Synthesis of 9-substituted 2,3,4,9-tetrahydro-1H-carbazole derivatives and evaluation of their anti-prion activity in TSE-infected cells

Article abstract of DOI:10.1016/j.ejmech.2011.08.039

2,3,4,9-Tetrahydro-9-[2-hydroxy-3-(1-piperidinyl)propyl] -6-methyl-1H-carbazol-1-one (GJP14) is a novel anti-prion compound that we previously discovered by in silico screening and cellular assay. In this study, a variety of GJP14 derivatives were prepared using pyrrole derivatives, (haloalkyl)oxiranes, and amines, and their anti-prion activity was evaluated in TSE-infected cells. It was found that the tricyclic aromatic ring, a hydroxy group at the 2-position and an amino group at the 3-position of the N-propyl group were the basic requirements for anti-prion activity. The derivatives bearing an N-ortho-halobenzyl group exhibited an improved activity, and the most potent derivative was 8 times as effective as the original lead compound, GJP14.

Full text of DOI:10.1016/j.ejmech.2011.08.039

European Journal of Medicinal Chemistry 46 (2011) 5675e5679
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Short communication
Synthesis of 9-substituted 2,3,4,9-tetrahydro-1H-carbazole derivatives
and evaluation of their anti-prion activity in TSE-infected cells
,
1
,
,c,
*
Tsutomu Kimuraa , Junji Hosokawa-Mutoa 2, Kenji Asamib, Toshiaki Muraib, Kazuo Kuwataa
a Center for Emerging Infectious Diseases, Gifu University, 1-1 Yanagido, Gifu 501-1194, Japan
b Department of Chemistry, Faculty of Engineering, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan
c United Graduate School of Drug Discovery and Medical Information Sciences, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
2,3,4,9-Tetrahydro-9-[2-hydroxy-3-(1-piperidinyl)propyl]-6-methyl-1H-carbazol-1-one (GJP14) is a novel
anti-prion compound that we previously discovered by in silico screening and cellular assay. In this study,
a variety of GJP14 derivatives were prepared using pyrrole derivatives, (haloalkyl)oxiranes, and amines, and
their anti-prion activity was evaluated in TSE-infected cells. It was found that the tricyclic aromatic ring,
a hydroxy group at the 2-position and an amino group at the 3-position of the N-propyl group were the basic
requirements for anti-prion activity. The derivatives bearing an N-ortho-halobenzyl group exhibited an
improved activity, and the most potent derivative was 8 times as effective as the original lead compound,
GJP14.
Received 10 June 2011
Received in revised form
24 August 2011
Accepted 26 August 2011
Available online 31 August 2011
Keywords:
Prion diseases
Transmissible spongiform encephalopathies
Anti-prion compound
Ó 2011 Elsevier Masson SAS. All rights reserved.
2,3,4,9-Tetrahydro-1H-carbazole
Ring-opening reaction
Structureeactivity relationships
1. Introduction
nervous system. The epidemic of BSE and the appearance of a new
variant CJD provoked great public concern and necessitated the
Prion diseases, also known as transmissible spongiform
encephalopathies (TSEs), are a family of invariably fatal neurode-
generative disorders that affect humans and animals [1e3].
CreutzfeldteJakob disease (CJD), GerstmanneSträusslereScheinker
syndrome (GSS), fatal familial insomnia, and kuru in humans,
scrapie in sheep and goat, bovine spongiform encephalopathy (BSE)
in cattle, and chronic wasting disease in deer and elk are well-
known representatives of prion diseases. These disorders are
characterized by the spongiform degeneration of the central
development of suitable treatments for prion diseases because no
effective curative therapy for the diseases exists thus far [4,5].
The causative agent of prion diseases is thought to be a patho-
genic isoform of cellular prion protein (PrPC), designated PrPSc
.
PrPSc is formed via the conformational conversion of PrPC and acts
as a seed that provides a template for converting PrPC to PrPSc
.
Therefore, PrPSc is one of the potential drug targets. However, PrPSc
forms heterogeneous aggregates, and the diverse prion strains
make rational drug design difficult [6]. The structurally well-
defined PrPC is an alternative target molecule that consists of
a flexible N-terminal region containing an octapeptide repeat
domain and
a
globular C-terminal region comprising three
-sheet. A chemical chaperone that
Abbreviations: TSEs, transmissible spongiform encephalopathies; CJD,
CreutzfeldteJakob disease; GSS, GerstmanneSträusslereScheinker syndrome; PrPC,
a-helices and one anti-parallel
b
cellular form of prion protein; PrPSc, infectious isoform of prion protein; PrPres
proteinase K-resistant prion protein.
,
binds to slowly fluctuating regions (hot spots) of PrPC and stabilizes
its conformation might be effective in inhibiting the accumulation
of PrPSc [7e9]. A virtual screening that was focused on the hot spots
of PrPC was performed in the ZINC database containing approxi-
mately 600 000 commercially available compounds using Auto-
Dock software, and we found a novel anti-prion compound, 2,3,4,9-
tetrahydro-9-[2-hydroxy-3-(1-piperidinyl)propyl]-6-methyl-1H-
carbazol-1-one (GJP14, Fig. 1) [10]. Surface plasmon resonance
* Corresponding author. Center for Emerging Infectious Diseases, Gifu University,
1-1 Yanagido, Gifu 501-1194, Japan. Tel.: þ81 58 230 6143; fax: þ81 58 230 6144.
E-mail address: kuwata@gifu-u.ac.jp (K. Kuwata).
1
Present address: Department of Chemistry, Faculty of Science Division II, Tokyo
University of Science, 12 Ichigaya-funagawara-machi, Shinjuku-ku, Tokyo 162-0826,
Japan.
2
Present address: First Department of Forensic Science, National Research
Institute of Police Science, 6-3-1 Kashiwanoha, Kashiwa, Chiba 277-0882, Japan.
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.08.039

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T. Kimura et al. / European Journal of Medicinal Chemistry 46 (2011) 5675e5679
3. Anti-prion activity assay
The anti-prion activities of compound 1 and its derivatives 2e5
were assayed in GT þ FK cells that are mouse neuronal cells (GT1-7)
persistently infected with mouse-adapted GSS agent (Fukuoka-1
strain) [13,14]. The ability of the compounds to inhibit the accu-
mulation of PrPSc was assessed by quantification of proteinase K-
resistant prion protein (PrPres) following treatment of the cells with
the compounds. The percentage of PrPres relative to the negative
control at a concentration of 10 mM is presented in Fig. 2. The IC50
Fig. 1. Chemical structure of 1 (GJP14).
value is the concentration of the compound that caused 50% inhi-
bition of PrPres accumulation. The original lead compound 1
inhibited the accumulation of PrPres with an IC50 value of
binding assay of GJP14 for PrPC revealed that it had a capacity for
binding to PrPC. Herein, we report the synthesis of GJP14 deriva-
tives and the evaluation of their anti-prion activity in TSE-infected
cells.
8.54 ꢀ 0.28
mM.
4. Results and discussion
Initially, the anti-prion activities of a series of derivatives 2 were
tested in GT þ FK cells (Scheme 1 and Fig. 2). When the 6-methyl-
2,3,4,9-tetrahydro-1H-carbazol-1-one scaffold of compound 1 was
replaced with pyrrole (2a), indole (2b), and 4,5,6,7-tetrahydro-1H-
indole (2c), anti-prion activity was lost. However, PrPres accumu-
lation was inhibited by the use of compounds derived from 2,3,4,9-
tetrahydro-1H-carbazole (2d) and carbazole (2e) to a similar extent
as compound 1. These results indicate that the presence of an
aromatic tricyclic component is crucial for anti-prion activity, while
a methyl group at the 6-position and carbonyl group at the
1-position in 1 are less important.
Second, the anti-prion activities of fragments 3aec and analogs
3d and 3e were examined (Scheme 2 and Fig. 2). 2,3,4,9-
Tetrahydro-1H-carbazole (3a) had no anti-prion activity. The
truncation of functional groups from the N-[2-hydroxy-3-(1-
piperidinyl)propyl] group of 2d led to a significant decrease in
activity. The above results suggest that both the amino group and
the hydroxy group are the essential structural features responsible
for anti-prion activity. The hydroxy group appeared to act as
a hydrogen bond donor because the replacement of the hydroxy
group of 2d with a methoxy group resulted in a loss of activity.
2. Chemistry
Lead compound 1 (GJP14) is composed of a 6-methyl-2,3,4,9-
tetrahydro-1H-carbazol-1-one scaffold, a piperidine unit, and a 2-
hydroxypropylene tether (Fig. 1). To identify the structural
features responsible for anti-prion activity and to improve the
activity of lead compound 1, a series of GJP14 derivatives 2e5 were
prepared, and their anti-prion activity was tested in GSS-infected
mouse neuronal cells. Our strategy for drug design mainly relies
on trial-and-error testing of a series of derivatives on TSE-infected
cells. Physicochemical properties of compounds such as molecular
weight and ClogP were also taken into account.
GJP14 derivatives 2, in which the 6-methyl-2,3,4,9-tetrahydro-
1H-carbazol-1-one scaffold was replaced with other units, were
prepared from a series of pyrrole derivatives, epichlorohydrin, and
piperidine (Scheme 1) [11,12]. The pyrrole derivatives were treated
with sodium hydride, and the resulting sodium amides were
reacted with epichlorohydrin to produce N-(2,3-epoxypropyl)are-
nes. Subsequently,
epoxypropyl)arenes with piperidine afforded the derivatives 2 in
a total yield of 42e71% for the entire process.
a ring-opening reaction of the N-(2,3-
GJP14 fragments 3b and 3c were prepared by alkylation of
2,3,4,9-tetrahydro-1H-carbazole 3a with 1-bromopropane and 2-
bromoethanol, respectively (Scheme 2). The alkyl chain-elongated
derivative 3d was synthesized from 3a, 1-bromo-3,4-
epoxybutane, and piperidine in a similar manner as employed for
the synthesis of 2. Alcohol 2d was converted to the corresponding
methyl ether 3e by methylation of sodium alkoxide with methyl
iodide. Finally, derivatives 4 and 5, in which the terminal 1-
piperidinyl group of 2d was replaced with other amino groups,
were prepared by reacting 1-(2,3-epoxypropyl)-2,3,4,9-tetrahydro-
1H-carbazole 2d0 with a variety of primary and secondary amines in
27e87% yield (Scheme 3).
Scheme 2. Synthesis of GJP14 derivatives 3. Reagents and conditions: (a) (i) NaH, DMF;
(ii) PrBr, DMF; (b) HOCH2CH2Br, KOH, DMSO; (c) (i) NaH, DMF; (ii) 1-bromo-3,4-
epoxybutane, DMF; (iii) piperidine, EtOH; (d) (i) NaH, THF; (ii) MeI, THF.
Scheme 1. Synthesis of 1 (GJP14) and its derivatives 2. Reagents and conditions: (a) (i)
NaH, DMF; (ii) epichlorohydrin, DMF; (b) piperidine, EtOH.

T. Kimura et al. / European Journal of Medicinal Chemistry 46 (2011) 5675e5679
5677
indicate that the hydrogen atom of the terminal amino group acts
as a hydrogen bond donor in the case of compounds derived from
primary amines and that the modest hydrophobic property of the
substituent on the nitrogen atom is required for activity. Of the
derivatives 4, the N-benzyl derivative 4d had the best terminal
amino group.
Finally, the benzyl group of compound 4d was refined
comprehensively (Scheme 3 and Fig. 2). The anti-prion activities of
derivatives bearing an electron-donating group such as a methyl
group (5a and 5b) or a methoxy group (5d) were comparable to that
of 4d. Intriguingly, derivatives bearing a fluoro, chloro, or bromo
group at the ortho-position of the phenyl group (5g, 5j, and 5m) had
improved activity, with relative PrPres levels of 10e12%, while the
introduction of such groups at the meta- or para-positions (5h, 5i,
5k, and 5n) did not lead to an increase in activity. The derivatives
bearing a (trifluoromethyl)phenyl group (5p and 5r), a pyridyl
group (5seu), or a 1-naphthyl group (5v) were less active than 4d.
The introduction of a second halo group to the aromatic ring
(5wez) led to retained activity or a slight loss in activity. The
derivatives 5e and 5q, and the para-substituted derivatives 5c, 5f,
5l, and 5o were cytotoxic at 10 mM.
Scheme 3. Synthesis of GJP14 derivatives 4 and 5. Reagents and conditions: (a) (i) NaH,
DMF; (ii) epichlorohydrin, DMF; (b) RR’NH, MeOH or EtOH.
The increase in activity observed in the case of ortho-halobenzyl
derivatives such as 5g, 5j, and 5m has been attributed to the
additional halogeneoxygen interaction between the ortho-halo
group and the biomolecule rather than the electronic effect of the
ortho-halo group on the aromatic ring [15]. In fact, the introduction
of a halo group at the meta- and para-positions did not necessarily
improve the activity of the compounds tested here. This is addi-
tionally supported by our results showing that derivatives with
a more electron-deficient aromatic ring (5wez) did not show
a further increase in activity.
Elongation of the alkyl chain led to a slight decrease in activity,
indicating that the propylene linker is adequate to maintain the
proper distance between the tricyclic ring and the functional
groups.
Third, the derivatives 4, in which the terminal 1-piperidinyl
group of 2d was replaced with various N-substituted and N,N-di-
substituted amino groups, were investigated (Scheme 3 and Fig. 2).
Compounds derived from primary amines (4a, 4b, and 4d) with the
exception of 4c were found to be more potent than those derived
from secondary amines (2d and 4fej). The morpholine derivative 4i
was less effective than the piperidine derivative 2d. These results
The GJP14 derivatives bearing a hydroxy group possess a chiral
center that gives rise to two enantiomers. To compare the anti-
prion activity of the R- and S-enantiomers, optically active R-5g
and S-5g were prepared from R- and S-epichlorohydrins, and their
Fig. 2. (a) Anti-prion activity of 1e4 in GT þ FK cells at 10
m
M. (b) Anti-prion activity of 1, 4d and 5 in GT þ FK cells at 10
M were as follows: 4e, 34 ꢀ 1%; 5c, 32 ꢀ 3%; 5e, 21 ꢀ 2%; 5f, 23 ꢀ 7%; 5l, 18 ꢀ 5%; 5o,
M were as follows: 4d, 28 ꢀ 3%; GN8, 34 ꢀ 3%.
m
M. Bars represent means ꢀ SD. The derivatives 4e, 5c, 5e,
5f, 5l, 5o, and 5q were cytotoxic at 10 m m
M. Relative PrPres levels for these compounds at 5
25 ꢀ 5%; 5q, 40 ꢀ 5%. Relative PrPres levels for 4d and GN8 at 5
m

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T. Kimura et al. / European Journal of Medicinal Chemistry 46 (2011) 5675e5679
Scheme 4. Synthesis of optically active GJP14 derivatives R-5g and S-5g. Reagents and conditions: (a) (i) NaH, DMF; (ii) (R)-epichlorohydrin, DMF; (b) 2-fluorobenzylamine, MeOH.
(c) (i) NaH, DMF; (ii) (S)-epichlorohydrin, DMF.
amines. The (S)-N-ortho-fluorobenzyl derivative S-5g was found to
be the most active compound among these derivatives, with an IC50
value of 1.11 mM. The derivative S-5g was 8-fold more effective than
the original lead compound 1. A tricyclic aromatic scaffold,
a hydroxy group, and a terminal amino group were determined to
be the basic requirements for anti-prion activity. Further investi-
gations in vitro and in vivo, as well as molecular modeling studies,
are currently ongoing and will be reported in due course.
6. Experimental protocols
6.1. Chemistry
Fig. 3. (a) Western blotting of PrPres in GT þ FK cells after treatment with GN8, 1, and
6.1.1. General methods
S-5g at 10
m
M. (b) Western blotting of PrPres in ScN2a-3-Ch cells after treatment with
All manipulations were performed under an argon atmosphere.
Argon gas was dried by passage through P2O5. Commercially
available reagents and solvents were purchased from Aldrich,
Tokyo Chemical Industry, Wako Pure Chemical Industries, and
Nacalai Tesque and were used without further purification. The
reactions were carried out in a temperature-controlled personal
organic synthesizer (PPS-1510, Eyela). The progress of the reactions
was monitored using thin-layer chromatography with a silica gel
60F254 TLC glass plate (Merck), with UV light as a visualizing agent.
Chromatographic purification of the compounds was performed by
medium-pressure liquid chromatography (MPLC) using a YFLC AI-
580 system (Yamazen) equipped with a silica gel high-flash
column or by gel permeation chromatography (GPC) using an LC-
9201 system (Japan Analytical Industry).
GN8, 1, S-5g, 3-(4-nitrophenyl)-1-phenyl-1H-pyrazol-5(4H)-one (Py), and chrysoidine
(Ch) at 5
m
M. Relative PrPres levels for these compounds were as follows: GN8, 6%; 1,
82%; S-5g, 39%; Py, 74%; Ch, 48%.
anti-prion activity was evaluated in GT þ FK cells (Scheme 4). Both
R-5g and S-5g have anti-prion activity, and S-5g (IC50
:
1.11 ꢀ 0.24
m
M) was more potent than R-5g (IC50: 2.23 ꢀ 0.20
mM),
with a eudysmic ratio of 2 (P < 0.01, Student’s t-test). Derivative
S-5g was 8 times more effective than the original lead compound 1,
and its anti-prion activity was comparable to that of GN8 (1.4 mM)
[16]. The expression level of PrPC remained intact in GT þ FK cells
treated with S-5g, as evidenced by a cellular assay without
proteinase K digestion.
In this study, we used GT þ FK cells as a cell culture model of
human prion disease. Unlike ScN2a cells, GT þ FK cells produce
significant amounts of PrPres as well as PrPC [14]. Hence, the assay
system using GT þ FK cells is reliable, reproducible and less
sensitive to minor perturbations such as those due to culture
conditions.
To date, a variety of compounds have been identified that inhibit
the accumulation of PrPres in TSE-infected cells [17,18]. However,
the strain-dependent activity of such compounds is a troublesome
matter in drug development for prion diseases. Indeed, anti-prion
compounds that were effective in scrapie-infected cells were not
necessarily effective in our assay system using GSS-infected cells
[19]. In contrast, the derivative S-5g exhibited anti-prion activity in
both GSS-infected cells and scrapie-infected cells (ScN2a-3-Ch
cells) [20], as shown in Fig. 3. It is noteworthy that the anti-prion
activity of S-5g in ScN2a-3-Ch cells was higher than those of an
edaravone derivative [21] and chrysoidine [22] that were reported
to be effective at low nanomolar concentrations in ScNB cells and
ScN2a cells.
6.1.2. Spectroscopic methods
NMR spectra were recorded on an Inova 500 spectrometer
(Varian) and an Avance 500 (Bruker) operating at 500 MHz for 1H
and 125 MHz for 13C acquisitions. The chemical shifts of protons
were reported in
d
values referred to Me4Si as an internal standard.
values referred to
The chemical shifts of 13C were reported in
d
CDCl3 (77.05 ppm) as an internal standard. IR spectra were obtained
on a Nicolet iS10 FT-IR (Thermo Scientific) in ATR mode. Mass
spectra (EI) and high-resolution mass spectra (EI) were obtained on
a JEOL JMS-700/GI spectrometer.
6.1.3. Analytical methods
Combustion analysis was performed at the laboratory for
organic elemental microanalysis of Kyoto University. Melting points
were measured on a Yanaco micro melting point apparatus (MP-J3)
and were left uncorrected.
6.1.4. General procedure for the synthesis of GJP14 and its derivatives
1e5. A representative procedure for the synthesis of 1-(1-piperidinyl)-
3-(1,2,3,4-tetrahydro-9H-carbazol-9-yl)-2-propanol (2d)
5. Conclusion
A solution of 2,3,4,9-tetrahydro-1H-carbazole (3a, 171 mg,
1.00 mmol) in DMF (1 mL) was added to a suspension of sodium
hydride (26 mg, 1.1 mmol) in DMF (4 mL) at 0 ꢁC, and the mixture
A total of 47 derivatives were prepared by the epoxyalkylation of
pyrrole derivatives and subsequent ring-opening reaction with

T. Kimura et al. / European Journal of Medicinal Chemistry 46 (2011) 5675e5679
5679
was stirred at 25 ꢁC for 1 h. Epichlorohydrin (102 mg, 1.10 mmol)
was added to the resulting solution at 0 ꢁC, and the mixture was
stirred at 25 ꢁC for 12 h. The reaction mixture was poured into
water and extracted with CH2Cl2. The organic layer was dried over
Na2SO4, filtered, and concentrated under reduced pressure. The
residue was purified by MPLC on a silica gel using hexane/CHCl3 as
the eluent and GPC using CHCl3 as the eluent to produce 1-(2,3-
epoxypropyl)-2,3,4,9-tetrahydro-1H-carbazole (2d0, 185 mg,
0.814 mmol, 81% yield) as a pale yellow oil. 1H NMR (CDCl3):
was used to detect PrPres. The signals were visualized using
a SuperSignal reagent (Thermo Fisher Scientific, Rockford, IL) and
scanned using a LAS-1000 UV mini analyzer (Fujifilm, Tokyo,
Japan). The total density of the PrPres bands in each sample was
measured and compared to that of the control treated with
medium containing solvent alone using Multi Gauge software
(Fujifilm, Tokyo, Japan). IC50 values were determined by densi-
tometry of four or five concentration points measured from the
immunoreactive bands observed by Western blot. Two or three
d
1.84e1.96 (m, 4H), 2.46 (dd, J ¼ 2.6, 4.9 Hz, 1H), 2.71e2.76 (m, 5H),
independent experiments were performed to determine the IC50.
3.19e3.22 (m, 1H), 4.14 (dd, J ¼ 4.8, 15.7 Hz, 1H), 4.30 (dd, J ¼ 3.6,
15.7 Hz, 1H), 7.08 (dt, J ¼ 0.9, 7.6 Hz, 1H), 7.08 (dt, J ¼ 1.1, 7.6 Hz, 1H),
7.29 (d, J ¼ 7.6 Hz, 1H), 7.47 (d, J ¼ 7.7 Hz, 1H). 13C NMR (CDCl3):
The anti-prion activity assay in ScN2a-3-Ch cells was performed
in a manner similar to that in GT þ FK cells. Approximately
1 ꢃ105 cells were plated in each well of a six-well plate in the assay
using ScN2a-3-Ch cells.
d
21.0, 22.2, 23.1, 23.3, 44.2, 45.5, 51.0, 108.6, 110.0, 117.9, 119.0,
120.9, 127.5, 135.6, 136.6. IR (ATR): 1256 cmꢂ1. MS (EI): m/z 227
(Mþ). HRMS calcd for C15H17NO: 227.1310. Found: 227.1323. Piper-
idine (104 mg, 1.22 mmol) was added to a solution of 2d0 (185 mg,
0.814 mmol) in EtOH (5 mL) at 25 ꢁC, and the mixture was stirred
under reflux for 12 h. After the solvent was evaporated, the residue
was purified by MPLC on a silica gel using CHCl3/MeOH as the
eluent to give 2d (221 mg, 0.707 mmol, 87% yield) as a colorless
Acknowledgments
This work was supported by the Program for the Promotion of
Fundamental Studies in Health Sciences of the National Institute of
Biomedical Innovation, Grants-in-Aid from the Research
Committee of Prion disease and Slow Virus Infection, the Ministry
of Health, Labour and Welfare of Japan, and the Molecular Imaging
Research Program of the Ministry of Education, Culture, Sports,
Science and Technology of Japan. We are grateful to Prof. Motohiro
Horiuchi (Hokkaido University, Sapporo, Japan) for providing
ScN2a-3-Ch cells. We also thank Ms. Tomomi Saeki and Ms. Miku
Yamada for outstanding research assistance.
solid. Mp: 65e67 ꢁC (dec). 1H NMR (CDCl3):
d 1.41e1.59 (m, 6H),
1.83e1.96 (m, 4H), 2.29e2.36 (m, 4H), 2.53 (br, 2H), 2.68e2.84 (m,
4H), 4.00e4.10 (m, 3H), 7.05 (dt, J ¼ 1.0, 7.7 Hz, 1H), 7.12 (dt, J ¼ 1.1,
7.7 Hz, 1H), 7.28 (d, J ¼ 7.7 Hz, 1H), 7.45 (d, J ¼ 7.7 Hz, 1H). 13C NMR
(CDCl3): d 21.1, 22.6, 23.2, 23.4, 24.2, 26.1, 47.1, 54.6, 62.5, 66.4,109.0,
109.6, 117.7, 118.7, 120.6, 127.5, 135.9, 136.7. IR (ATR): 3260 cmꢂ1. MS
(EI): m/z 312 (Mþ). HRMS calcd for C20H28N2O: 312.2202. Found:
312.2175.
Appendix. Supplementary data
6.2. Anti-prion activity assay
Supplementary data related to this article can be found online at
doi:10.1016/j.ejmech.2011.08.039.
The anti-prion activity assay was performed as described in our
preceding paper [10]. We used an immortalized neuronal mouse
cell line that was persistently infected with the human TSE agent
(Fukuoka-1 strain) [13]. This cell line was grown and maintained at
37 ꢁC under a 5% CO2 atmosphere in Dulbecco’s modified Eagle’s
medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal
bovine serum (Equitech-bio, Kerrville, TX), 50 U/mL penicillin G
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(150 mM NaCl, 0.5% Triton X-100, 0.5% sodium deoxycholate, and
50 mM TriseHCl [pH 7.5]) [14]. After 1 min of centrifugation at
11200ꢃg, the supernatant was collected, and its total protein
concentration was measured by the BCA protein assay (Thermo
Fisher Scientific, Rockford, IL) and adjusted with lysis buffer to
1 mg/mL. The samples were digested with 20 mg of proteinase K per
mL for 30 min at 37 ꢁC, and the digestion was stopped with 3 mM
Pefabloc SC (Roche Applied Science, Indianapolis, IN). The samples
were centrifuged at 21952ꢃg for 45 min at 4 ꢁC, and the pellets
were resuspended in sample buffer and then loaded onto a 15%
polyacrylamide gel just after boiling. Western blotting for PrPres
was performed as described previously [10]. As a primary antibody,
the PrP M-20 antibody (Santa Cruz Biotechnology, Santa Cruz, CA)