H. Liu, et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
Fig. 2. A) Compound concentration optimization. 3a, 3c and 3d were respectively incubated with 40 μM α-Syn solution in 0.1 M PBS (pH = 7.4) for 72 h at three
concentration gradients, 1 μM, 30 μM and 100 μM. Their relative fluorescence intensities were obtained after adding 20 μM ThT to the system. B) Fibrillation kinetics
of 40 μM α-Syn in the absence or presence of 3a, 3b and 3d at 30 μM monitored by relative fluorescence intensity in vitro. Each value is mean of three replicates. The
images on the right were TEM morphologies of α-Syn aggregation (500 nm). 40 μM α-Syn was incubated alone for 72 h (upper right), 40 μM α-Syn was incubated in
the presence of 30 μM 3b for 72 h (lower right).
appropriate concentration to apply in the following evaluations. Fur-
thermore, the inhibitory kinetic of these compounds to α-Syn fibrilla-
tion was investigated under the optimized conditions above. The re-
lative fluorescence intensity was measured in 40 μM α-Syn solution
incubated with 30 μM of 3a, 3b and 3d respectively (Fig. 2B). The
three phases: (i) lag phase, the formation of β-sheets nucleus during the
first 7 h, (ii) elongation phase, the logarithmic increase of β-sheets
during 7–48 h, and (iii) stationary phase, the saturation of β-sheets
during 48–72 h. The addition of compounds caused slower increase of
the relative fluorescence intensity over the time, which indicated the
inhibitory effect of these compounds on α-Syn fibril formation. To
confirm the relative fluorescence intensity results, the morphology of α-
Syn during the conformation transition with and without inhibitor were
observed by transmission electron microscope (TEM) (Fig. 2B right).
after 72 h incubation, which exhibit the typically bundled fibrils
formed. The lower right TEM image in Fig. 2B shown the image of α-
Syn after 72 h incubation with compound 3b at 30 μM. Compared to the
bundled fibrils above, the fibrils appeared thinner, revealing an effec-
tive inhibition of α-Syn fibrillation by 3b.
substituent at position 5, such as methyl and bromo groups slightly
reduce inhibitory activities. This trend was identified by comparing the
inhibition ratio of 3a, 3b and 3c (76.0%, 70.0% and 63.7%), as well as
3j, 3k and 3l (75.5%, 58.7% and 56.5%). Similarly, the substituent
groups at position 6, such as fluoro and chloro also reduced the in-
hibitory activities from the downtrend in 3a, 3d and 3e (76.0%, 71.7%
and 69.7%), as well as in 3j, 3n and 3m (75.5%, 39.7% and 37%).
Secondly, for the benzimidazole block, the inhibitory activities are
slightly reduced than benzoxazole block by comparing the inhibition
ratio of 3a and 3g (76.0% and 62.7%), as well as of 3j and 3p (75.5%
and 59.0%). And more, substituent at position 1 on the benzimidazole
block, such as methyl and tosyl, have little influence on their inhibitory
activities by comparing the inhibition ratio of 3g, 3h and 3i (62.7%,
56.5% and 62.7%). Interestingly, the compoud 3o, 5-nitro-1H-benzi-
midazole conjugated 2-pyridine has displayed an increased inhibitory
activity (73.7%).
Series 2 compounds are comprised of two building blocks of pyr-
idine and substituted benzene linked with full trans dienone and some
analogues showed medium to high inhibition activities (58.5% to
75.5%). Compound 5b has displayed higher activity (75.5%) and 5f,
5e, 5g and 5j shown the moderate activities as 60.0%, 65.0%, 65.0%
and 69.0% respectively. The SAR study revealed that benzamide block
is favorable to increase inhibition ratio, especially p-chlorobenzamide
(5b).
Under the same condition described above, most compounds of
series 1, series 2 and series 3 were evaluated for their relative fluor-
escence intensities. The results (Fig. 3) demonstrated that the relative
fluorescence intensities of these compounds are less than 100%, from
24.0% to 62.3%. Understandably, the lower relative fluorescence in-
tensity is corresponding to the higher inhibitory activity against α-Syn
aggregation. In order to describe the inhibition activity more con-
veniently, the relative fluorescence intensity was transformed to α-Syn
aggregation inhibition ratio by the formula: α-Syn aggregation inhibi-
tion ratio = 100% (blank group)-x% (compound group). The ratio
value intuitively reflected the inhibitory activity of compound
Series 3 compounds are consisted of two building blocks conjugated
by trans 1,4-divinylbenzene. One block is pyridine and the other block
is substituted benzene or benzoic acid derivative. Compound 8 has two
same blocks of pyridine. These compounds also displayed inhibitory
activities from 42.5% to 76.0%. Compound 8j possessing the allyl
benzoate block gave the highest ratio of 76%. On the other hand, 8k,
8e, and 8n, which possess benzoic acid, methyl benzoate and benza-
mide, showed lower ratio of 54.0%, 48.2% and 56.2%. In addition, 8d
(substituted benzene block) and 8 also showed moderate to low activ-
ities from 53.0% to 42.5%.
From Table 1, the series 1 conmpouds have shown the inhibitory
ratio towards α-Syn aggregation from 37.7% to 76.0%. Compounds 3a,
3b, 3d, 3j and 3o displayed more than 70% inhibition ratio. Series 1
compounds are comprised of benzoxazole or benzimidazole and pyr-
idine moieties, which were conjugated by trans diene. Firstly, 3a and 3j
displayed similar inhibition ratio (76.0% vs 75.5%), suggesting pyridin-
2-yl and pyridin-4-yl had similar effect. For the benzoxazole block,
Next, some representative analogues with higher inhibitory activ-
ities were chosen to perform IC50 study (Table 2). The results indicated
that the linkers did not demonstrate distinct differences for the in-
hibitory activities. This is in agreement with our hypothesis, that the
function groups on both sides play key roles for high activity. In ad-
dition, the calculated LogP values of 3a, 3b, 3e and 3o suggest their
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