Synthesis of benzamide derivatives and their evaluation as antiprion agents

Article abstract of DOI:10.1016/j.bmc.2012.06.026

A new set of 5-(2-(pyrrolidin-1-yl)acetamido)-N-butyl-2-(substituted) benzamide and 5-(2-(piperidin-1-yl)acetamido)-N-butyl-2-(substituted) benzamide derivatives were synthesized in which as structural features the 2-(1-pyrrolidinyl)- or 2-(1-piperidyl)acetylamino group or a diphenylether moiety are associated to a benzamide scaffold. Their binding affinity for human PrP^C and inhibition of its conversion into PrP^Sc were determined in vitro; moreover, the antiprion activity was assayed by inhibition of PrP^Sc accumulation in scrapie-infected mouse neuroblastoma cells (ScN2a) and scrapie mouse brain (SMB) cells. The results clearly indicate the benzamide derivatives as attractive lead compounds for the development of potential therapeutic agents against prion disease.

Full text of DOI:10.1016/j.bmc.2012.06.026

Bioorganic & Medicinal Chemistry 20 (2012) 5001–5011
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis of benzamide derivatives and their evaluation as antiprion agents
Ferdinando Fiorino a, Martin Eiden b, Armin Giese c, Beatrice Severino a, Antonella Esposito a,
Martin H. Groschup b, Elisa Perissutti a, Elisa Magli a, Giuseppina Maria Incisivo a, Antonio Ciano a,
Francesco Frecentese a, Hans A. Kretzschmar c, Jens Wagner c, , Vincenzo Santagada a, Giuseppe Caliendo a,
⇑
a Dipartimento di Chimica Farmaceutica e Tossicologica, Università di Napoli ‘Federico II’, Via D. Montesano 49, 80131 Napoli, Italy
b Institute for Novel and Emerging Infectious Diseases at the Friedrich-Loeffler-Institut, Südufer 10, 17493 Greifswald-Insel Riems, Germany
c Zentrum für Neuropathologie und Prionforschung, Ludwig-Maximilians-Universität, München Feodor Lynen Str., 23, 81377 München, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
A new set of 5-(2-(pyrrolidin-1-yl)acetamido)-N-butyl-2-(substituted)benzamide and 5-(2-(piperidin-1-
yl)acetamido)-N-butyl-2-(substituted) benzamide derivatives were synthesized in which as structural
features the 2-(1-pyrrolidinyl)- or 2-(1-piperidyl)acetylamino group or a diphenylether moiety are asso-
ciated to a benzamide scaffold. Their binding affinity for human PrPC and inhibition of its conversion into
PrPSc were determined in vitro; moreover, the antiprion activity was assayed by inhibition of PrPSc accu-
mulation in scrapie-infected mouse neuroblastoma cells (ScN2a) and scrapie mouse brain (SMB) cells.
The results clearly indicate the benzamide derivatives as attractive lead compounds for the development
of potential therapeutic agents against prion disease.
Received 16 February 2012
Revised 23 May 2012
Accepted 13 June 2012
Available online 20 June 2012
Keywords:
Synthesis
Benzamide derivatives
Antiprion agents
SIFT assay
Ó 2012 Elsevier Ltd. All rights reserved.
Cell based antiprion activity
1. Introduction
of PrPSc can take place. However, despite the identification of a
number of compounds that are capable of inhibiting PrPSc accumu-
Prion diseases are fatal neurodegenerative diseases afflicting
both humans and animals. The most known disease in humans is
the Creutzfeldt–Jakob disease (CJD); other examples include Gerst-
mann–Straussler–Scheincker syndrome (GSS), fatal familial insom-
nia, and kuru in humans, scrapie in sheep and goat, bovine
spongiform encephalopathy in cattle, and chronic wasting disease
in deer and elk.1,2 The crucial molecular event in the pathogenesis
of all prion diseases is the post-translational refolding of normal
cellular prion protein PrPC into the pathological-associated isoform
PrPSc. This b-sheet rich form of the protein readily generates insol-
uble aggregates, the deposition of which is thought to be central to
pathogenesis and disease progression.3 This conversion can occur
spontaneously as a result of inherited mutations in the PrPC gene.
Moreover, the available evidences suggest that PrPSc acts both as a
template for this conversion and as a neurotoxic agent causing
neuronal dysfunction and cell death.1,4 Based on this knowledge,
one of the most promising therapeutic approaches for prion dis-
eases appears to be interference with PrPSc amplification and accu-
mulation. In fact evidence deriving from cell culture and in vivo
studies suggests that once formation of PrPSc is inhibited, clearance
lation and cleaning PrPSc from infected cell lines, little therapeutic
efficacy in vivo has been achieved so far and no viable treatments
of these devastating disorders are currently available.5
Several classes of chemical compounds are already known to
possess antiprion properties and among these the acridines6,7
(e.g., quinacrine, and structurally related tricyclic antidepressants),
dimeric8 and chimeric9 analogues of statins,10 2,4-diphenylthiazole
and 2,4-diphenyloxazole amides,11 pyrazolones,12,13 indole-3-gly-
oxamides,14 pyridyl hydrazones15 and GN8,16 a molecule charac-
terized by
a
diphenylmethane scaffold and two 2-(1-
pyrrolidinyl)acetylamino groups at the 4,40-positions of diph-
enylmethane. In addition, larger molecules of the polyanionic (sur-
amin, pentosan polysulfate) or polycationic chemotype (dendritic
polyamines, cationic polysaccharides17) have been reported to ex-
hibit antiprion activity in cells, although it seems unlikely that
such species could be therapeutically useful. In fact, only a pyridyl
hydrazone derivative and GN8 (Fig. 1) have been reported to signif-
icantly extend survival in animals.15,16 A possible explanation for
this poor track record is that the majority of small molecules,
investigated to date, was originally designed for other purposes
(e.g., malaria, hyperlipidemia) and not optimized for either antipri-
on effects or for crossing the blood–brain barrier (BBB).18
⇑
Corresponding author. Tel./fax: +39 081 678649.
E-mail address: caliendo@unina.it (G. Caliendo).
In our laboratories, there has been an ongoing effort to develop
new small molecules that bind to human PrPC and stabilize it
against the conversion into PrPSc, with the aim of identifying novel
Present address: Deutsches Zentrum für Neurodegenerative Erkrankungen
(DZNE) BMZ 1, Gebäude 344 Sigmund-Freud-Str. 25, 53105 Bonn, Germany.
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.06.026

5002
F. Fiorino et al. / Bioorg. Med. Chem. 20 (2012) 5001–5011
H
R
R
O
O
O
O
O
N
N
N
H
N
H
N
N
Compound B
O
HN
HN
N
N
O
O
O
N
N
N
N
H
H
(6a-o)
(7a-o)
GN8
Figure 2. General structures of benzamide derivatives.
Figure 1. Structures of the small molecules compound 8 and GN8 with significant
antiprion properties in vivo.
PrPSc accumulation in scrapie-infected mouse neuroblastoma cells
(ScN2a) and scrapie mouse brain (SMB) cells. Taken together, the
results suggest that benzamide derivatives represent a promising
new class of lead compounds useful for further optimization in or-
der to obtain novel pharmacological tools for prion diseases.
pharmacological tools characterized by more efficient antiprion
activity and that could improve also our knowledge about the
mechanism of formation and clearance of PrPSc. In this context a
new set of 5-(2-(pyrrolidin-1-yl)acetamido)-N-butyl-2-(substi-
tuted)benzamide (6a–o) and 5-(2-(piperidin-1-yl)acetamido)-N-
butyl-2-(substituted)benzamide (7a–o) derivatives (Fig. 2) was
synthesized. These derivatives contain a 2-(1-pyrrolidinyl)acetyl-
amino group or a diphenylether moiety as in the GN8 structure
and in some GN8 derivatives, associated to a benzamide scaffold
that was already investigated in the synthesis of substituted benz-
amides as anti-inflammatory agents.19 All synthesized compounds
were screened in vitro for their binding affinity to human PrPC
(hPrPC), truncated human PrPC (t-hPrPC), and murine PrPC (rPrPC).
For this purpose a high-throughput screening assay was applied
which is based on scanning intensely fluorescent targets (SIFT)
with an inverted dual-color confocal microscope setup. This tech-
nique was chosen as it is well suited for in vitro screening of a li-
brary of synthetic compounds, in order to identify inhibitors of
the aggregation processes accompanying prion diseases.20 Pharma-
cological data allowed to identify compounds capable of inhibiting
2. Results and discussion
2.1. Chemistry
The synthetic strategy employed for the preparation of the tar-
get compounds is summarized in Scheme 1. The general procedure
is based on condensation of commercially available 2-chloro-5-
nitrobenzoic acid (1) with n-butylamine in anhydrous DMF in the
presence of DCC/HOBt, to produce N-butyl-2-chloro-5-nitro-benz-
amide (2). Subsequent dissolution of 2 in anhydrous dioxane and
treatment with a solution of the appropriate alcohol and NaH in
anhydrous dioxane afforded smoothly the corresponding interme-
diates 3a–o. Reduction of the nitro group with sodium borohydride
led to intermediates 4a–o which were acylated with bromoacetyl
R
O
O
Cl
Cl
O
COOH
N
N
DCC/ HOBT
n-butylamine
NaH/ R-OH
H
H
R
O
NO2
NO2
NO2
O
O
(2)
(3a-o)
(1)
N
H
HN
N
I
R
O
a
R
O
N
O
O
O
/
O
C
NH
NH
K
N
H
(6a-o)
N
NaBH4
BrCH2COBr
TEA
H
Pd/C 10%
K
2
R
C
HN
NH2
O
O
O
3
Br
/
(4a-o)
N
a
N
I
H
(5a-o)
HN
N
a
e
m
h
H3C(H2C)3O
F3C
O
(7a-o)
b
f
i
l
n
o
Cl
H3C
R =
c
CF3
g
F
H3CO
d
Scheme 1. Synthesis of compounds 6a–o and 7a–o.

F. Fiorino et al. / Bioorg. Med. Chem. 20 (2012) 5001–5011
5003
Table 1
Antiprion activities in the three screening steps of compounds 6a–o and 7a–o
R
O
O
N
H
HN
X
O
Compd
Substituents
SIFT EC50
M)
SMB cells (% control)
ScN2a cells (% control)
EC50 (lM)
(
l
R
X
20
l
M
2
lM
20
l
M
2
l
M
SMB
cells
ScN2a
cells
compound
compound
compound
compound
N
N
N
6a
6b
200
38
67
32
38
7.81
3.14
2.76
Inactive
48
59
24
42
10.77
6c
6d
6e
80
42
65
82
77
74
92
16
32
41
34
43
48
12.43
99.22
2.00
3.52
5.52
N
N
Inactive
200
>1000
12.32
>1000
5.81
N
N
N
N
Cl
6f
6g
6h
55
46
92
41
68
57
50
16
39
12
25
45
26
1.70
4.70
1.61
F
200
40
F3C
6i
25
54
61
15
16
16.49
1.42
CF3
N
N
N
N
6l
6m
6n
6o
7a
H3CO
198
30
75
58
55
48
65
72
36
66
74
82
61
73
76
58
54
70
31
19
13
20
22
29
12
37
36
52
32
32
37
78
27
76
866.34
53.65
18.95
16.27
108.22
160.60
5.22
2.92
2.98
1.83
2.08
2.39
6.86
1.65
9.28
H3C(H2C)3O
H3C
70
18
80
N
N
N
N
7b
7c
Inactive
50
7d
Inactive
97.25
(continued on next page)

5004
F. Fiorino et al. / Bioorg. Med. Chem. 20 (2012) 5001–5011
Table 1 (continued)
Compd
R
Substituents
SIFT EC50
M)
SMB cells (% control)
ScN2a cells (% control)
EC50 (lM)
(
l
X
20
l
M
2
lM
20
l
M
2
l
M
SMB
cells
ScN2a
cells
compound
compound
compound
compound
7e
200
20
75
65
30
50
501.35
15.04
>1000
30.04
3.86
2.25
2.93
2.08
N
N
N
N
N
7f
7g
7h
Cl
49
81
53
68
61
80
21
32
17
35
35
35
F
70
F3C
70
7i
70
55
75
12
39
30.55
2.04
CF3
H3CO
N
N
N
N
150
50
82
52
86
46
83
76
86
72
28
21
26
13
49
61
36
43
>1000
24.27
>1000
13.80
3.56
3.73
2.57
2.22
7l
7m
7n
H3C(H2C)3O
H3C
40
7o
15
All the compounds were validated in SIFT dilution series and whenever possible, EC50 values were determined.
bromide in the presence of triethylamine to generate 5a–o. Subse-
quent condensation of 5a–o with pyrrolidine or piperidine, per-
formed in CH3CN in the presence of K2CO3 and NaI under reflux,
provided the final compounds 6a–o and 7a–o. The final products
were purified by silica gel chromatography and crystallization
from appropriate solvents. All new compounds gave satisfactory
elemental analyses and were characterized by 1H NMR and mass
spectrometry. 1H NMR and MS data for all final compounds were
consistent with the proposed structures.
EC50 values in the range of 20–40
6n, 7a, 7c, 7g, 7h, 7i and 7m were less active with EC50 values
ranging from 50 M to 80 M.
lM while compounds 6c, 6f,
l
l
Concerning the influence of the substituent of the benzamide
moiety, the 4-isopropylphenoxy (6o and 7o), 2-trifluoromethyl-
phenoxy (6i) and p-chlorophenoxy group (7f) conferred the high-
est inhibitory activity on PrPC/PrPSc association. The presence of a
4-trifluoromethylphenoxy or 4-butoxyphenoxy group on the benz-
amide moiety, i.e. 6h and 6m, respectively led also to compounds
of high inhibitory activity. Non aromatic moieties as well as phen-
oxy, 4-fluorophenoxy, and 4-methoxyphenoxy groups afforded
compounds with a dramatic decrease in inhibitory activity com-
pared to other substituents at the position 2 of the benzamide
moiety.
2.2. In vitro antiprion activity
All the synthesized compounds were screened with the SIFT as-
say for antiprion activity. As reported by Bertsch et al.20 this antip-
rion assay represents a technique to determine the inhibitory effect
of compounds on the association of PrPC and PrPSc. The compounds
2.3. Cell based antiprion activity
were checked in a dilution series (0.1–100 lM) for dose-dependent
inhibition of PrPC/PrPSc association. Most of the compounds affor-
ded dose response curves that confirmed their concentration-
dependent inhibitory activity. Half-maximal inhibition of binding
of rPrP to PrPSc was observed at 50% effective concentration
Scrapie-infected mouse neuroblastoma cells (ScN2a) and scra-
pie mouse brain (SMB) cells as established cell culture models of
prion propagation were selected to evaluate the antiprion activity
compounds 6a–o and 7a–o (Figs. 3 and 4). To quantify the reduc-
tion of PrPSc obtained after the treatment with the new small mol-
ecules, the relative amount of PrPSc, present in control cells, was set
at 100% and reduction was calculated from three independent
experiments. In the ScN2a cell culture model numerous com-
pounds significantly interfered with the accumulation of PrPSc
(EC50) values in the range of 15–200
of 2,3-dioleoyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-di-
methyl-1-propanaminium trifluoroacetate (DOSPA), a cationic li-
pid, that previously used at concentration of 17 M, has been
lM compared to the effect
l
found to inhibit PrPSc formation in scrapie-infected mouse cells
(Table 1).20 Besides the excellent inhibitory activity of compounds
without showing any overt sign of cytotoxicity at 2
20
M concentrations. PrPSc levels were reduced in the ScN2a cells
by 22–84% at 2 M and by 59–88% at 20 M concentration (Fig. 5).
lM and
6o (EC50 = 18
lM) and 7o (EC50 = 15
l
M), other interesting activi-
l
ties were found for the compounds 6h, 6i, 6m, 7f, and 7n with
l
l

F. Fiorino et al. / Bioorg. Med. Chem. 20 (2012) 5001–5011
5005
Figure 3. Inhibition of PrPsc formation in mouse neuroblastoma (ScN2a) cells treated for 3 days with the synthetic compounds at 20
lM or 2
lM concentration compared to
infected control cells (K).
Figure 4. Inhibition of PrPsc formation in scrapie mouse brain (SMB) cells treated for 3 days with the synthetic compounds at 20
lM or 2 lM concentration compared to
infected control cells (K).
Beside the remarkable inhibitory activity of compounds 6h
(EC50 = 1.61 M), 7c (EC50 = 1.65 M) and 7i (EC50 = 2.04 M) with
12% residual PrPSc level at 20
M concentration, other interesting
with residual PrPSc levels ranging from 19% to 41%. At 2
centration compounds 6f (EC50 = 1.70 M), 6h (EC50 = 1.61
6i (EC50 = 1.42 M) and 7c (EC50 = 1.65 M) retained significant
inhibitory activity (Figs. 3 and 5).
Concerning the results obtained in the SMB cell culture model, as
a general trend, the compounds showed a weaker inhibitory effect
l
M con-
l
l
l
l
lM),
l
l
l
inhibitory effects with residual PrPsc levels in the range of 13–
17% were observed at this concentration for compounds 6n, 6o,
6i, 6c, 6f and 7h while the remaining compounds were less active

5006
F. Fiorino et al. / Bioorg. Med. Chem. 20 (2012) 5001–5011
Figure 5. Antiprion activities of 6a–o and 7a–o at 20 lM and 2 lM on ScN2a cells.
Figure 6. Antiprion activities of 6a–o and 7a–o at 20 lM and 2 lM on SMB-cells.
than that recorded on the ScN2a cells (Figs. 4 and 6). A reduction of
PrPSc levels in the range of 50–14% and 64–14% was observed at con-
potencies, whereas the aliphatic or aromatic nature of the residue
R led to different results both in SIFT assay and in biological sys-
tems (ScN2a and SMB cell lines). Therefore alkyl and cycloalkyl res-
idues as R substituents led to inactive or less potent compounds,
while, as already demonstrated in a series of aminothiazole deriv-
atives,18 electronic effects of the substituents appear to be of smal-
ler importance; analogues with electron rich (e.g. compounds 6f,
6h, 6i etc.) or electron deficient (e.g. compound 6n, 6o, 7o etc.)
groups showed similar activities. Rather the steric hindrance, rep-
resented for example by an isopropyl (6o) or a trifluoromethyl (6h)
group linked to the para positon of phenoxybenzamide moiety,
could be useful to determine a favorable inhibitory activity on
the PrPC/PrPSc association. The good correlation of the results from
the SIFT assay with the antiprion activity found in cell cultures
confirms that the cell activities are indeed due to interference of
the small molecules with PrPc/PrPSc interactions.
centrations of 2
best inhibitory activity against PrPSc propagation was observed with
compounds6a (residual PrPSc level of 67% at 2
M and 38% at 20 M;
EC50 = 7.81 M and 41% at
M), 6h (residual PrPSc level of 50% at 2
20 M; EC50 = 5.81 M and
M), 7c (residual PrPSc level of 54% at 2
36% at 20 M; EC50 = 5.22
M) and 7f (residual PrPSc level of 68%
at 2 M and 49% at 20 M; EC50 = 15.04 M).
lM and 20 lM, respectively. In this cell system the
l
l
l
l
l
l
l
l
l
l
l
l
The results obtained with neuronal (ScN2a) and scrapie mouse
brain (SMB) cells suggest an interesting selectivity towards ScN2a
cells. Moreover, the collected data would suggest that the new
scaffold benzamide may play a critical role in determining antipri-
on activity and allow for some preliminary structure–activity rela-
tionship considerations. As already demonstrated21 in a series of
GN8 derivatives, the combination of hydrophobic properties with
the presence of a structural feature like a diphenylether moiety,
recognizable also inside the structure of some of the most active
benzamide derivatives, seems to play a crucial role in the ligand–
protein interaction and in the inhibitory activity of the synthesized
compounds. The antiprion profile of these new derivatives, bearing
only one acylamino group linked to a basic cyclic group (pyrroli-
dine or piperidine) is apparently not affected by the size of the ba-
sic cycle. In fact, both series of compounds showed similar
3. Conclusions
In a screening of a new series of small molecules with benzam-
ide as core structure by the SIFT antiprion assay and successive
evaluation of their antiprion activity in an in vitro cell-based assay
the derivatives 6h, 6i, 7c and 7i were found to exhibit interesting
antiprion properties. Moreover, the good correlation of the SIFT

F. Fiorino et al. / Bioorg. Med. Chem. 20 (2012) 5001–5011
5007
assay and cell culture data confirms the working assumption that
antiprion activities may derive from interference of these small
1H NMR (CDCl3, d, ppm): 9.09 (s, 1H, ArH), 8.27 (d, 1H, J = 7.9 Hz,
ArH), 7.88 (br s, 1H, CONH), 7.05 (dd, 1H, J = 7.9 Hz, ArH), 4.62 (m,
1H, cyclohexyl), 3.49 (qd, 2H, J = 6.9 Hz,CH2NH), 2.10–1.85 (m, 8H,
cyclohexyl), 1.67 (qt, 2H, J = 6.9 Hz, CH2CH2NH), 1.58 (m, 2H, cyclo-
hexyl), 1.48 (m, 2H, CH2CH3,), 0.98 (t, 3H, J = 6.9 Hz, CH2CH3).
Anal. (C17H24N2O4): C, H, N.
Using the procedure described for preparing 3a the following
compounds were prepared: N-butyl-2-isopropoxy-5-nitrobenza-
mide (3b), N-butyl-2-adamantanoxy-5-nitrobenzamide (3c), N-bu-
tyl-2-propargyloxy-5-nitrobenzamide (3d), N-butyl-2-phenoxy-5-
nitrobenzamide (3e), N-butyl-2-p-chlorophenoxy-5-nitrobenza-
mide (3f), N-butyl-2-p-fluorophenoxy-5-nitrobenzamide (3g), N-
butyl-2-p-trifluoromethylphenoxy-5-nitrobenzamide (3h), N-bu-
tyl-2-o-trifluoromethylphenoxy-5-nitrobenzamide (3i), N-butyl-
2-p-methoxyphenoxy-5-nitrobenzamide (3l), N-butyl-2-p-butoxy-
phenoxy-5-nitrobenzamide (3m), N-butyl-2-p-tolyloxy-5-nitrob-
molecules with conversion of PrPc into PrPSc 20
However, further
.
studies are required to confirm this conclusion and to identify by
in vivo studies compounds with favorable physiochemical proper-
ties for crossing the blood–brain barrier (BBB). For this purpose the
benzamide core structure may be a promising new lead for antip-
rion agents.
4. Experimental section
4.1. Chemistry
Melting points were determined using a Kofler hot-stage appa-
ratus and are uncorrected. 1H NMR spectra were recorded on Var-
ian Mercury Plus 400 MHz instrument. Unless otherwise stated, all
spectra were recorded in CDCl3. Chemical shifts are reported in
ppm using Me4Si as internal standard. The following abbreviations
are used to describe peak patterns when appropriate: s (singlet), br
s (broad singlet), d (doublet), t (triplet), qd (quadruplet), qt (quin-
tuplet), hep (heptet), m (multiplet). Mass spectra of the final prod-
ucts were recorded on API 2000 Applied Biosystem mass
spectrometer. Where analyses are indicated only by the symbols
of the elements, results obtained are within 0.4% of the theoretical
values. All reactions were followed by TLC on Merck Silica Gel 60
F254 plates with fluorescent indicator and the plates were visual-
ized with UV light (254 nm). Preparative chromatographic purifi-
cations were performed on silica gel column (Kieselgel 60).
Solutions were dried over Na2SO4 and concentrated with Büchi ro-
tary evaporator at low pressure.
enzamide
(3n)
and
N-butyl-2-p-isopropylphenoxy-5-
nitrobenzamide (3o) in yields ranging between 70% and 80%. The
1H NMR spectra of the intermediates 3b–o were consistent with
the assigned structures.
4.1.3. 5-Amino-N-butyl-2-cyclohexyloxybenzamide (4a)
To a suspension of Pd/C (0.15 g) in 15 mL water was added a
suspension (20 mL) of NaBH4 (1.0 g, 26.4 mmol) in water. The
resulting reaction mixture was stirred at room temperature under
nitrogen atmosphere for 10 min. Then 3a (2.89 g, 8.1 mmol) in
100 mL methanol was added dropwise and the mixture was stirred
for 30 min. The reaction mixture was filtered through a Celite bed.
The solution was acidified with 1 N HCl to remove the excess of
NaBH4. The acidified solution was adjusted to alkaline pH with
2 N NaOH solution and the resulting suspension was extracted
with diethyl ether. The organic phase was dried over magnesium
sulfate, filtered, and evaporated to dryness to yield intermediate
4a as a white solid (2.70 g, 87%); 1H NMR data are reported as
hydrochloride salt.
4.1.1. N-Butyl-2-chloro-5-nitro-benzamide (2)
To a solution of 2-chloro-5-nitrobenzoic acid (1) (10.00 g,
50.0 mmol) in anhydrous DMF (100 ml) was added HOBt (7.30 g,
54.0 mmol) followed by DCC (11.14 g, 54.0 mmol) at 0 °C. The
resulting reaction mixture was stirred for 30 min and then n-butyl-
amine hydrochloride (4.08 g, 50.0 mmol) and TEA (5.06 g,
50.0 mmol) were added. The reaction mixture was stirred at 0 °C
for 2 h and overnight at room temperature. N,N0-Dicyclohexylurea
(DCU) was filtered off and the DMF was evaporated under reduced
pressure. The residue was dissolved in CH2Cl2, washed consecu-
tively with 1 N NaOH and 1 N HCl. The organic phase was dried
over anhydrous Na2SO4, filtered, concentrated and purified by crys-
tallization from diethyl ether affording 9.49 g of pure 2 as a yellow
solid, mp 134 °C.
1H NMR (CDCl3, d, ppm): 8.24 (br s, 1H, CONH), 7.53 (s, 1H, ArH),
6.81 (d, 1H, J = 7.9 Hz, ArH), 6.73 (dd, 1H, J = 7.9 Hz, ArH), 4.62 (m,
1H, cyclohexyl), 3.98 (br s, 2H, NH2), 3.49 (qd, 2H, J = 6.9 Hz,
CH2NH), 2.10–1.85 (m, 8H, cyclohexyl), 1.67 (qt, 2H, J = 6.9 Hz,
CH2CH2NH), 1.58 (m, 2H, cyclohexyl), 1.48 (m, 2H, CH2CH3,), 0.98
(t, 3H, J = 6.9 Hz, CH2CH3).
Anal. (C17H26N2O2): C, H, N.
Using the procedure described for preparing 4a the following
compounds were prepared: 5-amino-N-butyl-2-isopropoxybenza-
mide (4b), 5-amino-N-butyl-2-adamantaniloxybenzamide (4c), 5-
amino-N-butyl-2-propargyloxybenzamide (4d), 5-amino-N-butyl-
2-phenoxybenzamide (4e), 5-amino-N-butyl-2-p-chlorophenoxyb-
enzamide (4f), 5-amino-N-butyl-2-p-fluorophenoxybenzamide
1H NMR (400 MHz, CDCl3, d, ppm): 8.46 (s, 1H, ArH); 8.19 (dd,
1H, J = 7.9 Hz, ArH); 7.59 (d, 1H, J = 7.9 Hz, ArH); 6.23 (br s, 1H,
CONH); 3.48 (qd, 2H, J = 6.9 Hz, CH2NH); 1.69 (qt, 2H, J = 6.9 Hz,
CH2CH2NH); 1.44 (m, 2H, CH2CH3); 0.98 (t, 3H, J = 6.9 Hz, CH2CH3).
Anal. (C11H13ClN2O3): C, H, Cl, N.
(4g),
(4h),
5-amino-N-butyl-2-p-trifluoromethylphenoxybenzamide
5-amino-N-butyl-2-o-trifluoromethylphenoxybenzamide
(4i), 5-amino-N-butyl-2-p-methoxyphenoxybenzamide (4l), 5-
amino-N-butyl-2-p-butoxyphenoxybenzamide (4m), 5-amino-N-
butyl-2-p-tolyloxybenzamide (4n) and 5-amino-N-butyl-2-p-iso-
propylphenoxybenzamide (4o) in yields ranging between 70%
and 80%. The 1H NMR spectra of intermediates 4b–o are consistent
with the proposed structures.
4.1.2. N-Butyl-2-cyclohexyloxy-5-nitro-benzamide (3a)
A solution of sodium hydride (60% in mineral oil, 3.35 g,
84.0 mmol) in dry dioxane (150 mL) was added to cyclohexanol
(7.91 g, 79.0 mmol) in an ice-water bath under a nitrogen atmo-
sphere. After the evolution of hydrogen had ceased, the mixture
was stirred at 70 °C for 2 h. Then intermediate
2 (19.6 g,
4.1.4. 5-(2-Bromoacetamido)-N-butyl-2-
76.4 mmol) in anhydrous dioxane (100 mL) was added dropwise
and the resulting reaction mixture was stirred at 70 °C for 4 h
and overnight at room temperature. The solvent was removed un-
der reduced pressure and the residue was dissolved in ethyl ace-
tate and washed with brine. The organic phase was dried over
magnesium sulfate, filtered, concentrated and the residue purified
by chromatography on silica gel column (elution with diethyl
ether/hexane, 6:4 v/v) to afford 3a (9.4 g, 75%), mp 64–65 °C.
(cyclohexyloxy)benzamide (5a)
To a precooled solution (ꢀ10 °C) of 5-amino-N-butyl-2-cyclo-
hexyloxybenzamide (4a) (1.20 g, 4.16 mmol) and triethylamine
(0.50 g, 5.0 mmol) in CH2Cl2 (90 mL) and water (8 mL) a solution
of bromoacetyl bromide (1.68 g, 8.34 mmol) in CH2Cl2 (20 mL) was
added dropwise under stirring. Then the mixture was allowed to
warm up to room temperature within 3 h. The phases were sepa-
rated, the aqueous phase was washed with CH2Cl2, and the organic

5008
F. Fiorino et al. / Bioorg. Med. Chem. 20 (2012) 5001–5011
phase was reextracted with water. The organic phases were com-
bined and the solvent was evaporated to a small volume. Upon addi-
tion of hexane and cooling, the precipitate was filtered off, washed
with hexane and recrystallized from CH2Cl2/hexane; yield: 1.55 g
(91%), light yellow needles.
CH2CH2NH); 1.48 (m, 2H, CH2CH3); 1.46 (d, J = 5.8 Hz, 6H, 2 CH3-
Isopropyl); 0.98 (t, 3H, J = 6.9 Hz, CH2CH3).
ESI-MS: 362.3 [M+H]+; 384.3 [M+Na]+; 400.2 [M+K]+.
Anal. (C20H31N3O3): C, H, N.
1H NMR (CDCl3, d, ppm): 8.89 (s, 1H, ArH), 8.02 (br s, 1H, NHCO),
7.90 (d, 1H, J = 7.9 Hz, ArH), 7.88 (br s, 1H, CONH), 6.90 (dd, 1H,
J = 7.9 Hz, ArH), 4.62 (m, 1H, cyclohexyl), 4.20 (s, 2H, CH2Br), 3.49
(qd, 2H, J = 6.9 Hz,CH2NH), 2.10–1.85 (m, 8H, cyclohexyl), 1.67
(qt , 2H, J = 6.9 Hz, CH2CH2NH), 1.58 (m, 2H, cyclohexyl), 1.48 (m,
2H, CH2CH3), 0.98 (t, 3H, J = 6.9 Hz, CH2CH3).
4.1.5.3. 5-(2-(Pyrrolidin-1-yl)acetamido)-N-butyl-2-(adamanta-
nyloxy)benzamide (6c).
Yield: 81%; mp 126–128 °C; 1H NMR
(400 MHz, CDCl3) d: 9.23 (br s, 1H, CONH); 8.20 (dd, 1H, J = 8.8 Hz,
ArH); 8.05 (br s, 1H, NHCO); 7.78 (s, 1H, ArH); 7.06 (d, 1H,
J = 8.8 Hz, ArH); 3.47 (qd, 2H, J = 6.9 Hz, J = 5.8 Hz, CH2NH); 3.25
(s, 2H, COCH2); 2.67 (br s, 4H, Pyrr); 1.91 (m, 6H, Adamantane);
1.84 (br s, 4H, Pyrr); 1.66–1.55 (m, 9H, Adamantane); 1.47 (qt,
2H, J = 6.9 Hz, CH2CH2NH); 1.34 (m, 2H, CH2CH3); 0.97 (t, 3H,
J = 6.9 Hz, CH2CH3).
Anal. (C19H27BrN2O3): C, H, N.
Using the procedure described for preparing 5a the following
compounds were prepared: 5-(2-bromoacetamido)-2-N-butyl-iso-
propoxybenzamide (5b), 5-(2-bromoacetamido)-2-N-butyl-ada-
mantanoxybenzamide (5c), 5-(2-bromoacetamido)-2-N-butyl-
propargyloxybenzamide (5d), 5-(2-bromoacetamido)-2-N-butyl-
phenoxybenzamide (5e), 5-(2-bromoacetamido)-2-N-butyl-p-chlo-
rophenoxybenzamide(5f), 5-(2-bromoacetamido)-2-N-butyl-p-flu-
orophenoxybenzamide (5g), 5-(2-bromoacetamido)-2-N-butyl-p-
trifluoromethylphenoxybenzamide (5h), 5-(2-bromoacetamido)-
2-N-butyl-o-trifluoromethylphenoxybenzamide (5i), 5-(2-bromo-
acetamido)-2-N-butyl-p-methoxyphenoxybenzamide (5l), 5-(2-
bromoacetamido)-2-N-butyl-p-butoxyphenoxybenzamide (5m),
5-(2-bromoacetamido)-2-N-butyl-p-tolyloxybenzamide (5n) and
5-(2-bromoacetamido)-2-N-butyl-p-isopropylphenoxybenzamide
(5o) in yields ranging between 35% and 98%. The 1H NMR spectra of
intermediates 5b–o are consistent with the proposed structures.
ESI-MS: 454.2 [M+H]+; 476.4 [M+Na]+.
Anal. (C27H39N3O3): C, H, N.
4.1.5.4.
ynyloxy)benzamide (6d).
5-(2-(Pyrrolidin-1-yl)acetamido)-N-butyl-2-(prop-2-
Yield: 76%; mp 157–162 °C; 1H
NMR (400 MHz, CDCl3) d: 9.26 (br s, 1H, CONH); 8.28 (dd, 1H,
J = 8.8 Hz, ArH); 7.86 (s, 1H, ArH); 7.83 (br s, 1H, NHCO); 7.02 (d,
1H, J = 8.8 Hz, ArH); 4.79 (s, 1H, CH-Propargyl); 3.48 (qd, 2H,
J = 6.9 Hz, J = 5.8 Hz, CH2NH); 3.32 (s, 2H, COCH2); 2.72 (br s, 4H,
Pyrr); 2.59 (s, 2H, CH2-Propargyl); 1.87 (br s, 4H, Pyrr); 1.62 (qt,
2H, J = 6.9 Hz, CH2CH2NH); 1.46 (m, 2H, CH2CH3); 0.98 (t, 3H,
J = 6.9 Hz, CH2CH3).
ESI-MS: 358.3 [M+H]+; 380.4 [M+Na]+; 396.3 [M+K]+.
Anal. (C20H27N3O3): C, H, N.
4.1.5. General procedure for the preparation of 5-(2-(pyrrolidin-
1-yl)acetamido)-N-butyl-2-(substituted)benzamide (6a–o) and
5-(2-(piperidin-1-yl)acetamido)-N-butyl-2-
4.1.5.5. 5-(2-(Pyrrolidin-1-yl)acetamido)-N-butyl-2-phenoxyb-
enzamide (6e).
Yield: 40%; mp 111–112 °C; 1H NMR
(400 MHz, CDCl3) d: 9.30 (br s, 1H, CONH); 8.24 (dd, 1H,
J = 8.8 Hz, ArH); 7.88 (s, 1H, ArH); 7.69 (br s, 1H, NHCO); 7.38 (t,
2H, J = 7.6 Hz, ArH); 7.18 (t, 1H, J = 7.6 Hz, ArH); 7.01 (d, 2H,
J = 7.6 Hz, ArH); 6.88 (d, 1H, J = 8.8 Hz, ArH); 3.44 (qd, 2H,
J = 6.9 Hz, J = 5.8 Hz, CH2NH); 3.27 (s, 2H, COCH2); 2.69 (br s, 4H,
Pyrr); 1.86 (br s, 4H, Pyrr); 1.54 (qt, 2H, J = J = 6.9 Hz, CH2CH2NH);
1.34 (m, 2H, CH2CH3); 0.88 (t, 3H, J = 6.9 Hz, CH2CH3).
ESI-MS: 396,1 [M+H]+.
(substituted)benzamide (7a–o) derivatives
A
mixture
of
5-(2-bromoacetamido)-N-butyl-2-(substi-
tuted)benzamide (5a–o) (3.14 mmol) and NaI (0.70 g; 4.72 mmol)
in acetonitrile was stirred under reflux for 30 min. Then the appro-
priate pyrrolidine or piperidine derivative (0.22 g; 3.14 mmol and
0.27 g; 3.14 mmol respectively) and anhydrous K2CO3 (0.65 g;
4.72 mmol) were added. The reaction mixture was stirred under
reflux for 4 h. After cooling, the mixture was filtered, evaporated
to dryness and the residue was dissolved in water (50 mL). The
solution was extracted several times with CH2Cl2. The combined
organic layers were dried over anhydrous Na2SO4 and the solvent
was evaporated. The crude products were purified by silica gel col-
umn chromatography using diethyl ether/methanol 9:1 (v/v) as
eluent. The resulting products were recrystallized from diethyl
ether.
Anal. (C23H29N3O3): C, H, N.
4.1.5.6. 5-(2-(Pyrrolidin-1-yl)acetamido)-2-(4-chlorophenoxy)-
N-butylbenzamide (6f).
Yield: 64%; mp 118–121 °C; 1H
NMR (400 MHz, CDCl3) d: 9.31 (br s, 1H, CONH); 8.24 (dd, 1H,
J = 8.8 Hz, ArH); 7.87 (s, 1H, ArH); 7.50 (br s, 1H, NHCO); 7.33 (d,
2H, J = 8.4 Hz, ArH); 6.94 (d, 2H, J = 8.4 Hz, ArH); 6.87 (d, 1H,
J = 8.8 Hz; ArH); 3.43 (qd, 2H, J = 6.9 Hz, J = 5.8 Hz, CH2NH); 3.28
(s, 2H, COCH2); 2.69 (br s, 4H, Pyrr); 1.87 (br s, 4H, Pyrr); 1.50
(qt, 2H, J = 6.9 Hz, CH2CH2NH); 1.31 (m, 2H, CH2CH3); 0.89 (t, 3H,
J = 6.9 Hz, CH2CH3).
4.1.5.1. 5-(2-(Pyrrolidin-1-yl)acetamido)-N-butyl-2-(cyclohexyl-
oxy)benzamide (6a).
Yield: 85%; mp 83–84 °C; 1H NMR
(400 MHz, CDCl3) d: 9.17 (br s, 1H, CONH); 8.25 (m, 2H, ArH);
7.82 (br s, 1H, NHCO); 6.97 (d, 1H, J = 7.9 Hz, ArH); 4.72 (m, 1H,
cyclohexyl); 3.48 (qd, 2H, J = 6.9 Hz, J = 5.8 Hz, CH2NH); 3.25 (s,
2H, COCH2); 2.67 (br s, 4H, Pyrr); 2.04 (m, 8H, cyclohexyl); 1.85
(br s, 4H, Pyrr); 1.64 (m, 2H, cyclohexyl); 1.55 (m, 2H, CH2CH2NH);
1.45 (m, 2H, CH2CH3); 0.98 (t, 3H, J = 6.9 Hz, CH2CH3).
ESI-MS: 402.7 [M+H]+; 424.4 [M+Na]+.
ESI-MS: 430.1 [M+H]+; 452.4 [M+Na]+.
Anal. (C23H28ClN3O3): C, H, N.
4.1.5.7. 5-(2-(Pyrrolidin-1-yl)acetamido)-2-(4-fluorophenoxy)-
N-butylbenzamide (6g).
Yield: 85%; mp 66–69 °C; 1H NMR
(400 MHz, CDCl3) d: 9.28 (br s, 1H, CONH); 8.22(dd, 1H,
J = 8.8 Hz, ArH); 7.88 (s, 1H, ArH); 7.61 (br s, 1H, NHCO); 7.08 (t,
2H, J = 8.4 Hz, ArH); 6.99 (m, 2H, J = 8.4 Hz, ArH); 6.83 (d, 1H,
J = 8.8 Hz ArH); 3.45 (qd, 2H, J = 6.9 Hz, J = 5.8 Hz, CH2NH); 3.27
(s, 2H, COCH2); 2.69 (br s, 4H, Pyrr); 1.86 (br s, 4H, Pyrr); 1.54
(qt, 2H, J = 6.9 Hz, CH2CH2NH); 1.34 (m, 2H, CH2CH3); 0.90 (t, 3H,
J = 6.9 Hz, CH2CH3).
Anal. (C23H35N3O3): C, H, N.
4.1.5.2.
oxybenzamide (6b).
5-(2-(Pyrrolidin-1-yl)acetamido)-N-butyl-2-isoprop-
Yield: 87%; mp 56–59 °C; 1H NMR
(400 MHz, CDCl3) d: 9.18 (br s, 1H, CONH); 8.26 (dd, 1H,
J = 8.8 Hz, ArH); 8.17 (br s, 1H, NHCO); 7.83 (s, 1H, ArH); 6.96 (d,
1H, J = 8.8 Hz, ArH); 4.73 (hep, 1H, J = 5.8 Hz, CH-Isopropyl); 3.48
(qd, 2H, J = 6.9 Hz, J = 5.8 Hz, CH2NH); 3.26 (s, 2H, COCH2); 2.68
(br s, 4H, Pyrr); 1.85 (br s, 4H, Pyrr); 1.62 (qt, 2H, J = 6.9 Hz,
ESI-MS: 414.1 [M+H]+.
Anal. (C23H28FN3O3): C, H, N.

F. Fiorino et al. / Bioorg. Med. Chem. 20 (2012) 5001–5011
5009
4.1.5.8.
5-(2-(Pyrrolidin-1-yl)acetamido)-2-(4-(trifluoro-
Yield: 43%; mp
J = 8.8 Hz, ArH); 7.88 (s, 1H, ArH); 7.76 (br s, 1H, NHCO); 7.22 (d,
2H, J = 8.4 Hz, ArH); 6.94 (d, 2H, J = 8.4 Hz, ArH); 6.86 (d, 1H,
J = 8.8 Hz, ArH); 3.44 (qd, 2H, J = 6.9 Hz, J = 5.8 Hz, CH2NH); 3.26
(s, 2H, COCH2); 2.93 (hep, 1H, J = 6.9 Hz, CH-Isopropyl); 2.69 (br
s, 4H, Pyrr); 1.86 (br s, 4H, Pyrr); 1.54 (qt, 2H, J = 6.9 Hz,
CH2CH2NH); 1.31 (m, 2H, CH2CH3); 1.26 (d, 6H, J = 6.9 Hz, 2CH3-
Isopropyl); 0.89 (t, 3H, J = 6.9 Hz, CH2CH3).
methyl)phenoxy)-N-butylbenzamide (6h).
110–112 °C; 1H NMR (400 MHz, CDCl3) d: 9.33 (br s, 1H, CONH);
8.28 (dd, 1H, J = 8.8 Hz, ArH); 7.87 (s, 1H, ArH); 7.61 (d, 2H,
J = 8.4 Hz, ArH); 7.29 (br s, 1H, NHCO); 7.05 (d, 2H, J = 8.4 Hz,
ArH); 6.95 (d, 1H, J = 8.8 Hz, ArH); 3.40 (qd, 2H, J = 6.9 Hz,
J = 5.8 Hz, CH2NH); 3.28 (s, 2H, COCH2); 2.69 (br s, 4H, Pyrr); 1.87
(br s, 4H, Pyrr); 1.47 (qt, 2H, J = 6.9 Hz, CH2CH2NH); 1.28 (m, 2H,
CH2CH3); 0.85 (t, 3H, J = 6.9 Hz, CH2CH3).
ESI-MS: 438.2 [M+H]+.
Anal. (C26H35N3O3): C, H, N.
ESI-MS: 464.0 [M+H]+; 486.1 [M+Na]+; 502.2 [M+K]+.
Anal. (C24H28F3N3O3): C, H, N.
4.1.5.14.
hexyloxy)benzamide (7a).
5-(2-(Piperidin-1-yl)acetamido)-N-butyl-2-(cyclo-
Yield: 74%; mp 96–98 °C; 1H
4.1.5.9.
5-(2-(Pyrrolidin-1-yl)acetamido)-2-(2-(trifluoro-
Yield: 92%; mp
NMR (400 MHz, CDCl3) d: 9.27 (br s, 1H, CONH); 8.23 (dd, 1H,
J = 8.8 Hz, ArH); 8.05 (br s, 1H, NHCO); 7.82 (s, 1H, ArH); 6.97 (d,
1H, J = 8.8 Hz, ArH); 4.44 (m, 1H, Cyclohexyl); 3.48 (qd, 2H,
J = 6.9 Hz, J = 6.2 Hz, CH2NH); 3.04 (s, 2H, COCH2); 2.51 (br s, 4H,
Pip); 2.06 (m, 8H, Cyclohexyl); 1.78 (m, 2H, Cyclohexyl); 1.65 (m,
6H, Pip); 1.44 (m, 4H, –CH2CH2–); 0.94 (t, 3H, J = 6.9 Hz, CH2CH3).
ESI-MS: 416.4 [M+H]+.
methyl)phenoxy)-N-butylbenzamide (6i).
119–120 °C; 1H NMR (400 MHz, CDCl3 d: 9.33 (br s, 1H, CONH);
8.29 (d, 1H, J = 8.8 Hz, ArH); 7.87 (s, 1H, ArH); 7.71 (d, 1H,
J = 7.3 Hz, ArH); 7,48 (t, 1H, J = 7.3 Hz, ArH), 7.42 (br s, 1H, NHCO);
7.23 (t, 1H, J = 7.3 Hz, ArNH); 6.92 (d, 1H, J = 7.3 Hz, ArH); 6.86 (d,
1H, J = 8.8 Hz, ArH); 3.38 (qd, 2H, J = 6.9 Hz, J = 5.8 Hz, CH2NH);
3.28 (s, 2H, COCH2); 2.69 (br s, 4H, Pyrr); 1.87 (br s, 4H, Pyrr);
1.47 (qt, 2H, J = 6.9 Hz, CH2CH2NH); 1.27 (m, 2H, CH2CH3); 0.85
(t, 3H, J = 6.9 Hz, CH2CH3).
Anal. (C24H37N3O3): C, H, N.
4.1.5.15.
oxybenzamide (7b).
5-(2-(Piperidin-1-yl)acetamido)-N-butyl-2-isoprop-
Yield: 97%, mp 90–92 °C; 1H NMR
ESI-MS: 464.2 [M+H]+; 486.3 [M+Na]+.
Anal. (C24H28F3N3O3): C, H, N.
(400 MHz, CDCl3) d: 9.27 (br s, 1H, CONH); 8.25 (dd, 1H,
J = 8.8 Hz, ArH); 8.17 (br s, 1H, NHCO); 7.82 (s, 1H, ArH); 6.96 (d,
1H, J = 8.8 Hz, ArH); 4.71 (hep, 1H, J = 5.8 Hz, CH-Isopropyl); 3.48
(qd, 2H, J = 6.9 Hz, J = 6.2 Hz, CH2NH); 3.04 (s, 2H, COCH2); 2.51
(br s, 4H, Pip); 1.68–1.57 (m, 6H, Pip, 2H, CH2CH2NH); 1.46 (m,
2H, CH2CH3); 1.41 (d, 6H, J = 5.8 Hz, 2CH3-Isopropyl); 0.98 (t, 3H,
J = 6.9 Hz, CH2CH3).
4.1.5.10. 5-(2-(Pyrrolidin-1-yl)acetamido)-2-(4-methoxyphen-
oxy)-N-butylbenzamide (6l).
Yield: 97%; mp 64–66 °C; 1H
NMR (400 MHz, CDCl3) d: 9.26 (br s, 1H, CONH); 8.18 (dd, 1H,
J = 8.8 Hz, ArH); 7.87 (s, 1H, ArH); 7.83 (br s, 1H, NHCO); 6.98 (d,
2H, J = 8.7 Hz, ArH); 6.91 (d, 2H, J = 8.7 Hz, ArH); 6.78 (d, 1H,
J = 8.8 Hz, ArH); 3.81 (s, 3H, OCH3); 3.46 (qd, 2H, J = 6.9 Hz,
J = 5.8 Hz, CH2NH); 3.26 (s, 2H, COCH2); 2.68 (br s, 4H, Pyrr); 1.86
(br s, 4H, Pyrr); 1.55 (qt, 2H, J = 6.9 Hz, CH2CH2NH); 1.36 (m, 2H,
CH2CH3); 0.91 (t, 3H, J = 6.9 Hz, CH2CH3).
ESI-MS: 376.4 [M+H]+; 398.4 [M+Na]+; 414.3 [M+K]+.
Anal. (C21H33N3O3): C, H, N.
4.1.5.16. 5-(2-(Piperidin-1-yl)acetamido)-N-butyl-2-(adamanta-
ESI-MS: 426.2 [M+H]+.
nyloxy)benzamide (7c).
Yield: 84%; mp 99–100 °C; 1H NMR
Anal. (C24H31N3O4): C, H, N.
(400 MHz, CDCl3) d: 9.33 (br s, 1H, CONH); 8.18 (dd, 1H,
J = 8.8 Hz, ArH); 8.03 (br s, 1H, NHCO); 7.70 (s, 1H, ArH); 7.06 (d,
1H, J = 8.8 Hz, ArH); 3.47 (qd, 2H, J = 6.9 Hz, J = 6.2 Hz, CH2NH);
3.04 (s, 2H, COCH2); 2.51 (br s, 4H, Pip); 2.18 (m, 6H, Adamantane);
1.91 (m, 6H, Pip); 1.64–1.55 (m, 2H, CH2-Butyl, 9H, Adamantane);
1.45 (m, 2H, CH2CH3); 0.97 (t, 3H, J = 6.9 Hz, CH2CH3).
ESI-MS: 468.5 [M+H]+.
4.1.5.11.
oxy)-N-butylbenzamide (6m).
5-(2-(Pyrrolidin-1-yl)acetamido)-2-(4-butoxyphen-
Yield: 69%; mp 65–67 °C; 1H
NMR (400 MHz, CDCl3) d: 9.24 (br s, 1H, CONH); 8.17(dd, 1H,
J = 8.8 Hz, ArH); 7.87 (s, 1H, ArH); 7.83 (br s, 1H, NHCO); 6.96 (d,
2H, J = 8.7 Hz, ArH); 6.90 (d, 2H, J = 8.7 Hz, ArH); 6.79 (d, 1H,
J = 8.8 Hz ArH); 3.96 (t, 2H, J = 6.6 Hz, OCH2); 3.46 (qd, 2H,
J = 6.9 Hz, J = 5.8 Hz, CH2NH); 3.26 (s, 2H, COCH2); 2.68 (br s, 4H,
Pyrr); 1.86 (br s, 4H, Pyrr); 1.80 (qt, 2H, J = 6.6 Hz, OCH2CH2);
1.57 (m, 4H, 2CH2, OCH2CH2CH2, CH2CH2NH); 1.36 (m, 2H,
CH2CH3); 0.99 (t, 3H, J = 6.6 Hz, O(CH2)3CH3,); 0.91 (t, 3H,
J = 6.9 Hz, CH2CH3).
Anal. (C28H41N3O3): C, H, N.
4.1.5.17.
ynyloxy)benzamide (7d).
5-(2-(Piperidin-1-yl)acetamido)-N-butyl-2-(prop-2-
Yield: 71%; mp 96–99 °C; 1H NMR
(400 MHz, CDCl3) d: 9.31 (br s, 1H, CONH); 8.28 (dd, 1H,
J = 8.8 Hz, ArH); 8.03 (br s, 1H, NHCO); 7.83 (s, 1H, ArH); 7.02 (d,
1H, J = 8.8 Hz, ArH); 4,79 (s, 1H, CH-Propargyl); 3.49 (qd, 2H,
J = 6.9 Hz, J = 6.2 Hz, CH2NH); 3.04 (s, 2H, COCH2); 2.59 (s, 2H,
CH2-Propargyl); 2.51 (br s, 4H, Pip); 1.63 (m, 6H, Pip; 2H,
CH2CH2NH); 1.46 (m, 2H, CH2CH3); 0.96 (t, 3H, J = 6.9 Hz, CH2CH3).
ESI-MS: 372.3 [M+H]+; 394.5 [M+Na]+; 410.4 [M+K]+.
ESI-MS: 468.6 [M+H]+.
Anal. (C27H37N3O4): C, H, N.
4.1.5.12.
butylbenzamide (6n).
5-(2-(Pyrrolidin-1-yl)acetamido)-2-(p-tolyloxy)-N-
Yield: 71%; mp 82–84 °C; 1H NMR
(400 MHz, CDCl3) d: 9.26 (br s, 1H, CONH); 8.21(dd, 1H,
J = 8.8 Hz, ArH); 7.87 (s, 1H, ArH); 7.75 (br s, 1H, NHCO); 7.17 (d,
2H, J = 8.4 Hz, ArH); 6.91 (d, 2H, J = 8.4 Hz, ArH); 6.84 (d, 1H,
J = 8.8 Hz, ArH); 3.44 (qd, 2H, J = 6.9 Hz, J = 5.8 Hz, CH2NH); 3.26
(s, 2H, COCH2); 2.68 (br s, 4H, Pyrr); 2.34 (s, 3H, ArCH3); 1.86 (br
s, 4H, Pyrr); 1.54 (qt, 2H, J = 6.9 Hz, CH2CH2NH); 1.34 (m, 2H,
CH2CH3); 0.89 (t, 3H, J = 6.9 Hz, CH2CH3).
Anal. (C21H29N3O3): C, H, N.
4.1.5.18. 5-(2-(Piperidin-1-yl)acetamido)-N-butyl-2-phenoxyb-
enzamide (7e).
Yield 89%; mp 99–101 °C; 1H NMR
(400 MHz, CDCl3) d: 9.30 (br s, 1H, CONH); 8.22 (dd, 1H,
J = 8.8 Hz, ArH); 7.88 (s, 1H, ArH); 7.66 (br s, 1H, NHCO); 7.38 (t,
2H, J = 7.6 Hz, ArH); 7.18 (t, 1H, J = 7.6 Hz, ArH); 7.00 (d, 2H,
J = 7.6 Hz, ArH); 6.88 (d, 1H, J = 8.8 Hz, ArH); 3.44 (qd, 2H,
J = 6.9 Hz, J = 6.2 Hz, CH2NH); 3.06 (s, 2H, COCH2); 2.53 (br s, 4H,
Pip); 1.66 (m, 6H, Pip); 1.51 (qt, 2H, J = 6.9 Hz, CH2CH2NH); 1.32
(m, 2H, CH2CH3); 0.88 (t, 3H, J = 6.9 Hz, CH2CH3).
ESI-MS: 410.2 [M+H]+.
Anal. (C24H31N3O3): C, H, N.
4.1.5.13. 5-(2-(Pyrrolidin-1-yl)acetamido)-2-(4-isopropylphen-
oxy)-N-butylbenzamide (6o).
NMR (400 MHz, CDCl3) d: 9.27 (br s, 1H, CONH); 8.21(dd, 1H,
Yield 78%; mp 68–69 °C; 1H
ESI-MS: 410.5 [M+H]+; 432.3 [M+Na]+.
Anal. (C24H31N3O3): C, H, N.

5010
F. Fiorino et al. / Bioorg. Med. Chem. 20 (2012) 5001–5011
4.1.5.19. 5-(2-(Piperidin-1-yl)acetamido)-2-(4-chlorophenoxy)-
N-butylbenzamide (7f).
Yield: 78%; mp 122–123 °C; 1H
J = 8.8 Hz, ArH); 3.96 (t, 2H, J = 6.6 Hz OCH2); 3.46 (qd, 2H,
J = 6.9 Hz, J = 6.2 Hz, CH2NH); 3.05 (s, 2H, COCH2); 2.53 (br s, 4H,
Pip); 1.80 (m, 2H, J = 6.6 Hz OCH2CH2); 1.64 (m, 6H, Pip); 1.57
(m, 4H, 2CH2, OCH2CH2CH2, CH2CH2NH); 1.36 (m, 2H, CH2CH3);
0.99 (t, 3H, J = 6.9 Hz, O(CH2)3CH3,); 0.91 (t, 3H, J = 6.9 Hz, CH2CH3).
ESI-MS: 482.7 [M+H]+; 504.6 [M+Na]+.
NMR (400 MHz, CDCl3) d: 9.40 (br s, 1H, CONH); 8.22 (dd, 1H,
J = 8.8 Hz, ArH); 7.86 (s, 1H, ArH); 7.47 (br s, 1H, NHCO); 7.32 (d,
2H, J = 8.7 Hz, ArH); 6.93 (d, 2H, J = 8.7 Hz, ArH); 6.87 (d, 1H,
J = 8.8 Hz, ArH); 3.43 (qd, 2H, J = 6.9 Hz, J = 6.2 Hz, CH2NH); 3.06
(s, 2H, COCH2); 2.53 (br s, 4H, Pip); 1.66 (m, 6H, Pip); 1.52 (qt,
2H, J = 6.9 Hz, CH2CH2NH); 1.32 (m, 2H, CH2CH3); 0.89 (t, 3H,
J = 6.9 Hz, CH2CH3).
Anal. (C28H39N3O4): C, H, N.
4.1.5.25.
butylbenzamide (7n).
5-(2-(Piperidin-1-yl)acetamido)-2-(p-tolyloxy)-N-
Yield: 88%; mp 79–82 °C; 1H NMR
ESI-MS: 444.3 [M+H]+; 466.5 [M+Na]+.
Anal. (C24H30ClN3O3): C, H, N.
(400 MHz, CDCl3) d: 9.37 (br s, 1H, CONH); 8.19 (dd, 1H,
J = 8.8 Hz, ArH); 7.87 (s, 1H, ArH); 7.75 (br s, 1H, NHCO); 7.15 (d,
2H, J = 8.4 Hz, ArH); 6.91 (d, 2H, J = 8.4 Hz, ArH); 6.84 (d, 1H,
J = 8.8 Hz, ArH); 3.45 (qd, 2H, J = 6.9 Hz, J = 6.2 Hz, CH2NH); 3.05
(s, 2H, COCH2); 2.52 (br s, 4H, Pip); 2.34 (s, 3H, ArCH3); 1.66 (m,
6H, Pip); 1.54 (qt, 2H, J = 6.9 Hz, CH2CH2NH); 1.36 (m, 2H, CH2CH3);
0.89 (t, 3H, J = 6.9 Hz, CH2CH3).
4.1.5.20. 5-(2-(Piperidin-1-yl)acetamido)-2-(4-fluorophenoxy)-
N-butylbenzamide (7g).
Yield: 90%; mp 125–126 °C; 1H
NMR (400 MHz, CDCl3) d: 9.38 (br s, 1H, CONH); 8.20 (dd, 1H,
J = 8.8 Hz, ArH); 7.87 (s, 1H, ArH); 7.60 (br s, 1H, NHCO); 7.05 (d,
2H, J = 8.7 Hz, ArH); 6.98 (d, 2H, J = 8.7 Hz, ArH); 6.83 (d, 1H,
J = 8.8 Hz, ArH); 3.45 (qd, 2H, J = 6.9 Hz, J = 6.2 Hz, CH2NH); 3.05
(s, 2H, COCH2); 2.52 (br s, 4H, Pip); 1.67 (m, 6H, Pip); 1.53 (m,
2H, CH2CH2NH); 1.34 (m, 2H, CH2CH3); 0.90 (t, 3H, J = 6.9 Hz,
CH2CH3).
ESI-MS: 424.4 [M+H]+; 446.3 [M+Na]+; 462.2 [M+K]+.
Anal. (C25H33N3O3): C, H, N.
4.1.5.26. 5-(2-(Piperidin-1-yl)acetamido)-2-(4-isopropylphen-
ESI-MS: 428.4 [M+H]+; 450.5 [M+Na]+; 466.4 [M+K]+.
Anal. (C24H30FN3O3): C, H, N.
oxy)-N-butylbenzamide (7o).
Yield: 92%; mp 75–77 °C; 1H
NMR (400 MHz, CDCl3) d: 9.37 (br s, 1H, CONH); 8.20 (dd, 1H,
J = 8.8 Hz, ArH); 7.87 (s, 1H, J = 7.9 Hz, ArH); 7.75 (br s, 1H, NHCO);
7.22 (d, 2H, J = 8.4 Hz, ArH); 6.94 (d, 2H, J = 8.4 Hz, ArH); 6.86 (d,
1H, J = 8.8 Hz, ArH); 3.44 (qd, 2H, J = 6.9 Hz, J = 6.2 Hz, CH2NH);
3.05 (s, 2H, COCH2); 2.94 (hep, 1H, J = 6.9 Hz, CH-Isopropyl); 2.53
(br s, 4H, Pip); 1.66 (m, 6H, Pip); 1.52 (m, 2H, , CH2CH2NH); 1.33
(m, 2H, CH2CH3); 1.27 (d, 6H, J = 6.9 Hz, 2CH3-Isopropyl); 0.89 (t,
3H, J = 6.9 Hz, CH2CH3).
4.1.5.21.
5-(2-(Piperidin-1-yl)acetamido)-2-(4-(trifluoro-
Yield: 34%; mp
methyl)phenoxy)-N-butylbenzamide (7h).
62–64 °C; 1H NMR (400 MHz, CDCl3) d: 9.45 (br s, 1H, CONH);
8.24 (dd, 1H, J = 8.8 Hz, ArH); 7.87 (s, 1H, ArH); 7.61 (d, 2H,
J = 8.0 Hz, ArH); 7.39 (br s, 1H, NHCO); 7.04 (d, 2H, J = 8.0 Hz,
ArH); 6.96 (d, 1H, J = 8.8 Hz, ArH); 3.40 (qd, 2H, J = 6.9 Hz,
J = 6.2 Hz, CH2NH); 3.07 (s, 2H, COCH2); 2.54 (br s, 4H, Pip); 1.67
(m, 6H, Pip); 1.48 (m, 2H, CH2CH2NH); 1.27 (m, 2H, CH2CH3);
0.86 (t, 3H, J = 6.9 Hz, CH2CH3).
ESI-MS: 452.7 [M+H]+.
Anal. (C27H37N3O3): C, H, N.
ESI-MS: 478.5 [M+H]+; 500.6 [M+Na]+.
4.2. Pharmacology
Anal. (C25H30F3N3O3): C, H, N.
4.2.1. Production of recombinant mouse PrP 23–231
4.1.5.22.
5-(2-(Piperidin-1-yl)acetamido)-2-(2-(trifluoro-
Yield: 54%; mp
Recombinant PrP 23–231 was produced and purified essentially
as previously reported,19 except that for bacterial expression,
BL21(DE3) RIL Escherichia coli cells (Novagen) were transformed
with plasmid pET17b-MmPrP23–231WT31 for mouse PrP23–231.
methyl)phenoxy)-N-butylbenzamide (7i).
86–88 °C; 1H NMR (400 MHz, CDCl3) d: 9.44 (br s, 1H, CONH);
8.27 (dd, 1H, J = 8.8 Hz, ArH); 7.87 (s, 1H, ArH); 7.72 (d, 1H,
J = 7.6 Hz, ArH), 7,49 (t, 1H, J = 8.0 Hz, ArH), 7.41 (br s,1H, NHCO);
7.24 (t, 1H, J = 7.6 Hz, ArH); 6.92 (d, 1H, J = 9.1 Hz, ArH); 6.84 (d,
1H, J = 8.8 Hz, ArH); 3.41 (qd, 2H, J = 6.9 Hz, J = 6.2 Hz, CH2NH);
3.07 (s, 2H, COCH2); 2.54 (br s, 4H, Pip); 1.68 (m, 6H, Pip); 1.44
(m, 2H, CH2CH2NH); 1.27 (m, 2H, CH2CH3); 0.86 (t, 3H, J = 6.9 Hz,
CH2CH3).
4.2.2. Preparation of PrPSc
PrPSc was prepared from brain of CJD patients as described by
Bertsch et al.19 Aliquots of the final pellet resuspended in 1ꢁ phos-
phate-buffered saline (PBS) plus 0.1% sarcosyl solution were di-
luted fivefold into 20 mM potassium phosphate buffer at pH 6.0,
0.1% Nonidet P-40 and sonicated in a water bath sonicator for
60 s. After centrifugation at 1.000 rpm for 1 min, the supernatant
was diluted 100-fold in potassium phosphate buffer, pH 6, 0.1%
Nonidet P40 for the assay.
ESI-MS: 478.5 [M+H]+; 500.5 [M+Na]+; 516.6 [M+K]+.
Anal. (C25H30F3N3O3): C, H, N.
4.1.5.23.
oxy)-N-butylbenzamide (7l).
5-(2-(Piperidin-1-yl)acetamido)-2-(4-methoxyphen-
Yield 97%; mp 48–50 °C; 1H
NMR (400 MHz, CDCl3) d: 9.35 (br s, 1H, CONH); 8.15 (dd, 1H,
J = 8.8 Hz, ArH); 7.87 (s, 1H, ArH); 7.81 (br s, 1H, NHCO); 6.97 (d,
2H, J = 8.8 Hz, ArH); 6.90 (d, 2H, J = 8.8 Hz, ArH); 6.78 (d, 1H,
J = 8.8 Hz, ArH); 3.80 (s, 3H, OCH3); 3.46 (qd, 2H, J = 6.9 Hz,
J = 6.2 Hz, CH2NH); 3.04 (s, 2H, COCH2); 2.52 (br s, 4H, Pip);
1.70–1.63 (m, 6H, Pip); 1.55 (qt, 2H, J = 6.9 Hz, CH2CH2NH); 1.36
(m, 2H, CH2CH3); 0.90 (t, 3H, J = 6.9 Hz, CH2CH3).
4.2.3. Fluorescent labelling of antibodies and recombinant PrP
L42 monoclonal antibody (r-biopharm, Darmstadt, Germany)
was labelled with Alexa Fluor 647 (Alexa-647; Invitrogen, Eugene)
´
according to the manufacturers manual. Mouse rPrP was labelled
with the Alexa Fluor 488 (Alexa-488; Invitrogen, Eugene) in
20 mM potassium phosphate buffer, pH 6, 0.1% Nonidet P40,
40 mM sodium bicarbonate buffer, pH 8.3. Unbound fluorophores
were separated by gelfiltration on PD10 columns (GE Healthcare,
Freiburg, Germany) equilibrated with 20 mM potassium phosphate
buffer, pH 6, 0.1% Nonidet P40. Quality control of labeling reaction
and ratio was performed by fluorescence correlation spectroscopy
(FCS) measurements on an Insight Reader (Evotec Technologies,
Hamburg, Germany). The labeling ratio was approximately 1.3
fluorophores per rPrP molecule.
ESI-MS: 440.4 [M+H]+; 462.4 [M+Na]+.
Anal. (C25H33N3O4): C, H, N.
4.1.5.24. 5-(2-(Piperidin-1-yl)acetamido)-2-(4-butoxyphenoxy)-
N-butylbenzamide (7m).
Yield: 86%; mp 52–54 °C; 1H NMR
(400 MHz, CDCl3) d: 9.35 (br s, 1H, CONH); 8.14 (dd, 1H,
J = 8.8 Hz, ArH); 7.87 (s, 1H, ArH); 7.83 (br s, 1H, NHCO); 6.96 (d,
2H, J = 8.4 Hz, ArH); 6.90 (d, 2H, J = 8.4 Hz, ArH); 6.79 (d, 1H,

F. Fiorino et al. / Bioorg. Med. Chem. 20 (2012) 5001–5011
5011
4.2.4. Assay for PrPC-PrPSc association
antibody rabbit 1023 in blocking solution was incubated with the
membrane for 60 min. After PBS-T rinsing, the membrane was
For the assay, the synthetized compounds 6a–o and 7a–o
(approximately 10 mM in DMSO) were first diluted 10-fold into
DMSO. This dilution was again diluted 10-fold into potassium
phosphate buffer, pH 6, 0.1% Nonidet P40. A mixture of labelled
mouse rPrP and labeled L42 monoclonal antibody was prepared
in 20 mM potassium phosphate buffer, pH 6, 0.1% Nonidet P40 so
that the labeled molecules were approximately equally abundant
incubated in
a 0.2 lg/ml solution of Horseradish Peroxidase
(HRP) conjugated with anti-rabbit IgG antibody (Promega) in
PBST-milk for 1 h at room temperature. After additional PBS-T rins-
ing the bound antibodies were detected using a chemilumines-
cence reagent system (ECL, Amersham) and were visualized
directly in an image analysis system (Versa Doc, Quantity One,
Bio-Rad, Munich, Germany). Inhibition was calculated as relative
signal volume compared to the untreated control (100%).
at 2–6 nM. In a 20
l
L assay volume 8
lL of the rPrP/antibody mix-
ture, 2 L compound and 10
l
l
L of the diluted PrPSc preparation
were mixed. The samples were loaded onto 96-well plates with
cover-glass bottom (Evotec-Technologies, Hamburg, Germany)
and measured on an Insight Reader.
Acknowledgments
The NMR spectral data were provided by Centro di Ricerca
Interdipartimentale di Analisi Strumentale, Università degli Studi
di Napoli ‘Federico II’. The assistance of the staff is gratefully
appreciated.
4.2.5. Single-particle measurement and analysis
FIDA (fluorescence intensity distribution analysis) measure-
ments were performed at excitation energies of 200
488 nm laser and 300 W for the 633 nm laser. Scanning parame-
ters were set to 100 m scan path length, 50 Hz beamscanner fre-
quency, and 2000 positioning table movement. The
lW for the
l
l
Supplementary data
lm
measurement time was 10 s. Fluorescence from the two fluoro-
phores was recorded separately with single photon detectors and
photons were summed over time intervals of constant length
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2012.06.026.
(bins) using a bin length of 40 ls. The number of red and green
References and notes
fluorescent photon counts was measured and analysed in a two-
dimensional intensity distribution histogram, as previously de-
scribed .22
The fluorescence intensity data was evaluated using a 2D-SIFT
software module (Evotec-Technologies, Hamburg, Germany). Cut-
off values for bin intensities for each measurement series were ad-
justed manually according to the control measurements.
1. Prusiner, S. B.; Prions Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 13363.
2. Miller, G. Science 2009, 326, 1337.
3. Brazier, M. W.; Wall, V. A.; Brazier, B. W.; Masters, C. L.; Collins, S. J. Expert Rev.
Anti Infect. Ther. 2009, 7, 83.
4. Giese, A.; Kretzschmar, H. A. Curr. Top. Microbiol. Immunol. 2001, 253, 203.
5. Sim, V. L.; Caughey, B. Infect. Disord. Drug Targets 2009, 9, 81.
6. Korth, C.; May, B. C.; Cohen, F. E.; Prusiner, S. B. Proc. Natl. Acad. Sci. U.S.A. 2001,
98, 9836.
7. May, B. C.; Witkop, J.; Sherrill, J.; Anderson, M. O.; Madrid, P. B.; Zorn, J. A.;
Prusiner, S. B.; Cohen, F. E.; Guy, R. K. Bioorg. Med. Chem. Lett. 2006, 16, 4913.
8. May, B. C.; Fafarman, A. T.; Hong, S. B.; Rogers, M.; Deady, L. W.; Prusiner, S. B.;
Cohen, F. E. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 3416.
9. Dollinger, S.; Lober, S.; Klingenstein, R.; Korth, C.; Gmeiner, P. A. J. Med. Chem.
2006, 49, 6591.
10. Kempster, S.; Bate, C.; Williams, A. NeuroReport 2007, 18, 479.
11. Heal, W.; Thompson, M. J.; Mutter, R.; Cope, H.; Louth, J. C.; Chen, B. J. Med.
Chem. 2007, 50, 1347.
12. Kimata, A.; Nakagawa, H.; Ohyama, R.; Fukuuchi, T.; Ohta, S.; Suzuki, T.;
Miyata, N. J. Med. Chem. 2007, 50, 5053.
13. Kimata, A.; Nakagawa, H.; Ohyama, R.; Fukuuchi, T.; Ohta, S.; Doh-ura, K.;
Suzuki, T.; Miyata, N. J. Med. Chem. 2008, 51, 1503.
4.2.6. Inhibition of PrPSc accumulation in a cell based dot blot
model
Mouse neuroblastoma (ScN2a) and scrapie mouse brain (SMB)
cells, infected with the Rocky Mountain Laboratory (RML) scrapie
strain, were seeded in a concentration of 20.000 cells per well in
100 ll of medium at a Costar 3599 flat-bottom 96-well plate
(Corning Inc., Corning, N.Y.) prior to compound addition. Test com-
pounds were added to the cell medium in a final dilution of
20.0 lM and 2.0 lM, respectively. After an incubation period of
14. Thompson, M. J.; Borsenberger, V.; Louth, J. C.; Judd, K. E.; Chen, B. J. Med. Chem.
2009, 52, 7503.
15. Kawasaki, Y.; Kawagoe, K.; Chen, C. J.; Teruya, K.; Sakasegawa, Y.; Doh-ura, K. J.
Virol. 2007, 81, 12889.
three days at 37 °C (ScN2a cells) or at 35 °C (SMB cells) in a CO2
incubator the cultures were lysed and analyzed for PrPSc/PrPres
(protease resistant prion protein) formation. After cell medium re-
16. Kuwata, K.; Nishida, N.; Matsumoto, T.; Kamatari, Y. O.; Hosokawa-Muto, J.;
Kodama, K.; Nakamura, H. K.; Kimura, K.; Kawasaki, M.; Takakura, Y.; Shirabe,
S.; Takata, J.; Kataoka, Y.; Katamine, S. Proc. Natl. Acad. Sci. U.S.A. 2007, 104,
11921.
17. Yudovin-Farber, I.; Azzam, T.; Metzer, E.; Taraboulos, A.; Domb, A. J. J. Med.
Chem. 2005, 48, 1414.
18. Gallardo-Godoy, A.; Gever, J.; Fife, K. L.; Silber, B. M.; Prusiner, S. B.; Renslo, A.
R. J. Med. Chem. 2011, 54, 1010.
19. Caliendo, G.; Santagada, V.; Perissutti, E.; Severino, B.; Fiorino, F.; Warner, T. D.;
Wallace, J. L.; Ifad, D. R.; Antunes, E.; Cirino, G.; de Nucci, G. Eur. J. Med. Chem.
2001, 36, 517.
moval, 100 ll of lysis puffer (50 mM Tris HCl, pH 8.0, 150 mM
NaCl; 0.5% (w/v) DOC; 0,5% (v/v) Triton X-100) was added to each
well for 5 min at room temperature. Using a dot blot apparatus
(Sigma–Aldrich) cell lysate was transferred under vacuum to an
activated polyvinylidene difluoride (PVDF) membrane (Immobi-
lon-P; Milipore) and fixed to the membrane by incubation for 1 h
at 37 °C. The membrane was incubated in lysis puffer and treated
with Proteinase K solution (final dilution 25 lg/ml) for 90 min at
37 °C. Subsequently, the membrane was washed twice with pure
water, the denaturation solution (3 M guanidinium thiocyanate,
0.1 M Tris HCl pH 8) was added for 10 min at room temperature
and membranes were washed five times with pure water. After
the denaturation step the membrane was blocked with 5% (w/v)
non-fat milk-0.1% (v/v) tween-20 (Sigma) in phosphate buffered
saline (PBS-T) for 60 min. An appropriate dilution of polyclonal
20. Bertsch, U.; Winklhofer, K. F.; Hirschberger, T.; Bieschke, J.; Weber, P.; Hartl, F.
U.; Tavan, P.; Tatzelt, J.; Kretzschmar, H. A.; Giese, A. J. Virol. 2005, 79, 7785.
21. Kimura, T.; Hosokawa-Muto, J.; Kamatari, Y. O.; Kuwata, K. Bioorg. Med. Chem.
Lett. 2011, 21, 1502.
22. Bieschke, J.; Giese, A.; Schaeffer, W. S.; Zerr, I.; Poser, S.; Eigen, M.; Kretzschmar,
H. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 5468.
23. Groschup, M. H.; Harmeyer, S.; Pfaff, E. J. Immunol. Method 1997, 207, 89.