4
M. A. MacLean et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
Herein we report on the application of this novel cross-coupling
strategy toward the modular assembly of a small library of edar-
avone derivatives featuring variation in the N-aryl substituent,
and the subsequent assessment of the abilities of such N-aryl pyra-
zolones to inhibit Ab40/42 aggregation. The protocol employed in
preparing a small library of edaravone derivatives is depicted in
Figure 1.
has a strong affinity for proteins with high b-sheet content, and
as such may be used to visualize Ab aggregates. Unbound ThT
has fluorescence excitation (kex) and emission (kem) wavelengths
of 430 and 342 nm, respectively; upon binding to amyloid fibrils,
ThT undergoes a characteristic spectral shift (kex 442 nm, kex
482 nm), which can be used to differentiate bound and unbound
ThT. Using a kinetic aggregation assay format,16 compounds were
This procedure makes use of the palladium-catalyzed cross-
coupling of (hetero)aryl chlorides or tosylates with hydrazine
hydrate to afford the requisite monoaryl hydrazine intermediate,
followed by treatment with ethyl acetoacetate under acidic condi-
tions to afford the desired pyrazolone. In exploiting this modular
strategy, the expedient synthesis of edaravone derivatives featur-
ing a diversity of substitution patterns at the N-aryl position was
achieved (Fig. 1). Edaravone itself (1) was prepared in this manner
in good yield (76% from PhCl; 77% from PhOTs), as were isomeric
N-tolyl (2–4) and naphthyl (5) compounds. Ortho-ethyl substitu-
tion was well accommodated (6), as was 2,5-dimethyl substitution
(7, albeit in lower yield when employing the aryl tosylate).
Edaravone derivatives featuring N-phenyl groups comprising a
combination of methyl, fluoro, trifluoro and/or methoxy sub-
stituents (8–12) were each prepared in acceptable isolated yield,
as were derivatives featuring amine and ether addenda (13–16).
While heteroaryl (pseudo)halides in general proved to be challeng-
ing reaction partners in this cross-coupling chemistry (including
heteroaryl chlorides derived from thiophene, imidazole, or
quinoline), pyrazolones featuring pyridine or pyrimidine groups
at nitrogen were prepared successfully in good isolated yield
(17–18). Finally, given the privileged nature of the biaryl motif in
pharmaceutical chemistry,13,14 we directed our attention to the
preparation of such edaravone derivatives. N-(hetero) biaryl pyra-
zolones featuring methoxy, pyridyl, and indolyl functionalities
were all synthesized successfully (19–22). Notably, 15 of the edar-
avone derivatives prepared herein represent new compounds that
had not been reported previously in the chemical literature,
thereby underscoring the utility of the expedient palladium-
catalyzed methodology employed.
incubated at a single concentration of 25
lM with Ab40 (20 lM)
in the presence of ThT (8 M) and fluorescence measurements
l
were taken over a period of 72 h. Ab40 was chosen over Ab42 due
its ease of handling and the reproducibility of this particular assay
system. The percentage inhibition of Ab40 aggregation (measured
at 72 h) by the analog series, is shown in Table 1.
We next screened the compounds through a biochemical assay
specifically designed to monitor the formation of smaller,
neurotoxic-associated oligomers. This was achieved using an assay
system developed by Levine17 that measures sub-nanomolar con-
centrations of Ab42 oligomers utilizing biotin-labeled Ab42 protein.
This robust assay system specifically recognizes multimeric
(>20 kDa) oligomers of N-a-biotinyl-Ab(1–42) (bio-Ab42) but not
monomeric bio-Ab42 via a single-site NeutrAvidin capture/labeled
streptavidin detection configuration. Compounds 1–22 were
screened in this assay system using a concentration range of
1–50 lM to generate a dose response curve from which IC50 values
were determined.
Analyzing the data in Table 1, compounds 1–12, 17 and 18,
which lack aromatic functionality outside of the phenyl pyrazolone
core, show no effect on inhibiting oligomerization in the bio-Ab42
assay (IC50 > 50
ate activity in the Ab40-ThT assay system, with compounds 5, 9,
10 and 18 exhibiting in the range of 50% inhibition at 25 M. The
only outlier in this context is the pyrrolidine 14, which shows
47% inhibition at 25 M (Ab40-ThT) and IC50 = 16.8 M (bio-Ab42).
lM). The same compounds show weak to moder-
l
l
l
Most interestingly, increased potency is observed in both assays
when aromaticity is introduced in the para position of the N-aryl
pyrazolone. Compounds 19–21 all display strong inhibition with
IC50 values in both assay system in the low micromolar range
(see Table 1). The Ab40-ThT and bio-Ab42 dose response curves
for 20 are shown in Figure 2.
With a small focused library of edaravone analogs in hand, we
next investigated their ability to inhibit aggregation of Ab
in vitro. For this purpose, we selected two functional biochemical
assays to study the effect of each compound on both fibril and oli-
gomer formation. To monitor fibril or larger aggregate formations
that are rich in beta-sheet conformation, analogs were screened
using the fluorescent dye-binding agent Thioflavin T (ThT).15
Briefly, Ab40/42, in its monomeric form, is soluble in solution at
Similar activity profile was also observed with the N-linked
aromatic heterocycles 15 (Ab40-ThT, IC50 = 26.0
IC50 = 4.0 M) and 22 (Ab40-ThT, IC50 = 19.6
IC50 = 3.53
strated similar anti-oligomer activity (bio-Ab42, IC50 = 4.0
but considerably lower inhibition in the ThT aggregation system
(Ab40-ThT, IC50 > 50 M), indicating potential separation in
lM; bio-Ab42
,
l
lM; bio-Ab42
,
lM). Interestingly, the diphenyl aniline 13 demon-
lM),
physiological temperature and pH, and takes a predominantly
a
l
a
helical/random coil structure. Following the process of misfolding
and self-association, mature aggregates/fibrils can be formed that
are enriched in cross-b pleated sheets. ThT, a benzothiazole dye,
oligomer versus larger aggregate inhibitory effects. Adding further
evidence that additional aromaticity needs to be positioned further
out from the phenyl pyrazalone structure, the benzyl ether 16
120
100
80
60
40
20
0
120
100
80
60
40
20
0
0.1
1
10
100
0
1
10
100
Log [Compound 20] (μM
Log [Compound 20] (μM)
Figure 2. Dose–response curves of the percentage of inhibition of Ab40 and bio-Ab42 of compound 20.