Design, synthesis, and structure - Activity relationship of indole-3-glyoxylamide libraries possessing highly potent activity in a cell line model of prion disease

Article abstract of DOI:10.1021/jm900920x

Transmissible spongiform encephalopathies (TSEs) are a family of invariably fatal neurodegenerative disorders for which no effective curative therapy currently exists. We report here the synthesis of a library of indole-3-glyoxylamides and their evaluation as potential antiprion agents. A number of compounds demonstrated submicromolar activity in a cell line model of prion disease together with a defined structure-activity relationship, permitting the design of more potent compounds that effected clearance of scrapie in the low nanomolar range. Thus, the indole-3-glyoxylamides described herein constitute ideal candidates to progress to further development as potential therapeutics for the family of human prion disorders. 2009 American Chemical Society.

Full text of DOI:10.1021/jm900920x

J. Med. Chem. 2009, 52, 7503–7511 7503
DOI: 10.1021/jm900920x
Design, Synthesis, and Structure-Activity Relationship of Indole-3-glyoxylamide Libraries Possessing
Highly Potent Activity in a Cell Line Model of Prion Disease
Mark J. Thompson, Vinciane Borsenberger, Jennifer C. Louth, Katie E. Judd,† and Beining Chen*
Department of Chemistry, University of Sheffield, Brook Hill, Sheffield, S3 7HF, U.K. †Present address: Department of Pharmacy and
Pharmacology, University of Bath, Bath, BA2 7AY, U.K.
Received June 22, 2009
Transmissible spongiform encephalopathies (TSEs) are a family of invariably fatal neurodegenerative
disorders for which no effective curative therapy currently exists. We report here the synthesis of a
library of indole-3-glyoxylamides and their evaluation as potential antiprion agents. A number of
compounds demonstrated submicromolar activity in a cell line model of prion disease together with a
defined structure-activity relationship, permitting the design of more potent compounds that effected
clearance of scrapie in the low nanomolar range. Thus, the indole-3-glyoxylamides described herein
constitute ideal candidates to progress to further development as potential therapeutics for the family of
human prion disorders.
Introduction
species.8 Evidence for multifaceted normal function is emer-
ging, with apparent roles for PrPC in neuroprotection,8 cell
adhesion,9 and iron metabolism,10 among others.
Prion diseases, or transmissible spongiform encephalopa-
thies (TSEsa), are a group of rare neurodegenerative disorders
affecting both humans and animals. They may occur as
sporadic, inherited, or iatrogenic diseases, which progress
rapidly after onset of symptoms and are invariably fatal.
The most common such condition affecting humans is
Creutzfeldt-Jakob Disease (CJD),1 though other human
prion disorders have been characterized, most notably variant
Whereas exact mechanisms of infectivity and pathogenesis
in prion disease are far from fully elucidated, significant work
by Mallucci et al.11 confirmed the neurotoxicity of PrPSc and
demonstrated that expression of PrPC in host neurons is
required for PrPSc replication and, hence, disease progression.
As such, persistently infected cell lines acting as a host for
PrPSc are frequently used as an in vitro model of prion
disease.12 In these cells, active replication of the scrapie
protein occurs, leading to its accumulation in readily detect-
able amounts. An infected mouse neuroblastoma-derived cell
line, ScN2a,13 has gained widespread use for in vitro screening
of potential antiprion agents, as has a scrapie mouse brain
(SMB) cell line, cloned from culture of murine brain infected
with the Chandler scrapie strain.14
A handful of compounds showing potent in vitro clearance
of PrPSc have been evaluated clinically for treatment of TSEs
(Figure 1) but with little success to date. The antimalarial
quinacrine failed to demonstrate any clear beneficial effect
when administered compassionately to patients with CJD,15 a
finding confirmed by the more recent patient-preference trial
termed “PRION-1”,16 wherein it was also concluded that
quinacrine did not significantly affect the clinical course of
prion disease. These failings in the clinic have hypothetically
been attributed to an unfavorable pharmacokinetic profile of
the drug.17
2
3
€
CJD (vCJD), Gerstmann-Straussler-Scheinker syndrome
(GSS), and familial fatal insomnia4 (FFI). A number of TSEs
also affect various animal species, including scrapie5 in sheep,
bovine spongiform encephalopathy6 (BSE) in cattle, and
chronic wasting disease7 (CWD) in deer and elk.
A common feature of TSEs is the deposition of insoluble
aggregates of disease-associated prion protein (PrPSc), the
post-translationally refolded and partially protease-resistant
isoform of normal cellular prion protein (PrPC), a glycosyl-
phosphatidylinositol (GPI)-anchored cell surface protein.
These observed deposits of PrPSc are thought, either directly
or indirectly, to be the cause of neuronal cell death in TSEs, a
process that leads to formation of vacuoles and ultimately
results in the characteristic spongiform degeneration of brain
tissue. The normal biological function of PrPC remains in-
completely defined, but it is expressed predominantly in
neurons and is highly conserved across all mammalian
In other studies, a double-blind, placebo-controlled trial of
flupirtine suggested this compound slowed cognitive decline,
but it did not markedly extend survival.18 Preliminary results
with pentosan polysulfate (PPS) showed evidence for slowing
of disease progression,19 though further studies are necessary
to confirm these findings. Treatment with doxycycline was
found to reduce prion infectivity and offer the prospect of
significantly longer survival of CJD patients,20 though a
formal clinical trial has not yet concluded.21 As is apparent,
*To whom correspondence should be addressed. Phone: þ44 114 222
9467. Fax: þ44 114 222 9346. E -mail: b.chen@sheffield.ac.uk.
a Abbreviations: TSE, transmissible spongiform encephalopathy;
CJD, Creutzfeldt-Jakob disease; vCJD, variant Creutzfeldt-Jakob
€
disease; GSS, Gerstmann-Straussler-Scheinker syndrome; FFI, famil-
ial fatal insomnia; BSE, bovine spongiform encephalopathy; CWD,
chronic wasting disease; PrPC (or PrP-sen), normal cellular prion
protein; PrPSc (or PrP-res), disease-causing isoform; GPI, glycosylpho-
sphatidylinositol; SMB, scrapie-infected mouse brain; ScNB, scrapie
agent-infected neuroblastoma cells; PPS, pentosan polysulfate; SAR,
structure-activity relationship; SPR, surface plasmon resonance.
r
2009 American Chemical Society
Published on Web 10/20/2009
pubs.acs.org/jmc

7504 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23
Thompson et al.
Figure 2. (a) Indole-3-glyoxylamides that displayed antiprion
activity during cell-line screening of a commercially sourced com-
pound library. (b) Related indole-3-glyoxylamide derivatives in
ongoing clinical evaluation.
Figure 1. Existing drugs that have been investigated as potential
therapeutics in human prion disease.
Scheme 1. Preparation of Indole-3-glyoxylamide Library
Membersa
disappointingly little progress has been made in the therapy of
TSEs despite considerable effort.22 There is still a pressing
need for potent antiprion agents that retain their efficacy in
vivo and consequently exert an obvious clinical benefit.
Many classes of compound have exhibited in vitro activity,
including 9-aminoacridine derivatives related to quinacrine,23
2-aminopyridine-3,5-dicarbonitriles,24 azobenzene and its
analogues,25 N0-benzylidenebenzohydrazides,26 2,4-diphenyl-
thiazoles and oxazoles,27 and a small series of pyrazolone
derivatives,28 among others. Despite the relatively large num-
ber of potential therapeutics characterized by cell line screen-
ing, very few have been progressed to animal studies, and
those that were failed to retain any remedial efficacy in vivo.29
Indeed, the groups of compounds listed above are generally
not ideal as drug candidates, since they have insufficient
potency for in vivo activity (typically low-micromolar EC50
values in vitro), lack a discernible structure-activity relation-
ship (SAR), or exert cytotoxic effects at relatively low con-
centrations, narrowing the therapeutic window. A require-
ment evidently exists for access to potently active compounds
that are noncytotoxic, are suitably “druglike” in nature,30 and
exhibit a well-defined SAR, this being particularly desirable
because it would suggest a single, definite mode of action that
may be deduced by further study.
We herein present preliminary results regarding a library
of compounds with obvious potential to satisfy the above
criteria, in addition to comprising a novel structural class of
antiprion agents. During evaluation of a library of commer-
ciallysourced screeningcompoundsin the SMB cellline assay,
two indole-3-glyoxylamides 1 and 2 (Figure 2a) were con-
firmed as active inhibitors of PrPSc accumulation, displaying
EC50 values of 1.5 and 6.4 μM, respectively. The indole-
3-glyoxylamides are in fact a medicinally significant class of
compounds, represented clinically by GW 842470 (reportedly
under investigation as a treatment for atopic dermatitis31) and
indibulin (D-24851), a potent tubulin polymerization inhibi-
tor32 that recently completed a phase I clinical trial in patients
with solid tumors33 (Figure 2b).
a Reagents and conditions: (i) oxalyl chloride, Et2O or THF, room
temp, 1 h; (ii) R3R4NH, iPr2NEt, room temp, 3-18 h.
Chemistry
Synthesis of the library members was based upon known
protocols34 and proved relatively straightforward (Scheme 1).
Both acylation of the 3-position of indole by oxalyl chloride
andsubsequentreactionofthe glyoxylylchloride intermediate
with various amines were carried out in a one-pot format in
parallel with few problems. During the course of library
synthesis from unsubstituted indole (R1 = R2 = H), THF
was found to offer advantage over ether as solvent owing to
increased solubility of the amines and glyoxylamide products.
Recently, another group independently reported similar
results during synthesis of antibacterials containing indole-
3-glyoxylamide substituents;35 thus, most of the compounds
in the present study were synthesized using THF as solvent.
After workup, library members were purified as necessary by
recrystallization, column chromatography, or a combination
of both. Some of the products required two rounds of
recrystallization from different solvent systems in order to
achieve a good degree of purity.
Considering the structures of 1 and 2, we chose to focus
initial library design around varying substitution at positions
1-3 of the indole system, particularly with respect to glyoxyl-
amides derived from a range of amines at the 3-position.
Synthesis of 18, derived from 3-(hydroxymethyl)aniline,
required protection of the more nucleophilic alcohol function;

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23 7505
Scheme 2. Preparation of Hydroxy-Containing Compounds
Scheme 3. A More Expedient, Transient TMS Protection Stra-
tegy for Additional Library Members Derived from 3-(Hydro-
xymethyl)anilinea
Required a Suitable Protection Strategy for the Alcohola
a Reagents and conditions: (i) TBDMS-Cl, imidazole, DMF, 0 °C, 18
h, 70% (n = 1), or TBDMS-Cl, imidazole, THF, room temp, 30 min,
56% (n = 0); (ii) iPr2NEt, THF, room temp, 3 h, 19% (n = 1) or 23%
(n = 0); (iii) TBAF, THF, room temp, 1 h, 63% (n = 1) or 80% (n = 0, as
tetrabutylammonium salt).
a Reagents and conditions: (i) TMS-Cl, THF, room temp, 1 h; (ii)
iPr2NEt, THF, room temp, 3 h; (iii) 1 M HCl.
passaged using 0.05% trypsin and 0.002% EDTA at a split
ratio of 4. To assess the effects of compounds, cells were
distributed into 96-well cluster plates at 3 ꢀ 104 cells per well
and incubated for 24 h to allow for cell attachment. The
compounds were prepared at 400 times the required concen-
tration in DMSO as stock solutions and then transferred, at a
20-fold dilution, into Hank’s balanced salt solution. This
solution was then transferred at a further 20-fold dilution
into the cell medium. The cells were incubated with the
compound-containing medium for 5 days. After this time,
cell viability was assessed by the MTT assay following the
standard protocol supplied with the reagent (Sigma). For dot
blot analyses, cells were extracted using lysis buffer (10 mM
Tris-HCl [pH 7.6], 100 mM NaCl, 10 mM EDTA, 0.5% v/v
NP40, and 0.5% w/v sodium deoxycholate), and the content
of the well was loaded onto a nitrocellulose membrane
(0.45 μm) under gentle vacuum at a total cellular protein
concentration of approximately 30-40 μg/well (determined
bythe Bradfordassayfollowing the protocol supplied withthe
reagent; Sigma). The membrane was air-dried and subjected
to 75 μg mL-1 proteinase K digestion for 1 h at 37 °C. The
reaction was stopped with 1 mM phenylmethylsulfonyl fluor-
ide (PMSF) in 20 mM Tris-HCl-buffered saline (TBS) and the
membrane washed extensively with TBS and immersed in
1.8 M guanidine thiocyanate in TBS for 10 min at room
temperature. After further washing with TBS, the membrane
was blocked using 5% fat-free milk powder in phosphate-
buffered saline (PBS), processed with 0.2 μg mL-1 mouse
monoclonal anti-PrP 6H4 (Prionics), and developed using an
ECL kit (Amersham Pharmacia Biotech).
thus, O-TBDMS derivative 35a was prepared and utilized in
the glyoxylamide formation step (Scheme 2, n=1). Removal
of the silyl protecting group from 36a proceeded smoothly
giving the desired hydroxymethyl compound 18.
In order to investigate the effect of substitution at N-1 and
C-2, libraries derived from 1-methylindole (R1=Me, R2=H)
and 2-methylindole (R1=H, R2 =Me) were also prepared,
using a subset of the amines employed in the synthesis of the
initial library. Compounds containing a 2-methyl group were
typically isolated in relatively low yield (perhaps due to
increased steric demand on the amide coupling step), but
nonetheless, they were obtained in sufficient quantities for
screening.
Structures derived from 3-(hydroxymethyl)aniline were
again included among the library members synthesized using
1- and 2-methylindole, though this time a transient protection
approach was employed. The O-TMS protected aniline build-
ing block 37 was generated in situ and reacted immediately
with an indole-3-glyoxylyl chloride to afford either 38 or 39
(Scheme 3). Acidic workup cleaved the silyl group, providing
controlled access to screening compounds 39 and 40 without
the need for isolation of any synthetic intermediates.
Screening (SMB Cells) Methodology
Compounds were screened for inhibition of PrPSc forma-
tioninSMBcellsofmesodermal origin essentially asdescribed
previously.27 The procedure was based upon that reported by
Rudyk et al.36 A persistently infected mouse cell line (SMB),
cloned originally from scrapie infected mouse brain but of
non-neuronal origin,14a was used. Cells were grown in tissue
culture-treated plastic dishes in medium 199 (phenol red free),
supplemented with 10% newborn calf serum (heat inacti-
vated), 5% fetal calf serum (heat inactivated), and penicillin-
streptomycin at 10 mg L-1 at 37 °C in an atmosphere of 5%
CO2 in air at 95% relative humidity. Medium was changed
every third or fourth day, and every 7 days confluent cells were
Every experiment was carried out in triplicate and an
average value for PrPSc concentration calculated, relative to
an untreated control (DMSO only), together with a standard
deviation. Curcumin was employed as a positive control and
effected essentially complete clearance of PrPSc at the con-
centration used (10 μM). Test compounds were initially
screened at 1, 10, and 20 μM and were considered to be active
if PrPSc levels were reduced to less than 70% of that of the
untreated control after 5 days’ exposure. The amounts of

7506 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23
Thompson et al.
Table 1. Initial Screening Results and EC50 Values for Indole-3-glyoxylamides 1-34 in the SMB Cell Line Assay
a %PrPSc remaining, relative to an untreated control, after 5 days of exposure to the test compound at the concentration specified (μM). b Results are
listed for the lowest concentration showing activity (<70% of control). For inactive compounds, the result at the highest nontoxic concentration tested
is given. c Toxic at 20 μM. d Yield over two steps (see Scheme 2). e Each EC50 determination was carried out in triplicate and each compound assessed in at
least two independent experiments. Full details for each active compound presented in Tables 1-3 are included in the Supporting Information, including
individual dose-reponse curves and standard deviation of the reported EC50
.
PrPSc remaining as a percentage relative to the control are
given in Tables 1-3. Compounds showing activity were
rescreened over a range of concentrations to determine an
EC50 value, such experiments being repeated at least twice (in
triplicate) to validate the results so obtained. Two well
documented antiprion agents were also investigated as posi-
tive controls. Observed EC50 values of 0.95 ( 0.07 and 0.42 (
0.12 μM were obtained for curcumin and quinacrine, respec-
tively, in the SMB cells. Reported values are 0.01 μM for
curcumin (in ScNB cells)37 and range from 0.3 to 0.5 μM for
quinacrine (in ScN2a cells).38 As such, in the SMB cell model,
quinacrine displays the same degree of efficacy as in other
prion-infected cell lines whereas curcumin is 100-fold less
potent than described elsewhere.
(20-22) amines were all inactive. Furthermore, library
members synthesized from secondary amines (32-34) were
also ineffective. The potent activity of 3, derived from ani-
line, was completely abolished by introducing a methyl
group at the glyoxylamide position (32).
Indeed, perhaps the most unexpected result of all was the
potent activity of unsubstituted phenyl compound 3, ob-
served to have an EC50 of 0.32 μM. Meta-substituted deri-
vatives, including parent compounds 1 and 2, possessed
comparable or lower efficacy; only substitution at the
para-position improved upon the activity of phenyl com-
pound 3. Specifically, p-chloro analogue 10 (EC50=65 nM),
p-methoxy derivative 13 (EC50=11 nM), and p-fluoro deri-
vative 16 (EC50=64 nM) were the most potent candidates
identified within this initial screening set. Ortho-substitution
was clearly not tolerated, as compounds 4 (o-Me), 9 (o-Cl),
and 11 (o-OMe) all proved ineffective.
Results and Discussion
Elucidation of Structure-Activity Relationship. Screening
results for the initial compound library derived from unsub-
stituted indole are shown in Table 1. Examination of the data
quickly reveals some obvious trends. Only glyoxylamides
derived from aromatic amines displayed any cell line activity;
compounds prepared from aliphatic (28-31) or benzylic
Aspects of a clear SAR were thus emerging from this
first set of test compounds. Glyoxylamides derived from
primary aromatic amines and bearing a para-substituent
were revealed as requirements for optimum potency in the
SMB cell line assay. The only exception to this trend was

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23 7507
Table 2. Bioactivity of Indole-3-glyoxylamides with an Additional Methyl Group Introduced at Either the 1- or 2-Position
a %PrPSc remaining, relative to an untreated control, after 5 days of exposure to the test compound at the concentration specified (μM). b Results are
listed for the lowest concentration showing activity (<70% of control). For inactive compounds, the result at the highest nontoxic concentration tested
is given. c Some compounds showed activity in the initial screen, but meaningfuldose-response curves could not be obtained (i.e., false positives). d Toxic
at 20 μM. e Synthesis of this compound failed. f Very inconsistent results were obtained with 45, making an accurate EC50 measurement problematic.
3-aminoquinolyl compound 27, which was the only bicyclic
compound (24-27) displaying activity. Comparison of the
isosteric 2-naphthylamine derived analogue was excluded
during initial library design, however, owing to the well
established carcinogenicity of the parent amine.39
Having established some basic requirements for activity
regarding the glyoxylamide portion of the molecule, we were
keen to examine the effects of substitution at positions 1 and
2 of the indole system. To this end, a second library was
synthesized, derived from 1-methylindole and 2-methylin-
dole in combination with a subset of the amines depicted in
Table 1 (Scheme 1, Table 2).
makes a significant, but not critical, interaction with the
target at the site of action.
Additional requirements for antiprion activity had there-
fore been deduced from this second library. Indole-3-glyox-
ylamides unsubstituted at N-1 are preferred, and there must
be no substituent at C-2. Together with the requirements
already identified (i.e., glyoxylamides derived from para-
substituted anilines), these observations constitute a very
clearly defined SAR for antiprion activity of indole-3-glyo-
xylamides. In addition, the highly potent activity of 13
(p-OMe) and 16 (p-F) strongly suggested an advantage in
having a hydrogen-bond acceptor at this position.
All of the 2-methylindole-3-glyoxylamides (41, 61-79)
were devoid of cell line activity, including those where the
analogous 2-unsubstituted compounds had displayed
submicromolar EC50 values. Thus, substitution at the
2-position of the indole ring is clearly not tolerated in
terms of antiprion activity. Incorporation of a methyl
group at N-1 also produced a marked effect. 1-Methyl
derivatives of the most active indole-3-glyoxylamides re-
tained some activity, but EC50 values were about an order
of magnitude higher (compare 3, 0.32 μM, with 42,
6.90 μM; 13, 11 nM, with 46, 68 nM; 14, 0.24 μM, with
47, 3.77 μM). Changing the indole N-H to N-Me resulted
in a drop in activity, suggesting that this N-H group
In order to further refine the above conclusions, an addi-
tional set of compounds was prepared, derived from unsub-
stituted indole and again using the conditions detailed in
Scheme 1 (R1=R2=H). In the design of this final library
(Table 3), our intention was to further probe the effect of
varied para-substituents upon cell line activity, paying par-
ticular attention to differing hydrogen-bond acceptor char-
acter at this position. Library member 86 was prepared using
a protection strategy similar to that for some of the hydroxy-
containing compounds already described (Scheme 2, n=0).
Screening results from this final library (Table 3) revealed
that analogues of active compounds 3, 13, and 16 with a
nitrogen included at the 3-position of the aromatic ring

7508 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23
Thompson et al.
Table 3. Screening Results for Additional Indole-3-glyoxylamides Designed To Improve Understanding of Antiprion SAR
a %PrPSc remaining, relative to an untreated control, after 5 days of exposure to the test compound at the concentration specified (μM). b Results are
listed for the lowest concentration showing activity (<70% of control). For inactive compounds, the result at the highest nontoxic concentration tested
is given. c Toxic at 10 and 20 μM. d Yield over two steps.
(91-93) all displayed a reduction in potency by approxi-
mately an order of magnitude. The detrimental effect of
incorporating a ring nitrogen was also evident from the
results for 3- and 4-pyridyl compounds 91 (EC50 = 1.66
μM) and 94 (EC50=0.79 μM), though of these isomers, the
4-pyridyl derivative 94 was the more effective, reflecting the
general requirement for para- rather than meta-substitution
observed in the initial screening set (Table 1).
(EC50=11 nM) was completely eradicated in compounds 80
and 81, prepared from the analogous benzylamines.
Clear trends were apparent in the potencies of several
indole-3-glyoxylamides derived from additional subsets of
para-substituted anilines. The sequence of EC50 values ex-
hibited by p-OMe 13 (11 nM) < p-SMe 85 (40 nM) < p-OEt
83 (60 nM) < p-OCF3 90 (0.38 μM) < p-OPh 84 (1.03 μM)
does suggest an important role for the H-bonding character
of the para-substituent but also a binding pocket of limited
size or shape. The most effective group of compounds by far
was those possessing a p-amino substituent: p-piperidino 96
(72 nM) > p-NMe2 95 (26 nM) > p-morpholino 97 (9 nM)
> p-pyrrolyl 98 (6 nM) > p-1H-pyrazolyl 99 (1 nM). The
last three compounds, all showing an observed EC50 < 10
nM, contain an N-linked heterocycle at the key para-
position of the phenyl ring. In combination with the
similarly potent activity of p-oxazol-5-yl derivative 104
(EC50=19 nM), it is apparent that a (preferably aromatic)
heterocycle, containing at least one hydrogen-bond accep-
tor, should be present at the para-position of the aromatic
ring for optimal antiprion activity.
Mode of Action. Given the potent activity of many of the
indole-3-glyoxylamides above, it is evidently important to
understand their mode of action in the antiprion assay. They
do not appear to exert their effect through direct interaction
with PrPC, however, since in the surface plasmon resonance
(SPR) binding assay we have documented previously,24c,27,40
evaluation of a selection of the active compounds (3, 12-15,
and 27, all with EC50 < 1 μM) gave no evidence for
discernible binding to either the human or murine protein
at concentrations up to 40 μM. In addition, it is worth noting
Indeed, within this final library, the previously observed
preference for para-substitution gains added support. Com-
parison of 86 with 14 (m-OH, EC50=0.82 μM; p-OH, EC50=
0.24 μM) and of 103 with 104 (m-oxazol-5-yl, EC50
=
0.53 μM; p-oxazol-5-yl, EC50=19 nM) further underscores
the trend already identified for improved potency of para-
over meta-substituted compounds. In addition, library mem-
bers derived from a number of 3,4-disubstituted anilines
were prepared (3,4-dimethoxy-, 82; 3,4-difluoro-, 87; 3,4-
dimethyl-, 88). In all cases, these compounds displayed EC50
values closer to those of the corresponding meta-substituted
rather than para-substituted analogue; note, for example,
the activity of 3,4-difluorophenyl derivative 87 (EC50=0.32
μM) compared to its m- and p-fluoro analogues 15 (EC50=
0.64 μM) and 16 (EC50 = 64 nM). Clearly, there is no
complementary, additive effect between the activities of the
individually substituted compounds, emphasizing a require-
ment for para-substitution only; and as further evidence of
this, 3,4,5-trisubstituted screening subjects (100, trifluoro-;
101, trimethoxy-) showed a complete loss of activity.
Cell line activity was also abolished upon insertion of a
methylene bridge between the amide nitrogen and phenyl
ring; i.e., the potent activity of 12 (EC50=0.23 μM) and 13

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23 7509
that the clearly defined SAR described above does not
appear to correlate with those deduced for other modes of
action of related indole-3-glyoxylamides, for example, as
tubulin polymerization inhibitors41 or as ligands for the
benzodiazepine binding site of the GABAA receptor.34b,34c
Although it presents considerable challenges, elucidating the
cellular target of the presently described lead series is an
obvious priority and may offer some insight into the as
yet poorly understood mechanisms operating within prion
disease.
with additional ethyl acetate, a second extraction was carried
out with saturated NH4Cl (10 mL) in the same manner. Product
solutions were then evaporated to dryness giving the crude
indole-3-glyoxylamides, which were purified as necessary (see
Supporting Information for individual compound details).
Typically, two sequential recrystallizations, from ethyl aceta-
te-hexane followed by 2-propanol-water, afforded final pro-
ducts of good purity.
For products derived from poorly nucleophilic anilines (e.g.,
89-91, 93, 99, 100), a further modification was found helpful.
Immediately following introduction of the arylamine to the
reaction mixture, DMAP (24 mg, 0.2 mmol, 10 mol %) was
added to assist the final step, as its use was found necessary to
promote any meaningful conversion to product in such cases.
3-(tert-Butyldimethylsilyloxymethyl)aniline 35a. tert-Butyldi-
methylsilyl chloride (3.01 g, 20 mmol) was added to a solution of
3-aminobenzyl alcohol (2.46 g, 20 mmol) and imidazole (3.40 g,
50 mmol) in anhydrous DMF (25 mL) at 0 °C. After 2 h, cooling
was removed and the mixture allowed to warm to room
temperature overnight. The mixture was poured into water
(150 mL) and extracted into ether (3 ꢀ 100 mL), following
which the combined extracts were washed with brine, dried over
MgSO4, and evaporated to dryness. Flash column chromatog-
raphy on silica gel, eluted with ethyl acetate-hexane (1:4),
provided the title compound as a pale-yellow oil (3.37 g,
70%). δH (250 MHz, CDCl3) 7.01 (t, 1H, J=8.0), 6.64-6.56
(m, 2H), 6.50-6.44 (m, 1H), 4.56 (s, 2H), 3.51 (br s, 2H), 0.84 (s,
9H), 0.00 (s, 6H); δC (62.8 MHz, CDCl3) 146.3, 142.8, 129.1,
116.4, 113.7, 112.8, 64.9, 26.0, 18.4, -5.3; m/z (ES) 238 ([M þ
H]þ); HRMS, found 238.1625 (C13H24NOSi requires 238.1627).
N-(3-(Hydroxymethyl)phenyl)-2-(1H-indol-3-yl)-2-oxoaceta-
mide 18. The TBDMS-protected intermediate 36a (Scheme 2)
was prepared from indole and amine 35a according to the
general procedure above. δH (400 MHz, CDCl3) 9.41 (s, 1H),
9.18 (br s, 2H), 8.48 (d, 1H, J=7.5), 7.70-7.64 (m, 2H), 7.47 (d,
1H, J=7.5), 7.43-7.32 (m, 3H), 7.19 (d, 1H, J=7.5), 4.80 (s,
2H), 0.99 (s, 9H), 0.15 (s, 6H); δC (125 MHz, CDCl3) 180.5,
160.0, 142.8, 138.5, 136.8, 135.7, 129.1, 126.7, 124.4, 123.6,
122.7, 122.5, 118.5, 117.5, 113.2, 111.7, 64.7, 26.0, 18.5, -5.2;
m/z (ES) 409 ([M þ H]þ); HRMS, found 409.1934 (C23H29-
N2O3Si requires 409.1947). Compound 36a (166 mg, 0.41
mmol) was dissolved in THF (2.5 mL), followed by addition of
tetrabutylammonium fluoride (1.0 M in THF, 0.45 mL,
0.45 mmol). After 1 h the reaction mixture was evaporated
and the residue purified by flash column chromatography on
silica gel, eluting with methanol-DCM (0:100, then 2:98, then
1:19), affording the title compound as pale-yellow needles
(73 mg, 61%).
N-(3-(Hydroxymethyl)phenyl)-2-(1-methyl-1H-indol-3-yl)-2-
oxoacetamide 40 and N-(3-(Hydroxymethyl)phenyl)-2-(2-meth-
yl-1H-indol-3-yl)-2-oxoacetamide 41. Trimethylsilyl chloride
(253 μL, 217 mg, 2 mmol) was added to a solution of
3-aminobenzyl alcohol (205 mg, 1.67 mmol) in dry THF
(5 mL). After 1 h, this solution was introduced directly into a
reaction between the appropriate substituted indole (1 mmol)
and oxalyl chloride (100 μL, 146 mg, 1.2 mmol) in the same
solvent (5 mL) (which had already been stirring at room
temperature for 1 h) together with N,N-diisopropylethylamine
(610 μL, 452 mg, 3.5 mmol). After a further 3 h, the reaction
was quenched by addition of 1 M HCl (10 mL), and after the
mixture was stirred for 10 min, it was evaporated to dryness.
The crude product was taken up in ethyl acetate and washed
successively with 0.4 M K2CO3 (2 ꢀ 25 mL), saturated NH4Cl
(2 ꢀ 25 mL) and brine, then dried over MgSO4 and evaporated
once more. Purification by flash column chromatography on
silica gel, eluted with ethyl acetate-cyclohexane (1:1), gave
either compound 40 (79 mg, 25%; HPLC purity 99%) or 41 (36
mg, 11%; HPLC purity 88%) as required.
Conclusions and Future Perspective
Indole-3-glyoxylamides prepared from a variety of aryla-
mines have been established as a novel lead series against
prion disease, possessing in vitro activity down to nanomolar
concentrations with a tightly defined SAR. A survey of the
available literature reveals no class of antiprion compounds
has yet been reported with both this degree of potency and a
clear SAR, and as such, discovery of the indole-3-glyoxyla-
mide libraries detailed above may revive promise that small-
molecule therapeutics against prion disease can be found. It
would be desirable to further validate the most active indole
compounds in other cell models of prion infection, and the
results of such work will be reported in due course. Additional
investigations (to refine pharmacokinetic properties, gain
further understanding of the SAR, and elucidate possible
modes of action of the compounds) are similarly ongoing in
order to identify the best possible candidates for progression
into in vivo studies.
Experimental Section
General Procedures. Accurate mass and nominal mass mea-
surements were obtained using a Waters-Micromass LCT elec-
trospray mass spectrometer. Anhydrous grade THF was
obtained from an in-house “Grubbs” solvent purification sys-
tem; all other solvents and reagents were purchased from
commercial sources and used as supplied. All reactions were
performed under N2, and parallel synthesis was carried out on a
Radleys 12-position carousel. Evaporation of solvent from
€
reaction tubes was performed using a Buchi Multivapor P-12
system. Parallel extractions were carried out with liquid-liquid
extraction columns, 20 mL sample loading capacity, in conjunc-
tion with a carousel workup station. Where required, flash
column chromatography was carried out using prepacked silica
columns (20 g/70 mL). Purity of screening compounds was
assessed by HPLC (Waters SymmetryShield 3.5 μm C-18 col-
umn, 150 mm ꢀ 4.6 mm, 30-95% MeCN in water over 12 min
at 1.0 mL min-1, held 10 min; 15 μL injection; UV detection at
254 nm; run time 22 min). The large majority of compounds
were isolated in >95% purity; a complete list of HPLC purities,
along with full characterization data for each screening com-
pound, is provided in the Supporting Information.
Indole-3-glyoxylamides 1-34 and 40-79. General Procedure.
A dry carousel reaction tube was charged with indole (234 mg,
2 mmol), 1-methylindole (250 μL, 262 mg, 2 mmol), or
2-methylindole (262 mg, 2 mmol) as appropriate, and this
starting material was dissolved in dry THF (12 mL). Oxalyl
chloride (192 μL, 279 mg, 2.2 mmol) was added and the mixture
stirred at room temperature. After 1 h, N,N-diisopropylethyla-
mine (785 μL, 582 mg, 4.5 mmol) was introduced to the mixture,
followed by the relevant amine (2.4 mmol). The temperature was
raised to 45 °C, and heating continued for 18 h. The solvent was
evaporated, then the residue resuspended in a mixture of ethyl
acetate (20 mL) and brine (10 mL) and stirred vigorously for 30
min. After the sample was passed through a liquid-liquid
extraction column, ensuring washing through of the column
3-(tert-Butyldimethylsilyloxy)aniline 35b. 3-Aminophenol
(2.00 g, 18.3 mmol) and imidazole (2.00 g, 29.4 mmol) were

7510 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23
Thompson et al.
dissolved in anhydrous THF (50 mL). Then tert-butyldimethyl-
silyl chloride (3.60 g, 23.4 mmol) was added with vigorous
stirring. After 30 min, the reaction mixture was poured into
water (200 mL) and extracted into ether (3 ꢀ 50 mL). The
combined extracts were dried over MgSO4 and evaporated to
dryness giving the crude product as a viscous, colorless oil which
was used in the subsequent step without further purification. δH
(400 MHz, CDCl3) 7.02 (t, 1H, J=8.0), 6.35-6.27 (m, 2H), 6.23
(t, 1H, J=2.0), 3.17 (br s, 2H), 1.00 (s, 9H), 0.21 (s, 6H).
N-(3-Hydroxyphenyl)-2-(1H-indol-3-yl)-2-oxoacetamide 86.
Protected intermediate 36b (Scheme 2) was prepared from
indole and crude 3-(tert-butyldimethylsilyloxy)aniline 35b
(0.80 g, 3.6 mmol) according to the general procedure for
indole-3-glyoxylamides above. δH (400 MHz, CDCl3) 9.35 (s,
1H), 9.19 (d, 1H, J=3.0), 9.02 (br s, 1H), 8.51-8.47 (m, 1H),
7.50-7.46 (m, 1H), 7.43-7.34 (m, 3H), 7.31-7.24 (m, 2H), 6.71
(ddd, 1H, J=1.5, 2.5, 7.5), 1.03 (s, 9H), 0.26 (s, 6H); δC (62.8
MHz, CDCl3) 180.5, 159.9, 156.4, 138.4, 137.9, 135.7, 129.9,
126.7, 124.4, 123.6, 122.5, 116.8, 113.2, 112.9, 111.9, 111.7, 25.7,
18.2, -4.4; m/z (ES) 395 ([M þ H]þ); HRMS, found 395.1807
(C22H27N2O3Si requires 395.1791). Compound 36b (132 mg,
0.34 mmol) was then dissolved in THF (1.5 mL), and tetra-
butylammonium fluoride (1.0 M in THF, 0.37 mL, 0.37 mmol)
was added. A thick precipitate had appeared within a few
minutes. After 30 min, the product was isolated by filtration,
washed twice each with THF, DCM, and water, and dried
thoroughly. The title compound 86 was thereby obtained as its
tetrabutylammonium salt (140 mg, 80%).
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Acknowledgment. The authors thank the U.K. Depart-
ment of Health (Contract No. DH007/0102) and BBSRC
(Grant No. BB/E014119/1) for their generous funding.
Supporting Information Available: Spectroscopic data for all
library members (HRMS, IR, 1H NMR, 13C NMR); individual
compound purification details; compound purities by HPLC;
and dose-response curves for all cell line active compounds
(including the positive controls curcumin and quinacrine). This
material is available free of charge via the Internet at http://
pubs.acs.org.
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Faucheux, B. A.; Soubrie, C.; Boher, E.; Belorgey, C.; Hauw, J. J.;
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