 8.22 (s, 1H). 13C NMR (CDCl3, 100 MHz): δ 31.15, 35.45,
38.79, 122.53, 123.92, 128.30, 129.80, 130.99, 132.38, 135.29,
135.87, 140.51, 148.63, 164.80. HRMS calculated for C16H15-
Cl2N2O3S (M þ H)þ 385.01804, found 385.02043.
2,6-Dichloro-N-[2-(3-hydroxy-phenylmethanesulfinyl)-ethyl]-
benzamide (33). The compound was prepared from 29 using the
procedure described for 32: mp = 165-166 °C. 1H NMR
(CD3OD, 400 MHz): δ 2.91-2.95 (m, 1H), 3.13-3.20 (m,
1H), 3.68-3.75 (m, 1H), 3.81-3.85 (m, 1H), 4.06-4.16 (m,
2H), 6.76-6.84 (m, 3H), 7.19 (t, J = 8.0 Hz, 1H), 7.37-7.42 (m,
3H). 13C NMR (DMSO-d6, 100 MHz): δ 33.77, 50.96, 58.53,
115.70, 117.42, 121.16, 128.14, 129.99, 130.84, 131.73, 132.17,
136.66, 158.00, 164.89. HRMS calculated for C16H16Cl2NO3S
(M þ H)þ 372.02279, found 372.02187.
2-Chloro-6-methoxy-N-[2-(3-methoxy-benzylsulfanyl)-ethyl]-
benzamide (42). The compound was prepared using 2-chloro-6-
methoxy-benzoyl chloride as the acid chloride and following the
procedure described for 27: mp = 58-59 °C. 1H NMR (CDCl3,
400 MHz): δ 2.68 (t, J = 6.4 Hz, 2H), 3.59 (q, J = 6.4 Hz, 2H),
3.71 (s, 2H), 3.78 (s, 3H), 3.79 (s, 3H), 6.17 (s, 1H), 6.75-6.81 (m,
2H), 6.88-6.91 (m, 2H), 6.97 (d, J = 8.0 Hz, 1H), 7.18-7.27 (m,
2H). 13C NMR (CDCl3, 100 MHz): δ 31.13, 36.00, 38.74, 55.74,
56.30, 109.67, 113.02, 114.86, 121.41, 121.86, 126.38, 129.80,
130.89, 132.11, 139.85, 157.43, 159.97, 165.37. HRMS calcu-
lated for C18H21ClNO3S (M þ H)þ 366.09307, found 366.09276.
3-[2-(2,6-Dichloro-benzoylamino)-ethylsulfanylmethyl]-ben-
zoic Acid Ethyl Ester (43). The compound was prepared using
3-(2-amino-ethylsulfanylmethyl)-benzoic acid ethyl ester as the
amine component and following the procedure described for 27:
mp = 43-45 °C. 1H NMR (CDCl3, 400 MHz): δ 1.39 (t, J = 7.2
Hz, 3H), 2.71 (t, J=6.4 Hz, 2H), 3.62 (q, J=6.4 Hz, 2H), 3.79 (s,
2H), 4.37 (q, J=7.2 Hz, 2H) 6.22 (bs, 1H), 7.22-7.31 (m, 3H),
7.38 (t, J=7.6 Hz, 1H), 7.54 (d, J=7.6 Hz, 1H), 7.91 (d, J=8.0
Hz, 1H), 7.98 (s, 1H). 13C NMR (CDCl3, 100 MHz): δ 14.55,
31.14, 35.77, 38.69, 61.33, 128.26, 128.67, 128.93, 130.04, 130.90,
131.09, 132.43, 133.50, 135.99, 138.61, 164.73, 166.56. HRMS
calculated for C19H20Cl2NO3S (M þ H)þ 412.05409, found
412.05431.
2,6-Dimethoxy-N-[2-(3-methoxy-benzylsulfanyl)-ethyl]-ben-
zamide (34). The compound was prepared using 2,6-dimethoxy-
benzoyl chloride as acid chloride and following the procedure
described for 27: mp = 41-43 °C. 1H NMR (CDCl3, 400 MHz):
δ 2.69 (t, J = 6.4 Hz, 2H), 3.60 (q, J = 6.4 Hz, 2H), 3.73 (s, 2H),
3.79 (s, 9H), 6.13 (bs, 1H), 6.55 (d, J = 8.4 Hz, 2H), 6.78 (dd, J =
1.6 Hz, J = 7.6 Hz, 1H), 6.89-6.92 (m, 2H), 7.19-7.29 (m, 2H).
13C NMR (CDCl3, 100 MHz): δ 31.33, 35.99, 38.78, 55.45, 56.18,
104.22, 113.01, 114.49, 115.89, 121.46, 129.79, 130.96, 139.97,
157.66, 159.99, 166.17. HRMS calculated for C19H24NO4S (M þ
H)þ 362.14260, found 362.14429.
2,6-Dihydroxy-N-[2-(3-hydroxy-benzylsulfanyl)-ethyl]-benza-
mide (35). The compound was prepared from 34 using the pro-
cedure described for 29: mp = 61-62 °C. 1H NMR (acetone-d6,
400 MHz): δ 2.77 (t, J = 6.4 Hz, 2H), 3.84 (q, J = 6.4 Hz, 2H),
4.11 (s, 2H), 6.68-6.75 (m, 3H), 6.82 (d, J = 8.8 Hz, 1H), 6.93 (s,
1H), 7.02 (t, J = 8.0 Hz, 1H), 7.67 (t, J = 8.4 Hz, 1H), 10.49 (bs,
1H). 13C NMR (acetone-d6, 100 MHz): δ 35.39, 41.24, 57.24,
103.83, 112.16, 114.30, 116.14, 119.87, 129.47, 139.11, 139.71,
157.87, 159.86, 165.52. LRMS calculated for C16H18NO4S (M þ
Na)þ 342.1, found 342.2.
N-[2-(3-Chloro-benzylsulfanyl)-ethyl]-2,6-dimethoxy-benza-
mide (36). The compound was prepared following the proce-
dure described for 27. 2,6-Dimethoxybenzoyl chloride was
used as acid chloride and 2-(3-chloro-benzylsulfanyl)-ethyla-
mine as the amine component: mp = 90-91 °C. 1H NMR
(CDCl3, 400 MHz): δ 2.68 (t, J=6.4 Hz, 2H), 3.60 (q, J=6.4
Hz, 2H), 3.72 (s, 2H), 3.79 (s, 6H), 6.13 (bs, 1H), 6.55 (d, J=8.4
Hz, 2H), 7.19-7.29 (m, 4H), 7.33 (s, 1H). 13C NMR (CDCl3,
100 MHz): δ 31.38, 35.51, 38.81, 56.19, 104.23, 115.81, 127.34,
127.55, 129.15, 130.05, 130.99, 134.59, 140.55, 157.65, 166.18.
HRMS calculated for C18H21ClNO3S (M þ H)þ 366.09307,
found 366.09277.
2,6-Difluoro-N-[2-(3-fluoro-benzylsulfanyl)-ethyl]-benzamide
(46). The compound was prepared following the procedure
described for 27. 2,6-Difluorobenzoyl chloride was used as acid
chloride and 2-(3-fluoro-benzylsulfanyl)-ethylamine as the
1
amine component, 92% pure by HPLC: mp=39-41 °C. H
NMR (CDCl3, 400 MHz): δ 2.68 (t, J = 6.4 Hz, 2H), 3.59 (q,
J = 6.4 Hz, 2H), 3.74 (s, 2H), 6.39 (bs, 1H), 6.92-6.96 (m, 3H),
7.06-7.11 (m, 2H), 7.24-7.29 (m, 1H), 7.33-7.39 (m, 1H). 13C
NMR (CDCl3, 100 MHz): δ 31.09, 35.64, 38.77, 112.12, 112.17,
112.38, 114.33, 114.54, 115.79, 116.02, 124.73, 124.76, 130.24,
130.33, 131.88, 131.98, 132.08, 140.74, 140.82, 158.91, 158.97,
160.67, 161.42, 161.48, 161.89, 164.35. HRMS calculated for
C16H15F3NOS (M þ H)þ 326.08264, found 326.08419.
N-[2-(3-Methoxy-benzylsulfanyl)-ethyl]-2,6-dinitro-benzamide
(37). The compound was prepared using 2,6-dinitrobenzoyl
chloride as acid chloride and following the procedure described
for 27: mp = 69-71 °C. 1H NMR (CDCl3, 400 MHz): δ 2.71 (t,
J=6.4 Hz, 2H), 3.54 (q, J=6.0 Hz, 2H), 3.69 (s, 2H), 3.77 (s, 3H),
6.45 (bs, 1H), 6.71 (d, J=7.2 Hz, 1H), 6.85-6.89 (m, 2H), 7.17 (t,
J = 7.6 Hz, 1H), 7.73 (t, J = 8.4 Hz, 1H), 8.33 (d, J = 8.0 Hz,
2H). 13C NMR (CDCl3, 100 MHz): δ 30.61, 36.07, 39.15, 55.44,
113.09, 114.39, 121.39, 128.01, 129.68, 129.81, 130.91, 139.79,
147.53, 159.96, 161.90. LRMS calculated for C17H18N3O6S
(M þ H)þ 392.1, found 392.3.
2,6-Dichloro-N-[2-(3-fluoro-benzylsulfanyl)-ethyl]-benzamide
(47). The compound was prepared using 2-(3-fluoro-benzyl-
sulfanyl)-ethylamine as the amine component and following the
procedure described for 27: mp=70-72 °C. 1H NMR (CDCl3, 400
MHz): δ 2.70 (t, J=6.4 Hz, 2H), 3.61 (q, J=6.4 Hz, 2H), 3.74 (s,
2H), 6.18 (bs, 1H), 6.91-6.96 (m, 1H), 7.04-7.11 (m, 2H), 7.23-
7.32 (m, 4H). 13C NMR (CDCl3, 100 MHz): δ 31.05, 35.61,
35.62, 38.67, 114.35, 114.57, 115.82, 116.03, 124.74, 124.77,
128.28, 130.27, 130.35, 130.92, 132.43, 135.98, 140.71, 140.79,
161.89, 164.34, 164.74. HRMS calculated for C16H15Cl2FNOS
(M þ H)þ 358.02354, found 358.02334.

Article
2,6-Dichloro-N-[2-(3-methyl-benzylsulfanyl)-ethyl]-benzamide
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 14 5317
1
component respectively: mp =70-72 °C. H NMR (CDCl3,
(48). The compound was prepared using 2-(3-methyl-benzyl-
sulfanyl)-ethylamine as the amine component and following the
procedure described for 27: mp = 56-57 °C. 1H NMR (CDCl3,
400 MHz): δ 2.32 (s, 3H), 2.69 (t, J = 6.4 Hz, 2H), 3.59 (t, J =
6.4 Hz, 2H), 3.70 (s, 2H), 6.15 (bs, 1H), 7.02 (d, J = 7.2 Hz, 1H),
7.09-7.19 (m, 3H), 7.24-7.33 (m, 3H). 13C NMR (CDCl3, 100
MHz): δ 21.59, 31.02, 36.00, 38.70, 126.09, 128.22, 128.26,
128.74, 129.79, 130.87, 132.47, 136.09, 138.05, 138.59, 164.69.
HRMS calculated for C17H18Cl2NOS (M þ H)þ 354.04861,
found 354.04895.
2,6-Dichloro-N-[2-(3-trifluoromethyl-benzylsulfanyl)-ethyl]-ben-
zamide (50). The compound was prepared using 2-(3-trifluoro-
methyl-benzylsulfanyl)-ethylamine as the amine component
and following the procedure described for 27: mp =54-55 °C.
1H NMR (CDCl3, 400 MHz): δ 2.70 (t, J=6.4 Hz, 2H), 3.62 (q,
J = 6.4 Hz, 2H), 3.80 (s, 2H), 6.23 (bs, 1H), 7.23-7.31 (m, 3H),
7.43 (t, J = 7.6 Hz, 1H), 7.49-7.54 (m, 2H), 7.59 (s, 1H). 13C
NMR (CDCl3, 100 MHz): δ 31.00, 35.62, 38.76, 122.87, 124.23,
124.26, 125.58, 125.71, 125.75, 128.22, 129.31, 130.89, 130.96,
131.29, 132.34, 132.52, 135.92, 139.32, 164.83. HRMS calcu-
lated for C17H15Cl2F3NOS (M þ H)þ 408.02035, found
408.01914.
N-[2-(3-Bromo-benzylsulfanyl)-ethyl]-2,6-dichloro-benzamide
(51). The compound was prepared using 2-(3-bromo-benzyl-
sulfanyl)-ethylamine as the amine component and following the
procedure described for 27: mp = 74-76 °C. 1H NMR (CDCl3,
400 MHz): δ 2.69 (t, J = 6.4 Hz, 2H), 3.61 (q, J = 6.4 Hz, 2H),
3.71 (s, 2H), 6.19 (bs, 1H), 7.17 (t, J = 8.0 Hz, 1H), 7.23-7.38
(m, 5H), 7.49 (s, 1H). 13C NMR (CDCl3, 100 MHz): δ 31.07,
35.48, 38.67, 122.88, 127.75, 128.29, 130.37, 130.59, 130.93,
132.02, 132.43, 135.97, 140.55, 164.75. HRMS calculated for
C16H15BrCl2NOS (M þ H)þ 417.94347, found 417.94308.
2,6-Dichloro-N-[2-(2,3-dimethoxy-benzylsulfanyl)-ethyl]-ben-
zamide (52). The compound was prepared using 2-(2,3-di-
methoxy-benzylsulfanyl)-ethylamine as the amine component
and following the procedure described for 27: mp = 108-110
°C. 1H NMR (CDCl3, 400 MHz): δ 2.74 (t, J = 6.4 Hz, 2H), 3.66
(q, J = 6.4 Hz, 2H), 3.77 (s, 2H), 3.83 (s, 3H), 3.85 (s, 3H), 6.28
(bs, 1H), 6.80 (d, J = 8.0 Hz, 1H), 6.90 (d, J = 7.6 Hz, 1H), 6.99
(t, J = 8.0 Hz, 1H), 7.22-7.31 (m, 3H). 13C NMR (CDCl3, 100
MHz): δ 29.94, 31.09, 39.03, 55.93, 61.21, 111.68, 122.38,
124.32, 128.24, 130.84, 132.26, 132.47, 136.18, 147.27, 152.91,
164.69. HRMS calculated for C18H20Cl2NO3S (M þ H)þ
400.05409, found 400.05331.
2,6-Dichloro-N-[2-(3-iodo-benzylsulfanyl)-ethyl]-benzamide
(53). The compound was prepared using 2-(3-iodo-benzyl-
sulfanyl)-ethylamine as the amine component and following
the procedure described for 27: mp = 75-78 °C. 1H NMR
(CDCl3, 400 MHz): δ 2.69 (t, J=6.4 Hz, 2H), 3.61 (q, J=6.4
Hz, 2H), 3.67 (s, 2H), 6.20 (bs, 1H), 7.03 (t, J = 8.0 Hz, 1H),
7.03-7.32 (m, 4H), 7.56 (d, J=8.0 Hz, 1H), 7.69 (s, 1H). 13C
NMR (CDCl3, 100 MHz): δ 31.11, 35.38, 38.69, 94.75, 128.29,
128.38, 130.51, 130.93, 132.44, 135.99, 136.53, 137.94, 140.59,
164.73. HRMS calculated for C16H15Cl2INOS (M þ H)þ
465.92961, found 465.92940.
400 MHz): δ 2.68-2.73 (m, 2H), 3.57-3.62 (m, 2H), 3.71 (s,
2H), 6.23 (bs, 1H), 7.07-7.12 (m, 1H), 7.15-7.19 (m, 1H),
7.25-7.27 (m, 1H), 7.35-7.37 (m, 1H), 7.48-7.52 (m, 3H).
13C NMR (CDCl3, 100 MHz): δ 30.93, 35.49, 38.69, 120.65,
122.88, 127.78, 130.39, 130.58, 131.57, 131.96, 132.04, 139.74,
140.56, 166.57. HRMS calculated for C16H15Br3NOS (M þ H)þ
505.84244, found 505.83892.
N-[2-(3-Chloro-benzylsulfanyl)-ethyl]-2,6-dinitro-benzamide
(56). The compound was prepared following the procedure
described for 27. 2,6-Dinitrobenzoyl chloride was used as acid
chloride and 2-(3-chloro-benzylsulfanyl)-ethylamine as the
1
amine component: mp = 139-140 °C. H NMR (CDCl3, 400
MHz): δ 2.74 (t, J = 6.0 Hz, 2H), 3.61 (q, J = 6.4 Hz, 2H), 3.71
(s, 2H), 6.35 (bs, 1H), 7.19-7.25 (m, 3H), 7.33 (s, 1H), 7.77 (t,
J = 8.0 Hz, 1H), 8.38 (d, J = 8.0 Hz, 2H). 13C NMR (DMSO-d6,
100 MHz): δ 29.74, 34.54, 39.60, 127.50, 127.92, 128.29, 129.34,
130.09, 130.87, 132.01, 133.62, 141.97, 147.92, 161.90. HRMS
calculated for C16H15ClN3O5S (M þ H)þ 396.04209, found
396.04216.
2,6-Dichloro-N-[2-(2,3-dihydroxy-benzylsulfanyl)-ethyl]-ben-
zamide (58). The compound was prepared from 52 using the
procedure described for 29. Highly viscous liquid. 1H NMR
(CDCl3, 400 MHz): δ 2.66 (t, J = 6.4 Hz, 2H), 3.59 (q, J = 6.4
Hz, 2H), 3.76 (s, 2H), 6.48 (s, 1H), 6.58-6.72 (m, 4H), 7.04 (bs,
1H), 7.17-7.26 (m, 3H). 13C NMR (CDCl3, 100 MHz): δ 30.66,
39.15, 114.82, 120.82, 121.98, 124.30, 128.28, 131.15, 132.34,
135.43, 142.69, 144.86, 165.74. HRMS calculated for C16H16-
Cl2NO3S (M þ H)þ 372.02279, found 372.02221.
2,6-Dichloro-N-[2-(3,5-dihydroxy-benzylsulfanyl)-ethyl]-ben-
zamide (59). The compound was prepared from 60 using the
1
procedure described for 29: mp = 55-57 °C. H NMR (ace-
tone-d6, 400 MHz): δ 2.68-2.72 (m, 2H), 3.55-3.59 (m, 2H),
3.65 (s, 2H), 6.23 (t, J=2.4 Hz, 1H), 6.36 (d, J = 2.0 Hz, 2H),
7.35-7.42 (m, 3H), 7.93 (bs, 1H), 8.28 (s, 2H). 13C NMR (acetone-
d6, 400 MHz): δ 30.44, 35.62, 39.27, 107.64, 128.22, 130.99, 132.01,
136.89, 141.02, 158.73, 164.22. HRMS calculated for C16H16Cl2-
NO3S (M þ H)þ 372.02279, found 372.02313.
2,6-Dichloro-N-[2-(3,5-dimethoxy-benzylsulfanyl)-ethyl]-ben-
zamide (60). The compound was prepared using 2-(3,5-di-
methoxy-benzylsulfanyl)-ethylamine as the amine component
and following the procedure described for 27: mp = 46-47 °C.
1H NMR (CDCl3, 400 MHz): δ 2.72 (t, J = 6.4 Hz, 2H), 3.61 (q,
J = 6.4 Hz, 2H), 3.67 (s, 2H), 3.77 (s, 6H), 6.19 (bs, 1H), 6.31 (t,
J = 2.0 Hz, 1H), 6.48 (d, J = 2.0 Hz, 2H), 7.22-7.31 (m, 3H).
13C NMR (CDCl3, 100 MHz): δ 31.07, 36.32, 38.74, 55.56,
99.51, 106.93, 128.25, 130.88, 132.41, 136.05, 140.53, 161.10,
164.69. HRMS calculated for C18H20Cl2NO3S (M þ H)þ
400.05409, found 400.05415.
2-Chloro-6-((2-(2,6-dichlorophenyl)-2-oxoethylthio)methyl)-
benzamide (61). The compound was prepared from 62 using the
procedure described for 45: mp = 64-66 °C. 1H NMR (CDCl3,
400 MHz): δ 2.67 (t, J = 7.2 Hz, 2H), 3.45 (q, J = 7.2 Hz, 2H),
3.77 (s, 2H), 6.29 (bs, 1H), 6.74 (s, 2H), 7.21-7.29 (m, 6H),
7.38-7.39 (m, 1H). 13C NMR (CDCl3, 100 MHz): δ 30.89,
33.39, 39.63, 128.22, 128.63, 130.44, 130.77, 130.89, 132.29,
135.88, 136.07, 137.65, 164.99, 169.11. HRMS calculated for
C17H16Cl3N2O2S (M þ H)þ 416.99980, found 417.00006. 91%
pure by HPLC.
2,6-Dichloro-N-[2-(3-chloro-2-cyano-benzylsulfanyl)-ethyl]-ben-
zamide (62). The compound was prepared using 2-(2-amino-
ethylsulfanylmethyl)-6-chloro-benzonitrile as the amine com-
ponent and following the procedure described for 27: mp =
166-168 °C. 1H NMR (CDCl3, 400 MHz): δ 2.78 (t, J = 6.8 Hz,
2H), 3.71 (q, J = 6.8 Hz, 2H), 3.95 (s, 2H), 6.33 (bs, 1H),
7.25-7.33 (m, 3H), 7.41-7.49 (m, 3H). 13C NMR (CDCl3, 100
MHz): δ 31.39, 34.39, 38.92, 113.72, 115.11, 128.30, 128.51,
128.87, 130.99, 132.41, 133.73, 135.87, 137.86, 144.84, 164.89.
HRMS calculated for C17H14Cl3N2OS (M þ H)þ 398.98924,
found 398.98777.
2,6-Dibromo-N-[2-(3-chloro-benzylsulfanyl)-ethyl]-benzamide
(54). The compound was prepared using 2,6-dibromobenzoic
acid as the acid component and following the procedure described
for 75: mp = 55-57 °C. 1H NMR (CDCl3, 400 MHz): δ 2.72 (t,
J = 6.4 Hz, 2H), 3.62 (q, J = 6.4 Hz, 2H), 3.72 (s, 2H), 6.15 (bs,
1H), 7.10 (t, J = 8.0 Hz, 1H), 7.22-7.23 (m, 3H), 7.34 (s, 1H), 7.51
(d, J = 8.0 Hz, 2H). 13C NMR (CDCl3, 100 MHz): δ 30.95, 35.54,
38.65, 120.66, 127.31, 127.68, 129.15, 130.10, 131.58, 131.97, 134.68,
139.75, 140.27, 166.57. HRMS calculated for C16H15Br2ClNOS
(M þ H)þ 461.89296, found 461.88998.
2,6-Dibromo-N-[2-(3-bromo-benzylsulfanyl)-ethyl]-benzamide
(55). The compound was prepared following the procedure
described for 75. 2,6-Dibromobenzoic acid and 2-(3-bro-
mo-benzylsulfanyl)-ethylamine were used as acid and amine

5318 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 14
Chakrabarti et al.
N-[2-(3-Chloro-benzylsulfanyl)-ethyl]-benzamide (63). The com-
pound was prepared following the procedure described for 27.
Benzoyl chloride was used as acid chloride and 2-(3-chloro-ben-
zylsulfanyl)-ethylamine as the amine component: mp = 75-76 °C.
1H NMR (CDCl3, 400 MHz): δ 2.67 (t, J = 6.4 Hz, 2H), 3.59 (q,
J = 6.4 Hz, 2H), 3.69 (s, 2H), 6.70 (bs, 1H), 7.18-7.26 (m, 3H),
7.33 (s, 1H), 7.39-7.43 (m, 2H), 7.47-7.51 (m, 1H), 7.74-7.76 (m,
2H). 13C NMR (CDCl3, 100 MHz): δ 31.43, 35.55, 38.64, 127.18,
127.26, 127.64, 128.82, 129.11, 130.09, 131.80, 134.53, 134.64,
140.42, 167.78. HRMS calculated for C16H17ClNOS (M þ H)þ
306.07194, found 306.07133.
N-(2-Benzylsulfanyl-ethyl)-benzamide (64). The compound
was prepared following the procedure described for 27. Benzoyl
chloride was used as acid chloride and 2-benzylsulfanyl-ethyla-
mine as the amine component: mp = 65-67 °C. 1H NMR (CD-
Cl3, 400 MHz): δ 2.67 (t, J = 6.4 Hz, 2H), 3.58 (q, J = 6.4 Hz,
2H), 3.73 (s, 2H), 6.65 (bs, 1H), 7.21-7.25 (m, 1H), 7.28-7.33
(m, 4H), 7.39-7.45 (m, 2H), 7.46-7.50 (m, 1H), 7.73-7.75 (m,
2H). 13C NMR (CDCl3, 100 MHz): δ 31.33, 36.02, 38.59, 127.19,
127.44, 128.79, 128.88, 129.07, 131.75, 134.62, 138.29, 167.72.
HRMS calculated for C16H18NOS (M þ H)þ 272.11091, found
272.11101.
(DMSO-d6, 100 MHz): δ 33.60, 50.71, 56.49, 128.45, 128.77,
129.71, 130.69, 130.99, 131.78, 133.68, 134.44, 136.81, 164.55.
HRMS calculated for C16H15Cl3NO2S (M þ H)þ 389.98890,
found 389.98985.
2,6-Dichloro-N-[2-(3-chloro-benzyloxy)-ethyl]-benzamide (72).
The compound was prepared using 2-(3-chloro-benzyloxy)-
ethylamine as the amine component and following the proce-
dure described for 27: mp = 70-72 °C. 1H NMR (CDCl3, 400
MHz): δ 3.66-3.73 (m, 4H), 4.50 (s, 2H), 6.26 (bs, 1H), 7.18-
7.32 (m, 7H). 13C NMR (CDCl3, 100 MHz): δ 39.89, 69.18,
72.64, 125.94, 127.95, 128.17, 128.28, 129.98, 130.87, 132.43,
134.60, 136.14, 140.09, 164.75. HRMS calculated for C16H15-
Cl3NO2 (M þ H)þ 358.01684, found 358.01726.
2,6-Dichloro-N-[2-(3-chloro-5-methoxy-benzylsulfanyl)-ethyl]-
benzamide (73). The compound was prepared using 2-(3-chloro-
5-methoxy-benzyloxy)-ethylamine as the amine component and
following the procedure described for 27: mp = 83-84 °C. 1H
NMR (CDCl3, 400 MHz): δ 2.69 (bs, 2H), 3.59 (bs, 2H), 3.66 (s,
2H), 3.78 (s, 3H), 6.33 (bs, 1H), 6.76 (s, 2H), 6.91 (s, 1H), 7.27 (s,
3H). 13C NMR (CDCl3, 100 MHz): δ 31.05, 35.59, 38.61, 55.77,
113.27, 121.39, 128.15, 128.25, 130.90, 132.38, 135.16, 135.97,
141.15, 160.58, 164.76. HRMS calculated for C17H17Cl3NO2S
(M þ H)þ 404.00455, found 404.00305.
N-(2-Benzylsulfanyl-ethyl)-2,6-dichloro-benzamide (65). The
compound was prepared using 2-benzylsulfanyl-ethylamine as
the amine component and following the procedure described for
27: mp = 96-97 °C. 1H NMR (CDCl3, 400 MHz): δ 2.69 (t, J =
6.4 Hz, 2H), 3.59 (q, J = 6.4 Hz, 2H), 3.74 (s, 2H), 6.16 (bs, 1H),
7.19-7.33 (m, 8H). 13C NMR (CDCl3, 100 MHz): δ 30.95,
36.04, 38.70, 127.45, 128.26, 128.87, 129.09, 130.89, 132.45,
136.07, 138.16, 164.71. HRMS calculated for C16H16Cl2NOS
(M þ H)þ 340.03296, found 340.03318.
N-[2-(3-Chloro-benzylsulfanyl)-ethyl]-2,6-bis-trifluoromethyl-
benzamide (74). The compound was prepared following the
procedure described for 27. 2,6-Bis-trifluoromethylbenzoyl
chloride was used as acid chloride and 2-(3-chloro-benzylsul-
fanyl)-ethylamine as the amine component. The reaction was
carried out at refluxing condition. mp = 93-94 °C. 1H NMR
(CDCl3, 400 MHz): δ 2.64 (t, J = 6.8 Hz, 2H), 3.58 (q, J = 6.8
Hz, 2H), 3.69 (s, 2H), 6.28 (bs, 1H), 7.19-7.22 (m, 3H), 7.32 (s,
1H), 7.66 (t, J = 8.4 Hz, 1H), 7.89 (d, J = 8.0 Hz, 2H). 13C NMR
(CDCl3, 100 MHz): δ 30.44, 35.36, 38.86, 121.79, 124.53, 127.27,
127.65, 128.83, 129.08, 129.15, 129.47, 129.79, 130.02, 130.06,
130.12, 130.17, 134.02, 134.66, 140.18, 164.38. HRMS calculated
for C18H15ClF6NOS (M þ H)þ 442.04670, found 442.04687.
2,6-Dichloro-N-[2-(2-chloro-benzylsulfanyl)-ethyl]-benzamide
(76). The compound was prepared using 2-(2-chloro-benzyl-
sulfanyl)-ethylamine as the amine component and following the
procedure described for 27: mp = 110-112 °C. 1H NMR (CD-
Cl3, 400 MHz): δ 2.75 (t, J = 6.4 Hz, 2H), 3.65 (q, J = 6.4 Hz,
2H), 3.87 (s, 2H), 6.28 (bs, 1H), 7.16-7.32 (m, 5H), 7.34-7.39
(m, 2H). 13C NMR (CDCl3, 100 MHz): δ 31.31, 33.53, 38.89,
127.22, 128.26, 128.91, 130.09, 130.90, 131.12, 132.44, 134.14,
135.93, 136.01, 164.73. HRMS calculated for C16H15Cl3NOS
(M þ H)þ 373.99399, found 373.99368.
2,6-Dichloro-N-[2-(3-chloro-phenylmethanesulfonyl)-ethyl]-ben-
zamide (66). The compound was prepared from 1 following
1
the procedure described for 30: mp=156-158 °C. H NMR
(DMSO-d6, 400 MHz): δ 3.29 (t, J=6.8 Hz, 2H), 3.63 (q, J=6.4
Hz, 2H), 4.62 (s, 2H), 7.37-7.49 (m, 7H), 8.99 (t, J = 5.6 Hz,
1H). 13C NMR (DMSO-d6, 100 MHz): δ 33.14, 50.92, 58.15,
128.79, 129.17, 130.49, 131.06, 131.47, 131.77, 131.93, 133.69,
136.59, 164.64. HRMS calculated for C16H15Cl3NO3S (M þ
H)þ 405.98382, found 405.98338.
N-[2-(3-Chloro-benzylsulfanyl)-ethyl]-2,6-difluoro-benzamide
(67). The compound was prepared following the procedure
described for 27. 2,6-difluorobenzoyl chloride was used as
acid chloride and 2-(3-chloro-benzylsulfanyl)-ethylamine as
the amine component: mp = 41-43 °C. 1H NMR (CDCl3,
400 MHz): δ 2.67 (t, J=6.4 Hz, 2H), 3.59 (q, J=6.4 Hz, 2H),
3.71 (s, 2H), 6.44 (bs, 1H), 6.90-6.96 (m, 2H) 7.19-7.26 (m,
3H), 7.32-7.39 (m, 2H). 13C NMR (CDCl3, 100 MHz): δ 31.08,
35.34, 38.77, 112.11, 112.37, 114.27, 127.28, 127.64, 129.11,
130.06, 131.88, 131.99, 132.08, 134.64, 140.29, 158.88, 160.69,
161.40, 161.47. HRMS calculated for C16H15ClF2NOS (M þ
H)þ 342.05309, found 342.05252.
2,6-Dichloro-N-[2-(3-cyclopropylcarbamoyl-benzylsulfanyl)-
ethyl]-benzamide (68). The compound was prepared from 44
following the procedure described for 78: mp = 42-43 °C. 1H
NMR (CDCl3, 400 MHz): δ 0.35-0.38 (m, 2H), 0.47-0.49 (m,
2H), 2.48-2.52 (m, 2H), 2.71-2.73 (m, 1H), 3.58 (q, J = 6.4 Hz,
2H), 3.74 (s, 2H), 7.18-7.37 (m, 5H), 7.45 (d, J = 7.6 Hz, 1H),
7.67-7.75 (m, 3H). 13C NMR (CDCl3, 100 MHz): δ 5.80, 23.41,
28.25, 34.68, 39.41, 127.07, 127.69, 128.13, 129.35, 130.91,
132.03, 132.21, 134.36, 135.71, 138.21, 165.37, 169.05. HRMS
calculated for C20H21Cl2N2O2S (M þ H)þ 423.07008, found
423.07114.
2,6-Dichloro-N-[2-(4-chloro-benzylsulfanyl)-ethyl]-benzamide
(77). The compound was prepared using 2-(4-chloro-benzyl-
sulfanyl)-ethylamine as the amine component and following the
procedure described for 27: mp = 104-106 °C. 1H NMR (CD-
Cl3, 400 MHz): δ 2.67 (t, J = 6.8 Hz, 2H), 3.59 (q, J = 6.4 Hz,
2H), 3.71 (s, 2H), 6.25 (bs, 1H), 7.22-7.31 (m, 7H). 13C NMR
(CDCl3, 100 MHz): δ 30.86, 35.29, 38.71, 128.28, 128.99, 130.44,
130.93, 132.39, 133.19, 135.90, 136.71, 169.75. HRMS calcu-
lated for C16H15Cl3NOS (M þ H)þ 373.99399, found 373.99380.
Acknowledgment. We wish to thank the Cleveland Clinic
Small Molecule Screening Core for assistance with the drug
library screen. We thank Dr. Chris Eckman for supplying the
H4-APP cells. We thank David Cramp and Colin Agatisa-
Boyle, who provided excellent technical assistance. We thank
Dr. Bruce Lamb for help in preparing the primary mouse corti-
cal neuron cultures. We thank Dr. Vinod Labhasetwar for
assistance with the transepithelial resistance measurements
and Damir Janigro for assistance and discussion about penetra-
tion of the blood-brain barrier. The initial stages of this project
were supported by a grant to J.D.S form the Institute for the
Study of Aging (now known as the Alzheimer’s Drug Discovery
Foundation) in addition to a grant from the Alzheimer’s
2,6-Dichloro-N-[2-(3-chloro-phenylmethanesulfinyl)-ethyl]-ben-
zamide (71). The compound was prepared from 1 following
1
the procedure described for 32: mp=174-176 °C. H NMR
(DMSO-d6, 400 MHz): δ 2.74-2.79 (m, 1H), 2.97-3.00 (m, 1H),
3.52-3.55 (m, 1H), 3.59-3.61 (m, 1H), 4.07 (d, J=12.8 Hz, 1H),
4.23 (d, J=12.8 Hz, 1H), 7.27-7.29 (m, 1H), 7.38-7.41 (m,
4H), 7.45-7.47 (m, 2H), 8.98 (t, J=5.6 Hz, 1H) . 13C NMR

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 14 5319
Association to G.T. We dedicate this work to the memory of
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