Improved 2,4-diarylthiazole-based antiprion agents: Switching the sense of the Amide Group at C5 leads to an increase in potency

Article abstract of DOI:10.1002/cmdc.201000217

Amide derivatives of 2,4-diarylthiazole-5-carboxylic acids were synthesised and tested for efficacy in a cell line model of prion disease. A number of compounds demonstrating antiprion activity were thereby identified from the screening libraries, showing improved potency and reproducibility of results relative to amide derivatives of the related 2,4-diphenyl-5-aminothiazole, which have been documented previously. Thus, 'switching' the sense of the amide bond at thiazole C5 revealed a more promising lead series of potential prion disease therapeutics. Furthermore, 3,5-diaryl-1,2,4-thiadiazoles isolated as by-products during library synthesis provided a handful of additional examples possessing an antiprion effect, thereby augmenting the set of newly identified active compounds. Evaluation of binding to cellular prion protein (PrP^C) showed only weak affinities at best, suggesting that the newly identified antiprion agents do not mediate their biological effect through direct interaction with PrP^C.

Full text of DOI:10.1002/cmdc.201000217

MED
DOI: 10.1002/cmdc.201000217
Improved 2,4-Diarylthiazole-Based Antiprion Agents:
Switching the Sense of the Amide Group at C5 Leads to an
Increase in Potency
Mark J. Thompson,* Jennifer C. Louth, Gemma K. Greenwood, Fiona J. Sorrell,
Sandra G. Knight, Nathan B. P. Adams, and Beining Chen[a]
Amide derivatives of 2,4-diarylthiazole-5-carboxylic acids were
synthesised and tested for efficacy in a cell line model of prion
disease. A number of compounds demonstrating antiprion ac-
tivity were thereby identified from the screening libraries,
showing improved potency and reproducibility of results rela-
tive to amide derivatives of the related 2,4-diphenyl-5-amino-
thiazole, which have been documented previously. Thus,
’switching’ the sense of the amide bond at thiazole C5 re-
vealed a more promising lead series of potential prion disease
therapeutics. Furthermore, 3,5-diaryl-1,2,4-thiadiazoles isolated
as by-products during library synthesis provided a handful of
additional examples possessing an antiprion effect, thereby
augmenting the set of newly identified active compounds.
Evaluation of binding to cellular prion protein (PrPC) showed
only weak affinities at best, suggesting that the newly identi-
fied antiprion agents do not mediate their biological effect
through direct interaction with PrPC.
Introduction
Prion diseases, or transmissible spongiform encephalopathies
(TSEs), are a group of invariably fatal disorders that afflict both
humans and animals for which no effective therapy presently
exists. Although rare, their inevitable lethality renders the dis-
covery of suitable treatments a pressing clinical need.
distinct structural classes: 9-aminoacridines and related com-
pounds,[8]
pyridine-3,5-dicarbonitriles,[9]
indole-3-glyoxyla-
mides,[10] and 2,4-diphenyl- thiazoles and oxazoles
1
(Figure 1).[11] Within the latter class, six moderately active com-
pounds were identified, with EC50 values ranging from 1.5–
20 mm.
The principal such human condition is Creutzfeldt–Jakob dis-
ease (CJD), which accounts for a mortality rate of approximate-
ly one person per million per year across all populations stud-
ied to date.[1] The disease presents in sporadic, inherited, and
iatrogenic forms, and whereas the aetiology of sporadic CJD
remains uncertain, the familial form is associated with specific
pathogenic mutations in prnp, the gene that encodes normal
cellular prion protein (PrPC).[2] Similarly, the less prevalent
human prion diseases, Gerstmann–Strꢀussler–Scheinker syn-
drome (GSS) and familial fatal insomnia (FFI), are associated
with other defined prnp mutations.[3]
Figure 1. General structure of existing 2,4-diphenylazole-based antiprion
agents and their “reverse amide” analogues.
Examples of prion disease in animals include scrapie in
sheep and goats, chronic wasting disease (CWD) in cervids,
and bovine spongiform encephalopathy (BSE) in cattle. The
latter of these is thought to have given rise to the emergence
of variant CJD (vCJD)[4] in humans as a result of consumption
of contaminated meat products, and consequently triggered
much research into the mechanisms of, and potential treat-
ments for, human prion diseases.
Owing to poor reproducibility of these compounds’ antipri-
on effect across differing passage numbers, and also to clear
cytotoxicity of trifluoroacetyl compounds 1a and 1b close to
their active concentrations (EC50 ~1.5 mm; LD50 ~5 mm),[11] ana-
logues with improved potency and therapeutic window were
sought. Considering possible modifications to 1 that might
The central molecular event common to all TSEs is conver-
sion of native PrPC into a refolded, pathogenic isoform denot-
ed PrPSc. Thus, stably infected cell lines acting as a host for
PrPSc are routinely employed for in vitro screening of potential
antiprion agents.[5] We previously reported the use of a scrapie
mouse brain (SMB) cell line,[6,7] cloned from murine brain in-
fected with the Chandler scrapie strain, in the identification of
a number of active inhibitors of PrPSc accumulation across four
[a] Dr. M. J. Thompson, J. C. Louth, Dr. G. K. Greenwood, F. J. Sorrell,
S. G. Knight, N. B. P. Adams, Dr. B. Chen
Department of Chemistry, University of Sheffield
Brook Hill, Sheffield, S7 3HF (UK)
Fax: (+44)114-2229346
E-mail: m.j.thompson@sheffield.ac.uk
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201000217.
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2,4-Diarylthiazole-Based Antiprion Agents
achieve these aims, we devised a library of ’reverse amide’
thiazole analogues 2 (Figure 1), and set out to explore the in-
fluence of this structural change upon antiprion activity.
Table 1. Antiprion screening and cytotoxicity results for compounds 2a–
u.
Compd
R1
Yield [%]
(Method)
EC50 [mm]
LD50 [mm]
[a]
Results and Discussion
2a
2b
25 (B)
24 (B)
–
>20
>20
Synthesis and screening
–
A range of 2,4-diphenylthiazole-5-carboxamides 2a–u was pre-
pared via straightforward amide coupling reactions from either
the requisite carboxylic acid or acyl chloride (Scheme 1). These
2c
80 (B)
74 (B)
–
–
>20
>20
2d
2e
2 f
64 (B)
84 (B)
–
–
>20
>20
Scheme 1. Preparation of thiazole-5-carboxamide library members. Reagents
and conditions: a) R1NH2, benzotriazol-1-yl-oxytripyrrolidinophosphonium
hexafluorophosphate, iPr2NEt, CHCl3, RT, 3 h; b) R1NH2, iPr2NEt, CH2Cl2, RT,
3 h; c) R1NH2 (6 equiv), CH2Cl2, RT, 2 h.
2g
2h
46 (B)
65 (B)
–
–
>20
>20
compounds were screened for antiprion activity in the SMB
cell line as described,[10] and LD50 determination was carried
out where cytotoxicity was observed during the initial screen
(Table 1).
2i
2j
59 (B)
59 (C)
4.9ꢀ1.1
4.8ꢀ1.4
ambiguous[b]
ambiguous[b]
Two active library members were initially identified, includ-
ing 2-(diethylamino)ethyl derivative 2i, with an EC50 value of
4.9 mm (Figure 2a), which inspired the synthesis of analogues
2j–2l. As with the parent compound, dimethylamino deriva-
tive 2j showed borderline toxicity at higher concentrations
(>10 mm; Figure 2b), but similar potency with respect to PrPSc
clearance. Pyrrolidino analogue 2k was marginal in terms of
activity (EC50 =10.7 mm) and still showed some cytotoxicity,
whilst the final compound in the series—morpholino analogue
2l—displayed no discernible antiprion activity. Therefore, in
this 2-(dialkylamino)ethyl set of compounds 2i–2l, smaller sub-
stituents on nitrogen are evidently more favourable for activity.
Although an antiprion effect is evident in the case of 2i–2k,
ambiguous results were obtained for cell viability (Figure 2a–
c). Whereas clear-cut toxicity was not observed at higher con-
2k
2l
43 (C)
88 (B)
10.7ꢀ0.5
ambiguous[b]
–
>15
2m
2n
2o
79 (B)
53 (A)
63 (A)
0.43ꢀ0.19
>20
>20
>20
–
–
2p
2q
2r
78 (A)
77 (A)
57 (B)
42 (A)
–
–
–
–
>20
>20
>20
>20
centrations—as was observed for compounds 1a and 1b[11]
—
some decrease in cell number was apparent, and a distinct
change in morphology of the remaining cells was evident (Fig-
ure 2d–h). Cells exposed to 2j or 2k at 1 mm showed no differ-
ence in appearance from the untreated control, but at a test
compound concentration of 30 mm, a definite morphological
change became clear, with apparently increased cell adhesion
in these wells. The observed changes were noticeable gradual-
ly with increasing compound concentration, becoming evident
at ~7.5 mm for 2j or 2k (data not shown). Compound 2i did
show some such effect, but not as consistently, and proved
less toxic than its related analogues 2j and 2k (Figure 2a–c);
thus, of the 2-aminoalkyl series, 2i was selected for further op-
timisation.
2s
2t
53 (A)
35 (B)
–
–
>20
>20
2u
[a] No antiprion effect observed up to 20 mm. [b] Compounds 2i–2k
showed borderline toxicity at higher concentrations (cell viability ~50–
70%), but conclusive LD50 curves could not be obtained.
pound 2m, by far the most active antiprion agent of the 2,4-
diphenylthiazole class identified to date (EC50 =0.43 mm).
Through preparation and screening of a range of closely relat-
In terms of potency, however, the most promising lead from
the initial series of amides was furfurylamine-derived com-
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Scheme 2. Preparation of analogues incorporating variations at the 2-posi-
tion of the thiazole ring. Reagents and conditions: a) EtOH, reflux, 90 min;
b) R1NH2, TBD (30 mol%), 808C, 18 h.
ethanol at reflux. Identification of ethyl benzoylacetate 8 as an
additional side-product during thiazole synthesis offered a
plausible explanation: the a-bromoketone 3 functions as an
oxidant to promote dimerisation of the thioamide (in turn
being reduced to 8), in addition to acting as an electrophile en
route to desired product 4.
Only one previous report exists of 1,2,4-thiadiazole forma-
tion during the Hantzsch thiazole synthesis,[16] but interestingly,
a relatively hindered a-bromoketone (ethyl 2-bromo-2-(o-nitro-
benzoyl)acetate) was also employed in this case. As such, we
propose that the extent of formation of 1,2,4-thiadiazoles 7
during the Hantzsch reaction may generally be significant
where sterically demanding a-bromoketones are employed.
With such electrophiles of lower reactivity, the extent to which
they act as oxidising agents in a competing process to form 7
appears to become important, though as is evident from the
variable product distribution in the present examples, the
nature of the thioamide also plays a role.
Figure 2. Typical results for assessment of antiprion activity (black line)
versus cell viability (square symbols, as determined by MTT assay) for test
compounds a) 2i, b) 2j, and c) 2k. Cell images illustrate the changes in mor-
phology observed at higher concentrations of 2j and 2k: d) untreated con-
trol, e) 2j at 1 mm, f) 2j at 30 mm, g) 2k at 1 mm, and h) 2k at 30 mm. Scale
bar in image d) represents 100 mm; all panels are to the same scale, with
the exception of image g), in which the scale bar represents 40 mm. Images
were captured after exposure to the test compound for five days.
ed analogues of this structure, 2n–2u, it became disappoint-
ingly clear that variation of the furfuryl group is not tolerated,
as activity was abolished in all of the associated compounds.
Variation at the 2-position of the thiazole ring was then ex-
plored, whilst maintaining the best substituents at C5 identi-
fied above, in an attempt to improve activity. Hantzsch thiazole
synthesis[12] from ethyl 2-benzoyl-2-bromoacetate 3[13] and a
range of thioamides provided 4-phenylthiazole-5-carboxylic
esters 4a–c, which, following aminolysis mediated by 1,5,7-
triazabicyclo[4.4.0]dec-5-ene (TBD),[14] afforded a range of new
test compounds, 5a–c and 6a–c (Scheme 2), analogous to the
existing lead compounds 2i and 2m.
During the Hantzsch thiazole step, two other products were
unexpectedly detected in varying quantities, in addition to the
desired thiazole-5-carboxylic esters 4. Most intriguing was the
isolation of 1,2,4-thiadiazoles 7 in significant amounts (up to
41% yield). Formation of these species via dimerisation of thio-
amides is well established,[15] but normally requires the use of
an oxidising agent; thus, in line with expectations, no reaction
was detected upon holding a thioamide component alone in
Screening of the new thiazole sets 5 and 6 (Table 2) again
revealed that any modification to 2m is not tolerated, as activi-
ty was lost in all of its analogues 5a–c. Variation to (diethylami-
no)ethyl lead 2i gave more productive results, with p-me-
thoxy-substituted analogue 6b displaying similar activity
(EC50 =4.0 mm, versus 4.9 mm for its parent structure), but lower
toxicity towards the SMB cells (Figure 3a), that is, with no in-
duction of the morphological changes observed on exposure
to 2i–2k. Replacing the phenyl ring at C2 by a heterocycle, as
in 6a or 6c, compromised activity to a significant extent. Con-
sidering the full set of screening results thus far, it would
appear that analogues of 2i or 2j bearing substituted phenyl
rings at thiazole C2 (that is, the R2 position of 6) offer a promis-
ing avenue of exploration towards finding new compounds
that possess both improved antiprion activity and decreased
cytotoxicity.
Synthetic intermediates 4a–c and 1,2,4-thiadiazole by-prod-
ucts 7a–c were also screened for any antiprion effect, reveal-
ing 7b (R2 =p-methoxyphenyl) as a novel lead structure with
an EC50 value of 1.62 mm (Figure 3b; Table 3). In a similar trend
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2,4-Diarylthiazole-Based Antiprion Agents
Table 2. Antiprion screening and cytotoxicity results for compounds 4a–f
and 5a-f.
Table 3. Results for additional 1,2,4-thiadiazole compounds 7d–h.
Compd
R2
Yield [%][a]
25[b]
EC50 [mm]
LD50 [mm]
>20
Compd
R2
Yield [%][a]
EC50 [mm]
LD50 [mm]
>20
[e]
7a
–
[b]
5a
38
–
7b
7c
7d
12[b]
39[b]
9[c]
1.62ꢀ0.77
>20
>20
>20
5b
12
–
>20
–
–
5c
6a
14
37
–
>20
>30
~20
7e
63
2.94ꢀ0.02
>20
6b
6c
18
17
4.0ꢀ2.0
6.7ꢀ0.4
>10[c]
~30
7 f
7g
7h
71
78
8[d]
n/a[f]
borderline[g]
–
0.67
>20
>20
[a] Overall yield for the two-step procedure presented in Scheme 2.
[b] No antiprion effect observed up to 20 mm. [c] Partial decrease in cell
viability at higher concentrations, but not to the same extent observed
for 2i–2k.
[a] Yield for dimerisation of thioamide R2CSNH2 after column chromatog-
raphy; except: [b] Isolated during synthesis of 4a–c. [c] Synthesised in
the absence of Et4N+Brꢁ, resulting in poor conversion. [d] Low yield is
seemingly due to retention/decomposition of this compound on the
silica column during chromatography. [e] No antiprion effect observed up
to 20 mm. [f] Compound is too toxic to the cells to assess activity. [g] Bor-
derline antiprion activity observed towards the higher end of the concen-
tration range tested (PrPSc ~70% of control at 20 mm), but not sufficiently
potent to be considered a positive result.
offer insight into any potential structure–activity relationships
(SAR).
This premise was tested by preparing a small additional set
of 1,2,4-thiadiazoles 7d–h by oxidative dimerisation of appro-
priate thioamides (Scheme 3). Although the combination of o-
Scheme 3. Extra 1,2,4-thiadiazole derivatives prepared by thioamide dimeri-
sation. Reagents and conditions: a) PhI(OAc)2, Et4N+Brꢁ, CH2Cl2, reflux, 2 h
(8–78%).
Figure 3. Dose–response curves for a) compound 6b and b) compound 7b,
showing clear antiprion activity (solid line shows PrPSc concentration relative
to untreated control), but lower toxicity than compounds 2i–k. Points de-
noted by square symbols show cell viability, with the shaded area represent-
ing “nontoxic” (viability >70% of untreated control). The data above are
fused from multiple experiments; results for each individual experiment are
presented in the Supporting Information.
iodoxybenzoic acid (IBX) and tetraethylammonium bromide
has recently been described as an efficient method for this
process,[15] the considerable expense of IBX led us to substitute
an alternative hypervalent iodine reagent in its place; iodoben-
zene diacetate (IBDA) gave satisfactory results, and all reactions
were complete within 2 h at reflux in dichloromethane. IBDA
has been reported as an effective promoter of thioamide di-
merisation before, albeit using a polymer-supported reagent[17]
or reaction in an ionic liquid[18] rather than a conventional or-
ganic solvent.
to the thiazole series 6a–c, analogues 7a and 7c bearing het-
erocyclic substituents proved inactive, suggesting that a series
of 1,2,4-thiadiazoles 7 incorporating substituted phenyl groups
at the R2 positions would likewise be the best candidates to
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Upon screening of the new thiadiazoles, it was pleasing to
discover that two compounds possessed notable antiprion ac-
tivity (Table 3), although toxicity towards the SMB cells was ob-
served in one case (m-methoxy analogue 7 f). Whereas p-tolyl
derivative 7g only displayed a very weak effect, the 3,4-dime-
thoxyphenyl analogue 7e was more potent, demonstrating an
EC50 value close to that of 7b. Coupled with the ineffectiveness
of p-trifluoromethyl compound 7d, these results suggest that
3,5-diaryl-1,2,4-thiadiazoles bearing electron-donating groups
on the phenyl rings may constitute a worthwhile course of
future study, to refine the antiprion properties of this family of
compounds.
The active leads identified in the present work—all being
structures of type 2, 6, and 7—each incorporate a central five-
membered heterocycle bearing two aromatic substituents ar-
ranged in a 1,3-sense. Interestingly, other recent studies have
reported antiprion agents that contain very similar substructur-
al motifs, namely, 1,3- or 1,4-diphenyl-1H-pyrazol-5(4H)-one de-
rivatives[19] and 2-(N-pyridin-2-ylamino)-4-arylthiazoles.[20] In ad-
dition, a series of pyridine-3,5-dicarbonitrile antiprion com-
pounds bearing (2-aminoalkyl)thio groups[21] displayed marked
similarities to present analogues 2i–2l in terms of the relation-
ship between the nature of the amine side chain and antiprion
activity. Whereas no direct conclusions should be drawn imme-
diately, the possibility of a shared mechanism of action be-
tween the related compound classes detailed above is perhaps
worthy of further scrutiny.
Figure 4. Interaction of compounds 5b, 6a, and 6b with immobilised
human (hu-PrPC) and murine (mo-PrPC) forms of recombinant prion protein,
over a range of concentrations, as measured by surface plasmon resonance
(SPR). a) Binding of 5b approaches a limiting value, suggesting formation of
a specific 1:1 ligand–protein complex, although the measured SPR response
(RU) falls short of the calculated value, which would correspond to an equi-
molar ratio of ligand and immobilised protein (RUmax). b) Compounds 5b
and 6a each show evidence for specific binding to both forms of PrPC. c) 6b
shows similar affinity for the proteins, but appears to interact with them in a
nonspecific manner, that is, no approach towards a limiting response is ob-
served in this case.
Further characterisation of active leads
The 2,4-diarylthiazole-based compounds were originally identi-
fied as potential ligands for normal cellular prion protein (PrPC)
in a virtual screening programme, with the earlier effective an-
tiprion examples 1a–d all being found to bind to PrPC to some
extent.[11] Thus, the cell-line-active compounds described
above (and some of their closest structural analogues) were
also assessed for any interaction with PrPC, using the same sur-
face plasmon resonance (SPR)-based assay employed previous-
ly.[22]
giving a curve characteristic of 1:1 complex formation. The re-
sponse at saturation is considerably lower than expected, how-
ever, at only 40% RUmax. If this were a genuine binding event,
it is plausible that the ordering of the protein on the chip sur-
face is such that the ligand binding site is not accessible for all
of the immobilised PrPC, which would explain this apparent
discrepancy. Variation of binding response over time has al-
ready been noted during this assay,[22] and dynamic reordering
of the protein bound to the chip surface could certainly ex-
plain these changes in observed binding over the lifetime of
the chip.
Briefly, a solution of each test compound is passed over the
protein of interest immobilised on a sensor chip surface. The
SPR response, measured in the instrument’s response units
(RU), increases in proportion to any ligand binding that takes
place. Results are expressed in a normalised form—percent
RUmax—that is, as a percentage relative to the calculated value
of SPR response corresponding to formation of a 1:1 protein–
ligand complex. Compounds of interest are further character-
ised by screening over a range of concentrations to assess the
dose dependence of the observed binding. Interaction with
both human (hu-PrPC) and murine (mo-PrPC) forms of the pro-
tein was measured.
Considering compound 5b, fitting to a saturation binding
model gave apparent affinities (KD) of 27 and 87 mm against
hu-PrPC and mo-PrPC, respectively. Possible specific binding
was also observed for 6a (Figure 4b), although the interaction
seems considerably weaker, such that KD values could not be
computed. Lastly, a linear dose–response relationship was ob-
served during assessment of 6b (Figure 4c), suggesting non-
specific interaction with both forms of PrPC.
A number of members of the present test set showed weak
binding to both forms of the protein (full results are included
in table S2 in the Supporting Information), with three com-
pounds displaying a significant interaction (5b, 6a, and 6b;
Figure 4). The recorded response for 5b can be seen to ap-
proach a limiting value as concentration increases (Figure 4a),
Of the three ligands identified by SPR, however, only 6b dis-
played any significant activity during cell line screening. Also
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2,4-Diarylthiazole-Based Antiprion Agents
considering the weak binding affinities observed, it appears
likely that the antiprion effect of all cell-line-active compounds
identified above (2i–2k, 2m, 6a–c, and 7b) is not mediated
through interaction with PrPC. To reach a more firm conclusion,
binding of the three most potent leads (2m, 6b, and 7b) to
the human form (hu-PrPC) was further investigated by mea-
surement of fluorescence quenching, an approach that has
been applied successfully to quantify binding of Cu2+ to the
prion protein.[23]
mation were found to operate through an aggregation mecha-
nism.[25] Testing for aggregation behaviour is therefore an espe-
cially valuable tool in eliminating ’false positive’ hits from typi-
cal screening programmes, such that computational methods
for predicting such effects have recently been developed.[26]
Results for the present compound set (Figure 6), including the
known aggregator[24b] benzyl benzoate (BB) as a positive con-
Compound 6b did indeed show interaction with hu-PrPC by
this method (observed KD =3.8ꢀ0.8 mm; Figure 5a), suggest-
ing stronger binding of this ligand than was evident by SPR.
Figure 6. Assessment of the aggregation tendency of screening compounds
in aqueous solution. A high count rate indicates the presence of light-scat-
tering particles in solution. Compound 5b shows clear evidence for forma-
tion of aggregates under these conditions. KP: potassium phosphate buffer
(pH 7.4); ANS: 8-anilino-1-naphthalenesulfonic acid, a known non-aggrega-
tor;[23e] BB: benzyl benzoate.
trol, revealed 5b is a significant aggregator over the concen-
tration range examined in the SPR experiment. This tendency
precludes reliable assessment of the compound in any direct
binding assay, rendering the above SPR results (Figure 4a) in-
conclusive. Related structure 2m showed less tendency to ag-
gregate, apparently forming small particles (158ꢀ29 nm diam-
eter) at 10 mm, although it could not be assessed at higher
concentrations due to insolubility, which had not been en-
countered for the p-methoxy-substituted analogue 5b. In con-
trast to the preceding N-furfurylamides, 2-(diethylamino)ethyl-
containing compounds 6a and 6b both showed very low ag-
gregation tendency, together with full aqueous solubility up to
200 mm, as might be expected given the presence of this more
hydrophilic side chain. The interaction of 6b with PrPC ob-
served in both the SPR and fluorescence quenching assays
therefore appears to reflect genuine binding to the protein, as
defined particles were only detectable by DLS at 200 mm; simi-
larly, analogue 6a also appears to function as a ligand based
on the SPR results (Figure 4b), albeit with noticeably weaker
affinity.
Figure 5. Assessment of binding of 6b and 2m to PrPC by the fluorescence
quenching technique. Quenching of fluorescence of N-acetyl-l-tryptophana-
mide (NATA), at a concentration equivalent to the tryptophan residues in
the protein,[22a] was also measured to correct for collisional effects. It can be
seen that a) 6b shows some affinity for the protein, as was observed by SPR,
and b) 2m does not bind to PrPC, again reflecting the results obtained by
the alternative assay.
The SPR results were also reflected for 2m, with no discernible
binding evident from the fluorescence quenching data (Fig-
ure 2b); results for 7b were almost identical. Thus, there is
good correlation between assessment of binding to PrPC be-
tween the two assays, with the two most potent leads (2m
and 7b) emerging as clear non-binders, and 6b apparently
showing weak affinity for the protein.
The three ligands identified by SPR (5b, 6a, and 6b) were
also screened for their tendency to form colloid-like aggre-
gates in aqueous solution using the dynamic light scattering
(DLS) technique. Awareness of aggregation-based effects in
protein inhibition and binding assays has gained a high profile
in recent years,[24] and of particular relevance in the present
context, several small-molecule inhibitors of prion amyloid for-
When considering the suitability of a compound as a poten-
tial drug, it is advantageous to measure stability towards mi-
crosomal metabolism. In this way, compounds that are likely to
be metabolically labile may be excluded or modified at an
early stage of the discovery process. The cell-line-active antipri-
on agents in the present study were assessed by incubation
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with mouse liver microsome (MLM) preparation, with mixed re-
sults (Table 4). The most potent compound, 2,4-diphenylthia-
zole derivative 2m, showed significant depletion after 30 min;
1,2,4-thiadiazole series 7 fared better, though both analogues
Experimental Section
Materials and general methods: 2,4-Diphenylthiazole-5-carboxylic
acid and 2,4-diphenylthiazole-5-carbonyl chloride were purchased
from Maybridge (UK) and ABCR (Germany), respectively. Anhydrous
CH2Cl2 was obtained from an in-house ’Grubbs’ apparatus. ’Petro-
leum ether’ refers to light petroleum, bp: 40–608C. Reagents for
SPR screening: N-hydroxysuccinimide (NHS), N-ethyl-N’-(3-diethyl-
aminopropyl)carbodiimide hydrochloride (EDC), 1m ethanolamine,
HBS-EP buffer, surfactant P20, regeneration solution (10 mm glyci-
ne·HCl, pH 3.0) and CM-dextran (Mr =13 kDa), were all purchased
from GE Healthcare Bio-Sciences AB (Uppsala, Sweden). Recombi-
nant full-length human prion protein (hu-PrPC) and full-length
mouse prion protein (mo-PrPC) were generous gifts from Dr.
Andrew Gill (Roslin Institute, University of Edinburgh, UK). All other
reagents and solvents were obtained from appropriate commercial
sources and used as supplied. Melting points were determined
using a Gallenkamp melting point apparatus in capillary tubes, and
Table 4. Microsomal stability assessment of cell-line-active compounds.
Compd
EC50 [mm]
MLM Stability [%][a]
2m
6b
7b
0.43ꢀ0.19
4.0ꢀ2.0
1.62ꢀ0.77
2.94ꢀ0.02
n.d.
55.5ꢀ0.8
[b]
–
76.0ꢀ1.3
90.4ꢀ1.6
53.1ꢀ0.1
81.9ꢀ1.4
7e
testosterone[c]
diclofenac[c]
n.d.
[a] Percentage of compound remaining after 30 min incubation with
mouse liver microsome (MLM) preparation. [b] No result could be ob-
tained for compound 6b and related structures containing the 2-(diethy-
lamino)ethyl group due to unusual interaction with the C18 HPLC column;
these difficulties are addressed in greater detail in the main text discus-
sion. [c] Testosterone[27] and diclofenac[28] were used as positive controls,
as both compounds are known to be metabolised by cytochromes pres-
ent in microsomal preparations; n.d.: not determined.
1
are uncorrected. H and 13C NMR spectra were recorded at 400 and
100 Hz, respectively, on a Bruker AV-1400 spectrometer, or at 250
and 62.8 MHz, respectively, on a Bruker AV-1250 model. Mass spec-
tra were acquired using a Micromass LCT Premier XE system. Infra-
red spectra were recorded on a PerkinElmer Spectrum RX instru-
ment fitted with a SensIR Technologies DurasamplIRTM II device.
HPLC analysis of compound purity was carried out using a Genesis
4 mm C18 column, 4.6ꢂ150 mm, eluted with a gradient of MeCN/
H2O (30!100% over 12 min; hold at 100% MeCN for 10 min), flow
rate: 1 mLminꢁ1, with UV detection at l 254 nm. Optical rotation
values were measured with an Optical Activity Ltd AA-10 polarime-
ter. Fluorescence spectra were recorded at the steady state using a
PerkinElmer LS 50B Spectrometer, and test samples contained
within quartz cuvettes. Dynamic light scattering (DLS) measure-
ments were carried out using a Malvern Instruments Zetasizer
Nano ZS system, equipped with a 4 mW He–Ne laser at l 633 nm.
were metabolised to some degree. The challenge for future
work in this area will therefore be to develop compounds that
retain or improve cell line activity, whilst proving more robust
towards likely first-pass clearance.
Regrettably, data could not be obtained for the most active
member of the N,N-(dialkylamino)ethyl-containing thiazoles,
6b, and related analogues 2i–k, 6a, and 6c. For each of these
compounds, unsuitable or erratic behaviour was observed
under the HPLC conditions used for the assay, precluding accu-
rate determination of any extent of metabolism.[29]
Assessment of antiprion activity in SMB cells: Compounds were
screened for inhibition of PrPSc formation in the SMB.s15 cell line[7]
according to the protocol described previously.[9,10] 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 peni-
cillin–streptomycin at 10 mgLꢁ1, at 378C in an atmosphere of 5%
CO2 in air at 95% relative humidity. The medium was changed
every third or fourth day, and every seven 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 distribut-
ed into 96-well cluster plates at 3ꢂ104 cells per well and incubated
for 24 h to allow for cell attachment. The compounds were pre-
pared at 400 times the required concentration in DMSO as stock
solutions then transferred, at a 20-fold dilution, into Hank’s bal-
anced 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 five 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; 0.5% w/v
sodium deoxycholate), and the content of the well was loaded
onto a nitrocellulose membrane (0.45 mm) under gentle vacuum at
a total cellular protein concentration of ~30–40 mg per well (deter-
mined by the Bradford assay following the protocol supplied with
the reagent; Sigma). The membrane was air dried and subjected to
75 mgmLꢁ1 proteinase K digestion for 1 h at 378C. The reaction
was stopped with 1 mm phenylmethylsulfonyl fluoride (PMSF) in
Conclusions
A more coherent lead series of 2,4-diarylthiazole-based antipri-
on agents has been identified by “switching” the sense of the
amide bond at C5. Thiazole-5-carboxamides such as those of
type 2 and 6 showed more potent and reproducible cell line
activity than related amide structures 1 (X=S; Figure 1), de-
rived from 5-amino-2,4-diphenylthiazole. Of the present screen-
ing set, compounds 2m and 6b offer the best promise for fur-
ther development as potential prion disease therapeutics.
Binding of the biologically active library members to the
prion protein was largely found to be weak or negligible,
which, with the possible exception of 6b (KD =3.8 mm, EC50 =
4.0 mm), suggests that the antiprion activity of the present
compounds is not effected through interaction with PrPC.
Screening of a small number of 3,5-diaryl-1,2,4-thiadiazoles,
initially isolated as by-products during preparation of the thia-
zole libraries, revealed two members of this series (7b and 7e)
as novel inhibitors of PrPSc accumulation. Preliminary aspects
of a SAR with respect to antiprion activity have been deduced
for both the thiazole and thiadiazole series of compounds, par-
ticularly suggesting the importance of electron-donating
groups attached to the aromatic substituents.
1482
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ChemMedChem 2010, 5, 1476 – 1488

2,4-Diarylthiazole-Based Antiprion Agents
20 mm Tris·HCl-buffered saline (TBS) and the membrane was
washed extensively with TBS and immersed in 1.8m guanidine thi-
ocyanate 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 mgmLꢁ1 mouse monoclonal anti-PrP 6H4 (Prionics), and devel-
oped using an ECL kit (Amersham Pharmacia Biotech). Every ex-
periment was carried out in triplicate, and an average value for
PrPSc concentration was calculated, relative to an untreated control
(DMSO only), together with a standard deviation. Curcumin was
employed as a positive control, and effected complete clearance of
PrPSc at the concentration used (10 mm; EC50 =0.95 mm[10]). Test
compounds were initially screened at 1, 10, and 20 mm and were
considered to be active if PrPSc levels were decreased to <70% of
that of the untreated control after five days’ exposure. Compounds
showing activity were re-screened over a range of concentrations
to determine an EC50 value; such experiments were repeated at
least twice (in triplicate) to validate the results so obtained. LD50
values were assessed from a plot of cell viability (determined by
the MTT assay), after five days’ exposure, against test compound
concentration.
Assessment of binding to PrPC by fluorescence quenching: Re-
combinant human PrPc was prepared at a protein concentration of
5 mm in 25 mm NaOAc buffer (pH 5.5) then its initial fluorescence
spectrum was recorded (lex 290 nm; lem 310–500 nm; 5 nm slit;
1200 nmminꢁ1; eight scans). Additional samples were then pre-
pared in the same buffer, which in addition to PrPC, contained the
test compound of interest at various concentrations (for example,
17 concentrations ranging between 1–50 mm in the case of 6b),
added from a stock solution in DMSO (10 mm). After equilibration
at ambient temperature for 2 min, the fluorescence spectrum of
each test solution was recorded in the same manner as above. Cor-
rection for collisional quenching effects was applied by repeating
the measurements with 5 mm PrPC replaced by 28.6 mm N-acetyl-l-
tryptophanamide, equivalent to the concentration of Trp residues
in the protein,[23a] and subtracting the collisional fluorescence
quenching readings from those observed during assessment of
ligand binding. Raw data were collected using Winlab software,
converted into ASCII format, then imported into GraphPad Prism
5.02 for curve fitting and nonlinear regression (one-site binding
model), to determine observed KD values.
Microsomal stability assay: The procedure was modified from
that reported in a recent study of quinacrine metabolism.[30] A mix-
ture of test compound (5.0 mm stock solution in DMSO, 0.8 mL),
isocitrate dehydrogenase (31.4 mL, 2 U), 0.2m potassium phosphate
buffer (pH 7.0, containing 10 mm d,l-isocitric acid trisodium salt
and 10 mm MgCl2; 228 mL), and microsome preparation from
mouse liver (0.5 mgmLꢁ1 in the same buffer; 100 mL) was incubat-
ed in an Eppendorf tube for 5 min at 378C. NADPH (10 mm stock
solution; 40 mL) was then added to initiate the reaction. Contents
of the tube were mixed using aspiration/dispensation five times,
then a t=0 aliquot (150 mL) was drawn immediately and quenched
with ice-cold acetonitrile (300 mL), containing 8 mm 1-(2-methyl-1H-
indol-3-yl)-2-morpholinoethane-1,2-dione[10] as internal reference
standard. The remaining reaction mixture was maintained at 378C
for 30 min, then a second aliquot removed and diluted as above.
The quenched solutions were each vortexed for 30 s and centri-
fuged at 4000 rpm (458 fixed-angle rotor F-45-12-11) for 10 min,
then the supernatant analysed by HPLC: Genesis 4 mm C18 column,
4.6ꢂ150 mm; 5–95% MeCN/H2O over 4 min, hold 6 min at 95%
MeCN; flow rate 1 mLminꢁ1; UV detection at l 267 nm. Each analy-
sis was performed in triplicate, and metabolism results after 30 min
were compared with those at t=0. The percentage of drug re-
maining, together with the standard deviation, is reported for each
test compound. During assessment of testosterone, N-(4-(1H-
pyrrol-1-yl)phenyl)-2-(1H-indol-3-yl)-2-oxoacetamide[10] was used as
internal standard, due to the appearance of metabolite peaks over-
lapping the signal for the reference described above.
Assessment of binding to PrPC by SPR: The method used was es-
sentially as reported previously.[22] Experiments were carried out
using a Biacore 3000 instrument equipped with a CM5 sensor chip,
containing a CM-dextran surface. Prior to screening, PrPC was im-
mobilised on the chip surface according to the following protocol.
A 1:1 mixture of 100 mm NHS and 400 mm EDC was passed over
the sensor chip for 7 min at a flow rate of 5 mLminꢁ1. A solution of
PrPC, at a concentration of 2 mgmLꢁ1 in HBS-EP buffer, was then
passed over the chip surface for 7 min. Unreacted sites were
blocked by injection of 1m ethanolamine (pH 8.5) for 7 min, after
which the chip surface was prepared for use by three consecutive
injections (5 mL at 30 mLminꢁ1) of 25 mm NaOH/1m NaCl solution.
Finally, the sensor chip surface was equilibrated with running
buffer (10 mm Na3PO4, pH 7.4; 150 mm NaCl; 3.4 mm EDTA;
0.005% v/v surfactant P20) overnight prior to screening. The four
flow cells of the Biacore instrument were employed as follows:
flow cell 1 was used as a reference surface (no immobilised pro-
tein); flow cell 2 contained hu-PrPC; flow cell 3 contained mo-PrPC;
and flow cell 4 was not used. Test compounds were dissolved in
DMSO at 800 mm and diluted to the required concentration (40 mm
for routine screening; variable concentrations for KD determination)
with running buffer prior to injection. Assays were performed at
258C with a flow rate of 30 mLminꢁ1. To correct for solvent effects,
6.5% DMSO was added to the running buffer, and DMSO calibra-
tion sequences using buffer samples containing 5.5–7.5% DMSO
(in 0.5% intervals) were carried out at the start of each run, and
after every 10 test compounds. Each analytical cycle consisted of
running buffer for 1 min (stabilisation phase), sample injection for
1 min (association phase), and running buffer for 3 min (dissocia-
tion phase). Between cycles, surface regeneration was carried out
at 35 mLminꢁ1 by injecting 25 mm NaOH/1m NaCl/0.0005% SDS
pH 8.5 (for 30 s), then by 10 mm glycine·HCl pH 3.0 (for 35 s), fol-
lowed by a re-equilibration phase in running buffer for 1 min. Each
test compound was injected in triplicate in order to establish an
average response (recorded in resonance units, RU) and a standard
deviation. Binding affinities are expressed as percent RUmax, where
RUmax is the theoretical response for a 1:1 binding interaction be-
tween the ligand and PrPC, calculated according to the following
Equation (1), in which RUimmobilised protein is the response (in RU) ob-
served at the end of the immobilisation procedure.
Assessment of aggregation tendency by dynamic light scatter-
ing (DLS): Compounds were diluted from a 10 mm DMSO stock so-
lution to the required concentration in 50 mm potassium phos-
phate buffer, pH 7.4, at a final DMSO concentration of 2%. Three
measurements of the derived count rate (in kilocounts per second;
kcps) were taken, and a mean value is reported for each sample as-
sessed, together with the standard deviation over the three results.
Synthesis of 2,4-diphenylthiazole-5-carboxamides (2a–u). Meth-
od A: The relevant amine (R1NH2, 0.44 mmol) was added to a mix-
ture of 2,4-diphenylthiazole-5-carboxylic acid (125 mg, 0.44 mmol)
and Hꢃnig’s base (155 mL, 115 mg, 0.89 mmol) in CHCl3 (4 mL). Ben-
zotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate
(231 mg, 0.44 mmol) was introduced to the reaction mixture,
which was then stirred at room temperature for 3 h. After this
time, the solution was applied directly to a silica gel column and
RUmax ¼ ðRUimmobilised protein=Mr proteinÞ ꢂ Mr ligand
ð1Þ
ChemMedChem 2010, 5, 1476 – 1488
ꢁ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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1483

M. J. Thompson et al.
MED
eluted as indicated for each individual example to provide the
pure product.
8.0 Hz, 1H), 6.75–6.68 (m, 1H), 6.64–6.59 (m, 2H), 6.04 (t, J=5.0 Hz,
1H), 4.33 (d, J=6.0 Hz, 2H), 3.69 ppm (s, 3H); 13C NMR (62.8 MHz,
CDCl3): d=44.1, 55.3, 113.1, 113.4, 120.0, 126.8, 128.95, 129.04,
129.4, 129.5, 129.7, 130.9, 132.9, 133.9, 138.7, 154.6, 159.8, 161.1,
168.9 ppm; IR (solid): n˜ =3291, 1635, 1597, 1534, 1483, 1435, 1258,
1167, 1044, 760, 686 cmꢁ1; MS (ES): m/z (%): 401 (100) [M+H]+;
HRMS-ES: m/z [M+H]+ calcd for C24H21N2O2S: 401.1324, found:
401.1309.
Method B: The amine (R1NH2, 0.44 mmol) was dissolved in anhy-
drous CH2Cl2 (3 mL) under N2, then Hꢃnig’s base (77 mL, 57 mg,
0.44 mmol) and 2,4-diphenylthiazole-5-carbonyl chloride (133 mg,
0.44 mmol) were added, and the reaction mixture stirred at room
temperature for 3 h. The solution was applied directly to a silica
gel column and eluted as indicated for each individual example to
provide the pure product.
2,4-Diphenylthiazole-5-carboxylic acid 4-methoxybenzylamide
(2 f): After elution with 0!1% MeOH/CH2Cl2, obtained as a white
solid (148 mg, 84%): 1H NMR (250 MHz, CDCl3): d=8.02–7.95 (m,
2H), 7.66–7.59 (m, 2H), 7.49–7.35 (m, 6H), 7.07–7.00 (m, 2H), 6.83–
6.77 (m, 2H), 6.00 (brs, 1H), 4.37 (d, J=5.5 Hz, 2H), 3.78 ppm (s,
3H); 13C NMR (62.8 MHz, CDCl3): d=43.6, 55.3, 114.0, 126.8, 128.9,
129.0, 129.2, 129.3, 129.4, 129.5, 129.7, 130.8, 133.0, 133.9, 154.5,
159.1, 161.0, 168.9 ppm; IR (solid): n˜ =3316, 1617, 1532, 1509,
1478, 1250, 1234, 1023, 817, 760, 699, 681, 654 cmꢁ1; MS (ES): m/z
(%): 401 (100) [M+H]+; HRMS-ES: m/z [M+H]+ calcd for
C24H21N2O2S: 401.1324, found: 401.1306.
Method C: Excess amine (R1NH2, 2.64 mmol, 6 equiv) was added to
a solution of 2,4-diphenylthiazole-5-carbonyl chloride (133 mg,
0.44 mmol) in anhydrous CH2Cl2 (3 mL) under N2, then the reaction
mixture stirred at room temperature for 2 h. The solution was ap-
plied directly to a silica gel column and eluted as indicated for
each individual example to provide the pure product.
2,4-Diphenylthiazole-5-carboxylic acid tert-butylamide (2a):
After elution with CH2Cl2, obtained as a white solid (37 mg, 25%):
1H NMR (250 MHz, CDCl3): d=7.97–7.88 (m, 2H), 7.66–7.57 (m, 2H),
7.52–7.33 (m, 6H), 5.49 (s, 1H), 1.14 ppm (s, 9H); 13C NMR
(62.8 MHz, CDCl3): d=28.4, 51.9, 126.8, 128.9, 129.0, 129.5, 129.6,
130.7, 131.8, 133.1, 134.1, 153.9, 160.4, 168.5 ppm; IR (solid): n˜ =
2,4-Diphenylthiazole-5-carboxylic acid quinolin-3-ylamide (2g):
After elution with CH2Cl2/hexane (3:1) then 0!1!2.5% MeOH/
CH2Cl2, obtained as an off-white foam (82 mg, 46%): 1H NMR
(250 MHz, CDCl3): d=8.73 (d, J=2.0 Hz, 1H), 8.11–7.95 (m, 4H),
7.84–7.43 ppm (m, 12H); 13C NMR (62.8 MHz, CDCl3): d=123.5,
127.0, 127.4, 127.8, 128.1, 128.4, 129.07, 129.14, 129.6, 129.7, 130.0,
130.4, 130.7, 131.3, 132.8, 133.8, 143.3, 145.4, 155.5, 159.5,
170.4 ppm; IR (solid): n˜ =3293, 1668, 1641, 1533, 1482, 1364, 1312,
1256, 748, 726, 685, 639, 605 cmꢁ1; MS (ES): m/z (%): 408 (100)
[M+H]+; HRMS-ES: m/z [M+H]+ calcd for C25H18N3OS: 408.1171,
found: 408.1181.
3264, 3063, 2966, 1630, 1555, 1482, 1346, 1225, 978, 761, 684 cmꢁ1
;
MS (ES): m/z (%): 337 (100) [M+H]+; HRMS-ES: m/z [M+H]+ calcd
for C20H21N2OS: 337.1375, found: 337.1378.
2,4-Diphenylthiazole-5-carboxylic acid (2-chlorophenyl)amide
(2b): After elution with 7.5% EtOAc/hexane, obtained as a white
solid (41 mg, 24%): 1H NMR (250 MHz, CDCl3): d=8.49 (d, J=
8.0 Hz, 1H), 8.16–7.98 (m, 3H), 7.78–7.69 (m, 2H), 7.59–7.42 (m,
6H), 7.31–7.18 (m, 2H), 6.99 ppm (t, J=8.0 Hz, 1H); 13C NMR
(62.8 MHz, CDCl3): d=107.4, 121.3, 122.4, 124.8, 126.9, 127.7, 129.0,
129.1, 129.5, 129.7, 130.1, 131.1, 132.9, 133.7, 134.4, 155.5, 159.2,
169.9 ppm; IR (solid): n˜ =3360, 1656, 1590, 1525, 1438, 1421, 1328,
1313, 756, 747, 694, 681, 585, 572 cmꢁ1; MS (ES): m/z (%): 391 (100)
[M+H]+; HRMS-ES: m/z [M+H]+ calcd for C22H15ClN2OS: 391.0672,
found: 391.0671.
2,4-Diphenylthiazole-5-carboxylic acid benzothiazol-2-ylamide
(2h): After elution with CH2Cl2, obtained as a white, crystalline
solid (118 mg, 65%): 1H NMR (250 MHz, [D6]DMSO): d=13.28 (s,
1H), 8.09–8.01 (m, 2H), 7.95 (d, J=7.3 Hz, 1H), 7.86–7.79 (m, 2H),
7.66–7.41 (m, 8H), 7.36–7.28 ppm (m, 1H); 13C NMR (62.8 MHz,
[D6]DMSO): d=122.2, 123.8, 126.5, 126.7, 128.1, 128.9, 129.3, 129.4,
131.2, 132.3, 134.0, 156.6, 167.4 ppm; IR (solid): n˜ =2734, 1677,
1658, 1557, 1442, 1432, 1331, 1302, 1272, 859, 754, 741, 727, 681,
666 cmꢁ1; MS (ES): m/z (%): 414 (100) [M+H]+; HRMS-ES: m/z
[M+H]+ calcd for C23H16N3OS2: 414.0735, found: 414.0731.
2,4-Diphenylthiazole-5-carboxylic acid (3-methoxyphenyl)amide
(2c): After elution with 75!100% CH2Cl2/hexane, obtained as a
1
white solid (136 mg, 80%): H NMR (250 MHz, CDCl3): d=8.06–7.98
(m, 2H), 7.79–7.72 (m, 2H), 7.62–7.54 (m, 3H), 7.52–7.41 (m, 4H),
7.13 (t, J=8.0 Hz, 1H), 7.07 (t, J=2.5 Hz, 1H), 6.67–6.53 (m, 2H),
3.77 ppm (s, 3H); 13C NMR (62.8 MHz, CDCl3): d=55.3, 105.1, 110.5,
111.5, 126.9, 129.1, 129.4, 129.7, 130.1, 131.1, 132.9, 133.9, 138.4,
154.7, 159.0, 160.1, 169.8 ppm; IR (solid): n˜ =3298, 1638, 1600,
1493, 1412, 1258, 1162, 1040, 836, 779, 682 cmꢁ1; MS (ES): m/z (%):
387 (100) [M+H]+; HRMS-ES: m/z [M+H]+ calcd for C23H19N2O2S:
387.1167, found: 387.1160.
2,4-Diphenylthiazole-5-carboxylic acid (2-diethylaminoethyl)a-
mide (2i): After elution with 2.5!5% MeOH/CH2Cl2, obtained as a
1
thick, viscous oil (98 mg, 59%): H NMR (250 MHz, CDCl3): d=8.02–
7.93 (m, 2H), 7.74–7.66 (m, 2H), 7.51–7.38 (m, 6H), 6.62 (brs, 1H),
3.34 (q, J=5.5 Hz, 2H), 2.43 (t, J=6.0 Hz, 2H), 2.33 (q, J=7.0 Hz,
4H), 0.80 ppm (t, J=7.0 Hz, 6H); 13C NMR (62.8 MHz, CDCl3): d=
11.1, 37.4, 46.1, 50.6, 126.8, 128.7, 128.8, 129.0, 129.2, 129.4, 130.7,
133.1, 134.2, 154.5, 161.3, 168.4 ppm; IR (neat): n˜ =3276, 2967,
2926, 2810, 1634, 1542, 760, 729, 687 cmꢁ1; MS (ES): m/z (%): 380
(100) [M+H]+; HRMS-ES: m/z [M+H]+ calcd for C22H25N3OS:
380.1797, found: 380.1786.
2,4-Diphenylthiazole-5-carboxylic acid (4-methoxyphenyl)amide
(2d): After elution with CH2Cl2, obtained as a pale-yellow solid
(126 mg, 74%): 1H NMR (250 MHz, [D6]DMSO): d=10.47 (s, 1H),
8.10–8.00 (m, 2H), 7.88–7.78 (m, 2H), 7.61–7.36 (m, 8H), 6.92 (d,
J=8.9 Hz, 2H), 3.73 ppm (s, 3H); 13C NMR (125 MHz, [D6]DMSO):
d=55.2, 114.0, 121.5, 126.4, 127.4, 128.3, 128.5, 128.7, 129.4, 131.0,
131.5, 132.3, 133.6, 153.2, 155.9, 159.5, 165.7 ppm; IR (solid): n˜ =
3309, 1630, 1599, 1527, 1516, 1480, 1414, 1336, 1237, 1032, 810,
760, 684 cmꢁ1; MS (ES): m/z (%): 409 (100) [M+Na]+; HRMS-ES: m/z
[M+Na]+ calcd for C23H18N2O2SNa: 409.0987, found: 409.0995.
N-[2-(Dimethylamino)ethyl]-2,4-diphenylthiazole-5-carboxamide
(2j): After elution with 0!2.5!5!7.5% MeOH/CH2Cl2, obtained
as a thick, colourless oil (91 mg, 59%): 1H NMR (400 MHz, CDCl3):
d=8.04–7.99 (m, 2H), 7.72–7.68 (m, 2H), 7.54–7.43 (m, 6H), 6.46
(brm, 1H), 3.35 (q, J=6.0 Hz, 2H), 2.26 (t, J=6.0 Hz, 2H), 2.01 ppm
(s, 6H); 13C NMR (62.8 MHz, CDCl3): d=37.2, 44.8, 56.8, 126.8, 128.8,
129.0, 129.2, 129.4, 130.1, 130.7, 133.1, 134.3, 154.6, 161.2,
168.6 ppm; IR (neat): n˜ =3308, 2938, 2818, 2767, 1632, 1536, 760,
686 cmꢁ1; MS (ES): m/z (%): 352 (100) [M+H]+; HRMS-ES: m/z
[M+H]+ calcd for C20H22N3OS: 352.1484, found: 352.1478.
2,4-Diphenylthiazole-5-carboxylic acid 3-methoxybenzylamide
(2e): After elution with 0!1% MeOH/CH2Cl2, obtained as a pale-
yellow solid (113 mg, 64%): 1H NMR (250 MHz, CDCl3): d=7.94–
7.83 (m, 2H), 7.60–7.52 (m, 2H), 7.41–7.27 (m, 6H), 7.11 (t, J=
1484
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ChemMedChem 2010, 5, 1476 – 1488

2,4-Diarylthiazole-Based Antiprion Agents
2,4-Diphenyl-N-[2-(pyrrolidin-1-yl)ethyl]thiazole-5-carboxamide
(2k): After elution with 0!2.5!5!7.5% MeOH/CH2Cl2, obtained
as a thick, colourless oil (71 mg, 43%): 1H NMR (400 MHz, CDCl3):
d=8.04–7.99 (m, 2H), 7.73–7.69 (m, 2H), 7.53–7.43 (m, 6H), 6.52
(brm, 1H), 3.40 (q, J=5.5 Hz, 2H), 2.47 (t, J=6.0 Hz, 2H), 2.35–2.27
(m, 4H), 1.67–1.59 ppm (m, 4H); 13C NMR (100 MHz, CDCl3): d=
23.4, 38.4, 53.4, 53.6, 126.8, 128.8, 129.0, 129.3, 129.4, 130.1, 130.7,
133.1, 134.2, 154.5, 161.2, 168.6 ppm; IR (neat): n˜ =3304, 2965,
2796, 1627, 1532, 1480, 1434, 1310, 1266, 1144, 840, 760, 695,
684 cmꢁ1; MS (ES): m/z (%): 378 (100) [M+H]+; HRMS-ES: m/z
[M+H]+ calcd for C22H24N3OS: 378.1640, found: 378.1649.
(m, 1H), 3.62 (t, J=7.0 Hz, 2H), 3.54 (ddd, J=4.0 Hz, 5.5 Hz,
14.0 Hz, 1H), 3.30 (dt, J=6.0 Hz, 14.0 Hz, 1H), 1.95–1.71 (m, 3H),
1.51–1.41 ppm (m, 1H); 13C NMR (100 MHz, CDCl3): d=25.7, 28.4,
43.3, 67.9, 77.0, 126.8, 128.98, 129.01, 129.38, 129.43, 129.8, 130.8,
133.0, 134.2, 154.6, 161.4, 168.8 ppm; IR (solid): n˜ =3288, 2950,
2860, 1637, 1534, 1062, 761, 686 cmꢁ1; MS (ES): m/z (%): 365 (100)
[M+H]+; HRMS-ES: m/z [M+H]+ calcd for C21H21N2O2S: 365.1324,
found: 365.1325.
(R)-2,4-Diphenyl-N-[(tetrahydrofuran-2-yl)methyl]thiazole-5-car-
boxamide (2q): After elution with 20!30!40% EtOAc/isohexane,
obtained as a thick, colourless gum (124 mg, 77%): [a]2D0 =ꢁ12.08
(c=0.5 in acetone); 1H NMR (400 MHz, CDCl3): d=8.06–7.99 (m,
2H), 7.75–7.70 (m, 2H), 7.56–7.44 (m, 6H), 6.15 (brs, 1H), 3.91–3.84
(m, 1H), 3.62 (t, J=7.0 Hz, 2H), 3.54 (ddd, J=4.0 Hz, 5.5 Hz,
14.0 Hz, 1H), 3.30 (dt, J=6.0 Hz, 14.0 Hz, 1H), 1.95–1.71 (m, 3H),
1.51–1.41 ppm (m, 1H); 13C NMR (100 MHz, CDCl3): d=25.7, 28.4,
43.3, 67.9, 77.0, 126.8, 128.98, 129.01, 129.38, 129.43, 129.8, 130.8,
133.0, 134.2, 154.6, 161.4, 168.8 ppm; IR (solid): n˜ =3286, 2941,
2860, 1632, 1532, 1069, 761, 686 cmꢁ1; MS (ES): m/z (%): 365 (100)
[M+H]+; HRMS-ES: m/z [M+H]+ calcd for C21H21N2O2S: 365.1324,
found: 365.1335.
2,4-Diphenylthiazole-5-carboxylic acid (2-morpholin-4-ylethyl)a-
mide (2l): After elution with 1!2.5!5 MeOH/CH2Cl2, obtained as
1
a white, crystalline solid (152 mg, 88%): H NMR (250 MHz, CDCl3):
d=8.05–7.93 (m, 2H), 7.77–7.66 (m, 2H), 7.55–7.37 (m, 6H), 6.39
(brs, 1H), 3.51–3.32 (m, 6H), 2.31 (t, J=6.0 Hz, 2H), 2.27–2.15 ppm
(m, 4H); 13C NMR (62.8 MHz, CDCl3): d=36.0, 52.9, 56.2, 66.7, 126.8,
128.8, 129.0, 129.4, 129.5, 129.9, 130.8, 133.0, 134.3, 154.4, 161.2,
168.8 ppm; IR (solid): n˜ =3292, 2964, 2860, 2808, 1636, 1535, 1308,
1116, 866, 762, 693, 683 cmꢁ1; MS (ES): m/z (%): 809 (20) [2M+Na]+,
394 (100) [M+H]+; HRMS-ES: m/z [M+H]+ calcd for C22H24N3O2S:
394.1589, found: 394.1598.
N-[(3-Methylfuran-2-yl)methyl]-2,4-diphenylthiazole-5-carboxa-
mide (2r): After elution with 10!15!20!25% EtOAc/hexane,
2,4-Diphenylthiazole-5-carboxylic acid (furan-2-ylmethyl)amide
(2m): After elution with CH2Cl2, obtained as an off-white, crystalline
solid (125 mg, 79%): 1H NMR (250 MHz, CDCl3): d=7.99 (dd, J=
2.8 Hz, 6.1 Hz, 2H), 7.64 (dd, J=2.9 Hz, 6.3 Hz, 2H), 7.48–7.38 (m,
6H), 7.33–7.28 (m, 1H), 6.29 (dd, J=2.0 Hz, 3.2 Hz, 1H), 6.15–6.04
(m, 2H), 4.44 ppm (d, J=5.5 Hz, 2H); 13C NMR (62.8 MHz, CDCl3):
d=36.8, 107.7, 110.5, 126.8, 129.0, 129.1, 129.3, 129.4, 129.5, 130.9,
133.0, 133.8, 142.2, 150.4, 154.7, 161.0, 169.1 ppm; IR (solid): n˜ =
3294, 1637, 1512, 1477, 1433, 1331, 1256, 1193, 1148, 1073, 767,
744, 692, 599 cmꢁ1; MS (ES): m/z (%): 361 (100) [M+H]+; HRMS-ES:
m/z [M+H]+ calcd for C21H17N2O2S: 361.1011, found: 361.1021.
1
obtained as a white solid (94 mg, 57%): H NMR (400 MHz, CDCl3):
d=8.04–7.99 (m, 2H), 7.68–7.63 (m, 2H), 7.50–7.40 (m, 6H), 7.23
(d, J=2.0 Hz, 1H), 6.20 (d, J=1.5 Hz, 1H), 6.06 (t, J=5.0 Hz, 1H),
4.42 (d, J=5.5 Hz, 2H), 2.01 ppm (s, 3H); 13C NMR (100 MHz,
CDCl3): d=10.1, 35.2, 113.5, 117.7, 127.2, 129.3, 129.4, 129.71,
129.77, 129.83, 131.3, 133.4, 134.2, 141.7, 146.0, 155.1, 161.3,
169.4 ppm; IR (solid): n˜ =3222, 1629, 1550, 1483, 1438, 1270, 1107,
756, 730, 694, 681 cmꢁ1; MS (ES): m/z (%): 375 (100) [M+H]+;
HRMS-ES: m/z [M+H]+ calcd for C22H19N2O2S: 375.1167, found:
375.1159.
2,4-Diphenyl-N-(thiophen-2-ylmethyl)thiazole-5-carboxamide
(2n): After elution with CH2Cl2, obtained as a white solid (88 mg,
53%): 1H NMR (400 MHz, CDCl3): d=8.05–7.99 (m, 2H), 7.69–7.63
(m, 2H), 7.52–7.39 (m, 6H), 7.24 (dd, J=1.0 Hz, 5.0 Hz, 1H), 6.95
(dd, J=3.5 Hz, 5.0 Hz, 1H), 6.88–6.86 (m, 1H), 6.12 (t, J=5.5 Hz,
1H), 4.64 ppm (d, J=5.5 Hz, 2H); 13C NMR (100 MHz, CDCl3): d=
38.6, 125.4, 126.3, 126.8, 126.9, 128.99, 129.04, 129.4, 129.5, 130.9,
133.0, 133.8, 139.5, 154.8, 160.9, 169.1 ppm; IR (solid): n˜ =3230,
3049, 1621, 1545, 756, 692, 688 cmꢁ1; MS (ES): m/z (%): 377 (100)
[M+H]+; HRMS-ES: m/z [M+H]+ calcd for C21H17N2OS2: 377.0782,
found: 377.0787.
N-[(5-Methylfuran-2-yl)methyl]-2,4-diphenylthiazole-5-carboxa-
mide (2s): After elution with 0!1% MeOH/CH2Cl2, obtained as a
1
white solid (69 mg, 42%): H NMR (400 MHz, CDCl3): d=8.05–7.99
(m, 2H), 7.71–7.66 (m, 2H), 7.50–7.41 (m, 6H), 6.10 (t, J=6.0 Hz,
1H), 6.03 (d, J=3.0 Hz, 1H), 5.89 (dd, J=1.0 Hz, 3.0 Hz, 1H), 4.42
(d, J=5.5 Hz, 2H), 2.26 ppm (s, 3H); 13C NMR (100 MHz, CDCl3): d=
13.9, 37.4, 106.7, 108.9, 127.2, 129.3, 129.4, 129.8, 129.9, 131.3,
133.4, 134.2, 148.8, 152.4, 155.0, 161.3, 169.4 ppm; IR (solid): n˜ =
3238, 3061, 1619, 1543, 1480, 1020, 979, 772, 758, 683 cmꢁ1; MS
(ES): m/z (%): 375 (100) [M+H]+; HRMS-ES: m/z [M+H]+ calcd for
C22H19N2O2S: 375.1167, found: 375.1176.
2,4-Diphenyl-N-[(tetrahydrofuran-2-yl)methyl]thiazole-5-carboxa-
mide (2o): After elution with 50!70!90% EtOAc/hexane, ob-
tained as a white solid (101 mg, 63%): 1H NMR (400 MHz, CDCl3):
d=8.04–7.99 (m, 2H), 7.75–7.70 (m, 2H), 7.55–7.43 (m, 6H), 6.15 (t,
J=5.0 Hz, 1H), 3.91–3.84 (m, 1H), 3.62 (t, J=7.0 Hz, 2H), 3.53 (ddd,
J=4.0 Hz, 5.5 Hz, 14.0 Hz, 1H), 3.30 (dt, J=6.0 Hz, 14.0 Hz, 1H),
1.95–1.71 (m, 3H), 1.50–1.39 ppm (m, 1H); 13C NMR (100 MHz,
CDCl3): d=25.7, 28.4, 43.3, 67.9, 77.0, 126.8, 128.99, 129.02, 129.38,
129.43, 129.8, 130.8, 133.0, 134.2, 154.6, 161.4, 168.8 ppm; IR
N-[(3-Methylisoxazol-5-yl)methyl]-2,4-diphenylthiazole-5-carbox-
amide (2t): After elution with 0!1% MeOH/CH2Cl2, obtained as a
1
white solid (87 mg, 53%): H NMR (400 MHz, CDCl3): d=8.01–7.97
(m, 2H), 7.70–7.64 (m, 2H), 7.52–7.42 (m, 6H), 6.35 (t, J=6.0 Hz,
1H), 5.95 (s, 1H), 4.51 (d, J=6.0 Hz, 2H), 2.28 ppm (s, 3H); 13C NMR
(100 MHz, CDCl3): d=11.4, 35.5, 103.1, 126.8, 128.6, 129.07, 129.12,
129.3, 129.8, 131.0, 132.8, 133.8, 155.2, 159.9, 161.3, 167.6,
169.4 ppm; IR (solid): n˜ =3272, 1632, 1533, 1481, 1427, 1335, 1212,
760, 685 cmꢁ1; MS (ES): m/z (%): 375 (100) [M+H]+; HRMS-ES: m/z
[M+H]+ calcd for C21H18N3O2S: 375.1120, found: 375.1107.
(solid): n˜ =3268, 3054, 2945, 2868, 1644, 1074, 970, 762, 689 cmꢁ1
;
MS (ES): m/z (%): 365 (100) [M+H]+; HRMS-ES: m/z [M+H]+ calcd
N-[(5-Methylisoxazol-3-yl)methyl]-2,4-diphenylthiazole-5-carbox-
amide (2u): After elution with 0!1% MeOH/CH2Cl2, obtained as a
for C21H21N2O2S: 365.1324, found: 365.1322.
1
(S)-2,4-Diphenyl-N-[(tetrahydrofuran-2-yl)methyl]thiazole-5-car-
boxamide (2p): After elution with 20!30!40% EtOAc/isohexane,
obtained as a thick, colourless gum (126 mg, 78%): [a]2D0 =+14.08
(c=0.5 in acetone); 1H NMR (400 MHz, CDCl3): d=8.05–7.99 (m,
2H), 7.75–7.70 (m, 2H), 7.56–7.44 (m, 6H), 6.15 (brs, 1H), 3.92–3.84
white solid (58 mg, 35%): H NMR (400 MHz, CDCl3): d=8.04–7.99
(m, 2H), 7.71–7.66 (m, 2H), 7.52–7.44 (m, 6H), 6.31 (t, J=5.5 Hz,
1H), 5.95 (s, 1H), 4.48 (d, J=6.0 Hz, 2H), 2.41 ppm (s, 3H); 13C NMR
(100 MHz, CDCl3): d=12.2, 35.8, 101.0, 126.8, 128.9, 129.1, 129.4,
129.7, 131.0, 132.9, 133.8, 155.1, 160.4, 161.4, 169.2, 170.1 ppm; IR
ChemMedChem 2010, 5, 1476 – 1488
ꢁ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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1485

M. J. Thompson et al.
MED
(solid): n˜ =3308, 1631, 1606, 1536, 1478, 1435, 1266, 982, 817, 768,
686, 608 cmꢁ1; MS (ES): m/z (%): 375 (100) [M+H]+; HRMS-ES: m/z
[M+H]+ calcd for C21H18N3O2S: 375.1120, found: 375.1106.
4:1); 1H NMR (400 MHz, CDCl3): d=8.00–7.95 (m, 2H), 7.63 (t, J=
7.5 Hz, 1H), 7.51 (t, 2H, J=8.0 Hz), 4.24 (q, J=7.0 Hz, 2H), 4.02 (s,
2H), 1.28 ppm (t, 3H, J=7.0 Hz); MS (ES): m/z (%): 193 (100)
[M+H]+; HRMS-ES: m/z [M+H]+ calcd for C11H13O3: 193.0865,
found: 193.0864.
Ethyl 2-bromo-2-benzoylacetate (3):[13] A solution of ethyl benzoy-
lacetate (6.93 mL, 7.69 g, 40 mmol) in CH2Cl2 (180 mL) was cooled
to 08C, then bromine (1.84 mL, 5.75 g, 36 mmol) in the same sol-
vent (30 mL) was added dropwise over 15 min. The reaction mix-
ture was stirred for an additional 1 h then transferred to a separat-
ing funnel, washed with 10% K2CO3 (150 mL), dried over MgSO4
and evaporated giving a thick oily residue. The title compound
was obtained after flash column chromatography on silica, eluted
with 30!40!50!60% CH2Cl2/hexane, and obtained as a pale-
yellow oil (8.14 g, 75%): Rf =0.25 (hexane/CH2Cl2, 3:2); 1H NMR
(400 MHz, CDCl3): d=8.05–7.99 (m, 2H), 7.69–7.63 (m, 1H), 7.57–
7.50 (m, 2H), 5.69 (s, 1H), 4.32 (q, J=7.0 Hz, 2H), 1.28 ppm (t, J=
6.5 Hz, 3H); 13C NMR (100 MHz, CDCl3): d=14.3, 46.8, 63.7, 129.3,
129.6, 133.8, 134.7, 165.6, 188.5 ppm; IR (oil): n˜ =2981, 1758, 1736,
1684, 1448, 1300, 1256, 1183, 1138, 1023, 1000, 686 cmꢁ1; MS (ESꢁ):
m/z (%): 269 (100) [MꢁH]ꢁ; HRMS-ES: m/z [MꢁH]ꢁ calcd for
C11H10BrO3: 268.9813, found: 268.9824.
Ethyl 2-(thiophen-2-yl)-4-phenylthiazole-5-carboxylate (4c): After
elution with toluene, obtained as a white solid (0.31 g, 28%): Rf =
1
0.30 (hexane/EtOAc, 92:8); H NMR (400 MHz, CDCl3): d=7.86–7.81
(m, 2H), 7.67 (dd, J=1.0 Hz, 4.0 Hz, 1H), 7.51 (dd, J=1.0 Hz, 5.0 Hz,
1H), 7.50–7.45 (m, 3H), 7.15 (dd, J=4.0 Hz, 5.0 Hz, 1H), 4.32 (q, J=
7.0 Hz, 2H), 1.33 ppm (t, J=7.0 Hz, 3H); 13C NMR (100 MHz, CDCl3):
d=14.6, 62.0, 121.9, 128.2, 128.5, 128.6, 129.7, 129.9, 130.4, 134.3,
137.1, 161.1, 161.8, 163.9 ppm; IR (solid): n˜ =2976, 1720, 1442,
1249, 1227, 1136, 1083, 752, 685 cmꢁ1; MS (ES): m/z (%): 316 (100)
[M+H]+; HRMS-ES: m/z [M+H]+ calcd for C16H14NO2S2: 316.0466,
found: 316.0477. Ethyl benzoylacetate 8 was also isolated from this
reaction, as the last compound to elute from the column, and ob-
tained as a pale-yellow oil (162 mg, 24%).
Thiazole-5-carboxamides (5a-c, 6a-c). General procedure: The
thiazole-5-carboxylic ester 4a-c (0.5 mmol) and TBD (21 mg,
0.15 mmol) were mixed thoroughly in a 50 mL size Radleys carou-
sel reaction tube, then a small stirrer bar was introduced to ensure
good mixing over the course of the reaction. Either furfurylamine
(53 mL, 58 mg, 0.6 mmol) or N,N-diethylethylenediamine (84 mL,
70 mg, 0.6 mmol) was added, then the tube heated under N2 at
808C for 18 h with stirring of the reaction mixture. The resultant
material was allowed to cool back to ambient temperature, then
dissolved in a small volume of CH2Cl2, loaded directly to a silica
column, and eluted as described for each individual case to pro-
vide the pure amide product.
Thiazole-5-carboxylic esters (4a–c). General procedure: Ethyl 2-
bromo-2-benzoylacetate 3 (0.95 g, 3.5 mmol) and a thioamide
(3.5 mmol) were combined in EtOH (20 mL), and the mixture was
heated at reflux until TLC analysis showed complete consumption
of the bromide starting material (typically 90 min). The solvent was
evaporated, then the residue was taken up in CH2Cl2 and washed
with saturated NaHCO3, then dried over MgSO4 and evaporated.
Pure product was obtained after flash column chromatography on
silica, eluted with the solvent system detailed below for each indi-
vidual case.
Ethyl 4-phenyl-2-(pyridin-3-yl)thiazole-5-carboxylate (4a): After
elution with 10!20!30!40!50% EtOAc/hexane, obtained as a
pale-beige solid (0.63 g, 58%): Rf =0.30 (CH2Cl2/MeOH, 39:1);
1H NMR (400 MHz, CDCl3): d=9.26 (d, J=2.0 Hz, 1H), 8.74 (dd, J=
1.5 Hz, 4.5 Hz, 1H), 8.36 (dt, J=4.0 Hz, 8.0 Hz, 1H), 7.88–7.82 (m,
2H), 7.53–7.47 (m, 3H), 6.90 (ddd, J=0.5 Hz, 5.0 Hz, 8.0 Hz, 1H),
4.34 (q, J=7.5 Hz, 2H), 1.34 ppm (t, J=7.0 Hz, 3H); 13C NMR
(100 MHz, CDCl3): d=14.1, 61.8, 123.3, 123.9, 127.9, 129.0, 129.4,
129.9, 133.8, 134.1, 148.0, 151.7, 161.0, 161.3, 166.3 ppm; IR (solid):
n˜ =2976, 1722, 1520, 1479, 1408, 1246, 1233, 1138, 1084, 1020,
814, 774, 753, 688 cmꢁ1; MS (ES): m/z (%): 311 (100) [M+H]+;
HRMS-ES: m/z [M+H]+ calcd for C17H15N2O2S: 311.0854, found:
311.0847.
N-(Furan-2-ylmethyl)-4-phenyl-2-(pyridin-3-yl)thiazole-5-carboxa-
mide (5a): After elution with 50!70!90!100% EtOAc/hexane,
1
obtained as a pale-yellow, amorphous solid (119 mg, 66%): H NMR
(400 MHz, CDCl3): d=9.22 (s, 1H), 8.70 (d, J=4.0 Hz, 1H), 8.29 (dt,
J=2.0 Hz, 8.0 Hz, 1H), 7.69–7.64 (m, 2H), 7.49–7.40 (m, 4H), 7.34
(dd, J=0.5 Hz, 2.0 Hz, 1H), 6.33 (dd, J=2.0 Hz, 3.0 Hz, 1H), 6.20–
6.15 (m, 2H), 4.49 ppm (d, J=5.5 Hz, 2H); 13C NMR (100 MHz,
CDCl3): d=36.8, 107.8, 110.5, 123.8, 129.0, 129.3, 129.7, 130.2,
133.4, 134.0, 142.3, 147.8, 150.2, 151.5, 154.9, 160.6, 165.6 ppm; IR
(solid): n˜ =3288, 1623, 1536, 977, 744, 691 cmꢁ1; MS (ES): m/z (%):
362 (100) [M+H]+; HRMS-ES: m/z [M+H]+ calcd for C20H16N3O2S:
362.0963, found: 362.0957.
N-(Furan-2-ylmethyl)-2-(4-methoxyphenyl)-4-phenylthiazole-5-
carboxamide (5b): After elution with 10!20!30% EtOAc/
Ethyl 2-(4-methoxyphenyl)-4-phenylthiazole-5-carboxylate (4b):
Due to the close Rf values of 4b and 7b, some overlap occurred
during chromatography (elution with 5!7.5!10!12.5% EtOAc/
hexane); these ’mixed fractions’ were concentrated and subjected
to further purification using a second silica column. The pure title
compound was finally obtained as a microcrystalline, white solid
(0.33 g, 27%): Rf =0.43 (hexane/EtOAc, 4:1); 1H NMR (400 MHz,
CDCl3): d=8.01 (dt, J=2.5 Hz, 9.0 Hz, 2H), 7.86–7.82 (m, 2H), 7.52–
7.44 (m, 3H), 6.99 (dt, J=2.5 Hz, 9.0 Hz, 2H), 4.31 (q, J=7.0 Hz,
2H), 3.89 (s, 3H), 1.32 ppm (t, J=7.0 Hz, 3H); 13C NMR (100 MHz,
CDCl3): d=14.2, 55.5, 61.4, 114.4, 121.5, 125.8, 127.8, 128.6, 129.1,
129.9, 134.4, 160.8, 161.7, 162.1, 169.8 ppm; IR (solid): n˜ =3003,
2980, 2934, 2836, 1717, 1606, 1520, 1444, 1327, 1254, 1234, 1171,
1140, 1084, 1037, 1020, 828, 771, 752, 700, 686, 604 cmꢁ1; MS (ES):
m/z (%): 340 (100) [M+H]+; HRMS-ES: m/z [M+H]+ calcd for
C19H18NO3S: 340.1007, found: 340.0996. Ethyl benzoylacetate 8 was
also isolated from this reaction, as the highest-running spot, and
obtained as a colourless oil (258 mg, 38%): Rf ~0.55 (hexane/EtOAc,
1
hexane, obtained as a white solid (86 mg, 45%): H NMR (400 MHz,
CDCl3): d=7.95 (d, J=9.0 Hz, 2H), 7.68–7.62 (m, 2H), 7.48–7.40 (m,
3H), 7.35–7.32 (m, 1H), 6.97 (d, J=9.0 Hz, 2H), 6.32 (dd, J=2.0 Hz,
3.0 Hz, 1H), 6.15 (d, J=3.0 Hz, 1H), 6.07 (t, J=5.5 Hz, 1H), 4.46 (d,
J=5.5 Hz, 2H), 3.88 ppm (s, 3H); 13C NMR (100 MHz, CDCl3): d=
36.8, 55.5, 107.6, 110.4, 114.4, 125.9, 128.37, 128.42, 128.9, 129.35,
129.44, 133.9, 142.2, 150.4, 154.6, 161.1, 161.9, 169.0 ppm; IR
(solid): n˜ =3224, 3058, 1620, 1607, 1547, 1479, 1306, 1257, 1150,
1029, 828, 759, 739, 698, 645, 600 cmꢁ1; MS (ES): m/z (%): 391 (100)
[M+H]+; HRMS-ES: m/z [M+H]+ calcd for C22H19N2O3S: 391.1116,
found: 391.1130.
N-(Furan-2-ylmethyl)-4-phenyl-2-(thiophen-2-yl)thiazole-5-car-
boxamide (5c): After elution with 5!10!15!20!25% EtOAc/
hexane, obtained as a greasy, pale-orange solid (92 mg, 50%):
1H NMR (400 MHz, CDCl3): d=7.67–7.63 (m, 3H), 7.50–7.42 (m, 4H),
7.34 (dd, J=1.0 Hz, 2.0 Hz, 1H), 7.14 (dd, J=3.5 Hz, 5.0 Hz, 1H),
6.33 (dd, J=2.0 Hz, 3.0 Hz, 1H), 6.15 (dd, J=0.5 Hz, 3.0 Hz, 1H),
1486
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ChemMedChem 2010, 5, 1476 – 1488

2,4-Diarylthiazole-Based Antiprion Agents
6.04 (t, J=5.0 Hz, 1H), 4.48 ppm (d, J=5.5 Hz, 2H); 13C NMR
(100 MHz, CDCl3): d=36.8, 107.6, 110.4, 128.0, 128.2, 128.6, 129.0,
129.1, 129.4, 129.6, 133.5, 136.6, 142.2, 150.3, 154.5, 160.7,
162.9 ppm; IR (solid): n˜ =3324, 3107, 2927, 1640, 1524, 1502, 1147,
920, 754, 740, 692, 598 cmꢁ1; MS (ES): m/z (%): 367 (100) [M+H]+;
HRMS-ES: m/z [M+H]+ calcd for C19H15N2O2S2: 367.0575, found:
367.0588.
tions, 85:15 MeCN/H2O; flow rate 20 mLminꢁ1; UV detection at
l 254 nm) to remove remaining traces of 4b. The title compound
was obtained as white solid (63 mg, 12%): Rf =0.36 (hexane/EtOAc,
1
4:1); H NMR (400 MHz, CDCl3): d=8.34 (d, J=9.0 Hz, 2H), 8.00 (d,
J=9.0 Hz, 2H), 7.02 (dd, J=2.0 Hz, 9.0 Hz, 4H), 3.90 ppm (s, 6H);
13C NMR (100 MHz, CDCl3): d=55.4, 55.5, 114.0, 114.5, 123.7, 126.0,
129.2, 129.9, 161.3, 162.5, 173.4, 187.4 ppm; IR (solid): n˜ =2963,
2940, 2842, 1465, 1412, 1235, 1170, 1105, 1029, 1013, 987, 832,
748, 702 cmꢁ1; MS (ES): m/z (%): 299 (100) [M+H]+; HRMS-ES: m/z
[M+H]+ calcd for C16H15N2O2S: 299.0854, found: 299.0854.
N-(2-(Diethylamino)ethyl)-4-phenyl-2-(pyridin-3-yl)thiazole-5-car-
boxamide (6a): After elution with 0!5!10!15!20% MeOH/
CH2Cl2, obtained as a thick, sticky gum with a pale reddish-brown
tint (104 mg, 55%): 1H NMR (400 MHz, CDCl3): d=9.24 (dd, J=
0.5 Hz, 2.5 Hz, 1H), 8.71 (dd, J=1.5 Hz, 5.0 Hz, 1H), 8.30 (dt, J=
2.0 Hz, 8.0 Hz, 1H), 7.75–7.70 (m, 2H), 7.55–7.46 (m, 3H), 7.42 (ddd,
J=0.5 Hz, 5.0 Hz, 8.0 Hz, 1H), 6.62 (brs, 1H), 2.57–2.26 (m, 6H),
0.84 ppm (t, J=7.0 Hz, 6H); 13C NMR (100 MHz, CDCl3): d=11.1,
37.4, 46.2, 50.6, 123.7, 128.9, 129.1, 129.3, 129.5, 133.8, 147.9, 151.4,
154.7, 160.9, 165.1 ppm; IR (neat): n˜ =3276, 3057, 2968, 2811, 1639,
1525, 1504, 1475, 732, 697 cmꢁ1; MS (ES): m/z (%): 381 (100)
[M+H]+; HRMS-ES: m/z [M+H]+ calcd for C21H25N4OS: 381.1749,
found: 381.1744.
3,5-Bis(thiophen-2-yl)-1,2,4-thiadiazole (7c): Isolated during the
synthesis of 4c (higher-running spot) as a beige powder (171 mg,
1
39%): Rf =0.40 (hexane/EtOAc, 92:8); H NMR (400 MHz, CDCl3): d=
7.96 (dd, J=1.0 Hz, 3.5 Hz, 1H), 7.72 (dd, J=1.0 Hz, 3.5 Hz, 1H),
7.61 (dd, J=1.0 Hz, 5.0 Hz, 1H), 7.48 (dd, J=1.0 Hz, 5.0 Hz, 1H),
7.21–7.16 ppm (m, 2H); 13C NMR (100 MHz, CDCl3): d=127.9, 128.5,
128.9, 129.3, 129.9, 130.6, 133.1, 136.2, 168.4, 180.7 ppm; IR (solid):
n˜ =3097, 1540, 1462, 1413, 1312, 1285, 1221, 1091, 1070, 1036,
835, 705 cmꢁ1; MS (ES): m/z (%): 251 (100) [M+H]+; HRMS-ES: m/z
[M+H]+ calcd for C10H7N2S3: 251.9771, found: 251.9777.
N-[2-(Diethylamino)ethyl]-2-(4-methoxyphenyl)-4-phenylthia-
zole-5-carboxamide (6b): After elution with 2!4!6!8%
MeOH/CH2Cl2, obtained as a thick, colourless gum (132 mg, 65%):
1H NMR (400 MHz, CDCl3): d=7.92 (d, J=9.0 Hz, 2H), 7.71 (dd, J=
2.0 Hz, 8.0 Hz, 2H), 7.50–7.43 (m, 2H), 6.93 (d, J=9.0 Hz, 2H), 6.78
(brs, 1H), 3.85 (s, 3H), 3.40 (q, J=5.5 Hz, 2H), 2.53 (t, J=5.5 Hz,
2H), 2.48–2.39 (m, 4H), 0.88 ppm (t, J=7.0 Hz, 6H); 13C NMR
(100 MHz, CDCl3): d=11.2, 37.5, 46.6, 51.1, 55.8, 114.7, 126.4, 128.8,
129.2, 129.6, 129.8, 134.8, 155.1, 162.0, 162.1, 168.7 ppm; IR (neat):
n˜ =3269, 2967, 2931, 2832, 1634, 1605, 1256, 1172, 1031, 828, 728,
698 cmꢁ1; MS (ES): m/z (%): 410 (100) [M+H]+; HRMS-ES: m/z
[M+H]+ calcd for C23H28N3O2S: 410.1902, found: 410.1915.
1,2,4-Thiadiazoles by dimerisation of thioamides (7d–h). Gener-
al procedure: The thioamide (2.0 mmol), iodobenzene diacetate
(644 mg, 2.0 mmol) and tetraethylammonium bromide (420 mg,
2.0 mmol) were combined in CH2Cl2 (10 mL). The mixture was
heated at reflux; any solids initially apparent in suspension had dis-
solved within a few minutes of beginning heating. After 2 h at
reflux, the reaction mixture was diluted with additional CH2Cl2,
washed with saturated sodium bisulfite solution, then separated
and evaporated. The crude product was purified by flash column
chromatography on silica gel, eluted as indicated for each individu-
al case.
3,5-Bis(4-(trifluoromethyl)phenyl)-1,2,4-thiadiazole (7d): After
column elution with 2!4!6!8% EtOAc/petroleum ether, ob-
tained as an off-white, microcrystalline solid (32 mg, 9%): Rf =0.50
N-[2-(Diethylamino)ethyl]-4-phenyl-2-(thiophen-2-yl)thiazole-5-
carboxamide (6c): After elution with 0!2.5!5!7.5% MeOH/
CH2Cl2, obtained as a thick, pale-yellow gum (113 mg, 59%):
1H NMR (400 MHz, CDCl3): d=7.72–7.66 (m, 1H), 7.60 (d, J=3.5 Hz,
1H), 7.52–7.42 (m, 4H), 7.28 (s, 1H), 7.11 (dd, J=4.0 Hz, 5.0 Hz, 1H),
6.57 (brs, 1H), 3.37 (q, J=5.5 Hz, 2H), 2.51–2.30 (m, 6H), 0.84 ppm
(t, J=6.5 Hz, 6H); 13C NMR (100 MHz, CDCl3): d=11.1, 37.3, 46.1,
50.6, 127.7, 128.0, 128.78, 128.84, 129.3, 129.4, 133.9, 136.8, 154.2,
161.1, 162.3 ppm; IR (neat): n˜ =2970, 1641, 1527, 1480, 1447, 1332,
919, 844, 722, 698 cmꢁ1; MS (ES): m/z (%): 386 (100) [M+H]+;
HRMS-ES: m/z [M+H]+ calcd for C20H24N3OS2: 386.1361, found:
386.1370.
1
(hexane/EtOAc, 9:1); H NMR (400 MHz, [D6]DMSO): d=8.54 (d, J=
8.0 Hz, 2H), 8.20 (d, J=8.0 Hz, 2H), 7.86–7.77 ppm (m, 4H);
13C NMR (100 MHz, [D6]DMSO): d=125.8 (q, J=3.5 Hz), 126.4 (q, J=
3.5 Hz), 127.9, 128.7, 132.2 (q, J=32.5 Hz), 133.5, 133.7 (q, J=
32.5 Hz), 135.6, 172.7, 187.0 ppm; IR (solid): n˜ =1473, 1409, 1319,
1188, 1132, 1120, 1103, 1066, 1015, 990, 848, 712, 659 cmꢁ1; MS
(ES): m/z (%): 375 (100) [M+H]+; HRMS-ES: m/z [M+H]+ calcd for
C16H9F6N2S: 375.0391, found: 375.0383.
3,5-Bis(3,4-dimethoxyphenyl)-1,2,4-thiadiazole
(7e):
After
column elution with 20!30!40% EtOAc/petroleum ether, ob-
tained as a pale-pink solid (225 mg, 63%): Rf =0.15 (petroleum
ether/EtOAc, 2:1); 1H NMR (400 MHz, CDCl3): d=8.01 (dd, J=
2.0 Hz, 8.5 Hz, 1H), 7.88 (d, J=1.5 Hz, 1H), 7.60–7.55 (m, 2H), 6.99–
6.92 (m, 2H), 4.02 (s, 3H), 4.01 (s, 3H), 3.95 ppm (s, 6H); 13C NMR
(100 MHz, CDCl3): d=55.95, 56.03, 56.07, 56.14, 109.8, 110.8, 111.2,
121.2, 121.8, 123.7, 126.1, 149.0, 149.4, 150.9, 152.2, 173.3,
187.6 ppm; IR (solid): n˜ =2962, 2837, 1600, 1521, 1454, 1422, 1264,
3,5-Bis(pyridin-3-yl)-1,2,4-thiadiazole (7a): Isolated during the
synthesis of 4a as a lower-running spot, eluted with 3% MeOH/
CH2Cl2 after 4a had been recovered from the column, and ob-
tained as a pale-yellow powder (104 mg, 25%): Rf =0.15 (CH2Cl2/
1
MeOH, 39:1); H NMR (400 MHz, CDCl3): d=9.62 (d, J=1.5 Hz, 1H),
9.28 (d, J=1.5 Hz, 1H), 8.81 (dd, J=1.5 Hz, 5.0 Hz, 1H), 8.75 (dd,
J=1.5 Hz, 4.5 Hz, 1H), 8.66 (dt, J=2.0 Hz, 8.0 Hz, 1H), 8.37 (dt, J=
2.0 Hz, 8.0 Hz, 1H), 7.52 (ddd, J=0.5 Hz, 5.0 Hz, 8.0 Hz, 1H),
7.48 ppm (ddd, J=0.5 Hz, 5.0 Hz, 8.0 Hz, 1H); 13C NMR (100 MHz,
CDCl3): d=123.7, 124.1, 126.6, 128.4, 134.6, 135.6, 148.5, 149.7,
151.2, 152.9, 171.6, 185.6 ppm; IR (solid): n˜ =3046, 1589, 1574,
1477, 1399, 1338, 1297, 1126, 1023, 988, 899, 811, 726, 697,
616 cmꢁ1; MS (ES): m/z (%): 241 (100) [M+H]+; HRMS-ES: m/z
[M+H]+ calcd for C12H9N4S: 241.0548, found: 241.0556.
1244, 1228, 1171, 1138, 1108, 1020, 870, 834, 805, 739, 720 cmꢁ1
;
MS (ES): m/z (%): 359 (100) [M+H]+; HRMS-ES: m/z [M+H]+ calcd
for C18H19N2O4S: 359.1066, found: 359.1064.
3,5-Bis(3-methoxyphenyl)-1,2,4-thiadiazole (7 f): After column
elution with 2.5!5!7.5% EtOAc/petroleum ether, obtained as a
thick, colourless gum which crystallised on standing to give a pale-
1
yellow solid (212 mg, 71%): H NMR (400 MHz, CDCl3): d=8.02 (dt,
3,5-Bis(4-methoxyphenyl)-1,2,4-thiadiazole (7b): Isolated during
the synthesis of 4b (lower-running spot), then further purified by
preparative HPLC (Alltima HP C18 HL 5 mm column; isocratic condi-
J=1.0 Hz, 7.5 Hz, 1H), 7.97–7.94 (m, 1H), 7.64–7.59 (m, 2H), 7.44 (t,
J=8.0 Hz, 2H), 7.11 (ddd, J=1.0 Hz, 2.5 Hz, 8.5 Hz, 1H), 7.06 (ddd,
J=1.0 Hz, 2.5 Hz, 8.5 Hz, 1H), 3.94 (s, 3H), 3.93 ppm (s, 3H);
ChemMedChem 2010, 5, 1476 – 1488
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M. J. Thompson et al.
MED
13C NMR (100 MHz, CDCl3): d=55.5, 55.6, 112.2, 113.0, 116.8, 118.0,
120.1, 121.0, 129.8, 130.4, 131.8, 134.1, 159.9, 160.2, 173.6,
188.0 ppm; IR (solid): n˜ =3000, 2942, 2833, 1594, 1504, 1474, 1458,
1430, 1396, 1312, 1276, 1260, 1230, 1219, 1181, 1164, 1048, 1010,
866, 817, 789, 775, 733, 679 cmꢁ1; MS (ES): m/z (%): 299 (100)
[M+H]+; HRMS-ES: m/z [M+H]+ calcd for C16H15N2O2S: 299.0854,
found: 299.0848.
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Resource Centre at the Roslin Institute, University of Edinburgh (UK);
http://www.roslin.ed.ac.uk/tseresourcecentre/cell_lines.html (accessed
June 17, 2010).
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W. F. Cools, J. Goossens, D. Corens, A. de Groot, J. P. van Wauwe, J. Med.
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3,5-Bis(p-tolyl)-1,2,4-thiadiazole (7g): After column elution with
2!4!6% EtOAc/petroleum ether, obtained as a fine, off-white
solid (207 mg, 78%): 1H NMR (400 MHz, CDCl3): d=8.31 (d, J=
8.5 Hz, 2H), 7.96 (d, J=8.0 Hz, 2H), 7.33 (d, J=7.5 Hz, 4H),
2.45 ppm (s, 6H); 13C NMR (100 MHz, CDCl3): d=22.0, 22.1, 127.8,
128.6, 128.7, 129.8, 130.3, 130.8, 140.9, 142.9, 174.2, 188.4 ppm; IR
(solid): n˜ =3036, 2971, 2917, 1608, 1473, 1404, 1320, 1174, 1106,
984, 898, 827, 813, 736, 698 cmꢁ1; MS (ES): m/z (%): 267 (100)
[M+H]+; HRMS-ES: m/z [M+H]+ calcd for C16H15N2S: 267.0956,
found: 267.0954.
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3,5-Bis(pyridin-4-yl)-1,2,4-thiadiazole (7h): After column elution
with 50!75!100% EtOAc/petroleum ether, then 2.5!5% EtOH/
EtOAc, obtained the title compound as a pale-pink solid (19 mg,
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1
8%): H NMR (400 MHz, CDCl3): d=8.90–8.81 (m, 4H), 8.26–8.23 (m,
2H), 7.93–7.90 ppm (m, 2H); 13C NMR (100 MHz, CDCl3): d=121.4,
122.5, 137.2, 139.4, 151.2, 151.7, 172.6, 187.1 ppm; IR (solid): n˜ =
1600, 1465, 1407, 1344, 1291, 823, 732, 710, 676, 634 cmꢁ1; MS
(ES): m/z (%): 241 (100) [M+H]+; HRMS-ES: m/z [M+H]+ calcd for
C12H9N4S: 241.0548, found: 241.0541.
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The authors thank BBSRC (Grant No. BB/E014119/1) and The
Home Office (Contract No. 04-07-270) for their generous financial
support.
Keywords: drug discovery
· medicinal chemistry · prion
disease · structure–activity relationships · thiazoles
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Received: May 19, 2010
Revised: June 12, 2010
Published online on July 15, 2010
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ChemMedChem 2010, 5, 1476 – 1488