Library synthesis and screening: 2,4-Diphenylthiazoles and 2,4-diphenyloxazoles as potential novel prion disease therapeutics

Article abstract of DOI:10.1021/jm0612719

Transmissible spongiform encephalopathies (TSEs) are a family of invariably fatal neurodegenerative disorders for which no effective therapeutics are currently available. In this paper, we report on the synthesis and screening of a small library of 2,4-diphenylthiazol-5-ylamine and 2,4-diphenyloxazol-5- ylamine derivatives as potential novel prion disease therapeutics. Various synthetic strategies were investigated, including a novel phosgene-mediated cyclization of 2-N-benzoylphenylglycinonitrile, and a total of 45 compounds were synthesized. Library members were tested for both binding to prion protein (PrP^C) using the surface plasmon resonance technique and for inhibition of PrP^Sc formation in persistently infected SMB cells. Of the compounds prepared, 15 were found to bind to human PrP^C and six showed inhibition of PrP^Sc formation, displaying EC₅₀s between 1.5 and 20 μM.

Full text of DOI:10.1021/jm0612719

J. Med. Chem. 2007, 50, 1347-1353
1347
Library Synthesis and Screening: 2,4-Diphenylthiazoles and 2,4-Diphenyloxazoles as
Potential Novel Prion Disease Therapeutics
William Heal, Mark J. Thompson, Roger Mutter, Hannah Cope, Jenny C. Louth, and Beining Chen*
Department of Chemistry, UniVersity of Sheffield, Brook Hill, Sheffield, S3 7HF, UK
ReceiVed October 31, 2006
Transmissible spongiform encephalopathies (TSEs) are a family of invariably fatal neurodegenerative disorders
for which no effective therapeutics are currently available. In this paper, we report on the synthesis and
screening of a small library of 2,4-diphenylthiazol-5-ylamine and 2,4-diphenyloxazol-5-ylamine derivatives
as potential novel prion disease therapeutics. Various synthetic strategies were investigated, including a
novel phosgene-mediated cyclization of 2-N-benzoylphenylglycinonitrile, and a total of 45 compounds were
synthesized. Library members were tested for both binding to prion protein (PrPC) using the surface plasmon
resonance technique and for inhibition of PrPSc formation in persistently infected SMB cells. Of the compounds
prepared, 15 were found to bind to human PrPC and six showed inhibition of PrPSc formation, displaying
EC50s between 1.5 and 20 µM.
Introduction
Prion diseases, or transmissible spongiform encephalopathies
(TSEsa), are invariably fatal neurodegenerative disorders af-
fecting humans and animals. As yet, no effective curative or
prophylactic therapy exists.1 Prominent examples of prion
diseases include bovine spongiform encephalopathy (BSE,
cattle), scrapie (sheep), chronic wasting disorder (CWD, deer
and elk), and transmissible mink encephalopathy (TME).2 Since
a new variant of the human TSE Creutzfeldt-Jacob disease
(vCJD) was discovered, thought to have been triggered by the
consumption of contaminated beef products, prion diseases have
Figure 1. Hit compound 1a and early lead compounds.
ever, searching the literature for a general route allowing for
structural variation and based on readily available starting
materials gave limited results. Published routes to these
compounds are restricted with regard to substitution at the 2-
and 4-positions, sometimes involving many synthetic steps.11-13
To address this deficit we recently published a route to
5-aminothiazoles14 offering control over substitution at positions
2 and 4 in which we also reported the isolation of 5-aminoox-
azoles as a side product in some cases. When subjected to an
SPR binding assay, compounds 1c and 2a (Figure 1) both
showed binding to huPrPC.
Synthesis. Our initial goal was to prepare a library of
analogues of 1a with a range of amide functionalities at the
5-position. As such, synthesis of 2-nicotinyl-5-(2-thienyl)-
thiazol-5-ylamine 1b was pursued as a key intermediate for
further derivatization by a range of carboxylic acids. Access to
this compound proved difficult, however, and after approaching
it unsuccessfully by a number of routes, it was decided instead
to prepare the 2,4-diphenyl analogue 1d for synthetic expedi-
ency. This intermediate was prepared as reported previously14
and was purified further by flash column chromatography where
necessary prior to library synthesis.
5-Amino compound 1d proved unreactive toward carboxylic
acids under standard DCC-HOBT coupling conditions. Addi-
tion of an amine base (4-ethylmorpholine, NEM) to drive the
reaction resulted in formation of the bis-amide product. Use of
benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluoro-
phosphate as coupling agent in the presence of DIPEA also
resulted in bis-amide formation. Nonetheless, amide formation
was found to proceed smoothly by reaction of 1d with acid
chlorides in pyridine. A library of 18 such derivatives (1e-x)
been the focus of much research effort.3-5 They represent a
highly significant risk to public health due to transmission both
to and between humanssiatrogenic transmission having been
reported via contact with contaminated neurosurgical instru-
ments, grafts, and blood/tissue products6sand as such continue
to receive high profile media attention. TSEs are associated with
a post-translational conversion of the cell-surface glycosylphos-
phatidylinositol (GPI)-anchored protein PrPC (or PrPsen) to a
partially protease resistant isoform denoted PrPSc (or PrPres).
As part of a TSE-oriented medicinal chemistry program, we
are seeking small drug-like molecules that interact with human
PrPC (huPrPC) and/or reduce levels of PrPSc in persistently
infected cells, with the aim of identifying novel prion disease
therapeutics.
From a vHTS study,7 followed by the screening of sourced
compounds by surface plasmon resonance (SPR),8 structure 1a
(Figure 1) was identified as binding to huPrPC. As this
compound was a singleton and no suitable analogues were found
to be available commercially, a convenient synthetic route to
such structures was sought.
Specifically, we intended to first prepare a library of amides
at the 5-position through derivatization of the free 5-amino
compound 1b (Figure 1). 5-Aminothiazoles have received
attention ranging from antibiotics9 to photosensitisers;10 how-
* To whom correspondence should be addressed. Phone: +44 114 222
9467. Fax: +44 114 222 9346. E-mail: b.chen@sheffield.ac.uk.
a Abbreviations: PrPC, normal cellular prion protein or PrP-sen; PrPSc,
disease-causing isoform or PrP-res; huPrPC, human PrPC; moPrPC, murine
PrPC; SMB, scrapie-infected mouse brain; SPR, surface plasmon resonance;
TSE, transmissible spongiform encephalopathies; vCJD, variant Creutzfeldt-
Jakob disease; vHTS, virtual high-throughput screening.
10.1021/jm0612719 CCC: $37.00 © 2007 American Chemical Society
Published on Web 02/17/2007

1348 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 6
Heal et al.
Scheme 1. Synthesis of Thiazole Library by Amide Formationa
with sodium hydride in dry THF followed by treatment with
the acid chloride, though it was later found that milder conditions
were adequate.
N-Acylation of 2c in DCM, catalyzed by DMAP and in the
presence of DIPEA, was typically complete in 2 h at room
temperature. Removal of the N-Boc protecting group was
accomplished by addition of TFA to the reaction mixture to a
final dilution of 1:4 TFA-DCM followed by stirring for 18 h.
This method proved a particularly convenient protocol for
parallel synthesis of 5-amidooxazoles 2d-2w, which were
purified by recrystallization or column chromatography as
necessary.
a Reagents and conditions: (a) (COCl)2, DCM, room temp., 80 min; (b)
1d, pyridine, room temp., 18 h; (c) pyridine, DMAP, 1d, room temp., 18
h.
Scheme 2. Synthesis of (2,4-Diphenyloxazol-5-yl)-carbamic
Acid tert-Butyl Ester 2ca
Some of the 5-amidooxazoles prepared appeared to be
unstable, however; repeated HPLC analysis revealed that the
amide products containing a pyridine (2j and 2k), 5-nitrofuryl
(2n), or isoxazolyl (2o) ring all showed slow decomposition
over a period of days or weeks, even on storage at -20 °C.
These results naturally preclude the incorporation of such
substituents into any future libraries, though none of the unstable
compounds displayed any activity in the cell line assay.
a Reagents and conditions: (a) Triphosgene, DCM, room temp, 15 min,
then BuOH, DCM, room temp., 5 min.
t
Scheme 3. Synthesis of Oxazole Library by Bis-amide
Formation and Deprotectiona
Screening (SPR) Methodology. Surface plasmon resonance
(SPR) was carried out using a BIAcore 3000 (BIAcore, Uppsala,
Sweden) equipped with a CM5 sensor chip (carboxymethylated
dextran). The methodology was as reported previously.8 Interac-
tions were measured with two forms of prion protein, full length
human (huPrPC) and full length murine (moPrPC). Compounds
were screened at 40 µM in running buffer (10 mM sodium
phosphate, pH 7.4, 150 mM NaCl, 3.4 mM EDTA, 0.005% (v/
v) surfactant P20) containing 6.5% DMSO. DMSO calibration
using buffer samples containing 5.5-7.5% DMSO was carried
out to correct for solvent effects. Compounds producing a
response of more than 2.5 response units (RU) were considered
to be binders. Binding affinities are expressed as %RUmax, that
is, as a percentage of the theoretical maximum response for 1:1
protein-ligand binding. We have previously observed that the
numerical binding data obtained during the SPR assay varies
considerably with the age of the chip;8 therefore, direct
quantitative comparison is only relevant between compounds
included in the same screening run. Attempts to include all of
the compounds in this study within the same run proved
unsuccessful, however. Satisfactory results were obtained when
the compounds were split between runs: most were ultimately
screened together, but the rest of the structures were evaluated
separately as indicated in Tables 1 and 2. Direct comparison of
numerical binding data between all the compounds is immaterial
because of this, though the data presented does serve to indicate
a relative degree of binding for the various compounds. No data
is presented for compounds either seen to interfere with the chip
surface, or for those that could not be washed off the protein
(Tables 1 and 2).
a Reagents and conditions: (a) NaH, THF, room temp., 5 min, then
RCOCl, THF, room temp., 10 min; (b) RCOCl, DMAP, DIPEA, room
temp., 2 h; (c) TFA (20%) in DCM, room temp, 18 h.
was prepared by this method, with the exception of two cases
(1g, 1r) where the relevant acid chloride was not available
commercially. These amide derivatives were prepared from the
carboxylic acids, through in situ formation of the acid chloride
followed by reaction with amine 1d in pyridine (Scheme 1).
Since both N-(2,4-diphenylthiazol-5-yl)-2,2,2-trifluoroacet-
amide 1c and the analogous structure N-(2,4-diphenyloxazol-
5-yl)-2,2,2-trifluoroacetamide 2a had displayed binding to PrPC
in our SPR assay, a series of 5-amidooxazoles analogous to the
existing library of thiazole derivatives was desired to widen the
scope of this comparison.
2,4-Diphenyloxazol-5-ylamine, analogous to 5-aminothiazole
1b, has been reported in the literature.15 In our hands, however,
this compound could not be isolated, being found to exist in
equilibrium with its ring-opened form. A different approach to
the required 5-amidooxazoles was therefore necessary. We
confronted the problem by looking to trap the 2,4-diphenylox-
azol-5-ylamine as a suitably protected derivative as it was
formed. The solution to this problem was inspired by the work
of Verschave et al., in which oxalyl chloride is used in the
formation of oxazoyl-5-oxylamic acid esters16, which led us to
develop a novel phosgene-mediated cyclization of 2-N-ben-
zoylphenylglycinonitrile 3. Subsequent quenching of the chlo-
rocarbamate intermediate 4 with 2-methyl-2-propanol provided
the N-Boc-protected amine 2c in an acceptable overall yield
(Scheme 2). Preparation of 2c from 2-phenylglycinonitrile
hydrochloride and benzoyl chloride via a related one-pot
procedure was also found to be possible without the need for
isolation of the open-chain intermediate 3.
Screening (SMB Cells) Methodology. Compounds were
screened for inhibition of PrPSc formation in SMB cells of
mesodermal origin, the procedure used being based upon that
reported by Rudyk et al.17 A persistently infected mouse cell
line (SMB), cloned originally from scrapie infected mouse brain
but of non-neuronal origin,18 was used. Cells were grown in
tissue culture-treated plastic dishes in Medium 199 (phenol red
free), supplemented with 10% newborn calf serum (heat
inactivated), 5% fetal calf serum (heat inactivated), and penicil-
lin-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
passaged using 0.05% trypsin and 0.002% EDTA at a split ratio
of 4. To assess the effects of compounds, cells were distributed
The desired library of 5-amidooxazoles was accessed from
N-Boc protected intermediate 2c through acylation at N-5 with
the same series of acid chlorides detailed above followed by
TFA-mediated removal of the N-Boc group (Scheme 3). At first,
N-acylation reactions were carried out by deprotonation of 2c

Potential NoVel Prion Disease Therapeutics
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 6 1349
Table 1. Yields and Screening Results for the Thiazole Library
compd
no.
yield
[%]
%RUmax
(huPrPC)
%RUmax
PrPSc rel to control
[%] (( std dev)
concn
[µM]
R
(moPrPC)
1c
1e
1f
1g
1h
1i
1j
1k
1l
1m
1n
1o
1p
1q
1r
1s
CF3
Ph
4-F-Ph
-
9.5
0
18.7
0
13.7 (( 7.6)
97.7 (( 3.7)
109.3 (( 2.7)
69.4 (( 0.8)
102.0 (( 1.3)
105.1 (( 4.4)
112.5 (( 2.8)
93.9 (( 3.7)
99.8 (( 1.0)
93.4 (( 10.8)
98.7 (( 7.6)
95.2 (( 3.8)
86.2 (( 4.3)
86.3 (( 5.1)
83.6 (( 2.4)
101.3 (( 15.5)
88.2 (( 2.1)
60.2 (( 2.9)
87.4 (( 0.0)
74.6 (( 3.2)
76.8 (( 4.5)
2.5
10
10
10
10
10
10
10
10
10
10
10
1
74
86
12
83
83
79
69
75
65
51
9
a
a
-
-
a
a
4-OMe-Ph
2-CF3-Ph
3-CF3-Ph
4-CF3-Ph
3-pyridyl
4-pyridyl
2-quinoyl
2-furyl
5-NO2-2-furyl
5-isoxazoyl
thiophen-2-yl
5-Me-thiophen-2-yl
benzo[b]thiophen-2 -yl
benzofuran-2-yl
benzothiazol-2-yl
benzo[b]thiophen-5 -yl
cyclopropyl
-
-
0
0
65.0
0
0
0
0
0
5.0b
8.5b
a
a
-
-
6.5
0
8.4
0
74
82
67
83
86
27
59
61
74
0
0
a
a
-
-
1
19.8b
21.1
0b
0
10
10
10
1
10
1
a,b
a,b
1t
-
-
1u
1v
1w
1x
40.1b
0b
a,b
a,b
-
0
0
-
0
0
CH(n-propyl)2
1
a Compound interacted with the chip surface or could not be washed off the protein. b Binding data obtained from a different screening run.
Table 2. Yields and Screening Results for the Oxazole Library
compd
no.
yield
[%]
%RUmax
(huPrPC)
%RUmax
PrPSc rel to control
[%] (std dev [%])
concn
[µM]
R
(moPrPC)
2a
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
2m
2n
2o
2p
2q
2r
2s
CF3
O-tert-butyl
Ph
4-F-Ph
4-OMe-Ph
2-CF3-Ph
3-CF3-Ph
4-CF3-Ph
3-pyridyl
-
13.7
11.3
22
10.6
35.1 (( 11.2)
46.1 (( 7.0)
72.8 (( 26.5)
80.4 (( 14.3)
84.9 (( 7.8)
51.7 (( 1.6)
80.9 (( 1.9)
80.2 (( 1.2)
85.6 (( 7.4)
93.2 (( 1.2)
106.8 (( 8.7)
92.9 (( 2.1)
84.2 (( 7.9)
77.1 (( 1.0)
90.5 (( 12.8)
79.3 (( 1.8)
83.2 (( 10.8)
86.6 (( 4.2)
87.8 (( 2.5)
84.8 (( 3.8)
87.8 (( 0.2)
95.8 (( 0.4)
2.5
1
1
1
10
1
1
10
1
10
10
10
10
10
1
10
10
10
10
10
10
10
22
72
81
69
71
54
31
14
59
27
77
19
23
79
56
18
55
47
65
54
38
a
a
-
-
0
0
0b
0b
a
a
-
-
a
a
-
-
0
0
37.3
40.0
4-pyridyl
2-quinoyl
2-furyl
0b
0b
0
0
3.8b
0
0b
5-NO2-2-furyl
5-isoxazoyl
thiophen-2-yl
5-Me-thiophen-2-yl
benzo[b]thiophen-2 -yl
benzofuran-2-yl
benzothiazol-2-yl
benzo[b]thiophen-5 -yl
cyclopropyl
CH(n-propyl)2
0
21.7
37.4
18.3
27.5
a,b
a,b
-
-
48.0
59.6
48.9b
58.0b
a
a
2t
-
-
a
a
2u
2v
2w
-
-
0
-
0
a,b
a,b
-
a Compound interacted with the chip surface or could not be washed off the protein. b Binding data obtained from a different screening run.
into 96-well cluster plates at 3 × 104 cells per well and incubated
for 24 h to allow for cell attachment. The compounds were
diluted to 400 times the required concentration in DMSO as
stock solutions 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.
(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 (deter-
mined by the Bradford assay following the protocol supplied
with the reagent, Sigma). The membrane was air-dried and
subjected to 75 µg mL-1 proteinase K digestion for 1 h at
After 5 days, cell viability was assessed by the MTT assay
following the standard protocol supplied with the reagent

1350 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 6
Heal et al.
Figure 2. IC50 curves for compounds 1c, 1g, 1u, 2a, 2c, and 2g. Circles (graphs 1c and 2a) represent cell viability; other compounds not toxic at
concentrations shown.
37 °C. The reaction was stopped with 1 mM phenylmethylsul-
fonyl fluoride (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 mem-
brane 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).
Each experiment was carried out in triplicate and an average
value for PrPSc concentration calculated, relative to an untreated
control, together with a standard deviation. Compounds were
initially screened at 1 and 10 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
PrPSc remaining as a percentage relative to the control are given
in Tables 1 and 2. Where an acceptable dose-response curve
was observed, EC50 values were determined. However, where
the data was inconclusive, the lowest active concentration
supplanted the initial screening result in Tables 1 and 2.
active at 10 µM but demonstrated more effective clearance of
PrPSc at higher concentrations. The remainder of the compounds
were largely inactive, rendering a comprehensive SAR elusive
at this stage. In a similar manner to the binding behavior, cell
line activity was highly influenced by the presence of either O
or S in the azole ring. With the exception of the trifluoroacetates
1c and 2a, there is no common R group between the actives of
the thiazole and the oxazole libraries. Looking at the oxazole
library, the activity of 2-trifluoromethylphenyl compound 2g
when compared to the 3- and 4-trifluoromethyl derivatives 2h
and 2i suggests an interesting effect. If the role of the CF3 group
were purely to activate the amide carbonyl as a hydrogen bond
acceptor, the 4-substituted analogue 2i would be expected to
show some activity also. The activity of 1g suggests that this is
not a steric effect: the trifluoromethyl group of 2g may therefore
be involved in a protein-ligand interaction. However, as none
of the corresponding thiazoles (1h-j) were active, it cannot be
determined whether this is a general trend. Cyclopropyl
compound 1w reduced PrPSc levels to 74.6% of the control at
1 µM, which could be considered as borderline activity, whereas
the corresponding oxazole 2v showed no activity at 10 µM.
Conversely, phenyl compound 2d was a similar borderline active
at 1 µM (although with high standard deviation in this instance),
while the thiazole analogue 1e showed no activity at the higher
concentration. The primary amine 1d and the bis-amide
compounds formed as described earlier were not found to be
active in our cell-line assays.
Compounds 1c and 2a were both found to have EC50s of
∼1.5 µM, 1g ∼20 µM, 1u ∼13 µM, 2c ∼11 µM, and 2g ∼8
µM (Figure 2). Compounds 1u and 2c displayed an intriguing
biphasic dose-response. Known as hormesis, this phenomenon
is not fully understood,19 although it has been encountered in
some of our other screening subjects20 and may be indicative
of metabolism occurring at higher concentrations. In particular,
examination of the dose-response profile of N-Boc compound
2c reveals that this compound displays a possible initial EC50
in the range of 100 nM before PrPSc levels recover as drug
concentration increases. This apparent activity, coupled with
that of the trifluoroacetates 1c and 2a and the borderline activity
of cyclopropyl derivative 1w, suggests that related compounds
bearing small, lipophilic, aliphatic groups at the 5-position merit
further investigation in the search for more potent inhibitors of
PrPSc accumulation.
Results and Discussion
SPR Screening. Among the thiazole library (Table 1), it can
be seen that seven compounds bound to huPrPC and three to
moPrPC, a further six compounds being found to interact with
the chip surface. The oxazole library included eight binders to
huPrPC and seven to moPrPC, with seven compounds found to
interact with the chip surface (Table 2). Most compounds bound
to both or neither form of PrPC except for five compounds which
bound to huPrPC only, these mostly coming from the thiazole
library (1i, 1r, 1s, and 1u). Perhaps the most interesting
observation was the difference in behavior between the two
libraries. There were several examples where binding was
effected or lost by changing the azole heteroatom between sulfur
and oxygen, notably 1k and 2j; 1l and 2k; 1p and 2o. In other
cases the comparison between O and S was hampered by either
of both of the compounds interacting with the carboxymethyl-
ated dextran chip surface.
Cell Line Assay. The results from the remaining compounds
are shown in Tables 1 and 2. It was interesting to note that
synthetic intermediates 1c, 2a, and 2c were the most potent
compounds found in this investigation, with 1u and 2g also
found to be active at 1 µM. Compound 1g was a borderline

Potential NoVel Prion Disease Therapeutics
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 6 1351
N-(2,4-Diphenylthiazol-5-yl)-3-trifluoromethylbenzamide (1i):
m/z (ES), 425 ([M + H]+); HRMS, found 425.0926 (C23H16F3N2-
OS, [M + H]+, requires 425.0935).
N-(2,4-Diphenylthiazol-5-yl)-4-trifluoromethylbenzamide (1j):
m/z (ES), 425 ([M + H]+); HRMS, found 425.0949 (C23H16F3N2-
OS, [M + H]+, requires 425.0935).
N-(2,4-Diphenylthiazol-5-yl)nicotinamide (1k): m/z (ES), 358
([M + H]+); HRMS, found 358.1000 (C21H16N3OS, [M + H]+,
requires 358.1014).
N-(2,4-Diphenylthiazol-5-yl)isonicotinamide (1l): m/z (EI), 357
(M+); HRMS, found 357.0922 (C21H15N3OS, M+, requires 357.0936).
Quinoline-2-carboxylic acid (2,4-diphenylthiazol-5-yl)amide
(1m): m/z (ES), 408 ([M + H]+); HRMS, found 408.1155
(C25H18N3OS, [M + H]+, requires 408.1171).
Furan-2-carboxylic acid (2,4-diphenylthiazol-5-yl)amide (1n):
m/z (ES), 346 ([M + H]+); HRMS, found 347.0868 (C20H15N2O2S,
[M + H]+, requires 347.0854).
5-Nitrofuran-2-carboxylic acid (2,4-diphenylthiazol-5-yl)a-
mide (1o): m/z (ES), 392 ([M + H]+); HRMS, found 392.0710
(C20H14N3O4S, [M + H]+, requires 392.0705).
Although it has not been possible thus far to establish any
direct correlation between PrPC binding affinity and cell line
activity, it is encouraging to note that all of the active
compounds reported here for which data could be obtained
showed binding to the full-length human PrPC, and all but 1u
to the murine protein. It was not possible to establish whether
actives 1g and 2g were binders to PrPC using the SPR method.
From the results reported here it is evident that factors other
than PrPC binding alone are responsible for cell line activity,
though given that the mechanisms underlying prion disease are
not yet fully understood, any alternative explanation for these
compounds’ mode of action would merely be speculative in
nature. Our goal was primarily to identify new lead compounds
in the search for effective therapeutics against a presently
untreatable class of fatal diseases, and this goal has been realized
in the present work.
Conclusions
Thiazoles and oxazoles are frequently encountered in com-
pounds of biological interest. However, we present here the first
reported instance of antiprion activity in compounds of this type.
Out of 45 compounds synthesized, 15 were found to bind to
PrPC. Six of the 45 compounds showed potent inhibition of PrPSc
formation with EC50s in the range 1.5-20 µM. These structures
are therefore of considerable interest for further development,
and as such, variation of the groups at positions 2 and 4 of the
active azoles is under investigation, with a view to both
improving activity and establishing a more defined SAR.
Consequently, it is anticipated that new compounds such as
these, displaying significant antiprion activity, may eventually
address the lack of clinical effect found with compounds such
as quinacrine in treating the invariably fatal TSEs. To this end,
the results reported herein represent a useful advance in
addressing this pressing medical need, presenting compounds
with low-micromolar activity as valuable leads for further
development.
Isoxazole-5-carboxylic acid (2,4-diphenylthiazol-5-yl)amide
(1p): m/z (ES), 348 ([M + H]+); HRMS, found 348.0790
(C19H14N3O2S, [M + H]+, requires 348.0807).
Thiophene-2-carboxylic acid (2,4-diphenylthiazol-5-yl)amide
(1q): m/z (ES), 363 ([M + H]+); HRMS, found 363.0615
(C20H15N2OS2, [M + H]+, requires 363.0626).
Benzo[â]thiophene-2-carboxylic acid (2,4-diphenylthiazol-5-
yl)amide (1s): m/z (ES), 413 ([M + H]+); HRMS, found 413.0780
(C24H17N2OS2, [M + H]+, requires 413.0782).
Benzofuran-2-carboxylic acid (2,4-diphenylthiazol-5-yl)amide
(1t): m/z (ES), 397 ([M + H]+); HRMS, found 397.1005
(C24H17N2O2S, [M + H]+, requires 397.1011).
Benzothiazole-2-carboxylic acid (2,4-diphenylthiazol-5-yl)-
amide (1u): m/z (ES), 414 ([M + H]+); HRMS, found 414.0748
(C23H16N3OS2, [M + H]+, requires 414.0735).
Benzo[â]thiophene-5-carboxylic acid (2,4-diphenylthiazol-5-
yl)amide (1v): m/z (ES), 413 ([M + H]+); HRMS, found 413.0788
(C24H17N2OS2, [M + H]+, requires 413.0782).
Cyclopropanecarboxylic acid (2,4-diphenylthiazol-5-yl)amide
(1w): m/z (ES), 343 ([M + Na]+); HRMS, found 343.0893 (C19H16-
NaN2OS, [M + Na]+, requires 343.0881).
Experimental Section
General Procedures. Melting points were measured using a
Bibby-Sterilin SMP10 melting point apparatus and are uncorrected.
Accurate mass and nominal mass measurements were measured
using a Waters-Micromass LCT electrospray mass spectrometer.
Flash column chromatography was carried out using Fluorochem
silica gel 60 Å. All compounds were isolated in >95% purity as
determined by HPLC, unless otherwise stated. All reagents were
purchased directly from commercial sources and used as supplied.
Compounds 1c, 1d, and 2a have been reported previously.14 Full
characterization data for each new compound is provided in the
Supporting Information.
General Procedure for the Synthesis of Thiazoles. Acid
chloride (0.33 mmol) was added to a solution of 1d (76 mg, 0.30
mmol) and DMAP (10 mg) in pyridine (3 mL). The reaction mixture
was stirred at ambient temperature for 18 h. All volatiles were
removed under reduced pressure, and the residue was taken up in
DCM (30 mL). This solution was washed thoroughly with 1 M
HCl (4 × 30 mL) and then sat. NaHCO3 (2 × 30 mL), dried over
MgSO4, filtered, and evaporated to dryness to provide the product.
Where necessary, further purification was carried out by flash
column chromatography on silica gel.
N-(2,4-Diphenylthiazol-5-yl)-benzamide (1e): m/z (ES); HRMS,
357 ([M + H]+); HRMS, found 357.1047 (C22H17N2OS, [M + H]+,
requires 357.1062).
N-(2,4-Diphenylthiazol-5-yl)-4-fluorobenzamide (1f): m/z (ES),
375 ([M + H]+); HRMS, found 375.0961 (C22H16FN2OS, [M +
H]+, requires 375.0967).
N-(2,4-Diphenylthiazol-5-yl)-2-trifluoromethylbenzamide (1h):
m/z (ES), 425 ([M + H]+); HRMS, found 425.0938 (C23H16F3N2-
OS, [M + H]+, requires 425.0935).
N-(2,4-Diphenylthiazol-5-yl)-4-methoxybenzamide (1g). A stirred
suspension of p-anisic acid (44 mg, 0.29 mmol) in DCM (3.0 mL)
was treated with oxalyl chloride (26.2 µL, 0.30 mmol) and DMF
(40 µL), at which point effervescence was observed. A homoge-
neous solution was gradually obtained as the reaction took place.
After 80 min, the reaction mixture was concentrated under reduced
pressure and dried further under high vacuum (with periodical
warming) to yield the crystalline, crude acid chloride. 2,4-Diphenyl-
5-aminothiazole (70 mg, 0.28 mmol), pyridine (3.0 mL), and DMAP
(10 mg) were added, and the resultant orange solution was stirred
at ambient temperature for 18 h. The pyridine was removed under
vacuum and the residue taken up in DCM (40 mL) and then washed
with 1 M HCl (4 × 50 mL) followed by sat. NaHCO3 (2 × 40
mL). The organic layer was evaporated and the residue purified
by flash column chromatography on silica gel, eluted with 60-
75-90% DCM-hexane, to provide 1e as a yellowish solid (13
mg, 12%). m/z (ES), 409 ([M + Na]+); HRMS, found 409.1006
(C23H18N2NaO2S, [M + Na]+, requires 409.0987).
5-Methylthiophene-2-carboxylic Acid (2,4-diphenylthiazol-5-
yl)amide (1r). 5-Methylthiophene-2-carboxylic acid (213 mg, 1.50
mmol) was suspended in DCM (5.0 mL), and then oxalyl chloride
(131 µL, 1.50 mmol) and DMF-DCM (1:9, 20 µL) were added.
After the mixture was stirred at ambient temperature for 1 h, a
solution of 2,4-diphenyl-5-aminothiazole (252 mg, 1.00 mmol) in
pyridine (5.0 mL) was added and stirring of the resultant mixture
was continued at ambient temperature for 18 h. The reaction mixture
was diluted with DCM (60 mL) and washed successively with 1
M HCl (4 × 40 mL) and sat. NaHCO3 (2 × 40 mL). The organic
layer was dried over MgSO4, filtered, and evaporated giving crude
material, which was purified by flash column chromatography on

1352 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 6
Heal et al.
silica gel, eluting with 60-75% DCM-hexane and yielding 1r as
an off-white solid (252 mg, 67%). m/z (EI), 376 (M+); HRMS,
found 376.0695 (C21H16N2OS2, M+, requires 376.0704).
N-(2,4-Diphenyloxazol-5-yl)-2-trifluoromethylbenzamide (2g):
m/z (ES), 409 ([M + H]+); HRMS, found 409.1151 (C23H16F3N2O2,
[M + H]+, requires 409.1164).
2-Propylpentanoic Acid (2,4-diphenylthiazol-5-yl)amide (1x).
A stirred solution of 2-propylpentanoic acid (218 µL, 200 mg, 1.39
mmol) in DCM (3 mL) was treated with oxalyl chloride (122 µL,
177 mg, 1.39 mmol) and DMF-DCM (1:4, 20 µL). After being
stirred for 1 h, the reaction mixture was evaporated to dryness and
further dried under high vacuum to yield the crude acid chloride.
1d (70 mg, 0.28 mmol) in pyridine (3 mL) was added, followed
by DMAP (10 mg), and the solution stirred at ambient temperature
for 18 h. All volatiles were removed under reduced pressure, and
the residue was taken up in DCM (40 mL). This solution was
washed thoroughly with 1 M HCl (3 × 40 mL) and then sat.
NaHCO3 (3 × 40 mL) and evaporated. Purification by flash column
chromatography on silica gel, eluting with 40-50% DCM-hexane,
afforded the bis-amide product, 2,4-diphenyl-5-[N,N-bis(2-propyl-
pentanoyl)amino]thiazole (57 mg, 41%), which crystallized as a
pale orange solid upon scratching. A portion of this material (47
mg) was suspended in 2-propanol (2.0 mL), and then tetraethyl-
ammonium hydroxide, 20% w/v aqueous solution (146 µL, 2.0
mmol), was added. Dissolution of the starting material was observed
as hydrolysis took place. After 2.5 h, the mixture was diluted with
1 M HCl (50 mL) and DCM (50 mL). The organic layer was
separated, dried over MgSO4, filtered, and evaporated. Purification
by flash column chromatography on silica gel, eluted with 50-
65% DCM-hexane, yielded 1x as a white, crystalline solid (26
mg, 74%). m/z (ES), 379 ([M + H]+); HRMS, found 379.1841
(C23H27N2OS, [M + H]+, requires 379.1844).
(2,4-Diphenyloxazol-5-yl)-carbamic Acid tert-Butyl Ester (2c).
2-(Benzoylamino)-2-phenylglycinonitrile hydrochloride (3.89 g,
14.3 mmol) was partitioned between DCM (70 mL) and water (50
mL), and sodium carbonate was added portionwise, with thorough
mixing, until the aqueous layer was basic to universal indicator
paper (pH 10). The organic layer was separated, dried over MgSO4,
and filtered. The filtrate (∼100 mL) was added slowly to a solution
of triphosgene (4.23 g, 14.3 mmol) in DCM (30 mL). A precipitate
began to appear during this addition. After stirring for 15 min, tert-
butyl alcohol (30 mL) was added cautiously. A homogeneous
solution resulted after 2-3 min, at which point stirring was
continued for an additional 5 min. The reaction was quenched by
addition of 0.1 M K2CO3 (200 mL), and the organic layer was
separated, washed with further 0.1 M K2CO3 (2 × 150 mL), and
dried over MgSO4. The crude material was purified by flash column
chromatography on silica gel, eluted with 60-90-100% DCM-
hexane, giving 2c as an off-white foam (1.07 g, 22%). m/z (EI-),
335 (M-); HRMS, found 335.1396 (C20H20N2O3, M-, requires
335.1396).
N-(2,4-Diphenyloxazol-5-yl)-3-trifluoromethylbenzamide (2h):
m/z (ES), 409 ([M + H]+); HRMS, found 409.1180 (C23H16F3N2O2,
[M + H]+, requires 409.1164).
N-(2,4-Diphenyloxazol-5-yl)-4-trifluoromethylbenzamide (2i):
m/z (ES), 409 ([M + H]+); HRMS, found 409.1149 (C23H16F3N2O2,
[M + H]+, requires 409.1164).
N-(2,4-Diphenyloxazol-5-yl)nicotinamide (2j): m/z (ES), 342
([M + H]+); HRMS, found 342.1235 (C21H16N3O2, [M + H]+,
requires 342.1234).
Quinoline-2-carboxylic acid (2,4-diphenyloxazol-5-yl)amide
(2l): m/z (ES), 392 ([M + H]+); HRMS, found 392.1384
(C25H18N3O2, [M + H]+, requires 392.1399).
5-Nitrofuran-2-carboxylic acid (2,4-diphenyloxazol-5-yl)a-
mide (2n): m/z (ES), 376 ([M + H]+); HRMS, found 376.0934
(C20H14N3O5, [M + H]+, requires 376.0933).
Isoxazole-5-carboxylic Acid (2,4-diphenyloxazol-5-yl)amide
(2o): Purification by flash column chromatography (n-hexane-ethyl
acetate, 4:1) gave 2o (58.2 mg, 23%); m/z (ES), 332 ([M + H]+);
HRMS, found 332.1023 (C19H14N3O3, [M + H]+, requires 332.1035).
Thiophene-2-carboxylic acid (2,4-diphenyloxazol-5-yl)amide
(2p): m/z (ES), 347 ([M + H]+); HRMS, found 347.0851
(C20H15N2O2S, [M + H]+, requires 347.0854).
Benzo[â]thiophene-2-carboxylic acid (2,4-diphenyloxazol-5-
yl)amide (2r): m/z (ES), 397 ([M + H]+); HRMS, found 397.1001
(C24H17N2O2S, [M + H]+, requires 397.1011).
Cyclopropanecarboxylic acid (2,4-diphenyloxazol-5-yl)amide
(2v): m/z (ES), 305 ([M + H]+); HRMS, found 305.1278
(C19H17N2O2, [M + H]+, requires 305.1290).
N-(2,4-Diphenyloxazol-5-yl)-4-methoxybenzamide (2f) (DMAP
procedure). 2,4-Diphenyl-5-N-Boc-aminooxazole 2c (100 mg, 0.30
mmol) was dissolved in dry DCM (3.0 mL), and then N,N-
diisopropylethylamine (58 µL, 0.33 mmol) and DMAP (∼7 mg,
20 mol %) were added followed by 4-methoxybenzoyl chloride
(45 µL, 0.33 mmol). The reaction was deemed complete by TLC
after 150 min of stirring at ambient temperature. TFA (0.75 mL)
was then added and stirring continued at ambient temperature for
18 h. DCM (40 mL) was added and the solution washed with water
(2 × 40 mL) and then dried over MgSO4. Further purification was
achieved by flash column chromatography on silica gel, eluted with
0-1% MeOH-DCM, to provide 2f as an off-white foam (76 mg,
69%); m/z (ES), 371 ([M + H]+); HRMS, found 371.1383
(C23H19N2O3, [M + H]+, requires 371.1396).
N-(2,4-Diphenyloxazol-5-yl)isonicotinamide (2k): m/z (ES),
342 ([M + H]+); HRMS, found 342.1245 (C21H16N3O2, [M + H]+,
requires 342.1243).
General Procedure for the Synthesis of 5-Amidooxazoles
(NaH procedure). To a suspension of sodium hydride (60%
dispersion in mineral oil, 0.65 mmol) in dry THF (3.0 mL) was
added a solution of 2c (0.59 mmol) in dry THF (4.0 mL). Then
after 5 min, the reaction mixture was clear and a solution of acid
chloride (0.65 mmol) was added. The reaction was deemed complete
by TLC after 10 min. The reaction was quenched by the cautious
addition of water followed by the removal of all volatiles under
reduced pressure. The aqueous mixture remaining was extracted
thoroughly with DCM. The combined organic phases were com-
bined, dried over MgSO4, and concentrated to a volume of 50 mL
under reduced pressure. TFA was added to a concentration of 20%
and the mixture stirred at ambient temperature for 18 h. Removal
of all volatiles under reduced pressure gave the crude product, which
was recrystallized from hot n-hexane-ethyl acetate.
Furan-2-carboxylic acid (2,4-diphenyloxazol-5-yl)amide (2m):
m/z (ES), 331 ([M + H]+); HRMS, found 331.1093 (C20H15N2O3,
[M + H]+, requires 331.1083).
5-Methylthiophene-2-carboxylic acid (2,4-diphenyloxazol-5-
yl)amide (2q): m/z (ES), 361 ([M + H]+); HRMS, found 361.1007
(C21H17N2O2S, [M + H]+, requires 361.1011).
Benzofuran-2-carboxylic acid (2,4-diphenyloxazol-5-yl)amide
(2s): m/z (ES), 381 ([M + H]+); HRMS, found 381.1250
(C24H17N2O3, [M + H]+, requires 381.1239).
Benzothiazole-2-carboxylic acid (2,4-diphenyloxazol-5-yl)-
amide (2t): m/z (ES), 398 ([M + H]+); HRMS, found 398.0977
(C23H16N3O2S, [M + H]+, requires 398.0963).
Benzo[â]thiophene-5-carboxylic acid (2,4-diphenyloxazol-5-
yl)amide (2u): m/z (ES), 397 ([M + H]+); HRMS, found 397.1029
(C24H17N2O2S, [M + H]+, requires 397.1011).
2-Propylpentanoic acid (2,4-diphenyloxazol-5-yl)amide (2w):
m/z (ES), 363 ([M + H]+); HRMS, found 363.2066 (C23H27N2O2,
[M + H]+, requires 363.2073).
N-(2,4-Diphenyloxazol-5-yl)benzamide (2d): m/z (ES), 341
([M + H]+); HRMS, found 341.1289 (C22H17N2O2, [M + H]+,
requires 341.1290).
N-(2,4-Diphenyloxazol-5-yl)-4-fluorobenzamide (2e): m/z (ES),
359 ([M + H]+); HRMS, found 359.1188 (C22H16N2O2F, [M +
H]+, requires 359.1196).
Acknowledgment. We thank the Department of Health for
their generous funding (Contract No. DH007/0102).

Potential NoVel Prion Disease Therapeutics
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 6 1353
(10) Shimada, Y., Patent JP 02113070, 1990.
Supporting Information Available: Spectroscopic data for all
new compounds 1e-x, 2c-w (IR, 1H NMR, 13C NMR), and
analysis of compound purity by HPLC. This material is available
free of charge via the Internet at http://pubs.acs.org.
(11) Corrao, S. L.; Macielag, M. J.; Turchi, I. J. Rearrangement of
4-(Aminothiocarbonyl)oxazoles to 5-Aminothiazoles. Synthetic and
MINDO/3 MO Studies. J. Org. Chem. 1990, 55, 4484-4487.
(12) Burger, K.; Hoss, E.; Geith, K. Introduction of Amino-Groups
Adjacent to Trifluoromethyl Groups in Heterocyclic-Compounds -
Synthesis of 5-Amino-4-trifluoromethyl-thiazoles. Synthesis 1990,
357-360.
(13) Golankiewicz, B.; Januszczyk, P.; Gdaniec, M.; Kosturkiewicz, Z.
Reaction of Acylaminocyanoesters with 2,4-Bis(4-methoxyphenyl)-
1,3,2,4-dithiadiphosphetane 2,4-Disulfide Leading to Substituted
Aminothiazoles. Crystal Structure of 5-Aminothiazole-4-carboxylic
Acid Ethyl Ester. Tetrahedron 1985, 41, 5989-5994.
(14) Thompson, M. J.; Heal, W.; Chen, B. Synthesis of 5-aminothiazoles
as building blocks for library synthesis. Tetrahedron Lett. 2006, 47,
2361-2364.
(15) (a) Lichtenberger, J.; Fleury, J-P. Formation, constitution, and
properties of some derivatives of 2,4-disubstituted 5-aminooxazoles.
II. 2-Aryl(alkyl)-4-phenyl-5-arylideneaminooxazoles. Bull. Soc. Chim.
Fr. 1956, 1184-1192; (b) Kille, G.; Fleury, J-P. Heterocyclisation
of R-acylaminonitriles. II. Structure of 5-aminooxazoles and of their
salts. Bull. Soc. Chim. Fr. 1967, 4619-4624.
(16) Verschave, P.; Vekemans, J.; Hoornaert, G. N-Acylated Alpha-
Aminonitriles and their Conversion into 5-Aminooxazole, 5(4H)-
Oxazolone and 4(5H)-Imidazolone Derivatives. Tetrahedron 1984,
40, 2395-2404.
(17) Rudyk, H.; Vasiljevic, S.; Hennion, R. M.; Birkett, C. R.; Hope, J.;
Gilbert, I. H. Screening Congo Red and its analogues for their ability
to prevent the formation of PrP-res in scrapie-infected cells. J. Gen.
Virol. 2000, 81, 1155-1164.
(18) Clarke, M. C.; Haig, D. A. Evidence for the multiplication of scrapie
agent in cell culture. Nature 1970, 225, 100-101.
References
(1) Weissmann, C.; Aguzzi, A. Approaches to Therapy of Prion Diseases.
Annu. ReV. Med. 2005, 56, 321-344.
(2) Prusiner, S. B. Prions. Proc. Nat. Acad. Sci. U.S.A. 1998, 95, 13363-
13383.
(3) Will, R. G.; Ironside, J. W.; Zeidler, M.; Cousens, S. N.; Estibeiro,
K.; Alperovitch, A.; Poser, S.; Pocchiari, M.; Hofman, A.; Smith,
P. G. A new variant of Creutzfeldt-Jakob disease in the UK. Lancet
1996, 347, 921-925.
(4) Collinge, J.; Sidle, K. C. L.; Meads, J.; Ironside, J.; Hill, A. F.
Molecular analysis of prion strain variation and the aetiology of ‘new
variant’ CJD. Nature 1996, 383, 685-690.
(5) Anderson, R. M.; Donnelly, C. A.; Ferguson, N. M.; Woolhouse,
M. E. J.; Watt, C. J.; Udy, H. J.; MaWhinney, S.; Dunstan, S. P.;
Southwood, T. R. E.; Wilesmith, J. W.; Ryan, J. B. M.; Hoinville,
L. J.; Hillerton, J. E.; Austin, A. R.; Wells, G. A. H. Transmission
dynamics and epidemiology of BSE in British cattle. Nature 1996,
382, 779-788.
(6) Brown, P.; Preece, M.; Brandel, J. P.; Sato, T.; McShane, L.; Zerr,
I.; Fletcher, A.; Will, R. G.; Pocchiari, M.; Cashman, N. R.;
d’Aignaux, J. H.; Cervenakova, L.; Fradkin, J.; Schonberger, L. B.;
Collins, S. J. Iatrogenic Creutzfeldt-Jakob disease at the millennium.
Neurology 2000, 55, 1075-1081.
(7) Mpamhanga, C.; Chen, B.; Mutter, R.; Willett, P.; McClay, I. Virtual
high-throughput screening for the discovery of potential prion disease
therapeutics. Manuscript in preparation.
(8) Touil, F.; Pratt, S.; Mutter, R.; Chen, B. Screening a library of
potential prion disease therapeutics against cellular prion proteins
and studying their mode of biological activities by surface plasmon
resonance. J. Pharm. Biomed. Anal. 2006, 40, 822-832.
(9) Hoshino, K.; Ishida, H.; Omovskaya, O.; Dudley, M.; Rleger, R.;
Watkins, W. J.; Zhang, J. Z.; Renau, T. E.; Lee, V. J.; Ota, T.;
Nakayama, K.; Ishida, Y.; Otsuka, M.; Kawato, H., Patent JP
2002322054, 2002.
(19) Calabrese, E. J.; Baldwin, L. A. Hormesis: The dose-response
revolution. Annu. ReV. Pharmacol. Toxicol. 2003, 43, 175-197.
(20) Guo, K.; Mutter, R.; Heal, W.; Reddy, T. R. K.; Pratt, S.; Cope, H.;
Thompson, M. J.; Chen, B. Synthesis and evaluation of a focused
library of pyridine dicarbonitriles against prion disease. Eur. J. Med.
Chem., submitted.
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