Effects of novel acylhydrazones derived from 4-quinolone on the acetylcholinesterase activity and Aβ42 peptide fibrils formation

Article abstract of DOI:10.3109/14756366.2016.1144597

Acetylcholinesterase inhibitors and compounds that trigger Aβ amyloid oligomerization and fibrillization represent an opportunity to discover new drug candidates to treat Alzheimer’s disease. In this work, we synthesized nine new acylhydrazones and a known one, both employing 3-carboethoxy-4-quinolone derivatives as starting materials with chemical yields ranging from 63% to 90%. We evaluated the effect of these compounds on the acetylcholinesterase (AChE) activity and the fibrillization of Aβ₄₂ peptide. Except for one acylhydrazone, the compounds exhibited good inhibitory effect on AChE (1.2 μM < IC₅₀ values < 17 μM). They also showed a significant decrease in the thioflavin-T fluorescence emission, suggesting an inhibitory effect on the Aβ₄₂ fibril formation.

Full text of DOI:10.3109/14756366.2016.1144597

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: http://www.tandfonline.com/loi/ienz20
Effects of novel acylhydrazones derived from 4-
quinolone on the acetylcholinesterase activity and
Aβ42 peptide fibrils formation
Gisele S. da Silva, Micheli Figueiró, Claudio F. Tormena, Fernando Coelho &
Wanda P Almeida
To cite this article: Gisele S. da Silva, Micheli Figueiró, Claudio F. Tormena, Fernando Coelho
& Wanda P Almeida (2016): Effects of novel acylhydrazones derived from 4-quinolone on the
acetylcholinesterase activity and Aβ42 peptide fibrils formation, Journal of Enzyme Inhibition
and Medicinal Chemistry, DOI: 10.3109/14756366.2016.1144597
To link to this article: http://dx.doi.org/10.3109/14756366.2016.1144597
View supplementary material
Published online: 01 Mar 2016.
Submit your article to this journal
View related articles
View Crossmark data
Full Terms & Conditions of access and use can be found at
http://www.tandfonline.com/action/journalInformation?journalCode=ienz20
Download by: [New York University]
Date: 02 March 2016, At: 07:42

http://informahealthcare.com/enz
ISSN: 1475-6366 (print), 1475-6374 (electronic)
J Enzyme Inhib Med Chem, Early Online: 1–7
2016 Taylor & Francis. DOI: 10.3109/14756366.2016.1144597
!
RESEARCH ARTICLE
Effects of novel acylhydrazones derived from 4-quinolone on the
acetylcholinesterase activity and Ab42 peptide fibrils formation
1
2
1
1
Gisele S. da Silva , Micheli Figueiro , Claudio F. Tormena , Fernando Coelho , and Wanda P. Almeida
2
´
2
1Department of Organic Chemistry, Institute of Chemistry, University of Campinas, Campinas, Sa˜o Paulo, Brazil and Faculty of Pharmaceutical
Sciences, University of Campinas, Campinas, Sa˜o Paulo, Brazil
Abstract
Keywords
Acetylcholinesterase inhibitors and compounds that trigger Ab amyloid oligomerization and
fibrillization represent an opportunity to discover new drug candidates to treat Alzheimer’s
disease. In this work, we synthesized nine new acylhydrazones and a known one, both
employing 3-carboethoxy-4-quinolone derivatives as starting materials with chemical yields
ranging from 63% to 90%. We evaluated the effect of these compounds on the acetylcholin-
esterase (AChE) activity and the fibrillization of Ab42 peptide. Except for one acylhydrazone, the
compounds exhibited good inhibitory effect on AChE (1.2 mM5IC50 values517 mM). They also
showed a significant decrease in the thioflavin-T fluorescence emission, suggesting an
inhibitory effect on the Ab42 fibril formation.
Acetylcholinesterase, acylhydrazone,
Alzheimer’s disease, b-amyloid, quinolone
History
Received 21 December 2015
Revised 10 January 2016
Accepted 11 January 2016
Published online 26 February 2016
Introduction
In this work, we report the synthesis and effect
of 10 acylhydrazone-4-quinolone derivatives on the acetylcholin-
esterase activity. Since b-amyloid (Ab) peptide is also involved in
the pathogenesis of AD, we studied the effect of some
acylhydrazones on the Ab fibril formation.
Alzheimer’s disease (AD) is an age-related neurodegenerative
process with a limited therapeutic arsenal. The physiopathology
of AD is very complex, but a cognitive decline due to decreased
cholinergic activity is quite accepted1,2. Therefore, acetylcholin-
esterase inhibitors, such as donepezil, galantamine and rivastig-
mine, are currently indicated for the treatment of AD symptoms.
In the context of the development of AD drug candidates, we are
involved in the synthesis and biological evaluation of some
Experimental
Chemistry
Solvents and reagents were purchased from Sigma Aldrich (Sa˜o
Paulo, Brazil) and used without purification. The reaction
progress was monitored by thin-layer chromatography (TLC)
and revealed by UV-Vis. Compounds were purified by column
chromatography on silica gel (230–4000 mesh) and a mixture
of ethyl acetate and hexane as eluent. Uncorrected melting
points were determined in a Fisatom Mod.431 (115 V) device.
IR spectra were recorded on a BOMEN (MBSERIES model)
equipment. 1H and 13C NMR spectra (1D and 2D) were
acquired in a Bruker spectrometer operating at 400 or 500 MHz
3-carboethoxy-4-quinolone derivatives3,4
3-carboethoxy-4-quinolone inhibited
.
We noted that
acetylcholinesterase
(AChE), an important target for drugs action. However, the
effect was moderated (IC50 ¼ 70 mM). More recently, Pudlo et al.5
have found that quinolone–benzylpiperidine hybrids inhibited
AChE, with IC50 values ranging from 0.37 to 2.46 mM. Thus, we
were dedicated to find out more effective quinolone derivatives.
Hydrazides, hydrazones and acylhydrazones are a class of
nitrogen compounds, which are extensively studied and most of
them are bioactive compounds, including some naturally
occurring ones6. These organic functions have been pointed out
as the structural moiety related to the biological activity7.
For example, anticancer8, antimycobacterial9, analgesic and
anti-inflammatory10 activities are distinctive in this class of
compounds. In view of this, we studied some molecular hybrids
based on the structures of the 3-carboxy-4-quinolone and
hydrazones derived from aromatic aldehydes (Figure 1).
1
1
for H and 100 MHz or 125 MHz for 13C, respectively. H and
13C spectra at 600 MHz were obtained in Avance 600 as
specified for each compound. Tetramethylsilane was used as
internal standard. High-resolution mass spectrometry spectra
were obtained in a LTQ-Orbitrap Discovery Thermo Scientific.
Synthesis and characterization
The synthesis of the desired acylhydrazones started with the
preparation of 3-carboethoxy-4-quinolone 1 (Figure 1), which was
converted into the hydrazide 2. The next step consisted in the
coupling of hydrazide 2 with substituted aromatic aldehydes. The
synthesized acylhydrazones melt above 250 ꢀC, with
decomposition.
Address for correspondence: Wanda P. Almeida, Department of
Pharmacy, University of Campinas, Sergio Buarque De Holanda St,
250, Campinas, Sa˜o Paulo, 13083 859, Brazil. E-mail: wanda.almeida@
fcf.unicamp.br

2
G. S. da Silva et al.
J Enzyme Inhib Med Chem, Early Online: 1–7
General procedures for the preparation of acylhydrazones
derivatives
O
O
R
N
N
H
An equimolar mixture of the substituted benzaldehyde (4.3 mmol)
and hydrazide 2 (4.3 mmol) was stirred under reflux in ethanol
(3 mL) in the presence of 20 drops of piperidine for 12–18 h. The
solvent excess was removed under reduced pressure and the
collected solid washed with the minimum amount of ethanol
possible and then dried at room temperature.
N
H
Acylhydrazones
O
O
O
OHC
4-oxo-N0-[(1E)-phenylmethylidene]-1,4-dihydroquinoline-3-car-
bohydrazide (3)
R
N
H
1
Beige solid; yield: 68%. IR (KBr, cmꢁ 1): ꢀ 3267–3057 (HN–
C¼O and H2N–N), 1680 (C¼O hydrazide), 1617
(C¼O, quinolone); 1H NMR (d6-DMSO, 500 MHz) ꢁ (ppm):
13.58 (s, 1H, –CONH), 8.85 (s, 1H, H-2), 8.43 (s, 1H,
-N¼CH-14), 8.31 (dd, J ¼ 8.1 and 1.0 Hz, 1H, H-5), 7.81 (td,
J ¼ 8.1 and 1.0 Hz, 1H, H-6), 7.77 (dd, J ¼ 7.4 and 1.4 Hz,
2H, H-16 and H-20), 7.75 (d, J ¼ 7.8 Hz, 1H, H-7), 7.53 (td,
J ¼ 7.8 Hz and 1.0 Hz, 1H, H-8), 7.45 (m, 3H, H-17, H-18 and
Substituted benzaldehyde
IC50 = 70 µM (AChE)
3-Carboethoxy-4-quinolone
Figure 1. Structure of acylhydrazones derived from 3-carboethoxy-4-
quinolone and aromatic aldehydes.
H-19), 13C NMR (d6-DMSO, 150 MHz)
ꢁ (ppm): 176.1
Preparation of ethyl-4-oxo-1,4-dihydroquinoline-3-carboxylate
(1)
(C¼O), 161.5 (C¼ONH), 147.8 (CH-14), 144.4 (CH-2)
139.2 (C0–9), 134.5 (C0–15), 133.2 (CH-7), 130.1 (CH-18),
128.9 (CH-16 and CH-20), 127.3 (CH-17 and CH-19); 125.9
(CH-5), 125.6 (C0–10), 125.5 (CH-6), 119.3 (CH-8), 110.1
(C0–3); Calculated for C17H14N3O2 (M + H+): 292.1086,
Found 292.1086.
Beige solid, yield: 88%. mp 284–286 ꢀC. IR (KBr, cm ꢁ1): ꢀ
3101–3169 (–NH), 1701 (C¼O, hydrazide), 1622 (C¼O, 4-
carboxy); 1H NMR (CF3CO2D, 600 MHz), ꢁ (ppm): 10.09 (s, 1H,
H-2), 9.38 (dd, J ¼ 8.7 Hz and 0.6 Hz, 1H, H-5), 9.10 (t, J ¼
8.7 Hz, 1H, H-6), 8.97 (td, J ¼ 7.8 and 0.6 Hz, 1H, H-7), 8.71 (d,
J
¼ 7.8 Hz, 1H, H-8), 5.34 (q, J ¼ 7.2 Hz, 2H), 2.20 (t,
11
J ¼ 7.2 Hz, 3H);
C NMR (CF3CO2D, 150 MHz), ꢁ (ppm):
N0-[(1E)-(4-chlorophenyl)methylidene]-4-oxo-1,4-dihydroquino-
line-3-carbohydrazide (4)
173.8 (C¼O), 167.7 (C¼O), 139.7 (CH-2), 139.5 (C0–9), 138.5
(CH-7), 130.6 (CH-5), 130.3 (C0–10), 125.1 (CH-6), 124.9 (CH-
8), 119.9 (C0–3), 65.3 (CH2), 12.7 (CH3).
Beige solid, yield: 72%. IR (KBr, cm ꢁ1): ꢀ 3273–3026 (HN–
C¼O and H2N–N), 1645 (C¼O hydrazide), 1618 (C¼O,
These data are in accordance with the literature3,12
.
The numbering of H and C of was done as follows (Scheme 1):
1
quinolone); H NMR (d6-DMSO, 500 MHz) ꢁ (ppm): 13.40 (s,
1H, –CONH), 8.89 (s, 1H, H-2), 8.47 (s, 1H, –N¼CH-14),
8.34 (dd, J ¼ 8.0 and 1.0 Hz, 1H, H-5), 7.83 (d, J ¼ 8.6 Hz,
2H, H-16 and H-20), 7.81 (td, J ¼ 8.2 and 1.0 Hz, 1H, H-7),
7.78 (d, J ¼ 8.2 Hz, 1H, H-8), 7.57 (td, J ¼ 8.0 and 1.0 Hz, 1H,
H-6), 7.57 (d, J ¼ 8.6 Hz, 2H, H-17 and H-19); 13C NMR (d6-
DMSO, 125 MHz) ꢁ (ppm): 176.5 (C¼O), 162. 1 (C¼ONH),
146.9 (CH-14), 144.9 (CH-2) 139.7 (C0–9), 134.9 (C0–18),
133.8 (C0–15), 133.6 (CH-7), 129.4 (CH-17 and CH-19),
129.3 (CH-16 and CH-20), 126.3 (C0–10), 126.3 (CH-6),
125.9 (CH-5), 119.8 (CH-8), 110.3 (C0–3); TOF MS (ESI+)
Calculated for C17H13ClN3O2 (M + H+): 326.0696, Found
326.0716.
17
16
18
19
O
4
O
5
12
N
H
15
14
10
9
N
13
3
6
7
11
20
1
N
2
H
8
Scheme 1. Numbering of acylhydrazone structure.
Preparation of 4-oxo-1,4- dihydroquinolone-3-carbohydrazide (2)
N0-[(1E)-(3-chlorophenyl)methylidene]-4-oxo-1,4-dihydroquino-
line-3-carbohydrazide (5)
A stirred equimolar mixture of 3-carboethoxy-4-quinolone 1
(0.55 mmol) and 50% aqueous hydrazine monohydrate
(10.28 mmol) in ethanol (5 mL) was heated for 20 h. After this
period, the solvent was removed under reduced pressure. The
crude product was purified by recrystallization with ethanol to
afford hydrazide 2.
Yellow solid, yield: 63%.
IR (KBr, cmꢁ1): ꢀ 3265–3062 (HN–C¼O and H2N–N), 1646
(C¼O hydrazide), 1620 (C¼O, quinolone); 1H NMR (d6-DMSO,
400 MHz) ꢁ (ppm): 13.38 (s, 1H, –CONH), 8.86 (s, 1H, H-2), 8.43
(s, 1H, –N¼CH-14), 8.30 (dd, J ¼ 8.1 and 1.2 Hz, 1H, H-5), 7.82
(td, J ¼ 7.0 and 1.2 Hz, 1H, H-7), 7.81 (s, 1H, H-16), 7.74 (d, J ¼
7.0 Hz, 1H, H-8), 7.72 (dd, J ¼ 5.5 and 3.3 Hz, 1H, H-20), 7.54
(td, J ¼ 8.1 and 1.2 Hz, 1H, H-6), 7.50 (dd, J ¼ 5.5 and 3.3 Hz, 2H,
H-18 and H-19); 13C NMR (d6-DMSO, 100 MHz) ꢁ (ppm): 176.5
(C¼O), 162.1 (C¼ONH), 146.6 (CH-14), 144.9 (CH-2), 139.5
(C0–9), 137.1 (C0–15), 134.1 (C0–17), 133.6 (CH-7); 131.2 (CH-
18), 130.1 (CH-6), 126.9 (CH-16), 126.3 (C0–10), 126.3 (CH-19),
125.9 (CH-5), 125.9 (CH-20), 119.7 (CH-8), 110.4 (C0–3).
Calculated for C17H13ClN3O2 (M + H+): 326.0696. Found,
326.0716.
82% yield, yellow solid. Melting point 280–282 ꢀC (decom-
position). IR (KBr, cmꢁ 1): ꢀ 3448–3265 (HN–C¼O and H2N–N),
1
1676 (C¼O, hydrazide), 1631 (C¼O, 4-carboxy); H NMR (d6-
DMSO, 600 MHz) ꢁ (ppm): 10.72 (s, 1H, –CONH), 8.74 (s, 1H,
H-2), 8.27 (dd, J ¼ 8.4 and 1.2 Hz, 1H, H-5), 7.78 (td, J ¼ 7.5 and
1.2 Hz, 1H, H-6), 7.68 (dd, J ¼ 7.5 Hz, 1H, H-7), 7.47 (td, J ¼ 7.5
and 1.2 Hz, 1H, H-8). 13C NMR (d6-DMSO, 125 MHz) ꢁ (ppm):
176.2 (C¼O), 164.6 (C¼ONHNH2), 143.6 (CH-2), 139.5 (C0–9),
133.2 (CH-7), 126.4 (C0–10), 125.9 (CH-5), 125.4 (CH-6), 119.4
(CH-8), 110.8 (C0–3). These data are in accordance with the
literature13.

DOI: 10.3109/14756366.2016.1144597
Effects of novel acylhydrazones derived from 4-quinolone
3
N0-[(1E)-(2,3-dichlorophenyl)methylidene]-4-oxo-1,4-dihydroqui- (s, 4H, H-16, H17, H19 and H20), 7.81 (td, J ¼ 8.1 Hz, 1.0 Hz,
noline-3-carbohydrazide (6)
1H, H-7), 7.76 (d, J ¼ 8.1 Hz, 1H, H-8), 7.54 (td, J ¼ 8.1 and
1.0 Hz, 1H, H-6), 3.27 (s, 3H, CH3); 13C NMR (d6-DMSO,
125 MHz) ꢁ (ppm): 176.5 (C¼O), 162.2 (C¼ONH), 146.3
(CH-14), 145.0 (CH-2), 141.8 (C0–18), 139.8 (C0–15), 139.6
(C0–9), 133.6 (CH-7), 128.2 (CH-17 and 19), 127.9 (CH-16
and 20), 126.3 (C0–9), 125.9 (CH-7), 125.9 (CH-6), 119.8
(CH-8), 110.3 (C0–3), 43.9 (CH3); TOF MS (ESI+)
Calculated for C18H16N3O4S (M + H+): 370.0862. Found:
370.0889.
Yellow solid, yield: 65%.
IR (KBr, cm ꢁ1): ꢀ 3265–3067 (HN–C¼O and H2N–N), 1662
(C¼O hydrazide), 1621 (C¼O, quinolone); 1H NMR (d6-DMSO,
500 MHz) d (ppm): 13.59 (s, 1H, –CONH), 8.89 (s, 1H, H-2), 8.76
(s, 1H, –N¼CH-14), 8.32 (d, J ¼ 7.8 Hz, 1H, H-5), 8.00 (d, J ¼
7.0 Hz, 1H, H-8), 7.83 (s, 1H, H-18), 7.76 (m, 2H, H-19 and H-
20), 7.55 (t, J ¼ 7.0 Hz, 1H, H-7), 7.49 (t, J ¼ 7.8 Hz, 1H, H-6);
13C NMR (d6-DMSO, 125 MHz) d (ppm): 173.5 (C¼O), 161.9
(C¼ONH), 148.2 (C0–19), 145.5 (CH-14), 142.8 (CH-2), 142.6
(C0–16), 141.4 (C0–17), 139.9 (C0–9), 133.4 (CH-18), 131.9
(CH-7), 128.9 (CH-6), 126.9 (C0–10), 125.8 (CH-20), 126.3 (CH-
N0-[(1E)-[4-(methylsulfanyl)phenyl]methylidene]-4-oxo-1,4-dihy-
droquinoline-3-carbohydrazide (10)
19), 124.5 (CH-5), 116.8 (CH-8), 107.9 (C0–3); TOF MS (ESI+) White solid, yield: 85%.
Calculated for C17H12Cl2N3O2 (M + H+): 360.0307; Found:
361.1557.
IR (KBr): ꢀ 3265–2920 (HN-C¼O and H2N-N), 1670 (C¼O
hydrazide), 1630 (C¼O, quinolone). 1H NMR (d6-DMSO,
500 MHz) d (ppm): 13.33 (s, 1H, –CONH-12), 8.86 (s, 1H, H-
2), 8.38 (s, 1H, –N¼CH-14), 8.30 (dd, J ¼ 8.1 and 0.9 Hz, 1H,
H-5), 7.80 (td, 8.2 Hz and 1.0 Hz, 1H, H-7), 7.75 (dd, J ¼ 8.2 and
1.0 Hz, 1H, H-8), 7.69 (d, J ¼ 8.5 Hz, 2H, H-16 and H-20), 7.53
(td, 8.0 Hz and 0.9 Hz, 1H, H-6), 7.33 (d, J ¼ 8.5 Hz, 2H, H-17
and H-19), 2.52 (s, 3H, CH3); 13C NMR (d6-DMSO, 125 MHz) ꢁ
(ppm): 175.9 (C¼O), 161.9 (C¼ONH), 147.6 (CH-14), 144.99
(CH-2), 141.3 (C0–18), 139.9 (C0–9), 131.4 (C0–15), 128.1 (CH-
16 and 20), 126.4 (C0–10), 125.9 (CH-17 and 19), 125.7 (CH-5),
125.7 (CH-6), 133.4 (CH-7), 119.9 (CH-8), 110.5 (C0–3), 14.8
(CH3); TOF MS (ESI+) Calculated for C18H16N3O2S (M + H+):
338.0963. Found: 338.0988.
N0-[(1E)-(4-nitrophenyl)methylidene]-4-oxo-1,4-dihydroquino-
line-3-carbohydrazide (7)
Yellow solid, yield: 90%.
IR (KBr): ꢀ 3267–3067 (HN–C¼O and H2N–N), 1670 (C¼O
hydrazide), 1630 (C¼O, quinolone); 1H NMR (d6-DMSO,
500 MHz) ꢁ (ppm): 13.50 (s, 1H, –CONH-12), 8.84 (s, 1H, H-
2), 8.63 (s, 1H, –N¼CH-14), 8.34 (dd, J ¼ 8.0 and 1.0 Hz, 1H, H-
5), 8.03 (d, J ¼ 8.5 Hz, 2H, H-17 and H-19), 8.02 (d, J ¼ 8.5 Hz,
2H, H-16 and H-20), 7.80 (td, J ¼ 8.0 and 1.0 Hz, 1H, H-7), 7.75
(d, J ¼ 8.5 Hz, 1H, H-8), 7.53 (td, J ¼ 8.0 and 1.0 Hz, 1H, H-6);
13C NMR (d6-DMSO, 125 MHz) ꢁ (ppm): 175.9 (C¼O), 161.9
(C¼ONH), 147.8 (CH-14), 147.5 (C0–18), 145.2 (CH-2), 140.9
(C0–15), 139.7 (C0–9), 132.9 (CH-7), 128.0 (CH-17 and CH-19),
127.8 (CH-16 and CH-20), 125.9 (C0–10), 125.4 (CH-6), 124.1
4-oxo-N0-[(1E)-thiophen-2-ylmethylidene]-1,4-dihydroquinoline-
3-carbohydrazide (11)
(CH-5), 119.7 (CH-8), 109.6 (C0–3); 15N (d6-DMSO, 60 MHz) ꢁ White solid, yield: 79%.
(ppm) 145.40 (s, 1N, NH-1), 176.80 (s, 1H, –CONH-12), 329.70
(s, 1N, –N ¼ C-13), 369.80 (s, 1N, NO2–21). TOF MS (ESI+) hydrazide), 1620 (C¼O, quinolone).
IR (KBr): ꢀ 3264–3058 (HN–C¼O and H2N–N), 1645 (C¼O
Calculated for C17H13N4O4 (M + H+): 337.0937. Found:
337.0963.
1H NMR (d6-DMSO, 500 MHz) ꢁ (ppm): 13.20 (s, 1H,
-CONH-12), 8.84 (s, 1H, H-2), 8.66 (s, 1H, –N¼CH-14), 8.31 (d,
J ¼ 7.9 Hz, 1H, H-5), 7.80 (t, J ¼ 8.0 Hz, 1H, H-7), 7.74 (d,
J ¼ 8.0 Hz, 1H, H-8), 7.68 (d, J ¼ 4.0 Hz, 1H, H-16), 7.53 (t,
J ¼ 7.9 Hz, 1H, H-6), 7.47 (d, J ¼ 2.4 Hz, 1H, H-18), 7.16 (d, J ¼
4.4 Hz, 1H, H-17); 13C NMR (d6-DMSO, 125 MHz) ꢁ (ppm):
176.25 (C¼O), 162.12 (C¼ONH), 145.42 (CH-14), 143.19 (CH-
2), 140.51 (C0–9), 139.55 (C0–15), 133.25 (CH-7), 131.47 (CH-
16), 129.32 (CH-17), 128.37 (CH-18), 126.34 (C0–10), 125.86
(CH-5), 125.62 (CH-6), 120.40 (CH-8), 110.2 (C0–3); TOF MS
(ESI+) Calculated for C15H12N3O2S (M + H+): 298.0650. Found:
298.0672.
N0-[(1E)-(2-nitrophenyl)methylidene]-4-oxo-1,4-dihydroquino-
line-3-carbohydrazide (8)
Yellow solid, yield: 84%.
IR (KBr): ꢀ 3099–3029 (HN–C¼O and H2N–N), 1672
(C¼O hydrazide), 1608 (C¼O, quinolone); 1H NMR
(d6-DMSO, 500 M
(Hz) ꢁ (ppm): 13.48 (s, 1H, –CONH), 8.86 (s, 1H, H-2), 8.73
(s, 1H, –N¼CH-14), 8.30 (d, J ¼ 7.5 Hz, 1H, H-5), 8.09 (d,
J ¼ 8.0 Hz, 1H, H-17), 8.07 (d, J ¼ 8.0 Hz, 1H, H-20), 7.82 (t,
J ¼ 7.5 Hz, 2H, H-7 and H-18), 7.74 (t, J ¼ 7.5 Hz, 1H, H-8), 7.68
(t, J ¼ 7.5 Hz, 1H, H-19), 7.54 (t, J ¼ 7.5 Hz, 1H, H-6); 13C NMR
(d6-DMSO, 125 MHz) ꢁ (ppm): 176.5 (C¼O), 162.3 (C¼ONH),
148.7 (C0–16), 145.0 (CH-14), 143.8 (CH-2) 139.5 (C0–9), 134.2
(CH-19), 133.7 (CH-7), 131.2 (CH-18), 129.2 (C0–15), 128.9
(CH-17), 126.3 (C0–10), 126.0 (CH-7), 125.9 (CH-5), 125.2 (CH-
20), 119.7 (CH-8), 110.2 (C0–3); 15N (d6-DMSO, 60 MHz): ꢁ
(ppm) 143.40 (s, 1N, NH-1), 177.10 (s, 1H, –CONH-12), 325.29
(s, 1N, –N ¼ C-13); TOF MS (ESI+) Calculated for C17H13N4O4
(M + H+): 337.0937. Found: 337.0965.
N0-[(1E)-(4-methoxyphenyl)methylidene]-4-oxo-1,4-dihydroqui-
noline-3-carbohydrazide (12)
White solid, yield: 85%.
IR (KBr): ꢀ 3267–2894 (HN–C¼O and H2N–N), 1647 (C¼O
hydrazide), 1604 (C¼O, quinolone); 1H NMR (d6-DMSO,
500 MHz) ꢁ (ppm): 13.20 (s, 1H, –CONH-12), 12.98 (d,
J ¼ 6.1 Hz, 1H, –NH-1), 8.83 (d, J ¼ 6.1 Hz, 1H, H-2), 8.34 (s,
J ¼ 1 Hz, –N¼CH-14), 8.30 (d, J ¼ 7.7 Hz, 1H, H-5), 7.81 (d,
J ¼ 8.2 Hz, 1H, H-7), 7.73 (d, J ¼ 8.2 Hz, 3H, H-8, H-16 and H-
20), 7.53 (t, J ¼ 7.7 Hz, 1H, H-6), 7.02 (d, J ¼ 8.5 Hz, 2H, H-17
and H-19), 3.80 (s, 3H, CH3); 13C NMR (d6-DMSO, 125 MHz) ꢁ
(ppm): 176.5 (C¼O), 161.7 (C¼ONH), 161.3 (C0–18), 148.1
(CH-2), 144.6 (CH-14), 139.5 (C0–9), 133.6 (CH-7), 129.4 (CH-
16 and 20), 127.4 (C0–15), 126.3 (C0–10), 125.9 (CH-5), 125.8
(CH-6), 119.6 (CH-8), 114.8 (CH-17 and 19), 100.6 (C0–3), 55.8
(CH3). TOF MS (ESI+) Calculated for C18H16N3O3 (M + H+):
322.1192. Found: 322.1215.
N0[(1E)-(4-methanesulfonylphenyl)methylidene]-4-oxo-1,4-dihy-
droquinoline-3-carbohydrazide (9)
Yellow solid, yield: 63%.
IR (KBr): ꢀ 3261–3064 (HN–C¼O and H2N–N), 1654 (C¼O
hydrazide), 1637 (C¼O, quinolone); 1H NMR (d6-DMSO,
500 MHz) ꢁ (ppm): 13.51 (s, 1H, –CONH-12), 8.89 (s, 1H, H-
2), 8.56 (s, 1H, –N¼CH-14), 8.35 (d, J ¼ 8.1 Hz, 1H, H-5), 8.01

4
G. S. da Silva et al.
J Enzyme Inhib Med Chem, Early Online: 1–7
Biology
Results and discussion
Acetylcholinesterase activity
Chemistry
Acetylcholinesterase of electrophorus electricus (electric In a previous work3, quinolone 1 was prepared from a Morita–
b-amyloid hypothesis in Alzheimer’s disease: Seeing is Baylis–Hillman adduct derived from 2-nitrobenzaldehyde.
believing.) – Type VI-S, acid 5,50-dithio-bis(2-nitrobenzoic However, the methodology described by Moerdyk et al.14,
(DTNB), tacrine hydrochloride, 2,2-diphenyl-2-picrylhydrazyl proved to be more effective and practical. Thus, 1 was obtained
hydrate (DPPH) and acetylthiocholine iodide (ATC) were in 82% yield, through a condensation reaction involving diethyl
acquired from Sigma-Aldrich (St. Louis, MO); dimethyl (diethoxymethylene) malonate (DEM) and aniline, as described in
sulfoxide (DMSO) and ethanol from Labsynth (Brazil). the experimental section. After this, quinolone 1 was treated with
Acetylcholinesterase (AChE) activity was evaluated by the hydrazine monohydrate to furnish hydrazide 2, as outlined in
´
quantitative Ellmans colorimetric assay. Firstly, compounds Figure 2.
were tested at 200 mM concentration and tacrine, a known
AChE inhibitor, was used as positive control of inhibition. coupled with the appropriate aromatic aldehyde to produce
In order to accomplish our synthetic plan, hydrazide 2 was
After 15 min of incubation at 25 ꢀC, 30 mL of 0.8 mM acylhydrazones 3–12 in 63 to 90% yields, as presented in Table 1.
acetylthiocholine iodide (ATC) were added to microplates
The synthesized acylhydrazones were characterized by spec-
containing the solution of acylhydrazones. The microplate was troscopic methods, including infrared (IR), 1H and 13C NMR
read at 415 nm every 30 s for 5 min (Microplate Reader Model spectroscopy. The 1H-NMR spectra showed some duplicated
680, Bio-Rad Laboratories, Herts, UK). Experiments were signals. According to the literature15, such duplication is due to
performed in triplicate. AChE activities are expressed as % of the presence of rotamers of the amide bond. To confirm this
inhibition.
statement, we conducted 1H NMR experiments at higher
Hydrazide 2 and acylhydrazone 9 inhibited the AChE activity temperatures. An example of expanded spectrum is presented in
in 22% and 36%, respectively. In turn, the other acylhydrazones Figure 3. At room temperature (Figure 3a), we observed a
showed an inhibition percentage ranging from 58 to 80%. For duplication of the peaks attributed to H-2, H-12 and H-14
these compounds, the minimum concentration to inhibit the AChE
activity in 50% (IC50) was calculated by nonlinear regression
method.
Table 1. Synthesized acylhydrazones and chemical yields.
O
O
Effect of acylhydrazones on the ab fibril formation
N
R
Lyophilized Ab42 peptide was purchased from American
Peptide Co (Sunnyvale, CA). Films were prepared as previously
reported1,3 The effect of acylhydrazones on the Ab42 fibrilliza-
tion was evaluated by the thioflavin-T (ThT) fluorescence
N
H
N
H
3-12
emission using a Tecan InfiniteÕ (Mannedorf, Switzerland) 200
¨
Acylhydrazones
R
Yields (%)
PRO apparatus, at 450 nm and 485 nm, for excitation and
emission, respectively. For this purpose, in a 96-well plate,
0.25 mL of a solution of the peptide film (0.166 mg in 6 mL of
sterile DMSO), 0.25 mL of a 0.2 mM solution of acylhydrazone
in sterile DMSO, 1.25 mL of 10 mM HCl, to induce fibril
formation and 298.25 mL of a solution of ThT in phosphate
buffer, pH 7.4 (0.05 mmol/L) were added. After mixing,
samples were immediately analysed (t0) by fluorescence, and
then, the microplate was incubated at 37 ꢀC for 24 h and
another aliquot was analysed (t24). A positive control of
fibrillization was prepared in the same way as sample, but
without adding test-compounds.
3
4
5
6
7
8
9
10
11
12
Phenyl
4-Chlorophenyl
3-Chlorophenyl
2,3-Dichlorophenyl
4-Nitrophenyl
2-Nitrophenyl
4-(Methylsulfonyl) phenyl
4-(Methylthio) phenyl
4-(20-Thiophenyl)
68
72
63
65
90
84
63
85
79
84
4-Methoxyphenyl
Figure 2. Preparation of the desired acylhy-
drazones: reagents and conditions. (a) aniline,
O
O
O
O
O
O
O
NH
2
D, 1 h; then, diphenyl ether, 120 ꢀC, 24 h;
80% yield; (b) 50% aqueous NH2NH2, etha-
nol, D, 55 ꢀC, 12 h; 82% yield; (c) aromatic
aldehyde, ethanol, piperidine; D, 12–18 h;
63–90% yields.
O
N
b
a
H
O
O
N
H
N
H
1
2
DEM
DEM = diethyl (diethoxymethylene) malonate
c
O
O
R
N
N
H
N
H
3-12

DOI: 10.3109/14756366.2016.1144597
Effects of novel acylhydrazones derived from 4-quinolone
5
absorptions. As the temperature rises (Figure 3b), the intercon- promoted by the acylhydrazones 3–8, 10–12 and tacrine, at the
version between the conformational isomers derived from the minimum concentration (12.5 mM).
rotation around the amide bond occurs. In our study, a complete The minimum concentration to inhibit AChE in 50% (IC50)
coalescence of the signals was reached at 120 ꢀC (Figure 3c).
was determined for the most active compounds. In order to do
Analytical data of the synthesized compounds and full spectra this, they were tested at four different concentrations, that is, 200,
are available in the experimental section and supplementary 100, 50, 25 and 12.5 mmol/L and IC50 was calculated using
material, respectively.
nonlinear regression by the software GraphPad Prisma25.
Acylhydrazones inhibited AChE in a significant way, with IC50
values ranging from 1.2 to 17 mM as summarized in Table 2.
In general, we can state that the nature of the substituent of the
C-3 in the quinolonic ring influences on the inhibitory effect on
the AChE. When acylhydrazone derivatives are compared to the
quinolone 1 and hydrazide 2, we found a remarkable inhibitory
effect. This observation may be due a number of properties,
including size and shape. The electronic nature of the aromatic
substituent cannot be directly related to the observed activity.
Acetylcholinesterase activity
Acetylcholinesterase is a serine hydrolase11 involved in the
pathology of AD in two ways. Firstly, this enzyme is responsible
for the hydrolysis of acetylcholine, which plays an important role
in the cognitive process, such as memory and learning16,17
.
Additionally, the peripheral site in the AChE structure18,19 has
been implicated in the amyloidogenic process20. These evidences
have been contributing for the development of compounds able to
interact with the catalytic site and/or the peripheral one21.
The great interest in AChE inhibitors22,23 motivated us to
Effect on the Ab42 fibrils formation
study the effect of hydrazide 2 and acylhydrazones 3–12 on the The abnormal cleavage of amyloid protein precursor (APP)
acetylcholinesterase (AChE) activity. After complete character- promoted by b and g-secretases is one of the most important
ization, the synthesized compounds were evaluated by the events in AD, leading to the formation of toxic peptides,
quantitative Ellman’s colorimetric assay24, as detailed in the especially the one containing 42 aminoacid residues, known as
experimental section. Hydrazide 2 and the acylhydrazone derived Ab4226,27. This peptide triggers a self-aggregation and fibril
from 4-methylsulphonylbenzaldehyde 9 impairs the AChE activ- formation processes. Thus, compounds having antiaggregation
ity in 36 and 44%, respectively, at the maximum concentration and antifibrillization properties have been studied as potential
(200 mM). In contrast, the other acylhydrazones inhibited the drug candidates to treat AD28–30. In this context, acylhydrazones
enzyme in a significant way, even at the lowest concentration. 3–8 and 10–12 were tested for their antifibrillization effect. For
Figure 4 shows a comparative graph of % inhibition of AChE this purpose, commercially available Ab42 peptide was incubated
Figure 3. Expansion of the 1H NMR spectrum in d6-DMSO of acylhydrazone 7 in the 7.0 –14.0 ppm region. (a) at room temperature; (b) at 80 ꢀC; (c) at
120 ꢀC. The arrows indicate the peaks related to the presence of conformational isomers.

6
G. S. da Silva et al.
J Enzyme Inhib Med Chem, Early Online: 1–7
Figure 4. Per cent inhibition of AChE
promoted by the synthesized acylhydrazones
at 12.5 mM.
monitor the fibril formation. Measurements of ThT fluorescence
emission are taken at the beginning of experiment (t0) and after
24 h of incubation (t24). A positive control of fibrillization was
prepared by mixing Ab42 peptide and HCl, in the absence of
acylhydrazones. Results are presented in Figure 5.
As expected, after 24 h of incubation an increase in the ThT
fluorescence emission was observed in the control, due to fibrils
formation. Meanwhile, after 24 h, the emission of fluorescence in
samples containing acylhydrazones decreased at least 49%
(acylhydrazone 5) and a maximum inhibitory effect was promoted
by acylhydrazone 8 (67%).
Table 2. Results of the inhibition acetylcholinesterase by acylhydrazones.
O
O
N
R
N
H
N
H
3-12
Compounds
R
IC50 SD (mM)
3
4
5
6
7
8
9
10
11
12
Phenyl
4-Chlorophenyl
3-Chlorophenyl
2,3-Dichlorophenyl
4-Nitrophenyl
2-Nitrophenyl
4-(Methylsulfonyl) phenyl
4-(Methylthio) phenyl
4-(20-Thiophenyl)
4-Methoxyphenyl
–
4.1 0.1
13 0.2
1.3 (*)
17 0.3
9.8 0.2
1.2 (*)
Nd
These observations associated to the inhibitory effect on
AChE, make these synthesized acylhydrazones very promising
dual-targeting prototypes for the development of AD drug
candidates.
Conclusion
8
0.1
3.4 (*)
5.9 0.1
0.04 (*)
We synthesized 10 acylhydrazones having a 4-quinolone moiety,
and nine of them are not reported in literature. These compounds
were obtained in three steps from 2-nitrobenzaldehyde in
moderate to good yields. Acylhydrazones were fully characterized
by spectroscopic methods, and experiments to evaluate their effect
on the acetylcholinesterase activity and Ab42 fibrillization were
conducted. We found that the ability of the majority of
acylhydrazones in inhibiting AChE is interesting, with IC50
values ranging from 1.2 to 17 mM. These results encourage us to
search more potent analogues. With respect to the effect on the
Ab fibrils formation, except for acylhydrazone 9, the studied
compounds have an impressive inhibitory effect on the formation
of Ab42 fibrils. These findings encourage us to continue the
search for more potent analogues.
Tacrine
Experiments were performed in triplicate. (*) SD50.02; (nd): not
determined.
Acknowledgements
Authors are grateful to the Sa˜o Paulo Foundation for Science (Fapesp,
Proc. 2013/18203–5) and CNPq for GSS and MF fellowship.
Declaration of interest
Figure 5. Effect of acylhydrazones (3–8 and 10–12) on the ThT
fluorescence emission at the beginning of the experiment (t ¼ 0) and
after 24 h of incubation at 37 ꢀC (t ¼ 24 h). Control represents the Ab42
peptide in the presence of HCl, without acylhydrazones.
The authors report no conflicts of interest. The authors alone are
responsible for the content and writing of this article.
with each acylhydrazone, in the presence of 10 mM HCl to induce
fibril formation2. Details are provided in the experimental section.
Thioflavin-T (ThT) binds to Ab fibrils causing an increase in its
fluorescence emission31. Here, we selected this method to
References
1. Kung HF. The b-amyloid hypothesis in Alzheimer’s disease: seeing
is believing. ACS Med Chem Lett 2012;3:265–7.

DOI: 10.3109/14756366.2016.1144597
Effects of novel acylhydrazones derived from 4-quinolone
7
2. Dahlgren KK, Manelli AM, Stine Jr WB, et al. Oligomeric and 16. Blake MG, Krawczyk MC, Baratti CM, Boccia MM.
fibrillar species of amyloid-beta peptides differentially affect
neuronal viability. J Biol Chem 2002;277:32046–53.
3. Huber PC, Reis GP, Amstalden MC, Lancellotti M. Synthesis,
Neuropharmacology of memory consolidation and reconsolidation:
Insights on central cholinergic mechanisms. J Physiol Paris 2014;
108:286–91.
spectroscopic characterizations and antimicrobial activity of 17. Lombardo S, Maskos U. Role of the nicotinic acetylcholine receptor
treatment.
copper and zinc complexes of levofloxacin, ciprofloxacin and
3-carboethoxy-quinolone. Polyhedron 2013;57:14–19.
4. Huber PC, Benevento CE, Castilho RF, Almeida WP. Effect of 18. Johnson G, Moore SW. The peripheral anionic site of acetylcholin-
in
Alzheimer’s
disease
Neuropharmacology 2014;96:255–62.
pathology
and
levofloxacin and 3-carboethoxy-4-quinolone on the b-amyloid
fibrils formation. J Chem Pharm Res 2015;7:993–1000.
esterase: structure, functions and potential role in rational drug
design. Curr Pharm Des 2006;12:217–25.
5. Pudlo M, Luzet V, Ismaıli L, et al. Quinolone-benzylpiperidine 19. Dikersont TJ, Beuscher IV AE, Rogers CJ, et al. Discovery of
¨
derivatives as novel acetylcholinesterase inhibitor and antioxidante
hybrids for Alzheimer disease. Biooorg Med Chem 2014;22:
2496–507.
acetylcholinesterase peripheral anionic site ligands through compu-
tational refinement of a directed library. Biochemistry 2005;44:
14845–3.
6. Le Goff G, Ouazzani J. Natural hydrazine-containing compounds: 20. Inestros NC, Dinamarca MC, Alvarez A. Amyloid-cholinesterase
biosynthesis, isolation, biological activities and synthesis. Bioorg interactions. Implications for Alzheimer’s disease. FEBS J 2008;
Med Chem 2014;22:6529–44. 275:625–32.
7. Maia RD, Tesch R, Fraga CAM. Acylhydrazone derivatives: a patent 21. Kryger G, Silman I, Sussman JL. Structure of acetylcholinesterase
review. Expert Opin Ther Pat 2014;24:1161–70.
complexed with E2020 (Aricept): implications for the design of new
anti-Alzheimer drugs. Structure 1999;7:297–307.
hydrazone derivatives and their utilization in the synthesis of 22. Prakash A, Kalra J, Mani V, et al. Pharmacological approaches for
8. Mohareb RM, Fleita DH, Sakka OK. Novel synthesis of hydrazine-
coumarin, pyridine, thizole and thiophene derivatives with antitumor Alzheimer’s disease: neurotransmitter as drug targets. Expert Rev
activity. Molecules 2011;16:16–27. Neurother 2015;15:53–71.
9. Ellis S, Kalinowski DS, Leotta L, et al. Potent antimycobacterial 23. Singh M, Kaur M, Kukreja H, et al. Acetylcholinesterase inhibitors
activity of the pyridoxal isonicotinoyl hydrazone: a lipophilic
transport vehicle for isonicotinic acid hydrazide. Mol Pharmacol
2014;85:269–78.
as Alzheimer therapy: from nerve toxins to neuroprotection. Eur J
Med Chem 2013;70:165–88.
24. Ellman GL. Tissue sulfhydryl groups. Arch Biochem Biophys 1959;
82:70–7.
Synthesis, anti-inflammatory and analgesic activities of arylidene- 25. GraphPad Prism version 6.04 for Windows, GraphPad Software.
10. Navidpour L, Shafaroodi H, Saeedi-Motahar G, Shafiee A.
2-(3-chloroanilino)nicotinic acid hydrazyde. Med Chem Res 2014;
23:2793–802.
La Jolla, CA. Available from: www.graphpad.com.
26. Multhaup G, Huber O, Buee L, Galas MC. Amyloid precursor
protein (APP), metabolites APP, intracellular fragment (AICD), a
beta 42 and Tau in nuclear roles. J Biol Chem 2015;39:23515–22.
27. Puzzo D, Gulisano W, Arancio O, Palmeri A. The keystone of
Alzheimer pathogenesis might be sought in Ab physiology.
Neuroscience 2015;307:26–36.
28. Karran E, Mercken M, De Strooper B. The amyloid cascade
hypothesis for Alzheimer’s disease: an appraisal for the development
of therapeutics. Nat Rev Drug Discov 2011;10:698–712.
´
11. Massoulie J, Bon S. The C-terminal T peptide of cholinesterases:
structure, interactions, and influence on protein folding and
secretion. J Mol Neurosci 2006;30:233–6.
12. Amarante GW, Benassi M, Pascoal RN, et al. Mechanism and
synthesis of pharmacologically active quinolones from Morita–
Baylis–Hillman adducts. Tetrahedron 2010;66:4370–6.
13. Srivatava N, Kumar A. Synthesis of substituted-4-oxo-1, 4-dihydro-
3-[1-oxo-2-hydrazino-3-{p-toluenesulfon}] quinoline derivatives
and their biological activity against bacterial infections. Orient J 29. Mohamed T, Rao PPN. Alzheimer’s disease: emerging
Chem 2013;29:507–11.
14. Moerdyk JO, Speelman AL, Kuper III KE, et al. Synthesis and
trends in small molecule therapies. Curr Med Chem 2011;18:
4299–320.
photochemistry of two quinolines analogs of the perimidinespir- 30. Trippier PC, Labby KJ, Hawker DD, Mataka JJ. Target- and
ohexadienone family of photochromes. J Photochem Photobiol A mechanism-based therapeutics for neurodegenerative diseases:
2009;205:84–92. strength in numbers. J Med Chem 2013;56:3121–47.
15. Lopez AB, Miguez E, Kummerle AE, et al. Characterization of 31. Krebs MRH, Bromley EHC, Donald AM. The binding of thioflavin-
amide bond conformers for a novel heterocyclic template of
N-acylhydrazone derivatives. Molecules 2013;18:11683–704.
T to amyloid fibrils: localisation and implications. J Struct Biol
2005;149:30–7.
Supplementary material available online