Insights into structure-activity relationships of 3-arylhydrazonoindolin-2-one derivatives for their multitarget activity on β-amyloid aggregation and neurotoxicity

Article abstract of DOI:10.3390/molecules23071544

Despite the controversial outcomes of clinical trials executed so far, the prevention of β-amyloid (Aβ) deposition and neurotoxicity by small molecule inhibitors of Aβ aggregation remains a target intensively pursued in the search of effective drugs for treating Alzheimer’s disease (AD) and related neurodegeneration syndromes. As a continuation of previous studies, a series of new 3-(2-arylhydrazono)indolin-2-one derivatives was synthesized and assayed, investigating the effects of substitutions on both the indole core and arylhydrazone moiety. Compared with the reference compound 1, we disclosed equipotent derivatives bearing alkyl substituents at the indole nitrogen, and fairly tolerated bioisosteric replacements at the arylhydrazone moiety. For most of the investigated compounds, the inhibition of Aβ₄₀ aggregation (expressed as pIC₅₀) was found to be correlated with lipophilicity, as assessed by a reversed-phase HPLC method, through a bilinear relationship. The N¹-cyclopropyl derivative 28 was tested in cell-based assays of Aβ₄₂ oligomer toxicity and oxidative stress induced by hydrogen peroxide, showing significant cytoprotective effects. This study confirmed the versatility of isatin in preparing multitarget small molecules affecting different biochemical pathways involved in AD.

Full text of DOI:10.3390/molecules23071544

molecules
Article
Insights into Structure-Activity Relationships of
3-Arylhydrazonoindolin-2-One Derivatives for Their
Multitarget Activity on β-Amyloid Aggregation
and Neurotoxicity
Rosa Purgatorio 1, Modesto de Candia 1, Annalisa De Palma 2, Francesco De Santis 2,
Leonardo Pisani 1 ID , Francesco Campagna 1, Saverio Cellamare 1, Cosimo Damiano Altomare 1
and Marco Catto 1,
1
*
ID
Dipartimento di Farmacia-Scienze del Farmaco, Università degli Studi di Bari “Aldo Moro”,
via E. Orabona 4, I-70125 Bari, Italy; rosa.purgatorio@uniba.it (R.P.); modesto.decandia@uniba.it (M.d.C.);
leonardo.pisani@uniba.it (L.P.); francesco.campagna51@gmail.com (F.C.); saverio.cellamare@uniba.it (S.C.);
cosimodamiano.altomare@uniba.it (C.D.A.)
Dipartimento di Bioscienze, Biotecnologie e Biofarmaceutica, Università degli Studi di Bari “Aldo Moro”,
via E. Orabona 4, I-70125 Bari, Italy; annalisa.depalma@uniba.it (A.D.P.);
desantis.nutrizione@gmail.com (F.D.S.)
2
*
Correspondence: marco.catto@uniba.it; Tel.: +39-080-544-2780
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 11 May 2018; Accepted: 24 June 2018; Published: 26 June 2018
Abstract: Despite the controversial outcomes of clinical trials executed so far, the prevention of
β-amyloid (Aβ) deposition and neurotoxicity by small molecule inhibitors of Aβ aggregation
remains a target intensively pursued in the search of effective drugs for treating Alzheimer’s disease
(AD) and related neurodegeneration syndromes. As a continuation of previous studies, a series
of new 3-(2-arylhydrazono)indolin-2-one derivatives was synthesized and assayed, investigating
the effects of substitutions on both the indole core and arylhydrazone moiety. Compared with the
reference compound 1, we disclosed equipotent derivatives bearing alkyl substituents at the indole
nitrogen, and fairly tolerated bioisosteric replacements at the arylhydrazone moiety. For most of
the investigated compounds, the inhibition of Aβ40 aggregation (expressed as pIC50) was found to
be correlated with lipophilicity, as assessed by a reversed-phase HPLC method, through a bilinear
relationship. The N1-cyclopropyl derivative 28 was tested in cell-based assays of Aβ42 oligomer
toxicity and oxidative stress induced by hydrogen peroxide, showing significant cytoprotective
effects. This study confirmed the versatility of isatin in preparing multitarget small molecules
affecting different biochemical pathways involved in AD.
Keywords: beta-amyloid aggregation inhibitors; indolin-2-ones; Alzheimer’s disease; quantitative
structure-activity relationships; multitarget activity
1. Introduction
Alzheimer’s disease (AD) is the most common cause of age-related neurodegenerative pathologies.
AD represents a serious challenge for health systems, physicians and caregivers, because of its disabling
course and the limited efficacy of pharmacological therapies [
namely the restoration of cholinergic transmission by means of acetylcholinesterase inhibitors [
and the neuroprotection from glutamate excitotoxicity exerted by memantine [
1
]. The treatments approved for AD,
],
], are only symptomatic
2
3
and do not meet the clinical need for effective disease-modifying drugs.
A typical feature of AD consists in the deposition of extracellular
β-amyloid (Aβ) peptide
aggregates (amyloid plaques), starting from cholinergic neurons of hippocampus and then
Molecules 2018, 23, 1544; doi:10.3390/molecules23071544
www.mdpi.com/journal/molecules

Molecules 2018, 23, 1544
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progressively extending to the whole brain cortex [
4]. Aβ
peptide derives from proteolysis of amyloid
precursor protein (APP) catalyzed by
(Aβ42). A monomers in AD brains aggregate to soluble oligomers, that in turn lead to intermediate
protofibrils and finally to the deposition of amyloid plaques [ ]. The formation of such oligomeric
β- and γ-secretases, and is formed as 40- (Aβ40) or 42-mer
β
5
and prefibrillar species is correlated with the neurotoxicity in the AD brain, which represents a major
causal factor of the cognitive impairment and the synaptic loss in AD patients [6–8].
The amyloidogenesis of Aβ occurs since the early stages of the disease insurgence, so
that preventing the oligomerization and/or fibrillization process could represent a promising
disease-modifying treatment for AD. Despite the number of research findings in this field, only
a very small number of molecules have reached the preclinical stage, and no one entered therapy
so far [
9]. However, many small molecules acting as disruptors of protein-protein interactions have
demonstrated potential in inhibiting A
β
aggregation [10]. Among them, indole derivatives such
as melatonin [11], fluorinated indoles [12], hydroxyindoles [13] (Figure 1) displayed the structural
features for an efficient antiaggregating activity. Particularly, they act as intercalators in hydrophobic
interactions between A
shown by our structure-activity relationship (SAR) studies [16
taking place between A and many classes of small molecules could be reinforced by polar interactions
β
side chains, including aromatic
π
-stacking interactions [14
–17]. As recently
,18], these hydrophobic interactions
β
and/or hydrogen bond (HB) formation.
Figure 1. Structures of indole derivatives endowed with amyloid anti-aggregating properties.
From previous studies [19
identified as a promising inhibitor of A
the submicromolar and low M range, respectively. Herein, we synthesized a number of congeners
of compound , and evaluated the in vitro activity as inhibitors of Aβ40 aggregation. With the
–
22], 5-methoxyisatin 3-(4-isopropylphenyl)hydrazone (
1, Figure 1) was
β
aggregation and acetylcholinesterase (AChE), with IC50s in
µ
1
aim of extending the SAR exploration, the new derivatives were designed in order to undertake
a more systematic exploration (Figure 2) of: (i) the 3-arylhydrazone moiety, by introducing a
number of diverse substituents and/or modifying the aryl substituent (compounds
compounds 13 18, Scheme 2); (ii) the length and the chemical nature of the linker (compounds 19
and 21 23, Scheme 3); (iii) the substitution either on the nitrogen (compounds 24 26 28 32 34
Scheme 4) and the benzene moiety of the indole ring (compounds 36 39, Scheme 5). SARs, including
4–11, Scheme 1;
–
–
,
,
,
– ;
–
correlation of antiaggregating activity with lipophilicity, were investigated, and for one of the most
potent Aβ40 aggregation inhibitors (compound 28) cytoprotection from toxic Aβ42 oligomers in a
cell-based assay was evaluated. Moreover, taking into account the role of oxidative stress in AD and
other neurodegenerative diseases [23], the antioxidant property of 28 against hydrogen peroxide insult
in SH-SY5Y cells was also tested.

Molecules 2018, 23, 1544
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variation of substitution
in para position
(hetero)aromatic
rings
substituent(s) in
5, 6 and 7
position
NH
O
N
length and
chemical nature of linker
N
H
substitution on
indole nitrogen
Figure 2. Summary of the investigated structural modifications of isatin-3-arylhydrazones.
O
Ar
H3CO
i
O
N
CN
4
8
9
H
NH2
W
Ar
NH
CF3
5
6
N
N
iv
iii
ii
H3CO
O
CF3
N
H
N2
10
11
W
CF3
N
3a (W = CH)
3b (W = N)
H3CO
7
N
O
N
N
v
H
2
Cl
Scheme 1. Reagents and conditions: (
(iii) NaOH, EtOH, H2O, reflux; (iv) NaNO2, HCl, 0 C; (v) MeOH, CH3COONa, 0 C.
i
) ArNHNH2, MeOH, room temperature; (ii) NH2NH2 H2O, EtOH;
·
◦
◦

Molecules 2018, 23, 1544
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Scheme 2. Reagents and conditions:
(i) thiosemicarbazide, isopropanol, room temperature;
(ii) (bromomethyl) alkyl or aryl ketone, EtOH, room temperature.
HN
N
CHO
H3CO
H3CO
i
N
H
N
H
19
R
NH2
N
N
N
H3CO
H3CO
iii
ii
O
O
N
N
H
H
O
21 R = 4-CH(CH3)2
22 R = 3-Cl
20
H3CO
O
Cl
N
H
O
NH
O
N
iv
H3CO
N
H
23
Scheme 3.
MeOH, room temperature;
(iv) 3-chlorobenzohydrazide, MeOH, room temperature.
Reagents and conditions:
(
i
)
4-isopropylphenylhydrazine hydrochloride,
(
ii) NH2NH2, AcOH, reflux;
(iii) R-C6H4CHO, MeOH, reflux;

Molecules 2018, 23, 1544
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NH
O
N
N
N
H3CO
H3CO
i
O
N
N
H
1
24
O
NH
O
N
H3CO
ii
H3CO
O
N
N
26
i
25
NH
O
O
O
N
iii
H3CO
H3CO
H3CO
ii
O
O
N
N
N
H
27
28
iv
NH
N
O
H3CO
H3CO
ii
O
O
N
N
(CH2)n
(CH2)n
29 n = 1
30 n = 2
31 n = 3
32 n = 1
33 n = 2
34 n = 3
Scheme 4. Reagents and conditions:
(i) Methyl iodide, K2CO3, DMSO, room temperature;
(
ii) 4-isopropylphenylhydrazine hydrochloride, MeOH, room temperature; (iii) Cyclopropylboronic
acid, Na2CO3, Cu(OAc)2, 1,2-dichloroethane, bipyridine, 50 ◦C; (iv) Br(CH2)nC6H5, K2CO3, DMSO,
room temperature.
NH
O
O
N
i, ii
iii
O
N
N
NH2
R
R
R
H
H
36 R = 5-n-butyl
37 R = 7-Br
35a R = 5-n-butyl
35b R = 7-Br
38 R = 5-NaSO3
39 R = 5,6- OO
35c R = 5-NaSO3
35d R = 5,6- O
O
Scheme 5.
NH2OH HCl, reflux; (ii) H2SO4 conc., 80 C; (iii) 4-isopropylphenylhydrazine hydrochloride, MeOH,
room temperature.
Reagents and conditions:
(i
) CCl3CH(OH)2, Na2SO4 10H2O, H2O, HCl conc.,
·
◦
·

Molecules 2018, 23, 1544
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2. Results and Discussion
2.1. Chemistry
New 5-methoxy-3-arylhydrazonoindolin-2-ones
4–
11 were prepared by condensation of
5-methoxyisatin with the corresponding (hetero)arylhydrazines (compounds
4–
7,
9,
11) or alternatively
via azo coupling of aryldiazonium salts 3a,b with 5-methoxyindolin-2-one 2 [24] to yield the
compounds 8 and 10, respectively (Scheme 1).
Thiazolylhydrazones 13
ketones with 2-(5-methoxy-2-oxoindolin-3-ylidene)hydrazinecarbothioamide 12 [26] (Scheme 2).
Hydrazones 19 21, and 22 were prepared through condensation of the corresponding
–18 were synthesized through the Hantzsch reaction [25] of bromomethyl
,
carbaldehydes and hydrazines, while compound 23 was synthesized by condensation of
5-methoxyisatin with 3-chlorobenzohydrazide. The 3-arylidenehydrazono-5-methoxyindolin-2-one
derivatives 21 and 22 required the preparation of 5-methoxyisatin-3-hydrazone 20 [27], which was
then reacted with 4-isopropyl or 3-chlorobenzaldehyde, respectively (Scheme 3).
The alkylation of the indole and/or hydrazone nitrogens was accomplished through different
methods, starting from commercial 5-methoxyisatin or the previously reported compound
(Scheme 4), which provided a series of N1-alkyl-substituted derivatives 24, 26, 28, 32–34.
1 [20]
Finally, compounds 36
hydrochloride with isatins 35a
–
39 were synthesized by condensation of 4-isopropylphenylhydrazine
28] and 35d 29] (Scheme 5), previously prepared through the
[
[
Sandmeyer reaction [30], and commercial isatins 35b and 35c. Synthesis and characterization of
compounds 15 [31], 25 [32] and 29 [33] have been already reported.
2.2. Inhibition of Amyloid Aggregation
In vitro inhibition of Aβ aggregation was assessed through a ThT fluorescence-based method [18],
with the use of 2% 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) as aggregation enhancer. In this
medium-throughput assay, we preferred using Aβ40 peptide, being more manageable than Aβ42 and
less prone to the formation of preaggregates [18]. Samples of A
in PBS at 100 M concentration and the antiaggregating activities were measured after 2 h of incubation
at 25 ◦C. For compounds showing >80% Aβ40 aggregation inhibition, IC50s were determined.
β were co-incubated with test molecules
µ
The already reported antifibrillogenic activity of the indolylhydrazones [19
identify (3Z)-5-methoxy-1H-indole-2,3-dione 3-[(4-isopropylphenyl)hydrazone] (
compound, displaying strong inhibition of Aβ40 aggregation (IC50 0.43
to be quite stable at pH 7.4 and 37 ◦C (half-life 24 h) and to inhibit AChE (IC50 6.25 µM) [22].
An early objective of the present study was to replace the 4-iPr substituent with phenyl
groups bearing polar and apolar electron-withdrawing substituents (compounds , Table 1). Such
modifications determined a drop of the activity, and only the 30,50-bis(trifluoromethyl) derivative
,
20] allowed us to
1
, Figure 1) as a hit
µM) [20]. Compound 1 proved
4–6
6
showed IC50 in the low micromolar range (9.9 µM), which resulted however 25-fold less potent than
compound 1.
The replacement of the hydrazone phenyl ring with a number of other aromatic or heteroaromatic
rings was carried out for investigating the effects of bulkiness and/or additional interactions on the
anti-aggregating potency. The bioisosteric replacement of the phenyl group with pyrid-2-yl ( ) resulted
in a much lower activity (IC50 90 M), whereas the 1-naphthyl moiety in compound did recover a fair
potency (IC50 28 M), indicating that additional aromatic interactions may improve the antiaggregating
potency. In contrast, quinolyl analogues and 10, (similarly to the pyrid-2-yl congener ), showed
lower potency, and the 7-chloroquinolin-4-yl derivative 11 resulted a very weak inhibitor.
π-π
7
µ
8
µ
9
7

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Table 1. Aβ Antiaggregating Activity of Compounds 1 and 4–11.
r
A
NH
N
H
3CO
O
N
H
Comp.
Ar
IC50 (µM) or (% Inhibition @ 100 µM) 1
1
4
5
6
7
8
9
10
11
4-(CH(CH3)2)C6H4
4-CNC6H4
4-CF3C6H4
3,5-(CF3)2C6H3
Pyrid-2-yl
0.43 ± 0.04
(16 ± 3)
(57 ± 5)
9.9 ± 0.8
90 ± 5
1-Naphthyl
28 ± 2
Quinolin-2-yl
Quinolin-8-yl
7-Cl-quinolin-4-yl
(67 ± 4)
(58 ± 4)
(14 ± 2)
1
Data are mean ± SEM of three independent experiments.
Further information on the bioisosteric replacement of phenylhydrazone ring was achieved with
thiazolylhydrazones 13 18 (Table 2). An exploration of the substituents on the thiazole moiety clearly
indicated a preference for 4-phenylthiazole derivative 15, which indeed proved to be a potent inhibitor
of A aggregation (IC50 1.2 M), apparently due to additional hydrophobic/aromatic interactions
–
β
µ
attained by the phenyl group at the C4 position of the thiazole ring.
Table 2. Aβ Antiaggregating Activity of Compounds 13–18.
R
R1
S
N
NH
N
H
3CO
O
N
H
Comp.
R
R1
IC50 (µM) or (% Inhibition @ 100 µM) 1
13
14
15
16
17
18
H
CH3
H
H
H
CH2Cl
CH3
C6H5
13 ± 3
(43 ± 1)
1.2 ± 0.2
16 ± 2
0
3 -OHC6H4
3 -OCH3C6H4
3 -BrC6H4
0
34 ± 6
0
H
40 ± 2
1
Data are mean ± SEM of three independent experiments.
The 30-substituted congeners of 4-phenylthiazol-2-yl derivatives 16
–18, regardless the
physicochemical feature of the meta substituent, retained antiaggregating activity in the micromolar
range, but resulted 8-to-30-fold less potent than 15, thereby suggesting critical steric requirements
for these derivatives. In contrast, smaller alkyl substituents, namely methyl or chloromethyl in
compounds 13 and 14, respectively, displayed contrasting effects, with the 4-chloromethyl derivative 13
retaining a fair anti-aggregating potency (IC50 13
weak inhibitor.
µM), and 4,5-dimethyl analogue resulting a very
According to our investigation strategy (Figure 2), the following step was aimed at exploring the
effects on the inhibition of A aggregation of a few variations of the linker (length and the chemical
nature) between the two structural moieties (Table 3).
β

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Table 3. Aβ Antiaggregating Activity of Compounds 19 and 21–23.
Comp.
19
R
L
IC50 (µM) or (% inhibition @ 100 µM) 1
41 ± 3
N
N
21
22
4-CH(CH3)2
3-Cl
6.7 ± 0.2
11 ± 2
N
N
O
23
3-Cl
(31 ± 3)
NH
N
1
Data are mean ± SEM of three independent experiments.
A comparison of the data in Table 3 with that of the hit compound
of potency (from 0.43 to 41
1
revealed a 100-fold decrease
µ
M), when linker was changed in the 1H-indole derivative 19, likely due to
the lack of intramolecular hydrogen bond (IMHB) between the hydrazone NH and the carbonyl O
at position 2 of isatin, which favors co-planarity between the two aromatic moieties. The inhibitory
activity was maintained in the low micromolar range by the aza-derivatives 21 (IC50 6.7
a strict analog of , and 22 (IC50 11 M), that is the 3-chloro congener of 21, whereas a sharp drop
of activity was observed for the carboxyhydrazide derivative 23. The antiaggregating potency of
compounds 21 23 appears to be related to their lipophilicity, as assessed by logP values calculated
µM), that is
1
µ
–
with the ACDLab software (4.97, 4.43 and 3.53 for 21, 22 and 23, respectively).
The introduction of alkyl and phenylalkyl groups at the indole N and methylation of the
hydrazone NH in the hit structure
small and bulky alkyls on the 1H-indole nitrogen of
N-cyclopropyl (28) and N-phenylalkyl (32 34) derivatives resulted almost equipotent with the
hit compound in the submicromolar range (IC50s ranging from 0.53 to 0.83 M). A nonlinear
relation between the antifibrillogenic potency and lipophilicity of the N-alkyl groups exists for these
compounds, the N-benzyl derivative 32 being the most potent one within the subset (IC50 0.53 M).
1
were then investigated (Table 4). Inhibition data highlighted that
1
were tolerated, so that the N-methyl (26),
–
1
µ
µ
In contrast, methylation of the hydrazone nitrogen (24) resulted in a more than 10-fold decrease in
the inhibition potency compared with the respective non-methylated compound (26), which proves
that N-methylation of hydrazone N may prevent IMHB formation with carbonyl O at 2-position [22
]
and/or may introduce a steric effect that hinders hydrazone NH from acting as HB-donor (HBD) in
the interaction with the Aβ peptide.

Molecules 2018, 23, 1544
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Table 4. Aβ Antiaggregating Activity of Compounds 24, 26, 28 and 32–34.
N
N
R1
H
3CO
O
N
R
Comp.
R
R1
IC50 (µM) 1
24
26
28
32
33
34
1
CH3
CH3
Cyclopropyl
Benzyl
2-Phenylethyl
3-Phenylpropyl
CH3
H
H
H
H
11 ± 2
0.81 ± 0.11
0.80 ± 0.13
0.53 ± 0.10
0.61 ± 0.03
0.83 ± 0.20
H
Data are mean ± SEM of three independent experiments.
Finally, compounds 36
–39, bearing diverse lipophilic and hydrophilic substituents on the indole
ring (Table 5), were tested for the effects on A
β
aggregation. The introduction of n-butyl (36) or Br (37
)
at 5- and 7-position, respectively, resulted detrimental, leading in the latter case to complete loss of
activity. The evidence of an enhancing role of hydrophilic substituents (particularly 5,6-dihydroxy
derivatives) for an efficient inhibition of Aβ40 aggregation, emerging from our previous works [20,21],
was endeavored by introducing a net negative charge with the sulfonate salt 38 and a protecting
group for catechol derivatives, i.e., the 5,6-methylenedioxy substituent, in 39. Both modifications
resulted poorly effective, and the two compounds showed inhibitory potencies much lower than that
of compound 1.
Table 5. Aβ Antiaggregating Activity of Compounds 36–39.
NH
N
O
R
N
H
Comp.
R
% Inhibition @ 100 µM 1
36
37
38
39
1
5-n-Butyl
55 ± 1
7-Br
<5
−
5-SO3 Na+
35 ± 4
70 ± 4
5,6-methylenedioxy
Data are mean ± SEM of three independent experiments.
To place on a more quantitative basis the above SAR trends, the lipophilicity of a number of isatin
arylhydrazone derivatives was measured by an RP-HPLC method [34,35]. The polycratic capacity
factors (log k’w) were determined (details in the Experimental Section). Lipophilicity descriptors
(i.e., 1-octanol–water partition coefficients) were also calculated (CLOGPs) using two computational
tools (Bio-Loom software, v. 1.7 and ACDLabs software, release 10.0). The ACDLabs software was
used also for calculating descriptors of polarizability and size, namely molar refractivity (CMR) and
molar volume (MV). The experimental lipophilicity parameters (log k’w), along with CMR and MV, are
reported in Table 6 for 18 compounds achieving finite IC50s (<100 µM) in the Aβ aggregation assay.

Molecules 2018, 23, 1544
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Table 6. Aβ antiaggregating potencies and physicochemical descriptors of isatin hydrazones.
Comp.
pIC50
log k’w
CMR
MV
1
6
7
8
6.40
5.00
4.05
4.55
4.89
5.92
4.80
4.47
4.40
4.39
5.17
4.96
4.96
6.09
6.10
6.28
6.21
6.08
5.44
6.29
2.34
5.15
3.95
5.10
4.58
5.29
5.67
6.42
4.82
4.21
4.23
5.96
6.36
7.44
7.24
7.87
8.288
8.475
7.369
9.113
8.149
9.738
9.824
10.320
10.494
9.331
9.331
8.445
9.935
9.402
10.088
11.931
12.392
12.853
2.304
2.657
1.942
2.413
2.019
2.445
2.417
2.661
2.570
2.681
2.681
2.309
2.946
2.733
2.778
3.421
3.581
3.741
13
15
16
17
18
19
21
22
24
26
28
32
33
34
Log k’w values resulted reasonably correlated, over a range of about five log units, with CLOGPs
from ACDLabs software (r2 = 0.805; Supplementary Materials Figure S1). The linear equation
correlating log k’w and CLOGP has a slope close to one (+1.03) and an intercept of about +0.70, the
latter revealing a systematic positive deviation from linearity, likely due to the so-called “silanophilic”
interactions on the silica-based C18 stationary phase [36–38].
A biphasic relationship was observed for the majority of examined (thirteen out of eighteen)
compounds between pIC50 and log k’w (Figure 3).
7
F3C
CF3
6
6
5
4
3
S
N
8
17 18
19
N
N
H
O
O
N
H
0
2
4
6
8
10
log k'w
Figure 3. Left: Bilinear relationship (Kubinyi’s model) between A
(pIC50) and lipophilicity, as assessed by RP-HPLC (log k’w). Data points deviating from the bilinear
relationship are represented as unfilled circles ( 17 18) and filled square (the only non-isatin
derivative, (19). Right: Superposition of bidimensional structures of molecules 17 and 18 behaving
peptides, most likely because of steric effects
β aggregation inhibition potency
6,
8,
,
6, 8,
as strong outliers; moieties that disrupt binding to A
(ligands’ exceeding volumes), are colored in red.
β

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Omitting from the regression analysis the strong outliers
6
,
8
, and 17
–
19, the following bilinear
Equation (1) was obtained according to the Kubinyi’s model [39]:
pIC50 = 0.65 (±0.11) log k’w − 0.95 (±0.33) log (βk’w + 1) + 2.32
n = 13, r2 = 0.900, s = 0.271, logβ = −6.47, optimum log k’w = 6.80
(1)
In the above equation,
β is the nonlinear term of the Kubinyi’s equation; n represents the number
of data points, r2 the coefficient of determination (squared correlation coefficient), and s the standard
deviation of the regression equation; 95% confidence intervals of the regression coefficients are given
in parentheses. The optimum value of log k’w, as calculated from the above equation, is 6.80. Similar,
but statistically slightly poorer, bilinear correlation was obtained with CMR (r2 = 0.704; Supplementary
Materials Figure S3).
The pIC50 values of the outliers 6, 8, 17–19 resulted 1.2–1.5 log units lower than those expected by
the bilinear relationship with lipophilicity. These deviations may be reasonably explained as follows:
(i) compound 19 is the only phenylhydrazone of 1H-indole-3-carbaldehyde, and not of isatin, whose
carbonyl O at position 2 may play a critical role either in forming IMHB with the hydrazone NH or
in interacting with counterparts of the amyloid peptides through dipole-dipole interactions or HBs;
(ii) compounds
of lipophilicity, may undergo detrimental steric effects when approaching and interacting into the
binding site of A peptide because of bulky moieties around the phenylhydrazone (Figure 3). As for
6 and 8 (as well as the quinoline-bearing compounds 9–11), 17 and 18, regardless
β
the 4-phenylthiazol-2-yl-containing derivatives, compounds 17 and 18, bearing OMe or Br in meta
position of phenyl, resulted less active than the respective unsubstituted analog 15 and the less bulky
30-OH congener 16, which in fact fit the bilinear equation model.
Taken together, our data suggested that both the 5-methoxy substituent on the indolin-2-one
moiety and the 4-isopropyl substituent on the phenylhydrazone moiety play an essential role in
maintaining the Aβ40 antiaggregating potency in the submicromolar range. N-methylation of
hydrazone NH, elimination of 2-carbonyl, introduction of other substituents at positions 5, 6 and 7
of the indolin-2-one nucleus and replacement of the isopropyl group with (hetero)aromatic moieties
proved unfavorable, whereas alkylation of the indole nitrogen was tolerated at least up to an optimal
lipophilicity corresponding, in our case, to the N1-benzyl derivative 32.
Based on data in Tables 1–5, compound 28 was selected for further biological and biophysical
studies, aimed at elucidating the mechanism of disruption of Aβ aggregation, and evaluating the
activity in biochemical assays related to neurodegeneration, and cytoprotection in a cell model
of A
β toxicity. Starting from the evidence that a bulky substituent led to a net improvement of
antiaggregating activity (Table 5), we chose the N1-cyclopropyl derivative 28 as the best compromise
between in vitro activity and aqueous solubility. As expected, solubility of 28 resulted lower than lead
◦
compound
1
(21 vs. 71 µM
[
22] in Tris/HCl buffer, pH 7.4 at 25 C), but sufficient to allow performing
the co-incubation assays where 20 µM represents the upper concentration threshold to test.
2.3. Kinetics of Aβ42 Fibrillization
For ThT and circular dichroism (CD) time-course measures of Aβ aggregation (50 µM), alone
or in the presence of 28 (20
µ
M◦), samples were prepared in PBS containing 2% v/v ethanol as the
cosolvent, and incubated at 37 C. Aβ42 peptide was preferred, being the principal responsible of
amyloid burden in AD brain. Kinetics of amyloid aggregation of Aβ42 was followed by means of
ThT fluorescence and CD absorption. In the latter case, we monitored the increase of the negative
band at 215 nm, probing the random coil to β-sheet transition in folding peptide. Results depicted in
Figure 4 show a fast fibrillization of self-aggregating peptide (dotted black line), with fibrils already
detectable in large amount in the first 48 h of incubation. Soluble β-rich species are in turn massively
detected after two days (full black line), suggesting that the fibrillization process takes place by quickly
recruiting oligomer intermediates. On the other hand, samples co-incubated with 28 showed a sharply

Molecules 2018, 23, 1544
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reduced amount either of
β-sheet arranged and ThT-stained aggregates (red lines), reaching only after
one week a fibril content of about 30% compared with that of control peptide.
Figure 4. Time-course aggregation kinetics of 50 µM Aβ42 control (black lines) and Aβ42 with 20 µM
28 (red lines). Results are expressed as percentage of the maximal values of CD ellipticity at 215 nm
(circles, full lines) and ThT fluorescence (squares, dotted lines) of control peptide sample.
2.4. Protection Assays Against Oxidative and Cytotoxic Effects
Protection from Aβ42-induced cytotoxicity was measured for compound 28 in a conventional
cell-based assay, measuring cell viability by means of MTT reduction [40]. Antioxidant activity of 28
was assessed in an H2O2-induced oxidation cell model. Reactive oxygen species (ROS) production was
detected by means of a spectrofluorometric measure of the fluorescent probe 20,70-dichlorofluorescein
(DCF), formed by oxidation of 20,70-dichlorodihydrofluorescein (DCFH) [41]. In both assays, human
SH-SY5Y neuroblastoma cell line was used.
The aggregates formed by 5
death within two days, while cells co-incubated with equimolar 5
the same time frame (Figure 5, top). This result agreed with the observed activity in vitro and confirmed
that previously observed in a close set of congeners [21 22]. Furthermore, to better investigate the
µM Aβ42 in cultured SH-SY5Y cells produced around 50% of cell
µM Aβ42 and 28 were fully viable in
,
cytoprotection exerted by this class of compounds, the antioxidant activity of compound 28 was
investigated. Figure 5 (bottom) shows the radical-scavenging effects of increasing concentrations
(
0 to 20 µM) of 28 against oxidation induced by 100
natural antioxidant, was used as reference compound. ROS scavenging was exerted by 28 even at
lower concentrations and reached a maximum effect at 20 M concentration, which is about 50% of the
effect shown by 100 M quercetin. Thus, the antioxidant activity of 28 is supposed to be comparable to
that of quercetin in the assay conditions herein used.
µM hydrogen peroxide. Quercetin, a well-known
µ
µ

Molecules 2018, 23, 1544
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Figure 5. Cytoprotection of SH-SY5Y cells by compound 28. Top, viability of cells at 0, 24 and
48 h incubated with 5 M Aβ42 alone (grey bars) or in coincubation with equimolar 28 (white bars).
The percentage of MTT reduction is relative to control cells. Bottom, radical scavenging activity of
increasing concentrations of 28 (0 to 20 M) in the presence of 100 M hydrogen peroxide (DCFH-DA
assay; control ( ): non-treated cells; (+): positive control). Values are expressed as mean S.E.M. from
µ
µ
µ
−
±
six replicates, significantly different from control (untreated cells) as estimated by the Student’s t test
(* p < 0.01).
3. Materials and Methods
3.1. Chemistry
Commercial reagents and solvents were purchased from Sigma-Aldrich (Milan, Italy). Melting
points (mp) were determined by the capillary method on a Stuart SMP3 electrothermal apparatus
(Bibby Scientific, Milan, Italy). IR spectra were recorded using potassium bromide disks on a Spectrum
One FT-IR spectrophotometer (Perkin Elmer, Milan, Italy); only the most significant IR absorption
bands are reported. 1H-NMR spectra were recorded in DMSO-d6 on a Mercury 300 spectrometer
(Varian, Cernusco sul Naviglio, Italy). Chemical shifts are expressed in
δ (ppm) and the coupling
constants J in Hz. The following abbreviations were used: s, singlet; d, doublet; t, triplet; qn, quintuplet;
sx, sextuplet; ep, septuplet; dd, double doublet; m, multiplet; br s, broad singlet. Chromatographic
separations were performed on silica gel 63–200 (Merck, Milan, Italy). ESI-MS was performed with an
electrospray interface and an ion trap mass spectrometer (1100 Series LC/MSD Trap System, Agilent,
Palo Alto, CA, USA). The sample was infused via a KD Scientific syringe pump at a rate of 10 mL/min.
◦
The pressure of the nebulizer gas was 15 psi. The drying gas was heated to 350 C at a flow of 5 L/min.
Full-scan mass spectra were recorded in the mass/charge (m/z) range of 50–800 amu. For some
representative compounds, HRMS experiments were performed with a dual electrospray interface
(ESI) and a quadrupole time-of-flight mass spectrometer (Q-TOF, Agilent 6530 Series Accurate-Mass
Quadrupole Time-of-Flight LC/MS, Agilent Technologies Italia S.p.A., Cernusco sul Naviglio, Italy).

Molecules 2018, 23, 1544
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1
The H-NMR and HPLC purities of all the newly synthesized final products were always higher
than 95%. Compounds
2 [24], 12 [26], 15 [31], 20 [27], 25 [32], 29 [33], 35a [28] and 35d [29] have
been prepared according to quoted references. Their analytical data agreed with those reported in
the literature.
3.1.1. Synthesis of 5-methoxy-1H-indol-2,3-dione 3-arylhydrazones 4–7, 9 and 11
5-Methoxyisatin (1 mmol) was dissolved in methanol (5 mL) with gentle heating, then
arylhydrazine (2.4 mmol) was slowly added with stirring at room temperature. After stirring overnight,
the resulting precipitate was filtered and crystallized or the reaction mixture was evaporated to dryness
and the residue suitably crystallized.
1
(Z)-4-(2-(5-Methoxy-2-oxoindolin-3-ylidene)hydrazinyl)benzonitrile (
4
). Yield: 60%. H-NMR
δ
3.76 (s, 3H,
0
5-OCH3), 6.83–6.85 (m, 2H, H-6 and H-7), 7.44 (d, 1H, J = 2.2 Hz, H-4), 7.60 (d, 2H, J = 8.8 Hz, H-3 and
0
0
0
H-5 ), 7.78 (d, 2H, J = 8.8 Hz, H-2 and H-6 ), 10.92 (s, 1H, NH), 12.77 (s, 1H, NNH). ESI-MS m/z = 290.8
(89%) [M
from isopropanol.
−
H]−. IR (KBr): 3359, 3218, 2217, 1686, 1606, 1571, 1515, 1488, 1151 cm−1. Mp 246–249 ◦C
1
(Z)-5-Methoxy-3-(2-(4-(trifluoromethyl)phenyl)hydrazono)indolin-2-one (5). Yield: 50%. H-NMR δ 3.76
(s, 3H, 5-OCH3), 6.80–6.87 (m, 2H, H-6 and H-7), 7.15 (d, 1H, J = 1.1 Hz, H-4), 7.60 (d, 2H, J = 8.5 Hz,
H-20 and H-60), 7.68 (d, 2H, J = 8.5 Hz, H-30 and H-50), 10.90 (s, 1H, NH), 12.80 (s, 1H, NNH). ESI-MS
m/z = 333.8 (100%) [M
from methanol.
−
H]−. IR (KBr): 3166, 1684, 1563, 1487, 12887, 1161 cm−1. Mp 228–230 ◦C
1
(Z)-3-(2-(3,5-bis(trifluoromethyl)phenyl)hydrazono)-5-methoxyindolin-2-one (
6
). Yield: 84%. H-NMR
δ
3.77
(s, 3H, 5-OCH3), 6.82 (d, 1H, J = 8.4 Hz, H-7), 6.87 (dd, 1H, J = 8.4 Hz, J = 2.6 Hz, H-6), 7.24 (d, 1H,
J = 2.4 Hz, H-4), 7.61 (s, 1H, H-40), 8.12–8.14 (m, 2H, H-20 and H-60), 10.89 (s, 1H, NH), 12.86 (s, 1H,
−
NNH). ESI-MS m/z = 401.8 (100%) [M
−
H]−. HRMS (ESI) m/z [M
−
H]− calcd for C17H10F6N3O2
402.0683; found, 402.0685. IR (KBr): 3164, 1684, 1564, 1487, 1374, 1279, 1163 cm−1. Mp 240–242 ◦C
(dec.) from methanol.
1
(Z)-5-Methoxy-3-(2-(pyridin-2-yl)hydrazono)indolin-2-one (
7
). Yield: 80%. H-NMR
δ
3.78 (s, 3H, 5-OCH3),
6.76 (d, 1H, J = 8.5 Hz, H-7), 6.87 (d, 1H, J = 8.5 Hz, H-6), 7.02 (s, 1H, H-4), 7.43–7.45 (m, 1H, H-40),
7.77–7.80 (m, 2H, H-50, H-60), 8.27 (br s, 1H, H-30), 10.41 (s, 1H, NH), 11.07 (s, 1H, NNH). ESI-MS,
m/z = 266.8 (100%) [M
−
H]−, 291.0 (100%) [M + Na]+. IR (KBr): 1715, 1691, 1570, 1484, 1269,
1197 cm−1. Mp > 250 ◦C from methanol.
(Z)-5-Methoxy-3-(2-(quinolin-2-yl)hydrazono)indolin-2-one (9 δ 3.76 (s, 3H,
). Yield: 94%. 1H-NMR
5-OCH3), 6.73 (d, 1H, J = 8.4, H-7), 6.80–6.88 (m, 1H, H-6), 7.23–7.29 (m, 1H, H-4), 7.44–7.76 (m,
4H, H-50, H-60, H-70and H-80), 7.80–8.10 (m, 2H, H-30 and H-40), 10.24 (s, 1H, NH), 12.00 (br s, 1H,
−
NNH). ESI-MS m/z = 316.8 (100%) [M
−
H] , 341.0 (100%) [M + Na]+. IR (KBr): 3430, 2919, 1688, 1634,
1476, 1302, 1186 cm−1. Mp 227–234 ◦C from ethanol.
1
(Z)-3-(2-(7-Chloroquinolin-4-yl)hydrazono)-5-methoxy-indolin-2-one (11). Yield: 82%. H-NMR
δ
3.79 (s,
0
3H, 5-OCH3), 6.60–6.64 (m, 1H, H-7), 6.80–6.90 (m, 2H, H-4 and H-6), 7.25–7.26 (m, 1H, H-5 ), 7.68–7.76
(m, 2H, H-20 and H-30), 8.03–8.12 (m, 1H, H-60), 8.30 (s, 1H, H-80), 9.67 (br s, 1H, NH), 11.20 (br s, 1H,
NNH). ESI-MS m/z = 350.8 (100%) [M − H]−, 352.8 (34%) [M − H]− + 2, 353.0 (39%) [M + H]+, 355.0
(14%) [M + H]+ + 2. IR (KBr): 3403, 3166, 1703, 1630, 1381, 1185 cm−1. Mp > 250 ◦C from ethanol.
3.1.2. Synthesis of 3-arylhydrazono-indolin-2-ones 8 and 10
Compounds
5-methoxyindolin-2-one
to corresponding amines (21.5 mmol) in HCl 6N (11 mL) with water solution (7.5 mL) of sodium
8
and 10 were prepared from azo coupling of aryldiazonium salts 3a
,b with
2
, previously described [24]. Aryldiazonium salts were prepared starting

Molecules 2018, 23, 1544
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nitrite (23 mmol). This solution was added to solution of indol-2-one (21.5 mmol) and sodium acetate
(43 mmol) in methanol (59 mL) at 0 ◦C. The resulting precipitate was filtered and purified.
(Z)-5-Methoxy-3-(2-(naphthalen-1-yl)hydrazono)indolin-2-one (8 δ 3.78 (s, 3H,
). Yield: 40%. 1H-NMR
5-OCH3), 6.86 (s, 2H, H-6 and H-7), 7.20 (s, 1H, H-4), 7.53–7.68 (m, 4H, H-40, H-50, H-60 and H-70),
7.83–7.89 (m, 2H, H-20 and H-30), 7.97 (d, 1H, J = 8.1 Hz, H-80), 11.04 (s, 1H, NH), 13.83 (s, 1H, NNH).
ESI-MS m/z = 315.8 (100%) [M
from hexane/ethyl acetate 70:30 (v/v).
−
H]−. IR (KBr): 3014, 1672, 1562, 1484, 1194, 1156 cm−1. Mp > 250 ◦C
(Z)-5-Methoxy-3-(2-(quinolin-8-yl)hydrazono)indolin-2-one (10). Yield: 50%. 1H-NMR
δ 3.78 (s, 3H,
5-OCH3), 6.84–6.85 (m, 2H, H-6 and H-7), 7.21–7.22 (m, 1H, H-4), 7.61–7.67 (m, 3H, H-30, H-40 and
H-60), 7.96 (d, 1H, J = 7.0 Hz, H-50), 8.39 (d, 1H, J = 8.4 Hz, H-20), 8.91–8.93 (m, 1H, H-70), 10.88 (s, 1H,
NH), 13.92 (s, 1H, NNH). ESI-MS m/z = 341.0 (64%) [M + Na]+, 316.8 [M
−
H]–. IR (KBr): 3435, 1675,
1649, 1522, 1194 cm−1. Mp > 250 ◦C from dichloromethane/methanol 95:05 (v/v).
3.1.3. Synthesis of 5-Methoxy-3-(2-thiazolylhydrazono)indolin-2-ones 13–18
Compounds 13
(36 mL) 1 mmol of appropriate bromomethylketone (1-bromo-3-chloropropan-2-one for 13
3-bromo-2-butanone for 14 -bromoacetophenone for 15; 3-hydroxy- -bromoacetophenone for 16
3-methoxy- -bromoacetophenone for 17; 3,
–18 were prepared adding to a solution of 12 [26] (1 mmol) in ethanol
;
;
;
α
α
α
α
-dibromoacetophenone for 18) at room temperature.
After stirring overnight the resulting precipitate was filtered and purified by column chromatography
or crystallization.
1
(Z)-3-(2-(4-(Chloromethyl)thiazol-2-yl)hydrazono)-5-methoxyindolin-2-one (13). Yield: 40%. H-NMR
δ 3.76
(s, 3H, 5-OCH3), 4.67 (s, 2H, CH2Cl). 6.86 (d, 1H, J = 8.4 Hz, H-7), 6.91 (dd, 1H, J = 8.4 Hz, J = 2.2 Hz
H-6), 7.04 (d, 1H, J = 2.2 Hz, H-4), 7.25 (s, 1H, H-50), 11.05 (s, 1H, NH), 13.33 (s, 1H, NNH). ESI-MS
m/z = 320.7 (100%) [M
H]−, 322.7 (48%) [M H]− + 2. IR (KBr): 3436, 2946, 1683, 1556, 1476,
1168 cm−1. Mp 153–155 ◦C (dec.) from chloroform/methanol 96:04 (v/v).
,
−
−
1
(Z)-3-(2-(4,5-Dimethylthiazol-2-yl)hydrazono)-5-methoxyindolin-2-one (14). Yield: 40%. H-NMR
δ 2.12 (s,
3H, CH3), 2.25 (s, 3H, CH3), 3.76 (s, 3H, 5-OCH3), 6.85 (d, 1H, J = 8.4 Hz, H-7), 6.89 (dd, 1H, J = 8.4 Hz
,
J = 2.2 Hz, H-6), 7.00 (d, 1H, J = 2.2 Hz, H-4), 10.98 (s, 1H, NH), 13.19 (s, 1H, NNH). ESI-MS m/z = 300.8
(100%) [M
from ethanol.
−
H]−, 325.0 (94%) [M + Na]+. IR (KBr): 3446, 1686, 1544, 1486, 1162 cm−1. Mp 160–164 ◦C
1
(Z)-3-(2-(4-(3-Hydroxyphenyl)thiazol-2-yl)hydrazono)-5-methoxyindolin-2-one (16). Yield: 44%. H-NMR
δ
3.84 (s, 3H, 5-OCH3), 6.69–6.72 (m, 1H, H-4”), 6.86 (d, 1H, J = 8.4 Hz, H-7), 6.91 (dd, 1H, J = 8.4 Hz
,
J = 2.4 Hz, H-6), 7.06 (d, 1H, J = 2.4 Hz, H-4), 7.19 (t, 1H, J = 8.1 Hz, H-5”), 7.29–7.31 (m, 2H, H-2” and
0
H-6”) 7.53 (s, 1H, H-5 ), 9.47 (s, 1H, 3”-OH), 11.06 (s, 1H, NH), 13.41 (s, 1H, NNH). ESI-MS m/z = 364.8
(100%) [M
−
H]−. HRMS (ESI) m/z [M + H]+ calcd for C18H15N4O3S+ 367.0859; found, 367.0833. IR
(KBr): 3431, 1551, 1523, 1163 cm−1. Mp > 250 ◦C from ethanol.
1
(Z)-5-Methoxy-3-(2-(4-(3-methoxyphenyl)thiazol-2-yl)hydrazono)indolin-2-one (17). Yield: 91%. H-NMR
δ
3.78 (s, 3H, 3”-OCH3), 3.80 (s, 3H, 5-OCH3), 6.87–6.93 (m, 3H, H-6, H-7 and H-4”), 7.060(d, 1H,
J = 2.2 Hz, H-4), 7.32 (t, 1H, J = 7.9 Hz, H-5”), 7.43–7.48 (m, 2H, H-6” and H-2”), 7.66 (s, 1H, H-5 ), 11.07
(s, 1H, NH), 13.39 (s, 1H, NNH). ESI-MS m/z = 378.8 (100%) [M
−
H]−. IR (KBr): 3171, 2926, 1679,
1551, 1171 cm−1. Mp > 250 ◦C from ethanol.
1
(Z)-3-(2-(4-(3-Bromophenyl)thiazol-2-yl)hydrazono)-5-methoxyindolin-2-one (18). Yield: 30%. H-NMR
δ
3.77 (s, 3H, 5-OCH3), 6.87 (d, 1H, J = 8.4 Hz, H-7), 6.92 (dd, 1H, J = 2.4 Hz, J = 8.4 Hz, H-6), 7.06 (d, 1H,
J = 2.4 Hz, H-4), 7.37 (t, 1H, J = 7.9 Hz, H-5”), 7.49–7.53 (m, 1H, H-4”), 7.78 (s, 1H, H-50), 7.89–7.91 (m,
1H, H-6”), 8.07 (t, 1H, J = 1.6 Hz, H-2”), 11.07 (s, 1H, NH), 13.40 (s, 1H, NNH). ESI-MS m/z = 426.8
−
− ◦
H] + 2. IR (KBr): 1683, 1552, 1483, 1155, 718 cm−1. Mp 240–242 C
(99%) [M
−
H] , 428.7 (100%) [M
−
(dec.) from dichloromethane/methanol 92:8 (v/v).

Molecules 2018, 23, 1544
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3.1.4. Synthesis of Compound 19
4-isopropylphenylhydrazine hydrochloride (1.8 mmol) was added to a solution of
5-methoxy-indole-3-carbaldehyde (1 mmol) in methanol (5 mL). After stirring overnight at room
temperature, the resulting precipitate was filtered and crystallized.
(E)-3-(2(4-Isopropylphenyl)hydrazonomethyl))-5-methoxy-1H-indole (19). Yield: 30%; 1H-NMR
δ 1.23 (d,
6H, J = 6.7 Hz, CH(CH3)2), 2.97 (ep, 1H, J = 6.7 Hz, CH(CH3)2), 3.82 (s, 3H, 5-OCH3), 6.80 (dd, 1H,
J = 8.8 Hz, J = 2.4 Hz, H-6), 6.94 (d, 2H, J = 8.1 Hz, H-30 and H-50), 7.07 (d, 2H, J = 8.1 Hz, H-20 and
0
H-6 ), 7.28 (d, 1H, J = 8.8 Hz, H-7), 7.54 (d, 1H, J = 2.5 Hz, H-4), 7.78 (d, 1H, J = 2.5 Hz, H-2), 8.05 (s, 1H,
CH=N), 9.68 (s, 1H, NH), 11.14 (s, 1H, NNH). ESI-MS m/z = 305.9 (100%) [M
−
H]−. IR (KBr): 3340,
1485, 1296, 1196 cm−1. Mp 175–177 ◦C from ethanol.
3.1.5. Synthesis of 3-(Benzylidenehydrazono)-5-methoxyindolin-2-ones 21 and 22
Compound 20 27] (1 mmol) was dissolved in methanol (20 mL), then the suitable benzaldehyde
[
(1 mmol) was added and the mixture was heated to reflux. The resulting precipitate was filtered and
purified by column chromatography.
(Z)-3-((E)-(4-Isopropylbenzylidene)hydrazono)-5-methoxyindolin-2-one (21). Yield: 30%; 1H-NMR
δ
1.23
(d, 6H, J = 6.7 Hz, CH(CH3)2), 2.97 (ep, 1H, J = 6.7 Hz, C
H(CH3)2), 3.69 (s, 3H, 5-OCH3), 6.82 (d, 1H,
J = 8.4 Hz, H-7), 7.00 (dd, 1H, J = 3.0 Hz, J = 8.4 Hz, H-6), 7.45 (d, 2H, J = 8.1 Hz, H-30 and H-50), 7.54
(d, 1H, J = 3.0 Hz, H-4), 7.88 (d, 2H, J = 8.1 Hz, H-20 and H-60), 8.58 (s, 1H, N=CH), 10.62 (br s, 1H,
NH). ESI-MS m/z = 319.9 (61%) [M
−
H]−, 344.0 (100%) [M + Na]+. HRMS (ESI) m/z [M + H]+ calcd
+
for C19H20N3O2 322.1537; found, 322.1550. IR (KBr): 3390, 2956, 1733, 1485, 1296, 1196 cm−1. Mp
223–225 ◦C from chloroform/methanol 95:5 (v/v).
1
(Z)-3-((E)-(3-chlorobenzylidene)hydrazono)-5-methoxyindolin-2-one (22). Yield: 30%. H-NMR
δ 3.78 (s, 3H,
5-OCH3), 6.86 (d, 1H, J = 8.5 Hz, H-7), 6.95 (dd, 1H, J = 8.5 Hz, J = 2.2 Hz, H-6), 7.15 (d, 1H, J = 2.2 Hz
,
H-4), 7.64 (t, 1H, J = 7.5 Hz, H-50), 7.70–7.75 (m 1H, H-40), 7.80–7.87 (m, 1H, H-60), 7.90–7.93 (m, 1H,
H-20), 8.70 (s, 1H, N=CH), 10.20 (br s, 1H, NH). ESI-MS m/z = 313.05 (100%) [M
−
H]−, 315.04 (34%)
◦
[M
−
H]− + 2. IR (KBr): 3465, 1745, 1626, 1487 cm−1. Mp 230–235 C from ethyl acetate/hexane
60:40 (v/v).
3.1.6. Synthesis of Carboxyhydrazide 23
5-Methoxyisatin (1 mmol) was dissolved in methanol (5 mL), then 3-chloro-benzohydrazide
(2 mmol) was added at room temperature and, after stirring overnight, the reaction mixture was
evaporated to dryness and the residue purified by column chromatography.
1
3-Chloro-N’-(5-methoxy-2-oxoindolin-3-ylidene)benzohydrazide (23). Yield: 40%. H-NMR
δ 3.77 (s, 3H,
5-OCH3), 6.87 (d, 1H, J = 8.5 Hz, H-7), 6.97 (dd, 1H, J = 8.5 Hz, J = 2.5 Hz, H-6), 7.12 (d, 1H, J = 2.5 Hz
,
H-4), 7.64 (t, 1H, J = 7.8 Hz, H-50), 7.74–7.77 (m, 1H, H-40), 7.80–7.83 (m, 1H, H-60), 7.87–7.89 (m,
1H, H-20), 11.26 (br s, 1H, NH), 13.83 (br s, 1H, NNHCO). ESI-MS m/z = 351.9 (45%) [M + Na]+,
353.9 (15%) [M + Na]+ + 2. IR (KBr): 3246, 1681, 1540, 1486, 1252, 1152 cm−1. Mp 236–238 ◦C from
dichloromethane/methanol 96:4 (v/v).
3.1.7. Synthesis of N-alkylisatin Derivatives 24, 30 and 31
5-methoxyisatin (1 mmol) or compound
1 [20] were dissolved in DMSO (1.5 mL), then K2CO3
(1.5 mmol) and suitable alkyl halide (1 mmol; methyl iodide for 24, 1-bromo-2-phenylethane for
30, and 1-bromo-3-phenylpropane for 31) were added at room temperature. After addition of ice,
the resulting precipitate was filtered, washed with water and used for subsequently steps without
further purification.

Molecules 2018, 23, 1544
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(Z)-3-(2-(4-Isopropylphenyl)-2-methylhydrazono)-5-methoxy-1-methylindolin-2-one (24). Yield: 33%.
1H-NMR
1.19 (d, 6H, J = 6.9 Hz, CH(CH3)2), 2.28 (ep, 1H, J = 6.9 Hz, C (CH3)2), 3.17 (s, 3H,
δ
H
N-CH3), 3.76 (s, 3H, 5-OCH3), 3.89 (s, 3H, N-CH3) 6.84 (dd, 1H, J = 8.4 Hz, J = 2.5 Hz, H-6), 6.94 (d,
1H, J = 8.4 Hz, H-7), 7.07 (d, 1H, J = 2.5 Hz, H-4), 7.27 (d, 2H, J = 7.7 Hz, H-30 and H-50), 7.51 (d, 2H,
J = 7.7 Hz, H-20 and H-60). ESI-MS m/z = 360.0 (100%) [M + Na]+. IR (KBr): 2924, 1673, 1500, 1278,
1247, 1097 cm−1. Mp 66–69 ◦C from ethyl acetate/hexane 20:80 (v/v).
1
5-Methoxy-1-phenetylindolin-2,3-dione (30). Yield: 60%. H-NMR
δ
2.88 (t, 2H, J = 7.4 Hz, NCH2CH2),
3.75 (s, 3H, 5-OCH3), 3.85 (t, 2H, J = 7.4 Hz, NCH2CH2), 7.08–7.27 (m, 8H, H-4, H-6, H-7, H-20, H-30,
H-40, H-50 and H-60). ESI-MS m/z = 280.0 (30%) [M
−
H]−. IR (KBr): 3084, 2964, 1739, 1626, 1491,
1287 cm−1. Mp 122–127 ◦C.
5-Methoxy-1-(3-phenylpropyl)indolin-2,3-dione (31). Yield: 60%. 1H-NMR
δ
1.87 (qn, 2H, J = 7.7 Hz,
NCH2CH2CH2), 2.64 (t, 2H, J = 7.7 Hz, NCH2CH2CH2), 3.66 (t, 2H, J = 7.7 Hz, NCH2CH2CH2), 3.75 (s,
3H, 5-OCH3), 7.06–7.27 (m, 8H, H-4, H-6, H-7, H-20, H-30, H-40, H-50 and H-60). ESI-MS m/z = 318.0
(100%) [M + Na]+. IR (KBr): 2926, 1722, 1596, 1491 cm−1. Mp 130–137 ◦C.
3.1.8. Synthesis of N-cyclopropyl-5-methoxyindolin-2,3-dione (27)
5-Methoxyisatin (2 mmol), cyclopropylboronic acid (4 mmol) and sodium carbonate (4 mmol) were
mixed in dichloroethane (3 mL). Then a solution of copper(II) acetate (2 mmol) and bipyridine (2 mmol)
◦
in dichloroethane (6.6 mL) at 50 C was added and the mixture was heated for 4 h. After cooling,
a solution of ammonium chloride was added and the resulting aqueous phase was extracted with
1
dichloromethane. 1-Cyclopropyl-5-methoxyndolin-2,3-dione 27. Yield: 60%. H-NMR
δ 0.76–0.85 (m,
2H, CH2 cyclopr.), 0.94–1.04 (m, 2H, CH2 cyclopr.), 2.66 (qn, 1H, J = 3.4 Hz, CH cyclopr.), 3.75 (s,
3H, 5-OCH3), 7.09 (d, 1H, J = 2.6 Hz, H-4), 7.14 (d, 1H, J = 8.8 Hz, H-7), 7.26 (dd, 1H, J = 8.8 Hz,
J = 2.6 Hz, H-6). ESI-MS m/z = 240.0 (100%) [M + Na]+. IR (KBr): 2962, 2924, 1722, 1490, 1281 cm−1
.
Mp 121–123 ◦C.
3.1.9. Synthesis of N-alkyl-3-phenylhyhydrazonoindolin-2-ones 26, 28, 32–34
The suitable N-alkylisatins 25 [32], 27, and 29–31 (1 mmol) were dissolved in methanol (5 mL),
and 4-isopropylphenylidrazine hydrochloride (2 mmol) was then added. After stirring overnight,
the resulting precipitate was filtered and crystallized or the reaction mixture was evaporated to dryness
and the residue purified by column chromatography.
(Z)-3-(2-(4-Isopropylphenyl)hydrazono)-5-methoxy-1-methylindolin-2-one (26). Yield: 30%. 1H-NMR
1.18 (d, 6H, J = 7.0 Hz, CH(CH3)2), 2.86 (ep, 1H, J = 7.0 Hz, C (CH3)2), 3.21 (s, 3H, NCH3), 3.77 (s,
δ
H
3H, 5-OCH3), 6.88 (dd, 1H, J = 8.5 Hz, J = 2.5 Hz, H-6), 7.01 (d, 1H, J = 8.5 Hz, H-7), 7.14 (d, 1H,
J = 2.5 Hz, H-4), 7.23 (d, 2H, J = 8.5 Hz, H-30and H-50), 7.36 (d, 2H, J = 8.5 Hz, H-20and H-60), 12.74 (s,
1H, NNH). ESI-MS m/z = 321.9 (100%) [M
−
H]−. IR (KBr): 2952, 2872, 1683, 1487, 1246, 1223 cm−1
.
Mp 101–103 ◦C from ethanol.
1
(Z)-1-Cyclopropyl-3-(2-(4-isopropylphenyl)hydrazono)-5-methoxyindolin-2-one (28). Yield: 50%. H-NMR
δ
0.84–0.86 (m, 2H, CH2 cyclopr.), 1.00–1.02 (m, 2H, CH2 cyclopr.), 1.18 (d, 6H, J = 7.0 Hz, CH(CH3)2),
2.77 (qn, 1H, J = 3.6 Hz, CH cyclopr.), 2.85 (ep, 1H, J = 7.0 Hz, CH(CH3)2), 3.77 (s, 3H, 5-OCH3), 6.89
(dd, 1H, J = 8.6 Hz, J = 2.4 Hz, H-6), 7.09 (d, 1H, J = 8.6 Hz, H-7), 7.12 (d, 1H, J = 2.4 Hz, H-4), 7.23
(d, 2H, J = 8.6 Hz, H-30e H-50), 7.35 (d, 2H, J = 8.4 Hz, H-20and H-60), 12.74 (s, 1H, NNH). ESI-MS
−
−1
.
m/z = 347.9 (100%) [M
−
H] , 372.1 (82%) [M + Na]+. IR (KBr): 3436, 1668, 1553, 1480, 1248, 1031 cm
Mp 106–109 ◦C from ethanol.
1
(Z)-1-Benzyl-3-(2-(4-isopropylphenyl)hydrazono)-5-methoxyindolin-2-one (32). Yield: 80%. H-NMR
δ 1.19
(d, 6H, J = 7.0 Hz, CH(CH3)2), 2.86 (ep, 1H, J = 7.0 Hz, CH(CH3)2), 3.75 (s, 3H, 5-OCH3), 4.97 (s, 2H,
CH2-Ph), 6.80 (dd, 1H, J = 8.6 Hz, J = 2.6 Hz, H-6), 6.93 (d, 1H, J = 8.6 Hz, H-7), 7.16 (d, 1H, J = 2.6 Hz,
H-4), 7.25 (d, 2H, J = 8.6 Hz, H-30 e H-50), 7.31–7.33 (m, 5H, H-2”, H-3”, H-4”, H-5” and H-6”), 7.40 (d,

Molecules 2018, 23, 1544
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2H, J = 8.6 Hz, H-20and H-60), 12.74 (s, 1H, NNH). ESI-MS m/z = 397.9 (100%) [M
−
H]−, 422.1 (34%)
+
[M + Na]+. HRMS (ESI) m/z [M + H]+ calcd for C25H26N3O2 400.2020; found, 400.2028. IR (KBr):
3436, 2964, 1676, 1487, 1162 cm−1. Mp 105–109 ◦C from ethanol.
(Z)-3-(2-(4-Isopropylphenyl)hydrazono)-5-methoxy-1-phenethylindolin-2-one (33). Yield: 90%. 1H-NMR
δ
1.18 (d, 6H, J = 7.0 Hz, CH(CH3)2), 2.85 (ep, 1H, J = 7.0 Hz, CH(CH3)2), 2.92 (t, 2H, J = 7.3 Hz,
NCH2CH2), 3.77 (s, 3H, 5-OCH3), 3.96 (t, 2H, J = 7.3 Hz, NCH2CH2), 6.83 (dd, 1H, J = 8.4 Hz,
0
0
J = 2.6 Hz, H-6), 7.06 (d, 1H, J = 8.4 Hz, H-7), 7.14 (d, 1H, J = 2.6 Hz, H-4), 7.16–7.28 (m, 7H, H-3 , H-5 ,
H-2”, H-3”, H-4”, H-5” and−H-6”), 7.36 (d, 2H, J = 8.4 Hz, H-20and H-60), 12.68 (s, 1H, NNH). ESI-MS
m/z = 412.0 (100%) [M
−
H] , 436.1 (88%) [M + Na]+. IR (KBr): 3431, 2950, 1674, 1557, 1487, 1249, 1160,
1149 cm−1. Mp 96–98 ◦C from ethanol.
(Z)-3-(2-(4-Isopropylphenyl)hydrazono)-5-methoxy-1-(3-phenylpropyl)indolin-2-one (34). Yield: 70%.
1H-NMR
δ
1.18 (d, 6H, J = 7.0 Hz, CH(CH3)2), 1.92 (qn, 2H, J = 7.3 Hz, NCH2CH2CH2), 2.62 (t,
2H, J = 7.3 Hz, NCH2CH2C 2), 2.85 (ep, 1H, J = 7.0 Hz, C (CH3)2), 3.74–3.79 (m, 2H, NC 2CH2CH2),
H
H
H
3.77 (s, 3H, 5-OCH3), 6.85 (dd, 1H, J = 8.4 Hz, J = 2.4 Hz, H-6), 7.02 (d, 1H, J = 8.4 Hz, H-7), 7.14–7.28 (m,
8H, H-4, H-30, H-50, H-2”, H-3”, H-4”, H-5” and H-6”), 7.36 (d, 2H, J = 8.4 Hz, H-20and H-60), 12.73
(s, 1H, NNH). ESI-MS m/z = 426.0 (100%) [M
−
H]−, 450.1 (100%) [M + Na]+. HRMS (ESI) m/z
+
[M + H]+ calcd for C27H30N3O2 428.2333; found, 428.2332. IR (KBr): 3435, 2957, 1669, 1559, 1485,
1248, 1158 cm−1. Hygroscopic compound from dichloromethane/hexane 65:35 (v/v).
3.1.10. Synthesis of 4-isopropylphenylhydrazonoindolin-2-ones 36–39
4-isopropylphenylhydrazine hydrochloride (1 mmol) was added to a methanolic solution (5 mL) of
suitable isatins 35a–d (1 mmol) (prepared using the Sandmayer method [30]). After stirring overnight,
the precipitate was filtered and crystallized from ethanol or the reaction mixture was evaporated and
the residue purified by column chromatography.
1
5-Butyl-3-(2-(4-isopropylphenyl)hydrazono)indolin-2-one (36). Yield: 80%. H-NMR
δ
0.88 (t, 3H, J = 7.1,
C
H3CH2CH2CH2) 1.18 (d, 6H, J = 7.0 Hz, CH(CH3)2), 1.29 (sx, 2H, J = 7.1 Hz, CH3CH2CH2CH2),
1.54 (qn, 2H, J = 7.5 Hz, CH3CH2CH2CH2), 2.56 (t, 2H, J = 7.5 Hz, CH3CH2CH2CH2) 2.85 (ep, 1H,
J = 7.1 Hz, C (CH3)2), 6.80 (d, 1H, J = 7.9 Hz, H-7), 7.03 (d, 1H, J = 7.9, H-6), 7.21 (d, 2H, J = 8.7 Hz,
H-30 and H-50), 7.31–7.34 (m, 3H, H-20, H-60 and H-4), 10.88 (s, 1H, NH), 12.72 (s, 1H, NNH). ESI-MS
m/z = 334.0 (100%) [M
H]−. IR (KBr): 3298, 2952, 2915, 1683, 1649, 1557, 1513, 1240, 1159 cm−1. Mp
164–166 ◦C from ethanol.
H
−
7-Bromo-3-(2-(4-isopropylphenyl)hydrazono)indolin-2-one (37). Yield: 80%. 1H-NMR
δ 1.18 (d, 6H,
J = 7.0 Hz, CH(CH3)2), 2.86 (ep, 1H, J = 7.0 Hz, C0H(CH3)2), 6.98 (t, 1H, J = 7.7 Hz, H-5), 7.24 (d,
0
0
0
2H, J = 8.4 Hz, H-3 and H-5 ), 7.36–7.41 (m, 3H, H-2 , H-6 and H-6), 7.52 (d, 1H, J = 7.3 Hz, H-4), 11.26
(s, 1H, NH), 12.78 (s, 1H, NNH). ESI-MS m/z = 355.9 (95%) [M
−
H]−, 357.7 (100%) [M − H]− + 2
.
HRMS (ESI) m/z [M + Na]+ calcd for C17H16BrN3NaO+ 380.0369; found, 380.0378. IR (KBr): 3154,
2959, 1672, 1551, 1170 cm−1. Mp 240–242 ◦C from ethanol.
1
Sodium 3-(2-(4-isopropylphenyl)hydrazono)-2-oxoindolin-5-sulfonate (38). Yield: 60%. H-NMR
δ
1.18 (d,
6H, J = 7.0 Hz, CH(CH3)2), 2.85 (ep, 1H, J = 7.0 Hz, CH(CH3)2), 6.83 (d, 1H, J = 8.1 Hz, H-7), 7.22 (d, 2H,
J = 8.6, H-30 and H-50), 7.35 (d, 2H, J = 8.6 Hz, H-20 and H-60), 7.48 (dd, 1H, J = 8.1 Hz, J = 1.8 Hz, H-6),
7.73 (d, 1H, J = 1.1, H-4), 11.04 (s, 1H, NH), 12.69 (s, 1H, NNH). ESI-MS m/z = 357.8 (100%) [M − Na]−
.
IR (KBr): 3201, 2959, 1560, 1189 cm−1. Mp > 250 ◦C from ethanol.
1
(Z)-7-(2-(4-isopropylphenyl)hydrazono)-5H-[1,3]dioxolo[4,5-f]indol-6(7H)-one (39). Yield: 50%. H-NMR
δ
1.17 (d, 6H, J = 7.0 Hz, CH(CH3)2), 2.84 (ep, 1H, J = 7.0 Hz, CH(CH3)2), 5.98 (s, 2H, OCH2O), 6.55
(s, 1H, H-4), 7.07 (s, 1H, H-7), 7.20 (d, 2H, J = 8.6 Hz, H-30 and H-50), 7.29 (d, 2H, J = 8.6 Hz, H-20 and
H-60), 10.79 (s, 1H, NH), 12.59 (s, 1H, NNH). ESI-MS m/z = 321.9 (100%) [M
1675, 1557, 1464, 1156 cm−1. Mp 229–234 ◦C ethyl acetate/hexane 50:50 (v/v).
−
H]−. IR (KBr): 3177,

Molecules 2018, 23, 1544
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3.2. Determination of Llipophilicity by RP-HPLC
The lipophilicity parameters were determined by an RP-HPLC technique [34
,35]. Methanol
solutions of the investigated hydrazone compounds (0.5 mg/mL) were injected into an Agilent 1260
infinity HPLC system (Agilent Technologies Italia, Milan), equipped with a diode array detector
(DAD), and a Phenomenex Kinetex C18 column (150
Maggiore, BO, Italy), and eluted at different mobile phase compositions (0.05 increments of MeOH
×
4.6 mm, 5 µ, Phenomenex Italy s.r.l., Castel
volume fraction in 10 mM ammonium formate buffer at pH 4.5, ranging
ϕ between 0.85 and 0.30).
◦
All RP-HPLC measurements were carried out at 25
254 nm wavelength.
±
1 C, at a flow rate of 1.0 mL/min, and at
The logarithm of capacity factors (log k0 = log (tR
−
t0)/t0) of each compound at different mobile
phase compositions have been calculated; tR represents the retention time of the solute and t0 is the
column dead time, measured as the elution time of a KNO3 solution in MeOH. For each compound,
the log k0 values increased linearly with decreasing MeOH volume fraction. Logarithms of capacity
factors extrapolated to the 100% aqueous mobile phase (log k0w) were calculated from the linear
regressions on at least five data points (r2 > 0.975).
Lipophilicity was also computationally assessed with the ACDLabs software, release 10.0
(Advanced Chemistry Development, Inc., Toronto, ON, Canada), and BioLoom software, vers. 1.7
(BioByte Corp. Claremont, CA, USA). The SAR equation (1) has been obtained by using the Prism
software (ver. 5.01, GraphPad Software Inc., La Jolla, CA, USA).
3.3. Inhibition of Aβ40 Aggregation
The spectrofluorimetric assays, measuring ThT fluorescence in the presence of Aβ, were
done as previously described [22]. Briefly, coincubation samples were prepared as described
above (Sections 2.2 and 2.3) in 96-well black, non-binding microplates (Greiner Bio-One GmbH,
Frickenhausen, Germany). Fluorimetric reads were performed in a multiplate reader Infinite M1000
Pro (Tecan, Cernusco sul Naviglio, Italy). Each concentration point was run in triplicate. CD ellipticity
measures were performed as detailed in ref. [20], following the increase of the negative band at 215 nm
as indicative of β-sheet arrangement.
3.4. Cell-Based Assays
Cytoprotection from Aβ42-induced neurotoxicity and from ROS generation was assessed in
SH-SY5Y cells as already described [22]. At least three independent experiments with six replicates
(n
≥
18) were carried out, and the results were averaged. Briefly, for MTT assay, SH-SY5Y cells were
cultured in DMEM-Dulbecco’s modified Eagle’s medium (Sigma-Aldrich), grown to 70% confluence
and seeded for experiment in 96-well plates at a density of 10,000 cells/well. Cultured cells were
added with a DMEM solution of Aβ42, alone or in mixture with 28 (5
and incubated at 37 C in 5% CO2. At the time points of 24 and 48 h, the culture medium was replaced
µ
M final concentration of both)
◦
by DMEM supplemented with a solution of MTT in PBS (50 µg/mL final concentration). After removal
of solution and addition of DMSO, the absorbance of product formazan produced from MTT was
measured at 570 nm using a multilabel plate counter Victor3 V (Perkin Elmer, Milan, Italy), with DMSO
medium as the blank solution.
Intracellular ROS production was evaluated using an oxidation-sensitive fluorescent probe,
20,70-dichlorodihydrofluorescin diacetate (DCFH-DA; Sigma-Aldrich). Viable SHSY5Y cells were
seeded in a black 96-well cell culture plate for 24 h, then the cells were incubated in DMEM
supplemented with different concentration (0 to 20
(50
CO2 for 0.5 h. After washing, 100
µ
M) of 28 for 1 h. After washing, DCFH-DA
◦
µ
M final concentration) was added directly to each well, and the plate was incubated at 37 C in 5%
µ
M H2O2/well in DMEM was added and the cells were incubated
for an additional 30 min. The formation of fluorescent dichlorofluorescein (DCF) due to oxidation
of DCFH in the presence of ROS was read directly in each well at an excitation wavelength of 485

Molecules 2018, 23, 1544
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nm and an emission wavelength of 530 nm using a multilabel plate reader Victor3 V (Perkin Elmer),
with DMSO medium as the control.
4. Conclusions
The SAR studies on the indoline-2-one 3-arylhydrazone derivatives reported herein warranted
further gain of knowledge in our ongoing study of inhibition of A
β aggregation. Rooting on
previous results [19 21] and keeping phenylhydrazone 22] as a hit compound prone to optimization,
–
1 [
the focused exploration of substituents on the indole scaffold, the modifications on the hydrazone
linker, and the isosteric replacement of the phenyl of hydrazone moiety with thiazole, highlighted the
essential role of 5-methoxy substituent, the requisite of an optimal lipophilicity, and the opportunity
of alkylating the indole nitrogen for maintaining an efficient inhibition of A
β aggregation. In this
regard, a handful of derivatives (thiazolyl hydrazone 15 and N1-substituted indolin-2-ones 26
,
28
,
,
32–34) were found almost equipotent in vitro with the hit compound 1. Among them, compound 28
i.e., the N1-cyclopropyl derivative of 1, emerged from cell-based assays as a multitarget agent with
strong cytoprotective effects in both Aβ42 oligomer toxicity and oxidative stress. These results were in
full agreement with those already reported for congeners of 28, particularly for cytoprotection from
amyloid insult that resulted well comparable with reference compound
1 [22]. Aβ antifibrillogenic
data have been analyzed to disclose reliable QSARs, and a bilinear correlation between pIC50 and
log k’w determined by RP-HPLC was derived which hold for those compounds that do not undergo
detrimental steric effects in binding Aβ peptides.
The present work adds new information to the potential of indolin-2-one 3-arylhydrazones as
privileged structures for the inhibition of Aβ aggregation and neurotoxicity, and pleiotropic agents for
AD therapy. Despite the claims of disengagement of pharmaceutical industry from AD research [42],
because of the controversial results so far obtained in clinical trials, many programs from research
institutions continue to bet in the so-called amyloid hypothesis. In this light, the multitarget activity of
28 could deserve for this compound a preliminary pharmacological investigation in animal models of
Aβ-dependent neurodegeneration, provided that further efforts in ameliorating aqueous solubility
would be beneficial. The potency and versatility of indolin-2-one derivatives may still encounter the
interest of research and development of small molecules as pharmacological tools for treating AD and
related neurodegenerative syndromes.
Supplementary Materials: The following are available online at http://www.mdpi.com/1420-3049/23/7/1544/
s1, Table S1: Experimental lipophilicity as described by RP-HPLC log k’w, and predicted 1-octanol–water partition
coefficients of the new synthesized isatin hydrazone derivatives; Figure S1: Log k’w versus log P calculated values
by means of ACDLabs suite; Figure S2: Scatter plot of A
β aggregation inhibition potency (pIC50) versus calculated
molar refractivity.
Author Contributions: A.D.P., F.C., C.D.A. and M.C. conceived and designed the experiments; R.P., M.d.C., F.D.S.
and L.P. performed the experiments; R.P., M.d.C., A.D.P., C.D.A. and M.C. wrote the paper; all authors analyzed
the data and revised the manuscript.
Funding: This research received no external funding.
Acknowledgments: L.P. acknowledges financial support from APQ Research Apulian Region “FutureInResearch
(FKY7YJ5)—Regional program for smart specialization and social and environmental sustainability” Fondo di
Sviluppo e Coesione 2007–2013.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds 1, 4–11, 13–19, 21–24, 26, 28, 32–34, 36–39 are available from
the authors.
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2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).