Synthesis of conjugates of 6-aminophenanthridine and guanabenz, two structurally unrelated prion inhibitors, for the determination of their cellular targets by affinity chromatography

Article abstract of DOI:10.1021/bc900314n

The synthesis of affinity matrices for 6-aminophenanthridine (6AP) and 2,6-dichlorobenzylidenaminoguanidine (Guanabenz, GA), two unrelated prion inhibitors, is described. In both cases, the same simple spacer, fc-aminocaproylaminopentanol, was introduced by a Mitsunobu reaction and the choice of the anchoring position of the linker was determined by the study of the residual antiprion activity of the corresponding 6AP or GA conjugates. Very recently, these two affinity matrices were used for chromatography assays leading to the identification of ribosome (via the rRNA) as a common target of these two antiprion drugs. Here, we show, using competition experiments with Quinacrine (QC) and Chlorpromazine (CPZ), two other antiprion drugs, that QC, but not CPZ, may also directly target the rRNA.

Full text of DOI:10.1021/bc900314n

Bioconjugate Chem. 2010, 21, 279–288
279
Synthesis of Conjugates of 6-Aminophenanthridine and Guanabenz, Two
Structurally Unrelated Prion Inhibitors, for the Determination of Their
Cellular Targets by Affinity Chromatography
Fabienne Gug,† Nassima Oumata,† De´borah Tribouillard-Tanvier,‡,| Ce´cile Voisset,‡ Nathalie Desban,§
Ste´phane Bach,§ Marc Blondel,‡ and Herve´ Galons*,†
Laboratoire de Chimie Organique 2, INSERM U648, Universite´ Paris-Descartes, 4 avenue de l’Observatoire, 75270 Paris cedex
06, France, INSERM U613, Faculte´ de Me´decine et des Sciences de la Sante´, CHU Morvan, Universite´ de Bretagne
Occidentale, Brest, F-29200, France, and CNRS USR3151, Station Biologique, Protein Phosphorylation and Disease Laboratory,
place Georges Teissier, 29680 Roscoff, France. Received July 15, 2009; Revised Manuscript Received December 7, 2009
The synthesis of affinity matrices for 6-aminophenanthridine (6AP) and 2,6-dichlorobenzylidenaminoguanidine
(Guanabenz, GA), two unrelated prion inhibitors, is described. In both cases, the same simple spacer,
ε-aminocaproylaminopentanol, was introduced by a Mitsunobu reaction and the choice of the anchoring position
of the linker was determined by the study of the residual antiprion activity of the corresponding 6AP or GA
conjugates. Very recently, these two affinity matrices were used for chromatography assays leading to the
identification of ribosome (via the rRNA) as a common target of these two antiprion drugs. Here, we show, using
competition experiments with Quinacrine (QC) and Chlorpromazine (CPZ), two other antiprion drugs, that QC,
but not CPZ, may also directly target the rRNA.
neurophysiological dysfunctions (3, 4). Therefore, approaches
leading to reduction of endogenous PrPC and/or PrPSc levels
may well be effective after the appearance of the neurological
symptoms. Among these approaches are identifications of
pharmacological compounds promoting PrPSc clearance. Some
of these identifications are based on the use of cell-free
systems (5–8), whereas others are based on the use of mam-
malian cells chronically infected with prions (9–12) and
reviewed in ref 13. All these assays are time- and money-
consuming, in particular, because experiments have to be carried
out in highly secured laboratories. For these reasons, we recently
developed a rapid and economical budding yeast (Saccharo-
myces cereVisiae) based two-step assay to screen for antiprion
molecules (14, 15). This assay allows easy detection of
compounds active against two yeast prions and has led, among
others, to the isolation of 6-aminophenanthridine (6AP) 1a and
2,6-dichlorobenzylidenaminoguanidine (Guanabenz, GA) 1b
(Figure 1). This yeast-based assay was validated for the isolation
of drugs active against mammalian prions, since most of the
active compounds isolated, including 6AP and GA, turned out
to also be active against mammalian prions in mammalian cell-
based assays mentioned above (16). In addition, both 6AP and
GA exhibited significant activity in a mouse model for scrapie
(17).
INTRODUCTION
Prion-based diseases are transmissible and yet incurable fatal
neurodegenerative disorders (1). Among these diseases are
Creutzfeldt-Jakob disease (CJD) in humans, bovine spongiform
encephalopathies in cattle, and Scrapie in sheep and goats. These
diseases are associated with neuronal cell death, which leads
to characteristic “spongiform” vacuolation of the brain, and are
therefore termed spongiform encephalopathies. According to the
“protein-only” hypothesis, prions are mostly if not solely
composed of an abnormally folded form (PrPSc) of the PrP
protein (PrPC), a glycosyl-phosphatidyl inositol (GPI) anchored
protein normally expressed at the surface of a number of cell
types including in particular neurons. Transmissibility neces-
sitates the conversion of host PrPC by exogenous PrPSc. PrPSc
isoform displays a pronounced protease resistance, shows an
increase in ꢀ-sheet structures, and forms amyloid fibers. Despite
intensive works devoted to the study of prions and their
mechanisms of propagation, many fundamental questions related
to the mechanism of the infection and subsequently to the
mechanisms of action of known antiprion compounds remain
unanswered. On the basis of the assumption that PrPSc corre-
sponds to (or at least is part of) the pathogenic entity, various
approaches aimed at reducing PrPSc or PrPC levels for the
development of prion disease therapies are currently being
explored (comprehensively reviewed in ref 2). A proof of
concept for these approaches has been made recently by showing
that depleting PrPC from neurons of prion-infected mice in which
the Prnp gene can be turned off, not only prevented progression
of disease, but also reversed spongiosis and early cognitive and
Although direct interaction with the prion protein PrPC has
been established by NMR spectroscopy (18) or by surface
plasmon resonance (19) for some drugs, this has not been
evaluated with most of the known antiprion agents. Recently,
using immobilized 9-aminoacridine, Prusiner’s group was able
to discriminate PrPC and PrPSc in an affinity chromatography
assay (20). Moreover, even if some of the compounds are known
to bind PrP, it is not established whether their antiprion activities
exclusively rely on this binding ability. Therefore, the mecha-
nism of action of most antiprions remains unclear, and at least
two different mechanisms can be envisioned: drugs can either
act in cis directly on prion aggregates or in trans on cellular
elements required for prion propagation. In addition, identifica-
tion of these unknown biological mechanisms may provide new
* Corresponding author. E-mail: herve.galons@parisdescartes.fr,
Phone +33(0)153739684, Fax +33(0)143290598.
† INSERM U648.
‡ INSERM U613.
§ CNRS USR3151.
| Present address: Laboratory of Persistent Viral Diseases, Rocky
Mountain Laboratories, National Institute of Health, Hamilton, Montana
59840, USA.
10.1021/bc900314n  2010 American Chemical Society
Published on Web 01/21/2010

280 Bioconjugate Chem., Vol. 21, No. 2, 2010
Gug et al.
with free quinacrine (QC) and chlorpromazine (CPZ), two other
active antiprion compounds, we found that QC, but not CPZ,
may also target the ribosome directly.
EXPERIMENTAL PROCEDURES
Chemical Methods. Reactions were monitored by TLC using
Merk silica gel 60F-254 thin layer plates. Column chromatog-
raphy experiments were carried out on SDS Chromagel 60 ACC,
40-63 µm. Column chromatography (CC) was performed using
silica gel (70-200 µm). Melting points were determined on a
Kofler hot-stage (Reichert) and are uncorrected. NMR spectra
were recorded on Bruker Avance 400 MHz. Chemical shifts
are given in parts per million (ppm) downfield of tetramethyl-
silane (TMS) used as an internal standard. Infrared spectra were
recorded on a FTIR Schimadzu 8300. The HPLC analyses were
carried out on a system consisting of a Waters 600 system
controller, a Jones chromatography heater-chiller oven, and a
Waters 994 photodiode array detector. Mass spectra (electro-
spray ionization (ESI) were measured on a TSQ Quantum
(Thermo Electron Corporation) instrument. Elemental analyses
for C, H, and N were performed on a Thermoquest EA-110
apparatus. 6AP was prepared as described previously (32, 33).
Commercially available GA was used or obtained upon con-
densation of aminoguanidine with 2,6-dichlorobenzaldehyde.
(R)-6-(1-Hydroxybut-2-ylamino)phenanthridine, 6APi (1b). A
mixture of 6-chlorophenanthridine (2.14 g, 10 mmol), (R)-2-
aminobutanol (3 mL, 35 mmol), and NEt3 (3 mL, 40 mmol)
was heated at 110 °C for 2 h. After cooling to rt, 30 mL H2O
was added, and the mixture was extracted with CH2Cl2 (2 ×
10 mL). The solvent was evaporated and 1b crystallized from
EtOAc. Mp 176-178 °C. Anal. (C17H18N2O) C, H, N. 1H NMR
(CDCl3) δ 1.19 (t, J ) 7 Hz, 3H, CH3); 1.78 (p, 2H, CH2CH3);
3.76 (t, 1H, J ) 9 Hz, 1H, CH2OH); 3.98 (d, J ) 10.9 Hz, 1H,
CH2OH); 4.26 (m, 1H, CHCH2OH); 5.57 (br s, 1H, OH); 6.75
(br s, 1H, NH); 7.34 (t, J1-2 ) J2-3 ) 7.5 Hz, 1H, 2-H); 7.54
(t, J7-8 ) J8-9 ) 7.5 Hz, 1H, 8-H); 7.59 (t, 1H, 3-H); 7.70 (d,
J3-4 ) 8 Hz, 1H, 4-H); 7.77 (t, 1H, J9-10 ) 7.5 Hz, 9-H); 7.86
(d, 1H, 7-H); 8.31 (d, 1H, 1-H); 8.51 (d, 1H, 10-H). 13C NMR
(CDCl3) δ 10.8; 24.7; 56.8; 68.2; 118.5; 120.3; 121.5; 121.8;
122.5; 122.6; 125.7; 126.7; 128.7; 130.2; 133.6; 142.9; 153.7.
2-Chloro-6-fluoro-benzylideneaminoguanidine, GAi (2b). Ami-
noguanidine hydrochloride (2.20 g, 20 mmol) was added to a
solution of 2-chloro-2-fluorobenzaldehyde (3.16 g, 20 mmol)
in 50 mL EtOH followed by the addition of AcONa, 5H2O (2.72
g, 20 mmol). After 2 h reflux, the hot mixture was filtered, and
2b precipitated upon cooling to rt. Yield 87%. Mp 164-167
°C. Anal. (C8H8ClFN4) C, H, N. 1H NMR (DMSO) δ 5.50 (br
s, 4H, 2 NH + NH2); 7.15 (t, J3-4 ) J4-5 ) 8 Hz, 1H, 4-H);
7.50 (d, 1H, 3-H); 7.65 (dd, JF-5 ) 10 Hz, 1H, 5-H); 8.20 (s,
1H, NCH).
Figure 1. 1a, 6-aminophenanthridine (6AP) and 2a, 2,6-dichloroben-
zylidenaminoguanidine: Guanabenz (GA), two antiprion drugs active
against both yeast and mammalian prions and their respective inactive
controls, 1b (6APi) and 2b (GAi).
insights for other neurodegenerative disorders characterized by
the occurrence of protein misfolding as observed in prion
diseases (21).
For these reasons, identification of these mechanisms using
reverse screening approaches is of particular interest. Several
comprehensive methods exist to decipher cellular mechanisms
targeted by small chemical compounds, including affinity
chromatography on immobilized drugs.
Affinity chromatography has been used in the determination
of the molecular targets of several compounds. Most of them
are kinase inhibitors such as flavopiridol (22), purvalanol (23),
(R)-roscovitine (24), gefitinib (25), and SU6668 (26). Affinity
chromatography has also been performed with the immunosup-
pressor FK506 (27) and geldanamycin, which was therefore
shown to be a specific inhibitor of HSP 90 (28).
As a typical example, the determination of the molecular
targets of (R)-roscovitine led, apart from the expected cyclin-
dependent kinases, to the identification of pyridoxal kinase, an
unexpected target. This was confirmed by X-ray crystallography,
and surprisingly, roscovitine appeared to bind the pyridoxal
pocket rather than the ATP binding site (29).
In affinity chromatography experiments, a spacer arm is
introduced between the compound of interest and the solid
supports with the aim of avoiding steric hindrance. The structure
of the spacer arm matters considerably. Polyoxyethylenic
linkages are generally used. A carboxylic acid group is often
introduced on the active molecules. Therefore, the introduction
of the spacer on the bioactive compound is frequently achieved
by a multiple-step procedure. The position of the linkage is
critical, as the conjugate should, at least partially, retain
biological activity. Linkage should be introduced at several
positions of the molecule under study and the biological activity
of the resulting conjugates tested. If available, structure-activity
relationships provide useful information on the positions of
linkage which are more likely to allow molecular recognition
by the biological targets (for a review, see ref 30).
In this paper, we report the synthesis of affinity matrices for
the two unrelated antiprion drugs cited above: 6-aminophenan-
thridine (6AP) and 2,6-dichlorobenzylidenaminoguanidine (gua-
nabenz, GA) by immobilization of the conjugates on preacti-
vated Sepharose beads. The 6AP and GA affinity beads were
very recently used and allowed to determine that protein folding
activity of the rRNA (Ribosome-borne Protein Folding Activity,
RPFA) is a selective target of both 6AP and GA (31). Here,
using competition experiments on 6AP and GA affinity matrices
Benzyloxy-6-aminophenanthridines 1c-e (reaction in
liquid ammonia). Ammonia (200 mL) was introduced into a
flask connected with an efficient condenser followed by
Fe(NO3)3 (0.02 g) and piece by piece Na (0.23 g, 10 mmol)
additions. The solution was stirred for 0.5 h during this period;
the initial blue color of the solution of Na gradually turned to
gray indicating the complete conversion into NaNH2. To this
suspension, benzyloxyanilines (1.99, 10 mmol) dissolved in the
minimum amount of anhydrous Et2O were added. The mixture
was stirred for 0.5 h followed by the addition of 2-chloroben-
zonitrile (1.37 g, 10 mmol) in anhydrous Et2O. The mixture
was stirred for 2 h, and lithium (0.14 g, 20 mmol) was then
added. After 2 h stirring, NH4Cl (10 g) was gradually introduced
under vigorous stirring. Ammonia was evaporated overnight.
After addition of H2O (50 mL), the mixture was extracted with
CH2Cl2 (3 × 30 mL), and the organic layer was first washed
with brine (40 mL), then dried with Na2SO4 and evaporated.

Antiprion Conjugates
Bioconjugate Chem., Vol. 21, No. 2, 2010 281
The residue was applied to a silica gel column and eluted first
with CH2Cl2 and then with CH2Cl2-MeOH (99:1 to 95:5).
1-Benzyloxy-6-aminophenanthridine (1c) and 3-benzyloxy-
6-aminophenanthridine (1e). 3-Benzyloxyaniline led to a
mixture of compounds 1c and 1e. The two isomers could not
be separated by column chromatography. They were isolated
in 23% yield as a (1:1) mixture.
2-(3-Benzyloxyphenanthridin-6-yl)-isoindole-1,3-dione (3e).
Yield: 83%. Mp 121-123 °C. Anal. (C28H18N2O3) C, H, N. 1H
NMR (CDCl3) δ 5.24 (s, 2H, CH2); 7.35 (t, J3′-4′ ) J4′-5′ ) 7.2
Hz, 1H, 4′-H); 7.41 (t, J2′-3′ ) J5′-6′ ) 7.2 Hz, 2-H, 3′-H +
5′-H); 7.48 (dd, J1-2 ) 9.4 Hz, J2-4 ) 2.4 Hz; 1H, 2-H); 7.50
(d, 2H, 2′-H and 6′-H); 7.61 (t, J7-8 ) J8-9 ) 8 Hz, 1H, 8-H);
7.70 (d, 1H, 4-H); 7.87 (dd, J ) 3.2 Hz, J ) 5.6 Hz, 2H,
phthalyl); 7.88 (m, 2H, 7-H and 9-H); 8.04 (dd, 2H, phthalyl);
8.55 (d, 1H, 1-H); 8.61 (d, J9-10 ) 8.8 Hz, 10-H). 13C NMR
(CDCl3) δ 70.7; 111.4; 119.6; 120.4; 122.5; 123.3; 123.9; 124.5;
126.1; 127.4; 128.1; 128.6; 129.1; 132.1; 132.6; 135.1; 135.5;
136.7; 145.3; 146.4; 159.9; 167.7. IR (KBr) γ 537; 575; 631;
681; 700; 721; 755; 785; 814; 842; 884; 947; 1027; 1072; 1102;
1138; 1181; 1214; 1257; 1302; 1370; 1404; 1452; 1472; 1574;
1600; 1715; 1792; 2991; 3031; 3044; 3434; 3600; 3708; 3831;
3894 cm-1. MS (E+) m/z: 431 [M + H]+; 453 [M + Na]+.
2-Benzyloxy-6-aminophenanthridine (1d). Prepared from
4-benzyloxyaniline. Yield 45%. Mp 138-141 °C. Anal.
1
(C20H16N2O) C, H, N. H NMR (CDCl3) δ 5.22 (s, 2H, CH2);
5.47 (brs, 2H, NH2); 7.31 (dd, J3-4 ) 9 Hz, J1-3 ) 2.4 Hz, 1H,
3-H); 7.32 (t, J ) 7.2 Hz, 1H, C6H5); 7.37 (t, 2H, J ) 7.2 Hz,
C6H5); 7.41 (m, 3H, C6H5 + 8-H); 7.52 (d, J3-4 ) 8 Hz, 1H,
4-H); 7.69 (t, 1H, 9-H); 7.86 (s, 1H, 1-H); 8.09 (d, J7-8 ) J8-9
) 8 Hz, 1H, 7-H); 8.42 (d, J9-10 ) 8.2 Hz, 10-H).
3-Benzyloxy-6-aminophenanthridine (1e) (by Suzuki-
Miyaura cross coupling). A solution of 5-benzyloxy-2-bro-
moaniline (1 g, 3.6 mmol) in 20 mL dioxane and 6 mL 1 M
Na2CO3 was stirred under N2 for 5 min. Pd[P(C6H5)3]4 (0.2 g,
0.17 mmol) was then introduced followed by 2-(4,4,5,5-
tetramethyl-[1,3,2]-dioxaborolan-2-yl)-benzonitrile (3.6 mmol,
0.825 g). The mixture was refluxed for 12 h and then
concentrated. The residue was extracted with CH2Cl2 (3 × 10
mL). The organic solution was washed with H2O and dried with
Na2SO4 and evaporated. The final compound was purified by
column chromatography (CH2Cl2-EtOH, 98:2) and crystallized
from EtOAc. Yield: 63%. Mp 121-123 °C. Anal. (C20H16N2O)
C, H, N. 1H NMR (CDCl3) δ 5.20 (s, 2H, CH2); 5.32 (br s, 2H,
NH2); 7.11 (dd, J1-2 ) 9.2 Hz, J2-4 ) 2.4 Hz, 1H, 2-H); 7.26
(d, 1H, 4-H); 7.34 (t, J3′-4′ ) J4′-5′ ) 7.2 Hz, 1H, 4′-H); 7.41
(t, 2H, J2′-3′ ) J5′-6′ ) 7.2 Hz, 3′-H and 5′-H); 7.50 (d, 2H,
2′-H, and 6′-H); 7.55 (td, J8-10 ) 1 Hz, J8-9 ) 8 Hz, 1H, 8-H);
7.76 (td, J9-10 ) 0.8 Hz, 1H, 9-H); 7.86 (d, J7-8 ) 8 Hz, 1H,
7-H); 8.34 (d, 1H, 1-H); 8.44 (d, J9-10 ) 8.4 Hz, 1H, 10-H).
13C NMR (CDCl3) δ 70.5; 108.7; 114.6; 115.9; 117.9; 122.7;
123.4; 123.7; 126.4; 128.0; 128.4; 129.0; 131.2; 134.8; 137.3;
146.2; 155.5; 160.1. IR (KBr) γ 517; 621; 681; 718; 757; 811;
856; 890; 994; 1020; 1085; 1135; 1178; 1220; 1253; 1300; 1357;
1402; 1460; 1514; 1547; 1600; 1657; 1755; 1824; 1958; 2368;
2848; 2851; 3114; 3314; 3742.
Phthalylation of Benzyloxy-6-Aminophenanthridines. The
amines 1c-e (0.7 g, 2.3 mmol) were dissolved in THF (30 mL)
containing 1 mL NEt3. Phthalic anhydride (0.34 g, 2.3 mmol)
was then added, and the mixture was refluxed for 6 h. THF
was removed under reduced pressure. Water (20 mL) was added
and the mixture extracted with CH2Cl2 (3 × 10 mL). The organic
layer was dried (Na2SO4) and evaporated. The residue was
subjected to chromatography (CH2Cl2 100%) and crystallization
from i-PrOH.
2-(1-Benzyloxyphenanthridin-6-yl)-isoindole-1,3-dione (3c).
Yield: 80%. Mp 189-193 °C. Anal. (C28H18N2O3) C, H, N. 1H
NMR (CDCl3) δ 5.45 (s, 2H, CH2); 7.33 (d, J2-3 ) 8 Hz, 1H,
2-H); 7.42 (t, J3′-4′ ) J4′-5′ ) 7.2 Hz, 1H, 4′-H); 7.47 (t, J2′-3′
) J5′-6′ ) 7.2 Hz, 2H, 3′-H and 5′-H); 7.60 (d, 2H, 2′-H and
6′-H); 7.65 (t, J7-8 ) J8-9 ) 8 Hz, 1H, 8-H); 7.70 (t, J3-4 ) 8
Hz, 1H, 3-H); 7.80 (t, 1H, 9-H); 7.88 (m, 3H, phthalyl and 4-H);
7.93 (d, 1H, 7-H); 8.05 (dd, J ) 5.6 Hz, J ) 3.2 Hz, 2H,
phthalyl); 9.72 (d, J9-10 ) 8.8 Hz, 1H, 10-H). MS (E+) m/z:
431 [M + H]+; 453 [M + Na]+.
Debenzylation of 2-(Benzyloxyphenanthridin-6-yl)-isoindole-
1,3-diones. Preparation of Phenols 4c-e. 0.1 g of 10% Pd/C
and 0.5 mL of AcOH were added to a solution of 2-(benzy-
loxyphenanthridin-6-yl)-isoindole-1,3-dione (1.9 mmol, 0.65 g)
in 50 mL EtOH and 50 mL CH2Cl2. The mixture was stirred
under H2 atmosphere (AP) for 12 h. The catalyst was removed
by filtration through a Whatman filter. The filter was washed
once with 5 mL EtOH. The phenols crystallized upon concen-
tration of the ethanolic solution.
2-(1-Hydroxyphenanthridin-6-yl)-isoindole-1,3-dione (4c). Yield:
97%. Mp 189-193 °C. Anal. (C21H12N2O3) C, H, N.1H NMR
(CDCl3) δ 7.23 (d, J2-3 ) J3-4 ) 8 Hz, 1H, 2-H); 7.52 (t, 1H,
3-H); 7.59 (t, J7-8 ) J8-9 ) 8 Hz, 1H, 8-H); 7.68 (d, 1H, 4-H);
7.82 (m, 4H, phthalyl and 7-H and 9-H); 7.97 (dd, J ) 3.2 Hz,
J ) 5.6 Hz, phthalyl); 9.80 (d, J9-10 ) 8.4 Hz, 1H, 10-H); 10.9
(br s, 1H, OH).
2-(2-Hydroxyphenanthridin-6-yl)-isoindole-1,3-dione
(4d).
Yield: 82%, Mp 201-203 °C. Anal. (C21H12N2O3) C, H, N. 1H
NMR (CDCl3) δ 7.35 (t, 1H); 7.63 (dd, 1H, J3-4 ) 8.5 Hz,
J1-3 ) 3 Hz, 3-H); 7.71 (t, 1H); 7.95 (m, 5H, phthalyl + 1H);
8.10 (m, 4H, phthalyl); 8.18 (d, 1H, 7-H); 8.60 (d, 1H, 10-H);
8.75 (brs, OH).
2-(3-Hydroxyphenanthridin-6-yl)-isoindole-1,3-dione (4e). Yield:
98%. Mp 169-172 °C. Anal. (C21H12N2O3) C, H, N.1H NMR
(CDCl3) δ 7.30 (dd, J1-2 ) 8.8 Hz, J2-4 ) 2.4 Hz, 1H, 2-H);
7.49 (t, J7-8 ) J8-9 ) 8 Hz, 1H, 8-H); 7.52 (d, 1H, 4-H); 7.80
(m, 4H, phthalyl + 7-H + 9-H); 7.97 (dd, J ) 5.6 Hz, J ) 3.2
Hz, phthalyl); 8.41 (d, 1H, 1-H); 8.52 (d, J9-10 ) 8.2 Hz, 1H,
10-H); 9.50 (brs, 1H, OH).
Preparation of Spacers (5a,b). N-Hydroxybenzotriazole
(2.83 g, 21 mmol) and dicyclohexylcarbodiimide (4.14 g, 20
mmol) were added to a cooled (5 °C) solution of ε-Z-
aminocaproic acid (6.62 g, 25 mmol) in 50 mL EtOAc. The
mixture was left under stirring for 2 h at rt. The precipitated
dicyclohexylurea was filtrated and washed once with 10 mL
EtOAc. 5-Aminopentan-1-ol (2.16 g, 21 mmol) and NEt3 (5
mL) were added to the combined filtrates. After 3 h stirring,
the mixture was diluted in 150 mL EtOAc and washed with 20
mL 1 N HCl followed by 50 mL saturated NaHCO3 and then
with 20 mL H2O. Derivatives 5 crystallized upon concentration
of the solvent. Further purification could be achieved by
crystallization from EtOAc.
5-(6-Benzyloxycarbonylaminohexanoylamino)pentan-1-ol
(5a). Yield: 85%. Mp: 108-110 °C. Anal. (C19H30N2O4) C, H,
N. 1H NMR (CDCl3) δ 1.25-1.85 (m, 12H, 6 × CH2); 2.15 (t,
J ) 7.4 Hz, 2H, CH2CO); 3.19 (q, J ) 6.4 Hz, 2H, CH2NH);
3.26 (q, J ) 6.4 Hz, 2H, CH2NH); 3.64 (t, J ) 6.4 Hz, CH2OH);
4.88 (br s, 1H, NH); 5.08 (s, 2H, CH2C6H5); 5.56 (br s, 1H,
NH); 7.28-7.38 (m, 5H, C6H5). 13C NMR (CDCl3) δ 23.4; 25.3;
25.6; 25.9; 29.7; 30.0; 32.5; 36.9; 39.6; 41.2; 62.9; 67.0; 128.5;
128.9; 137.0; 156.9; 173.3. IR (KBr) γ 528; 586; 700; 636;
2-(2-Benzyloxy-phenanthridin-6-yl)-isoindole-1,3-dione
(3d). Yield: 81%. Mp 138-141 °C. Anal. (C28H18N2O3) C, H,
N. 1H NMR (CDCl3) δ 5.30 (s, 2H, CH2); 7.40 (t, J3′-4′ ) J4′-5′
) 7.2 Hz, 1H, 4′-H); 7.45 (t, J2′-3′ ) J5′-6′ ) 7.2 Hz, 2H, 3′-H
+ 5′-H); 7.50 (dd, J3-4 ) 9.4 Hz, J1-3 ) 2.4 Hz, 1H, 3-H);
7.57 (d, 2H, 2′-H and 6′-H); 7.69 (t, J7-8 ) J8-9 ) 8 Hz, 1H,
8-H); 7.90 (m, 4H, phthalyl, 9-H and 4-H); 8.05 (m, 3H, phthalyl
and 1-H); 8.15 (d, 1H, 7-H); 8.62 (d, J9-10 ) 8.8 Hz, 1H, 10-H).

282 Bioconjugate Chem., Vol. 21, No. 2, 2010
Gug et al.
779; 950; 986; 1042; 1200; 1236; 1214; 1235; 1258; 1329; 1434;
1474; 1528; 1621; 1679; 2857; 2871; 3015; 3331; 3365; 3378;
3600; 3729 cm-1. MS (E+) m/z: 351 [M + H]+; 373 [M +
Na]+.
phthalyl); 8.48 (d, 1H, 1-H); 8.55 (d, J9-10 ) 8.4 Hz, 10-H).
MS (E+) m/z: 673 [M + H]+; 695 [M + Na]+.
Removal of the Phthalyl Protecting Group: Preparation
of 7c-e. Hydrazine hydrate (0.2 mL) was added to a solution
of 6c-e (0.336 g, 0.5 mmoL) in DMF (1 mL). The mixture
was heated at 70 °C for 15 min and then cooled to rt. After
evaporation under vacuum, the solid residue was taken up with
CH2Cl2 (30 mL). The solution was washed with H2O (2 × 5
mL), dried, and concentrated. Compounds 7c-e were purified
by column chromatography (CH2Cl2-EtOH-NEt3, 95:5:0.1).
5-(6-tert-Butyloxycarbonylaminohexanoylamino)pentan-
1-ol (5b). Yield 67%. Mp 67-72 °C. Anal. (C15H30N2O4) C,
H, N. 1H NMR (CDCl3) δ 1.3 (m, 12 H, 6 × CH2); 1.5 (s, 9H,
(CH3)3); 2.1 (t, 2H, CH2CO); 3.03 (q, J ) 6.4 Hz, 2H, CH2NH);
3.20 (q, 2H, CH2NH); 3.55 (t, 2H, CH2OH); 4.55 (s, 1H,
NHCOO); 5.5 (s, 1H, CH2NHCO). MS (E+) m/z: 303[M + H]+;
325 [M + Na]+.
General Procedure for the Mitsunobu Reaction. Synthesis
of Conjugates (6c-e). Z-Aminocaproylaminopentanol (5a) (1.8
mmol, 0.63 g) and triphenylphosphine (1.8 mmol, 0.47 g) were
added to a solution of 2-(hydroxyphenanthridin-6-yl)-isoindole-
1,3-dione (4c-e) (1.8 mmol, 0.61 g) in THF (30 mL). The
mixture was cooled to 0 °C, and iso-propyl diazodicarboxylate
(1.8 mol, 0.378 g) was introduced under vigorous stirring. After
2 h at rt, the reaction mixture was concentrated and H2O (20
mL) was added. The aqueous suspension was extracted with
CH2Cl2 (3 × 15 mL), the organic layer was dried with Na2SO4,
and the solvent was evaporated. The final compound was
obtained after column chromatography (EtOAc-CH2Cl2-
toluene: 50-25-25).
(5-{5-[6-(1,3-Dioxo-1,3-dihydroisoindol-2-yl)-phenanthridin-
1-yloxy]-pentylcarbamoyl}-pentyl)-carbamic Acid Benzyl
Ester 6c. Yield 92%. Mp 134-137 °C. Anal. (C40 H40N4O6)
C, H, N. 1H NMR (CDCl3) δ 1.58 to 1.68 (m, 12H, (6 × CH2);
2.09 (t, J ) 7.4 Hz, 2H, CH2CO); 3.14 (q, J ) 6.4 Hz, 2H,
CH2NH); 3.32 (q, J ) 6.4 Hz, 2H, CH2NH); 4.32 (t, J ) 6.4
Hz, 2H, CH2O); 4.83 (br s, 1H, NH); 5.07 (s, 2H, CH2C6H5);
5.46 (br s, 1H, NH); 7.24 (d, J2-3 ) J3-4 ) 8 Hz, 1H, 2-H);
7.34 (m, 5H, C6H5); 7.68 (m, 2H, 3-H + 8-H); 7.82 (m, 4H,
phthalyl + 9-H + 4-H); 7.89 (d, J7-8 ) 8 Hz, 1H, 7-H); 8.02
(dd, J ) 5.6 Hz, J ) 3.2 Hz, phthalyl); 9.71 (d, J9-10 ) 8.4 Hz,
1H, 10-H). 13C NMR (CDCl3) δ 24.0; 25.4; 26.5; 29.2; 29.7;
29.8; 36.8; 39.6; 41.0; 66.8; 69.3; 110.4; 115.9; 123.3; 124.3;
124.4; 125.4; 127.6; 128.3; 128.5; 128.8; 129.0; 131.7; 132.4;
134.9; 135.3; 136.9; 145.4; 146.4; 156.7; 157.7; 167.6; 173.1.
IR (KBr) cm-1 478; 532; 671; 717; 771; 887; 964; 1033; 1080;
1157; 1273; 1373; 1458; 1535; 1635; 1689; 1728; 1913; 2314;
2862; 2931; 3063; 3302; 3749; 3865. MS (E+) m/z: 673 [M +
H]+; 695 [M + Na]+.
(5-{5-[6-(1,3-Dioxo-1,3-dihydroisoindol-2-yl)-phenanthridin-
2-yloxy]-pentylcarbamoyl}-pentyl)-carbamic Acid Benzyl
Ester (6d). Yield 65%. Mp 131-132 °C. Anal. (C40 H40N4O6)
C, H, N. 1H NMR (CDCl3) δ 1.50 to 1.52 (m, 12H, 6 × CH 2);
2.18 (t, J ) 7.4 Hz, 2H, CH2CO); 3.20 (q, J ) 6.4 Hz, 2H,
CH2NH); 3.25 (q, J ) 6.4 Hz, 2H, CH2NH); 4.22 (t, J ) 6.4
Hz, 2H, CH2O); 4.80 (brs, 1H, NH); 5.10 (s, 2H, CH2C6H5);
5.50 (brs, 1H, NH); 7.37 to 7.48 (m, 5H, C6H5); 7.54 (d, J3-4
) 8 Hz, 1H, 3-H); 7.68 (m, 2H, 4-H and 8-H); 7.89 (m, 3H,
phthalyl and 9-H); 7.95 (s, 1H, 1-H); 8.45 (dd, J ) 3.2 Hz, J )
5.6 Hz, phthalyl); 8.12 (d, J7-8 ) 8 Hz, 1H, 7-H); 8.64 (d, J9-10
) 8.2 Hz, 1H, 10-H). MS (E+) m/z: 673 [M + H]+; 695 [M +
Na]+.
6-Amino-1-(benzyloxycarbonylaminohexanoylaminopent-
yloxy)phenanthridine (7c). Yield 65%. Mp 147-151 °C. Anal.
1
(C32H38N4O4) C, H, N. H NMR (CDCl3) δ 1.51 to 1.72 (m,
12H, (6 × CH2); 2.05 (t, J ) 7.0 Hz, 2H, CH2CO); 3.20 (q, J
) 6.5 Hz, 2H, CH2NH); 3.38 (q, J ) 6.5 Hz, 2H, CH2NH);
4.23 (t, J ) 6.5 Hz, 2H, CH2O); 5.12 (s, 2H, CH2C6H5); 5.55
(br s, 3H, NH); 7.25 (d, J2-3 ) J3-4 ) 8 Hz, 1H, 2-H); 7.24
(m, 5H, C6H5); 7.73 (m, 2H, 3-H and 8-H); 7.82 (m, 2H, 9-H
and 4-H); 7.85 (d, J7-8 ) 8 Hz, 1H, 7-H); 9.45 (d, J9-10 ) 8.4
Hz, 1H, 10-H). MS (E+) m/z: 543 [M + H]+; 565 [M + Na]+.
6-Amino-2-(benzyloxycarbonylaminohexanoylaminopent-
yloxy)phenanthridine (7d). Yield 62%, Mp 118-122 °C. Anal.
1
(C32H38N4O4) C, H, N. H NMR (CDCl3) δ 1.47 to 1.62 (m,
12H, 6 × CH2); 2.23 (t, J ) 6.5 Hz, 2H, CH2CO); 3.20 (q, J )
6.5 Hz, 2H, CH2NH); 3.35 (q, J ) 6.5 Hz, 2H, CH2NH); 4.22
(t, J ) 6.4 Hz, 2H, CH2O); 4.85 (brs, 1H, NH); 5.12 (s, 2H,
CH2C6H5); 5.85 (br s, 2H, NH2); 7.32 (m, 5H, C6H5); 7.52 (d,
J3-4 ) 7.5 Hz, 1H, 3-H); 7.65 (m, 2H, 4-H + 8-H); 7.85 (m,
1H, 9-H); 7.96 (s, 1H, 1-H); 8.10 (d, J7-8 ) 8 Hz, 1H, 7-H);
8.54 (d, J9-10 ) 8.2 Hz, 1H, 10-H).
6-Amino-3-(benzyloxycarbonylaminohexanoylaminopent-
yloxy)phenanthridine (7e). Yield 48%. Mp: 123-125 °C. Anal.
(C32H38N4O4) C, H, N. 1H NMR (CDCl3) δ 1.42-1.69 (m, 12H,
6 CH2); 2.15 (t, J ) 7.3 Hz, 2H, CH2CO); 3.13 (q, J ) 6.4 Hz,
2H, CH2NH); 3.22 (q, J ) 6.4 Hz, 2H, CH2NH); 4.06 (t, J )
6.6 Hz, 2H, CH2OAr); 5.01 (s, 2H, CH2C6H5); 5.71 (br s, 2H,
NH2); 7.24 (m, 5H, C6H5); 7.39 (dd, J1-2 ) 8 Hz, J2-3 ) 2.4
Hz, 2-H); 7.52 (m, 2H, 4-H and 8-H); 7.81 (m, 2H, 7-H + 9-H);
8.38 (d, 1H, 1-H); 8.50 (d, J9-10 ) 8.2 Hz, 10-H). MS (E+)
m/z: 543 [M + H]+; 565 [M + Na]+.
Removal of the Z-group. Preparation of 3-(6-Amino-
caproylaminopentyloxy)-6-phtalimidophenanthridine (8e).
Pd/C 10% (0.1 g) and 35% hydrochloric acid (0.2 mL) were
added to a solution of 6e (0.336 g, 0.5 mmoL) in 10 mL EtOH
and 10 mL CH2Cl2. The mixture was stirred under H2
atmosphere (AP) for 12 h. The catalyst was removed by filtration
through a Whatman filter. The filter was rinsed once with 1
mL EtOH. The hydrochloride was isolated upon concentration
under vacuum and used in the immobilization step without
further purification.
{5-[5-(3,5-Dichloro-4-formylphenyloxy)-pentylcarbamoyl]-
pentyl}-carbamic Acid tert-Butyl Ester (11). Boc-aminocaproy-
laminopentanol (3 mmol, 0.906 g) and triphenylphosphine (3
mmol, 0.786 g) were added to a solution of 2,6-dichloro-4-
hydroxybenzaldehyde 10 (35) (3 mmol, 0.525 g) dissolved in
30 mL of THF. The mixture was cooled to 0 °C, and iso-propyl
diazodicarboxylate (3 mmol, 0.590 g) was introduced under
vigorous stirring. After 2 h at rt, the reaction mixture was
concentrated and H2O (20 mL) was added. The aqueous
suspension was extracted with CH2Cl2 (3 × 10 mL), and the
organic layer was dried with Na2SO4 and the solvent evaporated.
The final compound was obtained after column chromatography
(EtOAc-toluene: 50-50). Yield: 83%. Mp: 68-70 °C. Anal.
(5-{5-[6-(1,3-Dioxo-1,3-dihydroisoindol-2-yl)-phenanthridin-
3-yloxy]-pentylcarbamoyl}-pentyl)-carbamic Acid Benzyl
Ester (6e). Yield 97%. Mp 155-157 °C. Anal. (C40 H40N4O6)
C, H, N. 1H NMR (CDCl3) δ 1.41-1.67 (m, 12H, 6 CH2); 2.12
(t, J ) 7.4 Hz, 2H, CH2CO); 3.11 (q, J ) 6.4 Hz, 2H, CH2NH);
3.19 (q, J ) 6.4 Hz, 2H, CH2NH); 3.24 (t, J ) 6.4 Hz, 2H,
CH2OAr); 4.81 (br s, 1H, NH); 5.01 (s, 2H, CH2C6H5); 5.71
(brs, 1H, NH); 7.24 (m, 5H, C6H5); 7.32 (dd, J1-2 ) 8 Hz, J2-3
) 2.4 Hz, 2-H); 7.55 (m, 2H, 4-H and 8-H); 7.81 (m, 4H,
phthalyl and 7-H and 9-H); 7.98 (dd, J ) 3.2 Hz, J ) 5.6 Hz,
1
(C23H34Cl2N2O5) C, H, N. H NMR (CDCl3) δ 1.52 (s, 9H,
C(CH3)3); 1.36-1.71 (m, 10H, CH2); 1.86 (q, 2H, CH2); 2.20
(t, 2H, J ) 7.5 Hz, CH2CO); 3.12 (dd, 2H, J ) 6.3 Hz, CH2NH);
3.29 (dd, 2H, J ) 6.3 Hz, CH2NH); 4.04 (t, 2H, J ) 6.1 Hz,

Antiprion Conjugates
Bioconjugate Chem., Vol. 21, No. 2, 2010 283
Scheme 1. Preparation of Benzyloxy-6-aminophenanthridines 1c-ea
a (a) NaNH2, LiNH2, NH3; (b) Na2CO3, Pd[P(C6H5)3], dioxane, H2O.
CH2O); 4.56 (br s, 1H, NH); 5.54 (br s, 1H, NH); 6.92 (s, 2H,
H-3 + H-5); 10.44 (s, 1H, CHO).
pended in homogenization buffer (300 µL/100 mL of culture)
and lysed using acid-washed glass beads (purchased from Sigma,
300 µL/100 mL of culture). Homogenates were vortexed for
30 s followed by 30 s ice-cooling (six times) and then
centrifuged for 3 min at 3000 rpm at 4 °C. Supernatants were
recovered, assayed for protein content (using Bio-Rad protein
assay), and immediately loaded batchwise on the affinity
matrices. Just before use, 6AP and GA beads were washed with
1 mL of bead buffer (50 mM Tris-HCl (pH 7.4), 5 mM NaF,
250 mM NaCl, 5 mM EDTA, 5 mM EGTA, 0.1% Nonidet P-40,
10 µg/mL of leupeptin, aprotinin, and soybean trypsin inhibitor,
and 100 µM benzamidine) and diluted 4 times in this buffer.
200 µg of yeast protein extract were added to 40 µL of diluted
beads (10 µL of packed beads). The volume was adjusted to
600 µL by adding bead buffer, and the tubes were rotated at 4
°C for 30 min. Competitions with active or inactive drugs were
performed with free compounds (or the corresponding volume
of DMSO) at a final concentration of 1 mM incubated with
cell extracts for 3 min before the mixture was added to the
affinity matrix. Then, bead buffer containing the same quantity
(1 mM) of the corresponding free compound was added
immediately (as described previously) to reach a final volume
of 600 µL. After incubation, a brief spin at 10 000 g at 4 °C,
and removal of the supernatant, the beads were washed 4 times
with bead buffer before addition of 35 µL of 2× Laemmli
sample buffer. Following heat denaturation for 3 min at 95 °C,
bound proteins were analyzed by 10% SDS-PAGE as described
below.
{5-[5-(3,5-Dichloro-4-benzylidenaminoguanidine)-pentyl-
carbamoyl]-pentyl}-carbamic Acid tert-Butyl Ester (12).
Aminoguanidine hydrochloride (0.22 g, 2 mmol) was added to
a solution of conjugate 11 (0.98 g, 2 mmol) in 10 mL EtOH
followed by the addition of AcONa, 5H2O (0.27 g, 2 mmol).
After 2 h refluxing, the hot mixture was filtered, and 12
precipitated upon cooling to rt. Yield: 54%. Anal.
1
(C24H38Cl2N6O4) C, H, N. H NMR (CDCl3) δ 1.24-1.63 (m,
10H, 5 × CH2); 1.43 (s, 9H, C(CH3)3); 1.74 (q, 2H, CH2); 2.17
(t, J ) 7 Hz, 2H, CH2CO); 3.08 (q, J ) 6.03 Hz, 2H, CH2NH);
3.25 (q, 2H, CH2NH); 3.95 (t, J ) 6 Hz, 2H, CH2-O); 5.79
(br s, 2H, NH2); 6.35 (br s, 1H, NH); 6.88 (s, 2H, 3-H and
5-H); 8.36 (s, 1H, NCH). 13C NMR (CDCl3) δ 23.0; 25.1; 26.1;
28.2; 28.3; 29.0; 29.4; 36.1; 39.0; 68.0; 78.6; 115.2; 123.9;
134.8; 141.4; 155.9; 157.9; 160.6; 172.9.
2,6-Dichloro-4-(ε-aminocaproylaminopentyloxy)-benzy-
lidenaminoguanidine) Trifluoroacetate (13). CF3COOH (1
mL) was added to a solution of 12 (0.27 g, 0.5 mmol) in CH2Cl2
(10 mL). The solution was stirred for 2 h at rt and then
concentrated until dryness. The viscous oil crystallized upon
trituration with 2 mL cyclohexane followed by trituration with
2 mL Et2O. Yield 76%, mp 75-78 °C. 1H NMR (DMSO-d6) δ
1.21-1.88 (m, 12 H, 6 × CH2); 2.21 (t, J ) 7 Hz, 2H, CH2CO);
3.03 (m, 4H, 2CH2NH); 4.02 (t, J ) 6 Hz, 2H, CH2-O); 6.23
(m, 4H, NH); 6.84 (s, 2H, 3-H and 5-H); 8.25 (s, 1H, NCH).
Immobilization of Ligands on Activated Sepharose
Beads. Ligands were attached to CNBr-activated Sepharose 4
fast flow from Amersham Pharmacia according to the manu-
facturer’s directions. A solution of 0.1 mmol ligands 8e or 13
in 2 mL DMSO was added to 38 mL pH 8.3 solution (0.02 M
NaHCO3 + 0.1 M NaCl). This solution (0, 1, 2, 5 mL) was
added to approximately 1 mL of beads suspended in pH 8.3
solution (final volume in each cartridge 10 mL). The cartridges
were allowed to shake for 12 h at rt. The solutions were then
drained from the beads, and 10 mL ethanolamine buffered
solution (pH 8.5) was added to each cartridge, which was
allowed to shake for 3 h at rt. This quenching step was repeated
once. The phthalyl protecting group of 6AP-beads was then
removed. A 3 mL solution of H2O, DMF, and hydrazine hydrate
(1:1:1) was added to the cartridges. After 15 min shaking, the
solution was drained and the beads washed 3 times with 10
mL buffer. Finally, 10 mL buffer containing 1 g ·L-1 NaN3 was
added to each cartridge, which was stored at 4 °C.
Electrophoresis and Silver Staining. The proteins bound
to 6AP or GA matrices were separated by 10% SDS-PAGE
(precast NuPAGEsInVitrogens1 mm thick gel) followed by
silver staining using a GE Healthcare Life Sciences SDS-PAGE
silver staining kit.
RESULTS
Preparation of 6-Aminophenanthridine Beads. The syn-
thesis of the conjugates of 6AP is depicted in Schemes 1 and
2. Condensation of 2- or 3-benzyloxyaniline with 2-chloroben-
zonitrile in liquid ammonia using metal amides (32) led to
benzyloxy-6-aminophenanthridine 1c-e. The mixture of 1c and
1e could not be separated on column chromatography. Alter-
natively, 3-benzyloxy-6-aminophenanthridine 1e was obtained
in a Suzuki-Myaura coupling procedure (33). Phthalylation of
6-aminophenanthridines 1c-e afforded derivatives 3c-e. The
benzyl group was removed by catalytic hydrogenation to afford
phenols 4c-e. The spacer arms 5 were obtained by acylation
of 5-aminopentanol with Boc or Z-ε-aminocaproic acid. Con-
densation of phenols 4c-e with spacer 5a under Mitsunobu
reaction conditions led to conjugates 6c-e. Finally, the phtal-
imido protecting group was eliminated to afford 8c-e. Follow-
ing the evaluation of 8c-e in the antiprion test, the removal of
Affinity Chromatography Experiments on Immobilized
6AP and GA. These experiments were performed as previously
described (31). Briefly, yeast (Saccharomyces cereVisiae) cell
extracts were prepared using homogenization buffer (25 mM
Tris-HCl (pH 7.4), 100 mM NaCl, 0.2% Triton X-100, and 1
mM PMSF). Yeast protein extracts were obtained from a culture
growing at 30 °C (OD600
) 0.8). Cell pellets were resus-
nm

284 Bioconjugate Chem., Vol. 21, No. 2, 2010
Gug et al.
Scheme 2. Synthesis of 6AP Conjugatesa
a (a) Phthalic anhydride, NEt3, THF; (b) H2, Pd/C; (c) (C6H5)3P, di-iso-propyl diazodicarboxylate, THF; (d) hydrazine hydrate, H2O, DMF.
Scheme 3. Synthesis of 6AP Beadsa
a (a) H2 Pd/C, HCl, EtOH; (b) Sepharose preactivated beads; (c) hydrazine hydrate, H2O, DMF.
the Z-group was performed only on 6e, which bears the spacer
arm in position 3.
Preparation of Guanabenz Beads. The conjugate was easily
obtained from 2,6-dichloro-4-hydroxybenzaldehyde 10 (35).
Coupling of the Boc-protected spacer arm 5b was achieved
under Mitsunobu conditions leading to conjugate 11. This
aldehyde gave the protected GA conjugate 12 upon refluxing
with aminoguanidine in EtOH. Acidic cleavage of the Boc
protecting group led to the trifluoroacetate 13, which was
eventually coupled to Sepharose preactivated beads to obtain
GA-beads 14 (Scheme 4).
Elimination of the Z protecting group of 6e led to the
conjugate 8e. The immobilization of conjugate 8e was performed
on commercially available preactivated beads (CNBr). Several
ratios of ligand conjugated to the Sepharose beads were tested.
In the following affinity chromatography experiments, optimal
results were obtained with the matrices made using the bigger
volume of ligand solution (5 mL). Residual activated groups
were quenched by aminoethanol. Finally, the phthalyl protective
group was removed by hydrazinolysis. Optimization of reaction
conditions led to the selection of a DMF, H2O, hydrazine hydrate
(1:1:1) mixture (Scheme 3).
Affinity Chromatography Assays. The ability of CPZ and
QC to compete for ribosomal components binding on 6AP and
GA beads is depicted in Figure 2. As can be seen, QC (lanes

Antiprion Conjugates
Bioconjugate Chem., Vol. 21, No. 2, 2010 285
Scheme 4. Preparation of GA Beadsa
a (a) (C6H5)3P, di-iso-propyl diazodicarboxylate, THF; (b) aminoguanidine, EtOH; (c) CF3COOH, CH2Cl2; (d) Sepharose preactivated beads.
6), but not CPZ (lanes 5), was able to compete for the binding
of ribosomal components to both 6AP and GA beads.
First, both 6AP and GA showed specific interactions with
ribosomal components: most of the proteins specifically bound
to 6AP or GA beads turned out to be ribosomal proteins. The
binding of these ribosomal proteins was specific, as they were
absent from control beads (Figure 2, panel B, lane 1 in both
gels), present on 6AP or GA beads (lane 2) and respectively
competed away by free 6AP (left gel, lane 3) or by free GA
(right gel, lane 3) but not by free 6APi (left gel, lane 4) or by
free GAi (right gel, lane 4). The interaction of both 6AP and
GA with ribosomal components was then shown to be RNA-
dependent, as a treatment of the cellular extracts with RNase A
before affinity chromatography purification completely abolished
the binding of all ribosomal proteins. 6AP and GA were further
shown to selectively interact with the domain V of the large
rRNA (rRNA) of the large subunit of the ribosome. Therefore,
the binding of ribosomal components to 6AP or GA beads is
best explained by postulating that, in affinity chromatography
experiments, immobilized 6AP or GA retains the entire ribosome
due to the ability to specifically interact with domain V of the
large rRNA. Interestingly, in parallel to their ability to specif-
ically interact with domain V of the large rRNA, both 6AP and
GA were shown to specifically inhibit its protein folding activity.
Indeed, in addition, to bear the peptidyl transferase activity
involved in translation, domain V of the large rRNA of the large
subunit of the ribosome has also been shown to be able to assist
protein folding, at least in Vitro (36). We thus found that both
6AP and GA inhibit this ribosome-borne protein folding activity
(RPFA) without any effect on the peptidyl transferase activity
of the ribosome. Both 6APi (1b) and GAi (2b), which are
inactive as antiprion chemical derivatives of, respectively, 6AP
and GA, were unable to compete for the binding of ribosomal
components to both 6AP and GA beads and also to inhibit RPFA
(30). We thus proposed that the antiprion effect of both 6AP
and GA could be due to their ability to inhibit the RPFA, and
thus, RPFA could be involved in prion propagation. Therefore,
DISCUSSION
We have previously described two single-step routes for the
synthesis of 6APs (32, 33). In the present work, these two
approaches were used. In preliminary structure-activity studies,
we noticed that derivatives substituted on the amino group were
devoid of any antiprion activity. Therefore, 1b was selected as
an inactive control useful for the affinity chromatography assays.
This also indicates that the spacer arm should not be linked to
the 6-amino group. Further, this amino group of 6AP appeared
to be rather nucleophilic and then, if unprotected, would likely
react in the coupling procedure on the activated Sepharose beads.
In order to circumvent this major problem, a protective group
which could be eliminated after grafting the conjugate to the
beads was required. It implies that the deprotection of the
6-amino group of 6AP derivatives should be achieved under
mild conditions. The phthalyl protective group fulfilled this
requirement. The antiprion activity of conjugates 7c-e has been
evaluated in the yeast prion-based assay. Only the 7e derivative
substituted in position 3 retained some antiprion activity of 1a,
whereas conjugates 7c and 7d, substituted in position 1 or 2
respectively, were totally inactive (31). Therefore, 8e was
selected for the affinity chromatography study. Concerning GA
beads, we noticed that arginine-containing peptides have been
previously grafted on Sepharose beads. In these assays, the
unprotected amidine group did not interfere in the coupling
process (34). Therefore, in contrast to the preparation of 6AP
beads, it was estimated that the amidine group of Guanabenz
conjugate did not need to be protected.
Affinity chromatography experiments were performed with
both 6AP and GA beads. Interestingly, although the two
compounds are chemically unrelated, they were both found to
interact with the ribosome in an RNA-dependent manner (31).

286 Bioconjugate Chem., Vol. 21, No. 2, 2010
Gug et al.
Figure 2. Competition experiments with Chlorpromazine (CPZ) and Quinacrine (QC). A. Molecular structures of CPZ and QC were depicted. B.
Extracts from yeast were incubated with 6AP beads (left panel) or GA beads (right panel). The beads were then washed extensively and the bound
proteins analyzed by SDS-PAGE. Proteins were then silver-stained. Most of the bands were previously identified as ribosomal proteins from both
the small and large subunits of the ribosome. In both gels, lane 1 corresponds to chromatography using control beads without 6AP (left gel) or GA
(right gel), and lane 2 to chromatography using 6AP (left gel) or GA (right gel) beads. Note that, in both cases, the patterns of eluted ribosomal
proteins were rather similar. In both gels, lanes 3 to 6 correspond to competition experiments with the indicated free compounds. Note that QC, but
not CPZ, was able to compete for the binding of ribosomal components to both 6AP and GA beads.
we were interested in determining if other known antiprion drugs
could compete for the binding of ribosomal components on 6AP
and GA beads when added to a mix of 6AP or GA beads and
cellular extracts. This possibility is particularly appealing, as
free 6AP was shown to efficiently compete for the binding of
ribosomal components to GA beads, and vice versa (31), clearly
indicating that they share overlapping binding sites on rRNA,
which was indeed recently shown to be the case (37). Therefore,
the ability of other unrelated antiprion drugs to compete for
the binding of the ribosome to 6AP and GA beads would be an
indication that these compounds share at least some common
binding sites on domain V of the large rRNA of the large
ribosomal subunit with 6AP or GA. Hence, all these molecules
could share the same biological target accounting for their
antiprion effect. Quinacrine (QC) and Chlorpromazine (CPZ)
are two drugs already in use in humans for the treatment of
malaria and psychosis, respectively. They were recently isolated
as active ex ViVo against mammalian prion in a N2a cell-based
model (8) and the latter against yeast prions (13). The ability
of CPZ and QC to compete for ribosomal components binding
on 6AP and GA beads was determined (Figure 2). Interestingly,
whereas QC was clearly able to efficiently compete for the
binding of ribosomal components to both 6AP and GA beads
(at least as efficiently as 6AP and GA themselves), CPZ was
not, as observed for 6APi and GAi, derivatives of 6AP and GA
that are inactive as antiprion drugs. These results suggest that
QC, but not CPZ, could share at least some binding sites with
6AP and GA on the large rRNA. The fact that CPZ was unable
to compete for the binding of rRNA on 6AP and GA beads
suggests that either it has other biological target(s) or it also
targets the ribosome and possibly also the large rRNA but on
different and nonoverlapping binding sites than 6AP and GA.
CONCLUSION
In the present study, we have used a simple chemical spacer
arm easy to introduce by Mitsunobu reaction. Therefore, the
introduction of the linker requires the synthesis of phenols
obtained from their benzyl ether precursors compatible them-
selves with the various reaction conditions used in the synthesis
of the tricyclic phenanthridine moitie. This spacer arm can be
considered less hydrophilic than classical polyoxyethylenic
linkers, thus greatly facilitating the purification of the conjugates
by column chromatography. The two hydrophobic parts of the
spacer should not be able to establish interactions either with
the bioactive molecule or with the hydrophilic Sepharose beads.
The amide bond in the middle of the linker reduces the flexibility
of the spacer. To the best of our knowledge, the deprotection
of functional groups after grafting on the beads has not been
previously reported. Using this procedure, we synthesized
affinity chromatography matrices for both 6AP and GA, two
antiprion drugs active from yeast to mammals. The identification
of the protein folding activity of the ribosome as a common
target of both compounds suggests the intriguing possibility that
this largely unknown function borne by the large rRNA could
be involved in prion propagation. Finally, it has been recently
found that PrPC is an amyloid-ꢀ oligomers receptor (38).

Antiprion Conjugates
Bioconjugate Chem., Vol. 21, No. 2, 2010 287
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ACKNOWLEDGMENT
We thank Sophie Mairesse-Lebrun for the microanalyses
determinations of compounds and the ANR “blanche” from the
French Ministe`re de la Recherche for financial support. DTT
was supported by a PhD program from the French “Ministe`re
de la Recherche” and CV by a Junior Contract from INSERM.
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