A chimeric ligand approach leading to potent antiprion active acridine derivatives: Design, synthesis, and biological investigations

Article abstract of DOI:10.1021/jm060773j

Human transmissible neurodegenerations including Creutzfeldt-Jakob disease are unique, since they are caused by prions, an infectious agent that replicates without nucleic acids but instead by inducing conversion of a host-resident normal prion protein to a misfolded conformational isoform. For pharmacotherapy of these unusual diseases, tricyclic heterocyclic compounds such as quinacrine have been considered, but with ambiguous success in vivo, so far. On the basis of the synergistic antiprion effects of quinacrine and iminodibenzyl derivatives, we introduce a novel class of potential pharmaceuticals representing structural chimeras of quinacrine and imipramine analogues. We describe the chemical synthesis and bioassays of a focused library of these compounds. The most potent target compound 2a revealed an EC₅₀ value of 20 nM determined with a cell model of prion disease, thus substantially improving the antiprion efficacy of quinacrine.

Full text of DOI:10.1021/jm060773j

J. Med. Chem. 2006, 49, 6591-6595
6591
A Chimeric Ligand Approach Leading to Potent Antiprion Active Acridine Derivatives:
Design, Synthesis, and Biological Investigations
Silke Dollinger,† Stefan Lo¨ber,† Ralf Klingenstein,‡ Carsten Korth,‡ and Peter Gmeiner*,†
Department of Medicinal Chemistry, Emil Fischer Center, Friedrich-Alexander UniVersity, Schuhstrasse 19, D-91052 Erlangen, Germany, and
Institute for Neuropathology, Medical School, Heinrich Heine UniVersity Duesseldorf, Moorenstrasse 5, D-40225 Duesseldorf, Germany
ReceiVed June 29, 2006
Human transmissible neurodegenerations including Creutzfeldt-Jakob disease are unique, since they are
caused by prions, an infectious agent that replicates without nucleic acids but instead by inducing conversion
of a host-resident normal prion protein to a misfolded conformational isoform. For pharmacotherapy of
these unusual diseases, tricyclic heterocyclic compounds such as quinacrine have been considered, but with
ambiguous success in vivo, so far. On the basis of the synergistic antiprion effects of quinacrine and
iminodibenzyl derivatives, we introduce a novel class of potential pharmaceuticals representing structural
chimeras of quinacrine and imipramine analogues. We describe the chemical synthesis and bioassays of a
focused library of these compounds. The most potent target compound 2a revealed an EC50 value of 20 nM
determined with a cell model of prion disease, thus substantially improving the antiprion efficacy of quinacrine.
Introduction
compounds sparked a tremendous effort to enter clinical trials
applying the approved drug quinacrine to patients with CJD.6
Since animal experiments with quinacrine gave only modest or
ambiguous results,7 quinacrine pharmacotherapy could be
improved by introducing a combination of antiprion drugs.8
Structural modifications employing quinacrine as a lead com-
pound revealed that bis-acridines augmented antiprion activity
around 10-fold in vitro,9 but poor solubility is likely to limit
their in vivo use. The synergistic antiprion effects of quinacrine-
and iminodibenzyl-derived antidepressants have prompted us
to synthesize heterodimers incorporating both recognition ele-
ments with a piperazine unit,10 which is known as a privileged
substructure in a great number of bioactive agents, as the linking
element.
Prion diseases are transmissible neurodegenerative diseases
of humans and animals.1 The prion agent replicates without a
nucleic acid-encoded genome by converting host-resident normal
prion protein (PrPC)a to a pathogenic and infectious conforma-
tional isoform PrPSc. Human prion diseases such as the most
prevalent Creutzfeldt-Jakob disease (CJD) are rare diseases that
cansuniquelysbe of sporadic, genetic, or infectious origin.
Animal prion diseases are almost exclusively infectious in origin
in the form of scrapie of sheep, bovine spongiform encephal-
opathy (BSE) or mad cow disease of cattle, or chronic wasting
disease of American mule deer or elk. The emergence of a new
strain of human prions causing variant CJD (vCJD) through
2
the consumption of BSE-contaminated material led to 160
On the basis of preliminary investigations on the hybrid 1
(Figure 1) displaying a 5-fold improved antiprion potency over
quinacrine,8 we herein describe a novel class of antiprion agents
of type 2 defined by covalently linked acridine and imino-
dibenzyl moieties and variations of the particular molecular
subunits. SAR studies led to the discovery of the iminodibenzyl
derivative 2a as the most active antiprion compound yet
described.
deaths, primarily in Great Britain, and to concerns of an epidemy
of vCJD which, so far, has neither happened nor been
completely excluded.3 The new vCJD strain has different
characteristics, such as the occurrence of infectivity in blood
and peripheral organs, which increases the risk of horizontal
transmission, for example, during blood transfusions.4 These
issues have led to a focus on pharmacotherapeutic options of
prion diseases both for symptomatic, curative, and prophylactic
purposes or for decontamination of blood and transplanted
organs.
Although screening for antiprion compounds in scrapie-
infected mouse neuroblastoma cells (ScN2a) as a cell culture
model of prion disease has led to the identification of many
antiprion compounds, most of them proved to be ineffective
when applied to experimentally prion-infected rodents owing
to the lack of blood-brain barrier (BBB) permeability or
toxicity.5 The identification of BBB-permeable heterocyclic
Chemistry. Following our strategy to investigate the structure-
activity relationships of quinacrine-imipramine chimeras, we
intended to realize modifications within the different pharma-
cophoric substructures. Thus, the iminodibenzyl, the acridine,
and the spacer unit should be an object of diversification, leading
to a focused set of test compounds.
The synthesis of target compounds was performed starting
from iminodibenzyl (3a), phenothiazine (3b), and diphenylamine
(3c) (Scheme 1). The conversions into the respective N-
chloropropyl or N-bromopropyl derivatives were accomplished
by direct alkylation with 1-bromo-3-chloropropane or via
palladium-catalyzed allylation11 followed by hydroboration and
subsequent Appel reaction. The latter was established as an
alternative due to the fact that alkylation with the 1,3-
biselectrophile resulted in a mixture of N-chloropropyl and
N-allyl substituted products, which is obviously due to â-elim-
ination. The halopropyl derivatives 4a-c12-15 were converted
into the respective cyanoalkylpiperazines 5a-i16-18 by nucleo-
* To whom correspondence should be addressed. Phone:
+49(9131)8529383. Fax: +49(9131)8522585. E-mail: gmeiner@
pharmazie.uni-erlangen.de.
† Friedrich-Alexander University.
‡ Heinrich Heine University Duesseldorf.
a Abbreviations: BBB, blood-brain barrier; CJD, Creutzfeldt-Jakob
disease; FCS, fetal calf serum; MEM, minimal essential medium; PrPC,
host-resident normal prion protein; PrPSc, infectious conformational isoform
of prion protein; RML, Rocky Mountain Laboratory; Tris, tris[(hydroxy-
methyl)amino]methane; vCJD, variant Creutzfeldt-Jakob disease.
10.1021/jm060773j CCC: $33.50 © 2006 American Chemical Society
Published on Web 09/29/2006

6592 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 22
Dollinger et al.
Scheme 1a
Figure 1. Development of structural chimeras based on quinacrine
and imipramine.
philic substitution. Subsequent reduction with LiAlH4 gave the
primary amines 6a-i, which underwent SN reaction with
9-chloroacridine or 6,9-dichloro-2-methoxyacridine, yielding the
desired antiprion chimeras 1 and 2a-q.
Biological Investigations. The acridine derived chimeras 1
and 2a-q were investigated for their ability to inhibit the
pathogenic prion protein PrPSc in a cell-based assay employing
quinacrine and imipramine as reference agents (Table 1). All
test compounds revealed significant dose-dependent antiprion
activity. Our initial observations were directed to the test
compound 1 displaying the closest structural similarity to the
reference building blocks. In fact, the EC50 value of 80 nM
indicated a substantially higher antiprion activity when compared
to quinacrine (300 nM) and imipramine (5000 nM). Thus, the
dimerization concept presented by May et al.9 proved to be
fruitful also for heterodimeric agents. The methoxy- and chloro-
substituents, which were adopted from the quinacrine structure,
proved to be beneficial for antiprion activity, because the
unsubstituted analogue 2c showed reduced potency (EC50 ) 150
nM). To evaluate the relationship between the distance of the
heterocyclic core units and the biological activity, the shortened
ethylene and propylene analogues 2a,d and 2b,e, respectively,
were also evaluated for antiprion activity. Interestingly, shorten-
ing of the original four-carbon spacer between the aminoacridine
and the piperazine unit was associated by a reduction of
biological activity for 2b,d,e. However, the ethylene unit proved
to be superior for the unsubstituted acridines 2a-c, resulting
in an EC50 value of 20 nM for the test compound 2a (Figure
2). The same SAR pattern could be observed for the diphenyl-
amine-derived bioisosteres 2l-q when the ethylene-bridged
agent 2l incorporating an unsubstituted acridine moiety revealed
an EC50 value of 75 nM. Replacement of the iminodibenzyl
moiety by the phenothiazine scaffold provoked a reduction of
antiprion activity, as indicated by EC50 values between 140 and
a Reagents and conditions: (a) for 4a, 1-bromo-3-chloropropane, NaNH2,
toluene, rt, 21 h (crude 100%). For 4b, 1-bromo-3-chloropropane, NaH,
DMF, rt, 4 h (90%). (b) (1) allylmethyl carbonate, Pd(PPh3)4, THF, rt, 5 h
(90%); (2) 9-BBN, THF, 2 N NaOH, H2O2 30%, rt, 16 h (71%); (3) PPh3,
CBr4, acetonitrile, rt, 17 h (80%). (c) For 5a, (1) piperazine, K2CO3,
acetonitrile, 16 h; (2) bromoacetonitrile, K2CO3, acetonitrile, 16 h (57%).
For 5b, (1) piperazine, K2CO3, acetonitrile, 16 h; (2) bromopropionitrile,
K2CO3, acetonitrile, 16 h (74%), For 5c, (1) piperazine, K2CO3, acetonitrile,
16 h; (2) bromobutyronitrile, K2CO3, acetonitrile, 16 h (31%). (d) For 5d
and 5g, piperazinoacetonitrile, diisopropylethylamine, NaI, DMF, reflux,
18 h (54-70%). For 5e and 5h, piperazinopropionitrile, diisopropylethyl-
amine, NaI, DMF, reflux, 18 h (75-77%). For 5f and 5i, piperazinobutyro-
nitrile, diisopropylethylamine, NaI, DMF, reflux, 18 h (50-72%). (e)
LiAlH4, diethyl ether, 0 °C, 30 min (21-91%). (f) 6,9-Dichloro-2-
methoxyacridine and 9-chloroacridine (1.0 mmol), respectively, phenol (10.0
mmol), 100 °C, 18 h (30-64%).
830 nM for the test compounds 2f-k. Thus, taking all
derivatives into consideration, the iminodibenzyl framework
proved to be the most valuable bioisostere.
In addition to the recently reported hybrid 1 showing an EC50
value of 80 nM, the analogue 2a bearing an unsubstituted
acridine moiety in combination with an ethylene spacer to the
piperazine turned out to exhibit the strongest potency, with a
hitherto unprecedented EC50 value of 20 nM and a full antiprion
activity (FAA) of 75 nM. When compared to the lead structure
quinacrine, the construction of chimeric derivatives in most of

Antiprion ActiVe Chimeric Acridine DeriVatiVes
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 22 6593
Figure 2. Western blot (A) and densitometry quantitation (B) of compound 2a. (A) Nontreated (Sc) or quinacrine-treated (Q; 1 mM) ScN2a cells
were used as controls; increasing concentrations of compound 2a show the disappearance of protease-resistant PrP immunoreactivity (a monoclonal
antibody against mouse PrP was used). (B) Three independent experiments as in part A were quantified by densitometry. The EC50 can be extrapolated
to 20 nM. Bars represent standard deviations.
Table 1. Antiprion Activity of Test Compounds 2a-ra
bis-acridines in order to improve antiprion potency, assuming
that quinacrine binds to the prion protein and linking two
acridine moieties would increase affinity for PrP.9 Although
high-affinity binding of quinacrine or its derivatives has not
been demonstrated, bis-acridines exhibit up to 10-fold increase
in antiprion potency compared to quinacrine.
Our reasoning of synthesizing acridine and iminodibenzyl
moieties into structural chimeras started from a completely
different background: the observation of the additive antiprion
EC50
(nM)
FAA toxicity
(nM)b (nM)c
effects of quinacrine and iminodibenzyl derivatives in ScN2a
cells led us to merge both antiprion-active compounds into a
single molecule. The favorable BBB-permeability profile of the
iminodibenzyl derivatives such as the tricyclic antidepressant
imipramine added to the future in vivo potential of this class of
compounds. The best compound of this class so far, 2a, has a
15-fold increased antiprion potency when compared to quina-
crine, making it a new lead compound. Given the effects of
these compounds on cellular lipid metabolism and the intactness
of lipid raft compartments,8 we expect that this novel class of
compounds might also be applicable to diseases where these
cellular compartments play a decisive role.
compd
2a
2b
2c
2d
2e
1
2f
2g
2h
2i
2j
2k
2l
2m
2n
R1
R2
X
n
H
H
H
H
H
H
CH2CH2
CH2CH2
CH2CH2
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
20 ( 6
75
500
230 ( 30
150 ( 30
570 ( 60
260 ( 30
80 ( 5
500
400
1000
500
100
600
500
750
1000
750
2000
1500
400
3000
1000
4000
4000
2500
2000
1000
1500
1000
1500
2000
>1000
Cl OMe CH2CH2
Cl OMe CH2CH2
Cl OMe CH2CH2
H
H
H
Cl OMe
Cl OMe
Cl OMe
H
H
H
H
H
H
S
S
S
S
S
S
370 ( 70
140 ( 40
300 ( 50
830 ( 140 2000
200 ( 80
350 ( 50
75 ( 5
500
1000
200
500
400
500
500
500
500
H
H
H
H, H
H, H
H, H
120 ( 20
230 ( 30
290 ( 20
280 ( 60
250 ( 50
300 ( 30
Experimental Section
2o
2p
2q
Cl OMe H, H
Cl OMe H, H
Cl OMe H, H
Chemistry. All reactions were carried out under nitrogen
atmosphere unless otherwise stated. Solvents were purified and dried
by standard procedures. All reagents were of commercial quality
and used as purchased. MS were run on a Finnegan MAT TSQ 70
spectrometer by EI (70 eV) with solid inlet and a Bruker Esquire
quinacrine
imipramine
5000 ( 540 10000
a EC50 is defined as the concentration of the test compound leading to
50% reduction of protease-resistant PrP. b FAA (full antiprion activity) is
defined as the concentration of the test compound leading to complete
reduction of protease-resistant PrP. c Toxicity was defined as less than 80%
confluency of ScN2a cells after 1 week of treatment.
2000 spectrometer applying APC ionization. [R]20 values were
D
measured on a Perkin-Elmer 241 instrument. The 1H NMR spectra
were obtained on a Bruker AM 360 (360 MHz) and Bruker
AVANCE (600 MHz) spectrometer in CDCl3 relative to TMS (J
values in Hz). IR spectra were performed on a Jasco FT/IR 410
spectrometer. Purification by chromatography was performed using
silica gel 60, and TLC analyses were performed using Merck 60
the cases resulted in retention or even increase of potency,
clearly proving that the tricyclic or diphenylamine serves as an
additional pharmacophoric element, presumably addressing an
accessory binding site of thesyet still unknownsmolecular
target of these antiprion active agents.
F254 aluminum sheets and analyzed by UV light (254 nm) or in the
presence of iodine. Preparative and analytical HPLC was performed
on an Agilent 1100 preparative HPLC system employing a VWL
detector. C, H, N elementary analyses were performed at the
Department of Organic Chemistry of the Friedrich Alexander
University or at the microanalytical laboratory Ilse Beetz (Kronach,
Germany).
General Procedure for the Preparation of N-(3-Cyanoalkyl)-
piperazines 5a-c. A suspension of 4a (30.5 mmol), piperazine
(61.0 mmol), Na2CO3 (30.5 mmol), and NaI (6.1 mmol) in
acetonitrile (250 mL) was heated at reflux temperature for 16 h.
Addition of diethyl ether (250 mL) and Na2SO4 was followed by
filtration and evaporation of the solvent. The residue was resolved
in acetonitrile (10 mL), treated with the respective bromoalkylnitrile
(2 equiv), Na2CO3 (1 equiv), and NaI (0.2 equiv), and stirred at
reflux temperature (for bromopropionitrile and bromobutyronitrile)
Conclusions
We have introduced a novel family of structural hybrids
including the iminodibenzyl derivative 2a demonstrating an
unprecedented antiprion potency. SARs support the specific
action of this class of compounds, even though the exact
mechanism of antiprion action remains unclear. Redistribution
of cellular cholesterol from conversion-mandatory lipid rafts
on the plasma membrane has been proposed to account for the
antiprion actions of the heterocyclic compounds, including test
compound 1. Of note, May et al. synthesized quinacrine-based

6594 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 22
Dollinger et al.
or at ambient temperature (for bromoacetonitrile) for 16 h. After
addition of diethyl ether (30 mL) and Na2SO4, filtration, and
evaporation, the crude product was purified by flash chromatog-
raphy.
7.0 Hz, 2H), 2.23-2.62 (m, 16H), 3.14 (s, 4H), 3.75 (t, J ) 7.0
Hz, 2H), 6.87-6.93 (m, 2H), 7.04-7.14 (m, 6H). EIMS: m/z 392
(M+).
3-[4-(3-Phenothiazin-10-ylpropyl)piperazin-1-yl]propyl-
[3-(10,11-Dihydrodibenzo[b,f]azepin-5-yl)propyl]piperazin-1-
ylacetonitrile (5a). The title compound was prepared according to
the general procedure when the crude product was purified by flash
chromatography (hexane/ethyl acetate/methanol 7/2/1) to give 5a
amine (6e).20 The title compound was prepared according to the
1
general procedure to give 73% of 6e as a solid. H NMR (360
MHz): δ 1.63 (quint, J ) 6.9 Hz, 2H), 1.80 (bs, 2H), 1.95 (quint,
J ) 7.0 Hz, 2H), 2.33-2.53 (m, 10H), 2.74 (t, J ) 6.9 Hz, 2H),
3.91 (t, J ) 7.0 Hz, 2H), 6.85-6.93 (m, 4H), 7.09-7.16 (m, 4H).
2-[4-(3-Diphenylaminopropyl)piperazin-1-yl]ethylamine (6g).
The title compound was prepared according to the general
procedure, yielding 65% of 6g as a yellow oil. 1H NMR (600
MHz): δ 1.75 (bs, 2H), 1.82 (quint, J ) 7.2 Hz, 2H), 2.29-2.59
(m, 12H), 2.78 (t, J ) 6.2 Hz, 2H), 3.76 (t, J ) 7.2 Hz, 2H), 6.92
(dd, J ) 7.7 Hz, 7.7 Hz, 2H), 7.00 (d, J ) 7.9 Hz, 4H), 7.24 (dd,
J ) 7.9 Hz, 7.7 Hz, 4H). EIMS: m/z 338 (M+).
General Procedure for Preparation of 9-Aminoacridines 1
and 2a-q. The respective primary amine (1.0 equiv) and com-
mercially available 6,9-dichloro-2-methoxyacridine or 9-chloro-
acridine (1.0 equiv) were stirred with phenol (10.0 equiv) at 100
°C for 18 h. The mixture was basified with 2 N NaOH (50 mL)
and extracted with ethyl acetate (3 × 50 mL). After drying (Na2SO4)
and evaporation, the crude product was purified by gravitation
column chromatography.
1
(57%) as a yellow oil. H NMR (360 MHz): δ 1.73 (quint, J )
7.0 Hz, 2H), 2.32-2.60 (m, 10H), 3.15 (s, 4H), 3.45 (s, 2H), 3.76
(t, J ) 7.0 Hz, 2H), 6.90 (ddd, J ) 7.3 Hz, 7.2 Hz, 1.3 Hz, 2H),
7.05-7.15 (m, 6H). EIMS: m/z 360 (M+).
4-{4-[3-(10,11-Dihydrodibenzo[b,f]azepin-5-yl)propyl]piper-
azin-1-yl}butyronitrile (5c). The title compound was prepared
according to the general procedure when the crude product was
purified by flash chromatography (hexane/ethyl acetate/methanol
1
7/2/1 + 0.5% triethylamine) to give 5c (31%) as a yellow oil. H
NMR (600 MHz): δ 1.75 (quint, J ) 6.9 Hz, 2H), 1.78 (quint, J
) 6.9 Hz, 2H), 2.36-2.46 (m, 12H), 2.57 (t, J ) 6.9 Hz, 2H),
3.15 (s, 4H), 3.76 (t, J ) 6.9 Hz, 2H), 6.90 (ddd, J ) 7.4 Hz, 7.2
Hz, 1.1 Hz, 2H), 7.06 (dd, J ) 8.1 Hz, 1.1 Hz, 2H), 7.08 (dd, J )
7.4 Hz, 1.3 Hz, 2H), 7.11 (ddd, J ) 8.1 Hz, 7.2 Hz, 1.3 Hz, 2H).
EIMS: m/z 388 (M+).
General Procedure for the Preparation of N-(3-Cyanoalkyl)-
piperazines 5d-i. A solution of N-(3-chloropropyl)phenothiazine
or N-(3-bromopropyl)diphenylamine (5.0 mmol), the respective
piperazin-1-ylalkylnitrile (20.0 mmol), diisopropylethylamine (5.0
mmol), and NaI (1.0 mmol) was refluxed in DMF (10 mL) for 18
h. In the case of piperazinylpropionitrile, side reactions (retro-
Michael reaction and N-formylation) were observed that could be
avoided by shortening the reaction time to 2 h. After adding brine
(100 mL), the mixture was extracted with diethyl ether (3 × 50
mL). The combined organic layers were washed with brine and
water. After drying (Na2SO4) and evaporation, the crude product
was purified by flash chromatography.
3-[4-(3-Phenothiazin-10-ylpropyl)piperazin-1-yl]propio-
nitrile (5e).20 The title compound was prepared according to the
general procedure. The crude product was purified by flash
chromatography (hexane/ethyl acetate 7/3 + 0.5% TEA) to give
5e (75%) as a yellow oil. 1H NMR (360 MHz): δ 1.94 (quint, J )
6.9 Hz, 2H), 2.33-2.58 (m, 12H), 2.67 (t, J ) 6.8 Hz, 2H), 3.92
(t, J ) 6.9 Hz, 2H), 6.85-6.94 (m, 4H), 7.10-7.16 (m, 4H).
[4-(3-Diphenylaminopropyl)piperazin-1-yl]acetonitrile (5g).
The title compound was prepared according to the general
procedure. The crude product was purified by flash chromatography
(hexane/ethyl acetate 7/3 + 0.5% triethylamine) to give 5g (70%)
as a yellow solid. 1H NMR (600 MHz): δ 1.81 (quin, J ) 7.2 Hz,
2H), 2.33-2.67 (m, 10H), 3.49 (s, 2H), 3.77 (t, J ) 7.2 Hz, 2H),
6.93 (dd, J ) 7.6 Hz, 7.6 Hz, 2H), 7.00 (d, J ) 7.8 Hz, 4H), 7.25
(dd, J ) 7.8 Hz, 7.6 Hz, 4H). EIMS: m/z 334 (M+).
General Procedure for Preparation of Aminoalkylpiperazines
6a-i. The respective nitrile (1.0 mmol) was dissolved in dry diethyl
ether (10 mL) and cooled to 0 °C. LiAlH4 (1 M in diethyl ether;
2.5 mmol) was added dropwise and stirring was continued for 30
min at room temperature. In the case of the piperazinylpropionitriles,
0.5 mmol of LiAlH4 was used to avoid the formation of a retro-
Michael side product. The reaction mixture was cooled and
quenched with Na2CO3 solution. After addition of ethyl acetate (30
mL), Na2SO4, and Celite the mixture was filtered. Evaporation of
the solvent gave the respective primary amines. No further
purification was necessary.
2-{4-[3-(10,11-Dihydrodibenzo[b,f]azepin-5-yl)propyl]piper-
azin-1-yl}ethylamine (6a). The title compound was prepared
according to the general procedure, yielding 21% of 6a as a yellow
oil. 1H NMR (360 MHz): δ: 1.66-1.79 (m, 2H), 2.07-2.23 (bs,
2H), 2.24-2.59 (m, 14H), 3.15 (s, 4H), 3.76 (t, J ) 7.0 Hz, 2H),
6.86-6.94 (m, 2H), 7.05-7.15 (m, 6H). EIMS: m/z 364 (M+).
4-{4-[3-(10,11-Dihydrodibenzo[b,f]azepin-5-yl)propyl]piper-
azin-1-yl}butylamine (6c). The title compound was prepared
according to the general procedure, yielding 89% of 6c as a yellow
oil. 1H NMR (600 MHz): δ: 1.41-1.55 (m, 4H), 1.74 (quint, J )
N-Acridin-9-yl-N-{2-{4-[3-(10,11-dihydrodibenzo[b,f]azepin-
5-yl)propyl]piperazin-1-yl}ethyl}amine (2a). The title compound
was prepared according to the general procedure starting from 6a
(19.6 mg, 0.054 mmol) and 9-chloroacridine (11.5 mg, 0.054
mmol). The crude product was purified by gravitation column
chromatography (hexane/ethyl acetate/methanol 8/1.5/0.5 + 0.5%
triethylamine) to give 2a (16.9 mg, 58%) as yellow crystals. Mp:
1
120-122 °C. H NMR (600 MHz): δ 1.78 (quint, J ) 6.9 Hz,
2H), 2.41-2.65 (m, 10H), 2.69 (t, J ) 5.8 Hz, 2H), 3.17 (s, 4H),
3.76 (t, J ) 6.4 Hz, 2H), 3.79 (t, J ) 6.9 Hz, 2H), 3.86-3.95 (m,
2H), 6.88-6.94 (m, 2H), 7.06-7.16 (m, 6H), 7.36 (ddd, J ) 8.5
Hz, 6.8 Hz, 1.1 Hz, 2H), 7.69 (ddd, J ) 8.5 Hz, 6.8 Hz, 1.1 Hz,
2H), 8.11-8.20 (m, 4H). EIMS: m/z 541 (M+). Anal. (C36H39N5)
HRMS purity HPLC.
N-(2-Chloro-6-methoxyacridin-9-yl)-N-(3-{4-[3-(10,11-dihy-
drodibenzo[b,f]azepin-5-yl)propyl]piperazin-1-yl}butyl)amine (1).
The title compound was prepared according to the general procedure
from 6c (73.6 mg, 0.187 mmol) and 6,9-dichloro-2-methoxyacridine
(104.3 mg, 0.375 mmol). The crude product was purified by flash
chromatography (ethyl acetate/methanol 95/5 + 0.5% TEA) to give
1
1 (71.2 mg, 60%) as yellow crystals. Mp: 56-58 °C. H NMR
(360 MHz): δ 1.55 (quint, J ) 7.2 Hz, 2H), 1.75 (quint, J ) 7.2
Hz, 2H), 1.79 (quint, J ) 7.2 Hz, 2H), 2.29-2.50 (m, 12 H), 3.16
(s, 4H), 3.73 (t, J ) 7.1 Hz, 2H), 3.77 (t, J ) 7.1 Hz, 2H), 3.97 (s,
3H), 6.92 (ddd, J ) 7.3 Hz, 7.3 Hz, 1.2 Hz, 2H), 7.04-7.13 (m,
6H), 7.23 (d, J ) 2.7 Hz, 2H), 7.30 (dd, J ) 9.1 Hz, 2.0 Hz, 2H),
7.99-8.05 (m, 3H), 8.07 (d, J ) 2.0 Hz, 2H). APCI-MS: m/z 634
(M+ + 1). Anal. (C39H44ClN5O) C, H, N.
N-Acridin-9-yl-N-{3-[4-(3-phenothiazin-10-ylpropyl)piperazin-
1-yl]propyl}amine (2g). The title compound was prepared accord-
ing to the general procedure from 6e (68.2 mg, 0.178 mmol) and
9-chloroacridine (38.1 mg, 0.178 mmol). The crude product was
purified by gravitation column chromatography (hexane/ethyl
acetate/methanol 8/1.5/0.5) and crystallized in methanol to afford
2g (49.9 mg, 50%) as yellow needles. Mp: 115 °C. 1H NMR (600
MHz): δ 1.94 (quint, J ) 5.9 Hz, 2H), 1.99 (quint, J ) 7.0 Hz,
2H), 2.48-2.69 (m, 12H), 3.95 (t, J ) 7.0 Hz, 2H), 4.05 (t, J )
5.9 Hz, 2H), 6.87-6.95 (m, 4H), 7.11-7.17 (m, 4H), 7.22 (ddd, J
) 8.7 Hz, 6.7 Hz, 1.0 Hz, 2H), 7.59 (ddd, J ) 8.7 Hz, 6.7 Hz, 1.0
Hz, 2H), 8.10 (dd, J ) 8.7 Hz, 1.0 Hz, 2H), 8.19 (dd, J ) 8.7 Hz,
1.0 Hz, 2H). EIMS: m/z 559 (M+). Anal. (C35H37N5S) C, H, N.
N-Acridin-9-yl-N-{2-[4-(3-diphenylaminopropyl)piperazin-1-
yl]ethyl}amine (2l). The title compound was prepared according
to the general procedure from 6g (46.6 mg, 0.138 mmol) and
9-chloroacridine (29.4 mg, 0.138 mmol). The crude product was
purified by gravitation column chromatography (hexane/ethyl
acetate/methanol 7/2/1 + 0.5% triethylamine) to give 2l (21.2 mg,

Antiprion ActiVe Chimeric Acridine DeriVatiVes
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 22 6595
30%) as yellow crystal. Mp: 110 °C. 1H NMR (360 MHz): δ 1.86
(quint, J ) 7.2 Hz, 2H), 2.39-2.75 (m, 12H), 3.79 (t, J ) 7.2 Hz,
2H), 3.88 (t, J ) 5.6 Hz, 2H), 6.55 (bs, NH), 6.94 (dddd, J ) 7.4
Hz, 7.3 Hz, 1.0 Hz, 0.9 Hz, 2H), 7.02 (dd, J ) 8.6 Hz, 1.1 Hz,
4H), 7.21-7.29 (m, 4H), 7.36 (ddd, J ) 8.9 Hz, 6.6 Hz, 1.0 Hz,
2H), 7.67 (ddd, J ) 8.7 Hz, 6.6 Hz, 1.1 Hz, 2H), 8.09 (dd, J ) 8.9
Hz, 1.1 Hz, 2H), 8.18 (dd, J ) 8.7 Hz, 1.0 Hz, 2H). EIMS: m/z
515 (M+). Anal. (C34H37N5) C, H, N.
(6) Korth, C.; May, B. C. H.; Cohen, F. E.; Prusiner, S. B. Acridine and
phenothiazine derivatives as pharmacotherapeutics for prion disease.
Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 9836-9841.
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Baldwin, M. A.; Craig, J. C.; De Armond, S.; May, B. C.; Cohen, F.
E.; Lin, E. T.; Huang, Y.; Prusiner, S.; Legname, G. In vivo efficacy
of quinacrine in animal models of prion disease, in Paris International
NeuroPrion Meeting, 2004, p 169, Paris. (b) Doh-ura, K.; Ishikawa,
K.; Murakami-Kubo, I.; Sasaki, K.; Mohri, S.; Race, R.; Iwaki, T.
Treatment of transmissible spongiform encephalopathy by intra-
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PrPSc Inhibition Assay in ScN2a Cells. ScN2a cells are an
established cell model of prion disease. Mouse neuroblastoma cells
(ATCC CCL131) that express PrPC on their cell surface can be
infected with mouse-adapted scrapie strains. In this case, the RML
strain was used to infect N2a cells that subsequently were subcloned
for selecting highest PrPSc-bearing cells.21 The advantage of this
model is that candidate substances can be directly assayed by adding
them into the cell culture medium. A confluent 10-cm dish was
split and a drop of cells (ca 100 µL) was pipetted into a 60-mm
dish of 4 mL of MEM containing 10% (vol/vol) FCS, penicillin-
streptomycin, and L-glutamine. Medium was exchanged every
second day, together with the drug. Cells were lysed (lysis buffer:
10 mM Tris, pH 8.0, 150 mM NaCl, 0.5% Triton X-100, 0.5%
deoxycholate) on the seventh day, having achieved about 80%
confluency. All experiments were repeated three times. Cell lysates
were digested with proteinase K at 20 µg/mL for 30 min at 37 °C.
The reaction was stopped with 2 mM PMSF, and the lysates were
centrifuged for 45 min at 100 000g in an ultracentrifuge (Beckman-
Coulter). Pellets were resuspended in sample buffer, and SDS-
PAGE/immunoblotting was performed according to standard tech-
niques. Immunoblots were incubated with an R-PrP-antibody and
developed with enhanced chemiluminescence (Amersham Phar-
macia). Effective concentrations where half the antiprion activity
was achieved (EC50) were determined by densitometry of five
concentration points measured from immunoreactive bands on
Western blot using NIH image software.
(9) May, B. C.; Fafarman, A. T.; Hong, S. B.; Rogers, M.; Deady, L.
W.; Prusiner, S. B.; Cohen, F. E. Potent inhibition of scrapie prion
replication in cultured cells by bis-acridines. Proc. Natl. Acad. Sci.
U.S.A. 2003, 100, 3416-3421.
(10) For very recent examples, see: (a) Bettinetti, L.; Schlotter, K.;
Hu¨bner, H.; Gmeiner, P. Interactive SAR studies: Rational discovery
of super-affine and highly selective dopamine D3 receptor antagonists
and partial agonists. J. Med. Chem. 2002, 45, 4594-4597. (b)
Schlotter, K.; Boeckler, F.; Hu¨bner, H.; Gmeiner, P. Fancy bio-
isosteres: Metallocene-derived G-protein-coupled receptor ligands
with subnanomolar binding affinity and novel selectivity profiles. J.
Med. Chem. 2005, 48, 3696-3699. (c) Leopoldo, M.; Berardi, F.;
Colabufo, N. A.; De Giorgio, P.; Lacivita, E.; Perrone, R.; Tortorella,
V. Structure-affinity relationship study on N-[4-(4-arylpiperazin-1-
yl)butyl]arylcarboxamides as potent and selective dopamine D3
receptor ligands. J. Med. Chem. 2002, 45, 5727-5735.
(11) Moreno-Manas, M.; Morral, L.; Pleixats, R. Palladium(0)-catalyzed
allylation of highly acidic and nonnucleophilic anilines. The origin
of stereochemical scrambling when using allilic carbonates. J. Org.
Chem. 1998, 63, 6160-6166.
(12) Davis, M. A.; Herr, F.; Thomas, R. A.; Charest, M.-P. New
psychotropic agents. VIII. Analogs of amitriptyline containing the
normeperidine group. J. Med. Chem. 1967, 10, 627-635.
(13) Gilman, H.; Shirley, D. A. Some derivatives of phenothiazine. J.
Am. Chem. Soc. 1944, 66, 888-892
(14) Liu, Z.; Larock, R. C. Facile N-Arylation of Amines and Sulfon-
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Acknowledgment. The authors wish to thank the VW
foundation for financial support.
Supporting Information Available: Experimental procedures
and analytical data for compounds 2b-f,i-k,m-q, 4a-c, 5b,d,
f,h,i, and 6b,d,f,h,i, elemental analysis results, high-resolution mass
spectrometry data, and HPLC purities. This material is available
free of charge via the Internet at http://pubs.acs.org.
(15) Husbands, S. M.; Izenwasser, S.; Kopajtic, T.; Bowen, W. D.; Vilner,
B. J.; Katz, J. L.; Newman, A. H. Structure-activity relationships at
the monoamine transporters and σ receptors for a novel series of
9-[3-(cis-3,5-dimethyl-1-piperazinyl)-propyl]carbazole (rimcazole)
analogues. J. Med. Chem. 1999, 42, 4446-4455.
(16) Sandefur, L. O.; Slusarek, W.; Wilson, B. D.; Maggiulli, C. A.
Cyanoalkylpiperazines and their methods for their preparation and
use. WO8400753, 1984.
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