Synthesis and in silico and in vitro evaluation of trimethoxy-benzamides designed as anti-prion derivatives

Article abstract of DOI:10.1007/s00044-019-02441-2

Transmissible spongiform encephalopathies (TSEs), also known as prion diseases, are neurodegenerative disorders which affect mammals, including the human species, and arise after the conversion of the monomeric cellular prion protein (PrP^C) int

Full text of DOI:10.1007/s00044-019-02441-2

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research
https://doi.org/10.1007/s00044-019-02441-2
ORIGINAL RESEARCH
Synthesis and in silico and in vitro evaluation of trimethoxy-
benzamides designed as anti-prion derivatives
Raissa A. Conceição1 Lucas M. Ascari Natália C. Ferreira
1 ●
1,2 ●
Carolina F. Goes Carolina O. Matos
1 ●
3 ●
●
3 ●
4 ●
1 ●
5 ●
2 ●
Anderson S. Pinheiro Marina A. Alves Alessandra M. T. Souza Rodolfo C. Maia Byron Caughey
1 ●
1,5
Yraima Cordeiro
Maria Letícia C. Barbosa
Received: 18 March 2019 / Accepted: 4 September 2019
Springer Science+Business Media, LLC, part of Springer Nature 2019
©
Abstract
Transmissible spongiform encephalopathies (TSEs), also known as prion diseases, are neurodegenerative disorders
which affect mammals, including the human species, and arise after the conversion of the monomeric cellular prion
C
Sc
protein (PrP ) into the aggregated scrapie form (PrP ). There is no therapy to treat TSEs and the identification of
C
Sc
compounds that bind PrP , preventing its conversion into PrP , is a viable therapeutic strategy. We designed and
synthesized six novel trimethoxy-benzamide compounds as anti-prion drug candidates. Molecular docking analyses
predicted that all the derivatives bind to a hotspot region located in the PrP globular domain with very similar spatial
orientation and interaction mode. Although none of the analogs inhibited in vitro-aggregation of recombinant PrP (rPrP)
in a cell-free conversion assay, the RT-QuIC, compound 8a accelerated rPrP conversion into PrP -like species. STD-
NMR and ITC analyses indicated that both 8a and 8b bind to rPrP
Sc
9
0–231
Sc
. These analogs were toxic to PrP -infected cell
lines, hence we could not assess their anti-prion activity by using this cellular approach, although this toxicity was cell
line-dependent. These results point out that the 4-amino-quinoline trimethoxy-benzamide scaffold described herein
represents a novel chemical pattern useful as a starting point for future structural optimization in the design of PrP
ligands with improved affinity and safety profiles.
●
●
●
●
●
Keywords Prion protein Anti-prion drug Therapy Anti-scrapie compounds Molecular hybridization Prion strains
Introduction
(Prusiner 1998), denoting its putative property of acting as
an unusual causative infectious agent in the pathogenesis of
transmissible spongiform encephalopathies (TSEs), also
known as prion diseases (Prusiner 1998). TSEs comprise a
collection of genetic, infectious, or sporadic neurodegen-
erative disorders that affect humans and other mammals and
are triggered by the conversion of the native cellular prion
The term prion, coined by Stanley Prusiner, was originated
from the expression proteinaceous infectious particle
These authors contributed equally: Raissa A. Conceição,
Lucas M. Ascari
C
protein (PrP ) into its pathogenic, misfolded form, which is
Sc
commonly referred as prion scrapie (PrP ) (Caughey et al.
Supplementary information The online version of this article (https://
doi.org/10.1007/s00044-019-02441-2) contains supplementary
material, which is available to authorized users.
2009).
*
*
Yraima Cordeiro
yraima@pharma.ufrj.br
National Institutes of Health, Hamilton, MT, USA
3
Department of Biochemistry, Institute of Chemistry, Federal
University of Rio de Janeiro (UFRJ), Rio de Janeiro, RJ, Brazil
Maria Letícia C. Barbosa
marialeticia@pharma.ufrj.br
4
Laboratory for the Support of Technological Development
(
LADETEC), Institute of Chemistry, Federal University of Rio de
1
2
Faculty of Pharmacy, Federal University of Rio de Janeiro (UFRJ),
Rio de Janeiro, RJ, Brazil
Janeiro (UFRJ), Rio de Janeiro, RJ, Brazil
5
Laboratory of Evaluation and Synthesis of Bioactive Substances
(LASSBio), Institute of Biomedical Sciences, Federal University
of Rio de Janeiro (UFRJ), Rio de Janeiro, RJ, Brazil
Laboratory of Persistent Viral Diseases, Rocky Mountain
Laboratories, National Institute of Allergy and Infectious Diseases,

Medicinal Chemistry Research
C
The constitutive, α-helix-rich PrP is specially abundant
Several promising bioactive compounds from different
structural classes, like 4-amino-quinolines and trimethoxy-
chalcones, have been described in the literature for their
anti-prion effects in in vitro and/or in vivo assays. However,
none of them have proven to be effective in preventing the
progress of TSEs in clinical trials so far (reviewed in Cor-
deiro and Ferreira 2015; Forloni et al. 2013). Among the
previously identified anti-prion drug candidates, some
already approved drugs—e.g., quinacrine (1) (Geschwind
et al. 2013), flupirtine (2) (Otto et al. 2004), and doxycy-
cline (3) (Haïk et al. 2014)—stood out during preclinical
studies, suggesting the possibility of their repositioning for
TSE treatment (Fig. 1). However, these drugs were also not
effective in further clinical stages of development (Forloni
et al. 2013; Ghaemmaghami et al. 2014).
in the nervous and immune systems and is typically located
on the cell surface via a glycosyl-phosphatidyl inositol
GPI) anchor (Caughey et al. 2009). According to the
protein-only hypothesis, the aberrant, β-sheet-rich PrP
induces PrP misfolding and promotes the propagation and
amplification of PrP aggregates, which, in turn, induce
(
Sc
C
Sc
C
widespread neurotoxicity (Caughey et al. 2009). While PrP
is protease-sensitive, PrPSc is partially protease-resistant
Res
and, for that reason, is also referred as PrP
998; Caughey et al. 2009).
The precise mechanism of transmission and propagation of
TSEs is still controversial, especially regarding the potential
involvement of cofactors assisting PrP to PrP conversion
Abid et al. 2010; Supattapone 2014; Macedo and Cordeiro
017). In addition, evidences indicate that subversion of PrP
normal function by PrP is a key process in the pathophy-
siology of TSEs (Westergard et al. 2007). In normal condi-
(Prusiner
1
C
Sc
(
2
C
C
A hotspot region within PrP structure, which is sus-
Sc
ceptible to undergo conformational fluctuations, comprises
specific amino acid residues, such as Asn159 at the α1–β2
loop; His187, Val189, Thr192, and Lys194 at the α2; and
Glu196 at the α2–α3 loop (Langella et al. 2004; Kuwata
C
tions, PrP is thought to play a physiological role as a scaffold
protein in cell signaling (Linden 2017) and to take part in cell
survival, cell differentiation, cell adhesion, Cu2 homeostasis,
synaptic transmission, myelin maintenance, and neuroprotec-
tion (Castle and Gill 2017).
Prion diseases are usually associated with long silent
incubation periods, ranging from years to decades. How-
ever, when the clinical signs become detectable, disease
usually progresses in a fast and aggressive manner, leading
to severe disability and death (Prusiner 1998). Unfortu-
nately, there is no effective therapy available in the clinics,
highlighting the relevance of searching for novel alter-
natives for the treatment of TSEs.
+
C
et al. 2007). In an attempt to identify novel PrP -stabilizing
ligands, one of the putative molecular mechanisms for small
anti-prion molecules, these authors conducted in vitro, in
silico, and in vivo assays and have reported the anti-prion
activity of prototype GN8 (4) (Kuwata et al. 2007; Yama-
moto and Kuwata 2009; Ishikawa et al. 2009) (Fig. 1).
Kuwata and coworkers reported that GN8 (4) was able to
Res
reduce the PrP content in prion-infected cells and prolong
the survival of TSE-infected mice. Using nuclear magnetic
resonance (NMR) and computational simulations, this
molecule was shown to interact with the hotspot region
Fig. 1 Anti-prion prototypes
previously described in the
literature

Medicinal Chemistry Research
within the major cavity of PrP globular domain. The
reporting of this key hotspot pocket emphasized its rele-
vance for the structural stabilization of PrPC and thus
offered the basis for further efforts in order to find novel
anti-prion drug candidates (Hyeon et al. 2015; Ishibashi
et al. 2016).
Our research group has contributed to this field with the
description of anti-prion derivatives bearing different che-
mical scaffolds, including 4-amino-quinolines (Macedo
et al. 2010), trimethoxy-chalcones (Ferreira et al.
Material and methods
Chemistry
All commercially available reagents and solvents were used
without further purification. Reactions were routinely
monitored by thin-layer chromatography (TLC) in silica gel
(F245 Merck plates), and the products were visualized with
1
13
ultraviolet light (254 and 365 nm). H and C NMR spectra
were determined in DMSO-d , D O, TFA-d, acetone-d , or
6
2
6
2
014, 2018), and oxadiazoles (Ferreira et al. 2014), as
CDCl solutions using a Varian 400-MR (IPPN-UFRJ, BR)
3
exemplified by compounds 5–7 depicted in Fig. 1. Herein
we describe the synthesis and the in silico and in vitro
evaluation of new trimethoxy-benzamide analogs, designed
by molecular hybridization from the 4-amino-quinoline 5
and the trimethoxy-chalcone 6, as novel anti-prion candi-
dates (Fig. 2).
spectrometer. The chemical shifts are given in parts per
million (δ) from solvent residual peaks, and the coupling
constant values (J) are given in Hz. Signal multiplicities are
represented by s (singlet), d (doublet), dd (double doublet),
t (triplet), dt (double triplet), q (quadruplet), qu (quintuplet),
m (multiplet) and/or br (broad signal). Saturation transfer
Fig. 2 Structural design of the
target trimethoxy-benzamide
analogs, designed by molecular
hybridization from the 4-amino-
quinoline 5 and the trimethoxy-
chalcone 6, as novel anti-prion
candidates

Medicinal Chemistry Research
difference NMR (STD-NMR) spectra were collected at
5 °C on a 600 MHz Bruker Avance III spectrometer
Synthesis of trimethoxy-benzamide intermediates 13a and
13b
2
equipped with a room temperature inverse-detection 5-mm
TXI probe.
A solution of acyl-chloride 14 (0.544 g, 2.36 mmol) in
dichloromethane (50 mL) was slowly added dropwise into a
250 mL flask containing a 10 eq solution (23.6 mmol) of the
corresponding amine (ethylenediamine or piperazine) in
20 mL of dichloromethane, under constant stirring at room
temperature. The resulting reaction mixture was stirred at room
temperature for around 20 min, when the end of the reaction
was detected by TLC (10% methanol: 90% dichloromethane).
The obtained trimethoxy-benzamide intermediates were iso-
lated by the addition of 50 mL distilled water, followed by
extraction in a separating funnel. These intermediates could be
easily purified by acid–base extraction. Initially, basic deri-
vatives 13a and 13b were extracted from the organic layer
with an aqueous solution of HCl (pH = 3). Afterwards, pH of
the aqueous layer was adjusted to neutrality and the pure
benzamides 13a and 13b were extracted back to the organic
layer. The final organic layers containing derivatives 13a and
13b were then dried over anhydrous sodium sulfate, filtered,
and concentrated under reduced pressure (Scheme 2d).
Mass spectra were obtained using a QExactive™ Hybrid
Quadrupole Orbitrap Mass Spectrometer (Thermo Fisher
Scientific, Waltham, USA) using electrospray ionization.
High-resolution mass spectrometry (HRMS) data were
obtained at the Laboratory for the Support of Technological
Development of the Federal University of Rio de Janeiro
(
LADETEC-UFRJ, BR).
The purity of the testing compounds was determined
by HPLC on a Shimadzu Prominence HPLC system
Shimadzu, Tokio, Japan) consisting of a vacuum
(
degasser (DGU-20A5), a binary pump (LC-20AD), an
autosampler (SIL-20A), UV/VIS Photodiode Array
Detector (SPD-M20A) and fitted with a guard column
(
(
CLC G-ODS) and a Shimadzu (CLC-ODS, M) column
250 × 4.6 mm ID) running at room temperature. The
mobile phase was prepared with Milli-Q water (A) and
with acetonitrile (B) for the compounds 8a, 8b, 9b, 10a,
and 10b; and with Milli-Q water containing 0.1% formic
acid (A) and with acetonitrile (B) for the compound 9a.
Isocratic elution was performed with 1:1 or 6:4 acetoni-
trile:water, at a flow rate set at 1 mL/min, and a 20 µL
injection volume was used. The detection was carried out
in the range of 294–330 nm wavelengths.
N-(2-aminoethyl)-2,4,5-trimethoxybenzamide (13a) Beige
1
oil; yield: 75%; H NMR (DMSO-d , 400 MHz): δ = 2.74
6
(2H, t, J = 6.4 Hz, CH NH ), 3.33 (2H, q, J = 5.96 Hz,
2
2
CONHCH ), 3.63 (2H, br, NH ), 3.72 (3H, s, ArOCH ),
2
2
3
All testing compounds, i.e., trimethoxy-benzamide deri-
vatives (8a, 8b, 9a, 9b, 10a, and 10b), were characterized
by H and C NMR and HRMS, showing spectral data
3.85 (3H, s, ArOCH ), 3.91 (3H, s, ArOCH ), 6.74 (1H, s,
3 3
H-3), 7.44 (1H, s, H-6), 8.29 (1H, br, CONH); C NMR
1
3
1
13
and APT (DMSO-d6, 100 MHz): δ = 40.6 (CH ), 41.0
2
according to the assigned structures. Compounds 8a, 8b,
(CH ), 55.0 (CH , ArOCH ), 55.9 (CH , ArOCH ), 56.7
2
3
3
3
3
9
a, 9b, 10a, and 10b presented >95% purity, as assessed
(CH , ArOCH ), 97.7 (CH, C-3), 112.9 (C, C-1), 113.6
3 3
by HPLC.
(CH, C-6), 142.4 (C, C-2), 152.1 (C, C-5), 152.5 (C, C-4),
We used the following recombinant PrP (rPrP) con-
164.5 (C, C=O).
23–231
90–231
structs: hamster rPrP
, hamster rPrP
, and mouse
9
0–231
rPrP
. They were expressed in E. coli and purified by
Piperazin-1-yl(2,4,5-trimethoxyphenyl)methanone (13b)
Beige oil; yield: 95%; H NMR (DMSO-d , 400 MHz): δ =
1
nickel affinity chromatography and refolded in-column, as
6
previously described (Wilham et al. 2010).
2.61 (2H, br, HNCH R), 2.72 (2H, t, J = 4.9 Hz, HNCH R),
2
2
3
.08 (2H, br, CONCH R), 3.52 (2H, br, CONCH R), 3.69
2
2
Synthesis of 2,4,5-trimethoxybenzoyl chloride (14)
(3H, s, ArOCH ), 3.77 (3H, s, ArOCH ), 3.81 (3H, s,
3 3
1
3
ArOCH ), 6.71 (1H, s, H-3), 6.75 (1H, s, H-6); C NMR
3
In a 100 mL flask, 2,4,5-trimethoxybenzoic acid 15 (0.50 g,
and APT (DMSO-d6, 100 MHz): δ = 42.1 (CH ), 45.2
2
2
.36 mmol) was dissolved in dichloromethane (50 mL).
(CH ), 45.7 (CH ), 47.4 (CH ), 55.9 (CH , ArOCH ), 55.9
2
2
2
3
3
Next, oxalyl chloride (0.66 g, 477 μL, 5.21 mmol) and 2
drops of dimethylformamide were added. The resulting
solution was stirred at room temperature for 2–4 h and the
end of the reaction was detected by TLC (70% ethyl acetate:
(CH , ArOCH ), 56.3 (CH , ArOCH ), 97.9 (CH, C-3),
3 3 3 3
112.0 (CH, C-6), 116.4 (C, C-1), 142.8 (C, C-2), 149.5 (C,
C-5), 150.3 (C, C-4), 166.5 (C, C=O).
3
0% hexane). The desired acyl-chloride 14 was isolated
Synthesis of the designed bis-trimethoxy-benzamide
analogs 10a and 10b
after solvent evaporation under reduced pressure with a
yield of 100% and was used as crude product for the
synthesis of benzamide derivatives 10a, 10b, 13a, and 13b
The acyl-chloride 14 (0.110 g, 0.48 mmol) was initially
(
Scheme 2c).
dissolved in dichloromethane (5 mL) and stirred at room

Medicinal Chemistry Research
temperature, followed by the gradual addition of 1–4
equivalents of the corresponding amines (ethylenediamine
or piperazine) until the end of the reaction, detected by
TLC. The work-up consisted of adding 10 mL of dichlor-
omethane to the flask and then washing the organic phase
with 10 mL of 10% HCl aqueous solution. Then, the
organic phase was dried with sodium sulfate and con-
centrated under reduced pressure. The resulting residues
were purified in chromatographic columns, using dichlor-
omethane as eluent, to provide the desired bis-trimethoxy-
benzamide analogs 10a and 10b (Scheme 2e).
N,N′-(ethane-1,2-diyl)bis(2,4,5-trimethoxybenzamide)
1
(
10a) White solid; yield: 82%; H NMR (DMSO-d6,
4
00 MHz) (Fig. S1): δ = 3.48 (4H, br NCH ), 3.71 (6H, s,
2
ArOCH ), 3.84 (6H, s, ArOCH ), 3.88 (6H, s, ArOCH ),
3
3
3
6
(
.72 (2H, s, H-3 and H-3′), 7.46 (2H, s, H-6 and H-6′), 8.30
3
2H, s, CONH); 1 C NMR (DMSO-d , 100 MHz) (Fig. S2):
Scheme 1 Reagents and conditions: (a) isopropyl alcohol, 82 °C,
8–72 h, 45–56%; (b) isopropyl alcohol, Et3N, 82 °C, 48–72 h,
54–72%
6
4
δ = 39.3 (CH ), 55.9 (CH , ArOCH ), 55.9 (CH ,ArOCH ),
2
3
3
3
3
5
6.5 (CH ,ArOCH ), 97.8 (CH, C-3), 112.7 (C, C-1), 113.8
3 3
(
1
CH, C-6), 142.5 (C, C-2), 152.2 (C, C-5), 152.6 (C, C-4),
1
64.7 (C, C=O); H NMR (CDCl , 400 MHz): δ = 3.69
corresponding benzamide intermediate (13a or 13b) were
added. The resulting reaction mixture was stirred and heated
at 82 °C until completion of the reaction, verified by TLC
(10% methanol: 90% dichloromethane). The resulting
precipitate was directly isolated by vacuum filtration
(Scheme 1a).
3
(
3
2H, br, CH ), 3.70 (2H, br CH ), 3.89 (6H, s, ArOCH ),
2 2 3
.90 (6H, s, ArOCH ), 3.92 (6H, s, ArOCH ), 6.49 (2H, s,
3 3
H-3 and H-3′), 7.75 (2H, s, H-6 and H-6′), 8.18 (2H, s,
CONH); C NMR and APT (CDCl , 100 MHz): δ = 39.8
1
3
3
(
(
(
CH ), 56.2 (CH , ArOCH ), 56.3 (CH , ArOCH ), 56.7
2 3 3 3 3
CH , ArOCH ), 96.6 (CH, C-3), 113.0 (C, C-1), 114.2
3
3
CH, C-6), 143.3 (C, C-2), 152.5 (C, C-5), 152.9 (C, C4),
N-(2-((7-chloroquinolin-4-yl)amino)ethyl)-2,4,5-trimethoxy-
+
1
1
4
65.8 (C, C=O); HRESIMS m/z (Fig. S3): [M+Na] :
benzamide (8a) Beige powder; yield: 45%; H NMR
71.17368 C H N NaO (calcd. 471.17378).
(D O, 600 MHz) (Fig. S7): δ = 3.51 (3H, s, ArOCH ), 3.52
2
2
28
2
8
2
3
(
3H, s, ArOCH ), 3.61 (2H, t, CH ), 3.68 (2H, t, CH ), 3.72
3 2 2
Piperazine-1,4-diylbis((2,4,5-trimethoxyphenyl)methanone)
(3H, s, ArOCH ), 6.41 (1H, s, H3′), 6.63 (1H, d, Jortho =
3
1
(
4
10b) White solid; yield: 87%; H NMR (DMSO-d6,
00 MHz) (Fig. S4): δ = 3.16 (4H, br, CH ), 3.54 (4H, br,
7.4 Hz, H-3), 6.87 (1H, s, H6′), 7.46 (1H, d, Jortho =
9.3 Hz, Jmeta = 2 Hz, H-6), 7.89 (1H, d, Jortho = 7.4 Hz,
2
1
CH ), 3.67–3.82 (18H, m, ArOCH ), 6.67 (1H, s, H-3), 6.73
H-2), 7.90 (1H, d, Jortho = 9,3 Hz, H-5); H NMR (TFA,
2
3
13
(
(
(
(
1H, s, H-3′), 6.80 (1H, s, H-6 and H-6′); C NMR
DMSO-d , 100 MHz) (Fig. S5): δ = 40.0 (CH ), 40.4
400 MHz) (Fig. S8): δ = 4.36 (13H, m, CH and ArOCH ),
2
3
7.13 (1H, s, H-3′), 7.23 (1H, s, H-6′), 7.96 (1H, br, H-3),
6
2
CH ), 45.2 (CH ), 45.6 (CH ), 53.8 (CH , ArOCH ), 54.7
8.03 (1H, br, H-6), 8.21 (1H, s, H-8), 8.53 (1H, br, H-5),
2
2
2
3
3
1
3
CH , ArOCH ), 54.9 (CH , ArOCH ), 55.0 (CH ,
8.63 (1H, br, H-2); C NMR (TFA, 100 MHz) (Figs. S9
3
3
3
3
3
ArOCH ), 55.0 (CH , ArOCH ), 96.5 (CH, C-3), 96.6 (CH,
and S10): δ = 39.6 (CH ), 43.8 (CH ), 55.8 (CH ,
3
3
3
2
2
3
C-3′), 110.9 (CH, C-6), 111.0 (CH, C-6′), 114.7 (C, C-1),
14.7 (C, C-1′), 141.5 (C, C-2), 141.6 (C, C-2′), 148.3 (C,
ArOCH ), 56.5 (CH , ArOCH ), 56.7 (CH , ArOCH ), 96.8
3 3 3 3 3
1
(CH, C-3′), 98.3 (CH, C-6′), 106.6 (C, C-1′), 115.4 (CH,
C-3), 119.6 (C, C-4a), 123.6 (CH, C-6), 129.4 (CH, C-5),
138.5 (CH, C-8), 142.2 (C, C-7), 142.6 (C, C-2′), 143.3
(CH, C-2), 156.6 (C, C-5′), 156.9 (C, C-4′), 157.2 (C,
C-8a), 171.1 (C, C-4), 181.4 (C, C=O); HRESIMS m/z
(Fig. S11): [M+H]+ 416.13677 C H ClN O (calcd.
C-5), 148.3 (C, C-5′), 149.2 (C, C-4), 149.2 (C, C-4′),
1
4
65.3 (C, C=O); HRESIMS m/z (Fig. S6): [M+Na]+
97.18888 C H N NaO (calcd. 497.18943).
2
4
30
2
8
Synthesis of the designed 7-chloro-4-amino-quinoline
analogs 8a and 8b
2
1
23
3
4
416.13716).
In a 50 mL flask equipped with a reflux condenser, the 4,7-
dichloroquinoline 11 (80 mg, 0.40 mmol) was dissolved in
(4-(7-chloroquinolin-4-yl)piperazin-1-yl)(2,4,5-trimethoxy-
phenyl)-methanone (8b) Beige powder; yield: 56%; H
1
8
mL of isopropyl alcohol. Next, 1.1 equivalents of the
NMR (D O, 400 MHz) (Fig. S12): δ = 3.62 (2H, br,
2

Medicinal Chemistry Research
RNCH R), 3.75 (3H, s, ArOCH ), 3.81 (3H, s, ArOCH ),
159.6 (C, C-4), 164.7 (C, C=O); HRESIMS m/z (Fig. S17):
2
3
3
+
3
.83 (3H, s, ArOCH ), 3.97 (2H, br, RNCH R), 6.68 (1H, s,
[M+H] 383.17094 C H N O (calcd. 383.17138).
3
2
20 23
4
4
H-3′), 6.88 (1H, s, H-6′), 7.05 (1H, d, Jortho = 7.00 Hz, H-
3
7
9
), 7.53 (1H, dd, Jortho = 9.1 Hz and Jmeta = 1.1 Hz, H-6),
(4-(quinazolin-4-yl)piperazin-1-yl)(2,4,5-trimethoxyphenyl)
methanone (9b) Compound 9b was obtained from the
benzamide intermediate 13b and isolated after
.82 (1H, d, Jortho = 1.1 Hz, H-8), 8.00 (1H, d, Jortho =
1
3
.1 Hz, H-5), 8.39 (1H, d, Jortho = 7.00 Hz, H-2);
C
a
NMR (D O, 100 MHz) (Fig. S13): δ = 41.5 (CH ), 45.8
liquid–liquid extraction employing an aqueous phase pH
around 9, followed by an additional purification acid-base
extraction for removing an undesired side product. For that,
product 9b was extracted from the organic layer with an
aqueous solution of HCl (pH = 3). Afterwards, pH of the
aqueous layer was adjusted to neutrality and the pure
derivative 9b was extracted back to the organic layer. The
final organic layers containing 9b were then dried over
anhydrous sodium sulfate, filtered and concentrated under
2
2
(
CH ), 50.1 (CH ), 51.0 (CH ), 55.7 (CH , ArOCH ), 56.1
2 2 2 3 3
(
1
CH , ArOCH ), 56.2 (CH , ArOCH ), 97.6 (CH, C-3′),
3
3
3
3
05.2 (CH, C-6′), 111.1 (CH, C-3), 114.0 (C, C-1′), 117.5
(
8
(
1
C, C-4a), 119.1 (C, C-6), 126.8 (CH, C-5), 127.8 (CH, C-
), 139.2 (C, C-2′), 139.5 (C, C-7), 141.3 (CH, C-2), 142.2
C, C-5′), 150.0 (C, C-4′), 150.6 (C, C-8a), 161.1 (C, C-4)+,
69.8 (C, C=O); HRESIMS m/z (Fig. S14): [M+H]
4
42.15255 C H ClN O (calcd. 442.15281).
2
3
25
3
4
reduced pressure, giving 9b as a pure beige powder; yield:
1
Synthesis of the designed 4-amino-quinazoline analogs 9a
and 9b
54%; H NMR (acetone-d , 400 MHz) (Fig. S18): δ = 3.58
6
(4H, br, CH ), 3.82 (3H, s, ArOCH ), 3.90 (3H, s,
2
3
ArOCH ), 3.92 (3H, s, ArOCH ), 3.98 (4HH, br, CH ), 6.81
3
3
2
In a 50 mL flask equipped with a reflux condenser, the 4-
chloroquinazoline 12 (180 mg, 1.09 mmol) was dissolved
in 20 mL of isopropyl alcohol. Next, 1.1 equivalents of
the corresponding benzamide intermediate (13a or 13b)
(1H, s, H-3′), 6.91 (1H, s, H-6′), 7.57 (1H, t, Jortho =
7.5 Hz, H-6), 7.87 (2H, m, H-5 and H-7), 8.13 (1H, d,
1
3
Jortho = 8.3 Hz, H-8), 8.71 (1H, s, H-2); C NMR (acet-
one-d , 100 MHz) (Fig. S19): δ = 42.1 (CH , ArNCH ),
6
2
2
and 5 drops of triethylamine (Et N) were added. The
47.3 (CH , ArNCH ), 50.4 (CH , CONCH ), 50.9 (CH ,
3
2 2 2 2 2
resulting reaction mixture was stirred and heated at 82 °C
until completion of the reaction, verified by TLC (10%
methanol: 90% dichloromethane). The reaction medium
was concentrated under reduced pressure and redissolved
in ethyl acetate. The resulting products were isolated and
purified by extraction in a separation funnel. The organic
layer was then dried over anhydrous sodium sulfate,
filtered and concentrated under reduced pressure
CONCH ), 56.3 (CH , ArOCH ), 56.7 (CH , ArOCH ),
2 3 3 3 3
56.9 (CH , ArOCH ), 98.6 (CH, C-3′), 113.7 (CH, C-6′),
3
3
117.5 (C, C-1′), 117.8 (C, C-4a), 126.1 (CH, C-6), 126.5
(CH, C-8), 129.4 (CH, C-5), 133.4 (CH, C-7), 144.4 (C, C-
2′), 151.0 (C, C-5′), 152.0 (C, C-4′), 152.9 (C, C-8a), 154.7
(CH, C-2), 165.4 (C, C-4), 167.6 (C, C=O); HRESIMS m/z
(Fig. S20): [M+H]+ 409.18656 C H N O (calcd.
2
2
25
4
4
409.18703).
(
Scheme 1b).
Molecular docking
2
,4,5-trimethoxy-N-(2-(quinazolin-4-ylamino)ethyl)benza-
mide (9a) This compound was obtained from the benza-
mide intermediate 13a and isolated after liquid–liquid
extraction employing an aqueous phase pH around 6 as a
pure white powder; yield: 72%; 1H NMR (DMSO-d6,
Initially, the three-dimensional structures of the compounds
were constructed on Avogadro software (version 1.2.0)
(Hanwell et al. 2012), and their energy was minimized
using Merck molecular force field (MMFF94) (Halgren
1996). We then performed the blind docking between the
compounds and mouse PrP globular domain (PDB: 1AG2)
using AutoDock software (version 4.2.6) (Morris et al.
2009). The protein was set rigid, and the ligands were set
flexible. The search space was centered on x-, y- and z-
coordinates of −3.000, 0.752, and −1.572, respectively,
and defined in a grid of 126 × 126 × 126 points with default
0.375 Å spacing. We considered the neutral ε-tautomeric
state for His177 and His187 and the neutral δ-tautomeric
state for His140, according to previous work (Langella et al.
2006; Malevanets et al. 2017). A total of 50 docking runs
were performed using the Lamarckian Genetic Algorithm
(LGA) and default parameters. In the next step, we per-
formed a site-directed molecular docking based on a
4
00 MHz) (Fig. S15): δ = 3.61 (2H, t, J = 5.50 Hz,
ArHNCH ), 3.74 (5H, m, CONHCH and ArOCH ), 3.78
2
2
3
(
7
3H, s, ArOCH ), 3.83 (3H, s, ArOCH ), 6.71 (1H, s, H-3′),
3
3
.43 (1H, s, H-6′), 7.52 (1H, t, Jortho1 = 7.5 Hz and Jor-
tho2 = 8.10 Hz, H-6), 7.68 (1H, d, Jortho = 8.10 Hz, H-5),
7
8
.76 (1H, t, Jortho1 = 7.50 Hz and Jortho2 = 8.0 Hz, H-7),
.23 (1H, t, Jortho = 8.0 Hz, H-8), 8.30 (1H, t, J = 5.50 Hz,
NHAr), 8.42 (1H, t, J = 5.20 Hz, CONH), 8.47 (1H, s, H2);
1
3
C NMR (DMSO-d , 100 MHz) (Fig. S16): δ = 38.7
6
(
CH ), 40.3 (CH ), 55.9 (CH , ArOCH ), 56.5 (CH ,
2 2 3 3 3
ArOCH ), 97.7 (CH, C-3′), 112.8 (C, C-1′), 113.6 (CH, C-
3
6′), 115.0 (C, C-8a), 122.6 (CH, C-6), 125.7 (CH, C-8),
127.5 (CH, C-5), 132.6 (CH, C-7), 142.4 (C, C-4a), 149.1
(
C, C-2′), 152.1 (C, C-5′), 152.5 (C, C-4′), 155.1 (CH, C-2),

Medicinal Chemistry Research
previously described method (Ferreira et al. 2018). The
search space was centered on x-, y- and z-coordinates of
residual water suppression. A 40 ms spinlock filter was applied
to suppress protein background signals. A 3 Hz line broad-
ening was applied to FID prior to Fourier transformation.
STD-NMR spectra were obtained by subtracting the on- from
the off-resonance spectra.
4
.913, 7.305, and 2.540, respectively, and defined in a grid
of 64 × 58 × 54 points. LGA parameters were the same used
in the blind docking. The selected conformer was the one
that presented the lowest Gibbs free energy (ΔG) of bind-
ing. GN8 (4) was used to validate the site-directed mole-
cular docking.
Isothermal titration calorimetry (ITC)
Binding studies between rPrP90–231 and the compounds
were performed at 25 °C on an ITC-200 microcalorimeter
Real-time quaking induced conversion (RT-QuIC)
(Malvern MicroCal, USA). Both protein and ligand work-
The RT-QuIC assay was performed as previously described
ing solutions were prepared in 10 mM phosphate buffer (pH
7.4) containing 150 mM NaCl. The syringe was filled with
compound working solutions, while 200 μL of rPrP work-
ing solution was placed into the sample cell. The reference
cell was loaded with degassed ultrapure water. Then, we
performed a single injection of 20 μL at an interval of 180 s,
during which the solution was stirred at 750 rpm with the
syringe inside the sample cell with no foaming. For 8a
analysis, the 8a solution in the syringe was at 1 mM and the
rPrP solution in the sample cell was at 10 μM. For 8b
analysis, the 8b solution in the syringe was at 0.5 mM and
the rPrP solution in the sample cell was at 5 μM. In both
cases, the protein:ligand molar ratio was 1:10. The heat of
dilution of the compounds in buffer was subtracted from the
heat of dilution of the compounds in rPrP solution. Analysis
of ITC data was carried out on Origin 7.0.
(
Wilham et al. 2010). The brain homogenate was diluted in
PBS containing 0.1% SDS and N2 medium supplement
GIBCO) (SDS–PBS–N2). We loaded each well of a black-
(
walled, clear-bottom 96-well plate (Nunc) with 90 μL of the
reaction medium, which had the following composition:
1
0 mM phosphate buffer (pH 7.4); 170 mM NaCl; 10 μM
90–231
EDTA; 10 μM ThT; and hamster rPrP
at 0.1 mg/mL
(
6 μM) or hamster rPrP23–231 at 0.1 mg/mL (4.3 μM). Then,
5
we added 2 μL of brain homogenate (1 × 10 dilution) and
μL of stock solutions of the compounds to reach the final
8
concentrations of 40, 50, 100, and 200 μM. The plates were
sealed with a plate sealer film (Nalgene Nunc International)
and incubated on a BMG FLUOstar plate reader (BMG
Biotech), at 42 °C, under continuous cycles of 1 min of
double-orbital shaking at 700 rpm and 1 min of rest. ThT
fluorescence readings were performed every 15 min (exci-
tation at 450 ± 10 nm and emission at 480 ± 10 nm). Post-
mortem-collected sporadic Creutzfeldt-Jakob disease
Dot blot
(
sCJD) brain tissue was obtained from Prof. Pierluigi
The dot blot assay was performed as previously described
(Ferreira et al. 2018) with ScN2a cells infected with prion
strains RML or 22 L. Each treatment was tested in quad-
ruplicate. The vehicle for the compounds at 0.1, 0.5, 1, and
Gambetti at Case Western Reserve University and was used
under Exemption #11197 from the NIH Office of Human
Subjects Research Protections.
10 μM corresponded to PBS (pH 7.4) containing DMSO at
Saturation transfer difference nuclear magnetic
resonance (STD-NMR)
0.001%, 0.005%, 0.01%, and 0.1%, respectively. We used
the primary antibody 6D11 (1:7,500; sc-58581) and the
alkaline phosphatase-conjugated secondary antibody.
NMR spectra were collected at 25 °C on a 600 MHz Bruker
Avance III spectrometer equipped with a room temperature
MTT reduction
inverse-detection 5-mm TXI probe. The mixture contained 8a
or 8b at 1 mM and murine rPrP9
(
0–231
at 10 μM in 100% D O
Cell viability in the presence of the compounds was fol-
lowed by the MTT reduction assay as previously described
(Ferreira et al. 2014) using ScN2a cells infected with prion
strains RML or 22 L. Each treatment was tested in quad-
ruplicate. The vehicle for the compounds at 0.1, 0.5, 1, and
10 μM corresponded to PBS (pH 7.4) containing DMSO at
0.001%, 0.005%, 0.01%, and 0.1%, respectively.
2
1:100 protein:ligand molar ratio). Protein signals were selec-
tively saturated using 60 Gaussian-shaped pulses of 50 ms
each, resulting in a total saturation time of 3 s. A relaxation
delay of 3 s was used during the STD-NMR experiment,
taking into consideration the long T1 values of the ligands.
Selective protein saturation occurred at different frequencies.
On-resonance saturation occurred at –0.19, 0.12, 0.76, 6.52,
6
7
.62 ppm for 8b–rPrP interaction and at –0.11, 0.21, 0.38,
.28, 8.45 ppm for 8a–rPrP interaction, while off-resonance
Statistical analysis
saturation occurred at –10 ppm. STD-NMR experiments were
Data from dot blot and MTT reduction assays were ana-
acquired with 48 scans and Watergate sequence was used for
lyzed using one-way ANOVA with Dunnett’s multiple

Medicinal Chemistry Research
comparisons test. All statistical evaluations were performed
with GraphPad Prism 8 software.
Chemical synthesis
The target heterocyclic trimethoxy-benzamide derivatives
a, 8b, 9a, and 9b were synthesized via nucleophilic aro-
8
Results and discussion
matic substitution (S Ar) by a key condensation step
N
between the 4-chloro-heterocycles (11 or 12) and the cor-
responding amidic intermediates (13a or 13b) (Sashidhara
et al. 2012; Kershaw et al. 2013; Braga et al. 2017), as
depicted in Scheme 1. 7-chloro-4-amino-quinoline deriva-
tives 8a and 8b precipitated directly from the reaction
media, which was favorable for shifting the reaction balance
to their formation. In the case of the synthesis of 4-amino-
quinazolines 9a and 9b, no precipitation was observed and
Structural design
Starting from the anti-prion prototypes 4-amino-quinoline 5
(
Macedo et al. 2010) and trimethoxy-chalcone 6 (Ferreira
et al. 2014), the first heterocyclic trimethoxy-benzamide
analog 8a was designed by molecular hybridization, linking
the structural fragments A and B from compound 5 to the
fragment C from compound 6 in the novel hybrid structure
of 8a, as depicted in Fig. 2. Aiming to evaluate the possible
influence of conformational flexibility on the ability to
interact with the target, i.e., the prion protein, the con-
formationally restricted analog 8b was also planned.
Moreover, to assess the relevance of 7-chloro-4-amino-
quinoline heterocyclic moiety A for the anti-prion activity
of this novel hybrid compounds, derivatives 9a and 9b,
bearing a 4-amino-quinazoline A’ core, were designed. The
heterocyclic nucleus A’ was selected due to its well-known
property of acting as a privileged scaffold in Medicinal
Chemistry, since the 4-amino-quinazoline nucleus has been
extensively studied for its wide range of pharmacological
and therapeutic effects (Kamal et al. 2006; Waiker et al.
the addition of triethylamine (Et N) was necessary to absorb
3
the hydrochloric acid by-product, favoring product
formation.
All heterocyclic trimethoxy-benzamide derivatives 8a,
1
13
8b, 9a, and 9b were characterized by H and C NMR and
HRMS and showed spectral data consistent with the
assigned structures. As expected, the mass spectra of the 7-
chloro-4-amino-quinoline products 8a and 8b revealed the
isotopic clusters predicted for monochlorinated molecules.
The HRMS spectra of 8a and 8b displayed ions with a 3:1
35
relative abundance, which is the m/z expected for the Cl
3
7
and Cl isotopic ratio (Trivedi et al. 2016).
For the synthesis of trimethoxy-benzamide intermediates
13a and 13b, as well as for obtaining the target bis-
trimethoxy-benzamide analogs 10a and 10b, the acyl-
chloride 14 was employed as starting material (Ahmad et al.
2010; Mallikarjuna et al. 2016) (Scheme 2). The favored
formation of the monoamides 13a and 13b or the bis-
amides 10a and 10b was easily managed with the proper
adjustments in the synthetic methodology in terms of
number of amine equivalents and order of addition of the
reagents. In other words, for the synthesis of trimethoxy-
benzamide intermediates 13a and 13b, a solution of the
acyl-chloride 14 in dichloromethane was slowly added
dropwise into a dichloromethane solution containing a great
excess of ethylenediamine or piperazine (10 eq). In turn, for
2
014; Khan et al. 2015).
In turn, the bis-trimethoxy-benzamide analog 10a was
designed by molecular hybridization, combining the frag-
ment C from trimethoxy-chalcone 6 (Ferreira et al. 2014)
with the ethylenediamine B from 4-amino-quinoline pro-
totype 5 (Macedo et al. 2010), furnishing the hybrid deri-
vative 10a, which encloses two trimethoxy-phenyl rings
separated by a spacer, in a direct analogy to the structure of
the anti-prion prototype 6 (Fig. 2). Once again, the influence
of the conformational flexibility was considered for the
design of the conformationally restricted piperazine analog
1
0b.
Scheme 2 Reagents and
conditions: c oxalyl chloride,
CH2Cl2, cat. DMF, r.t., 2 h,
1
00%; d A solution of the acyl-
chloride 14 in CH Cl was
2
2
slowly added dropwise into a
piperazine or ethylenediamine
1
7
0eq solution, CH2Cl2, r.t., 20’,
5–95%; e piperazine or
ethylenediamine (1–4 eq) were
slowly added into a CH2Cl2
solution of 14 until its complete
consumption was observed, r.t.,
2
h, 82–87%

Medicinal Chemistry Research
the synthesis of the designed bis-trimethoxy-benzamides
rPrP is used as substrate, and the infectious particles in the
biological samples seed the conversion of monomeric rPrP
into rPrP fibrils under specific shaking and temperature
conditions. The conversion is monitored in real-time by
thioflavin T (ThT), an amyloidophilic dye which binds to
newly formed rPrP fibrils (Wilham et al. 2010). Besides its
usefulness for diagnostic purposes, this assay has been
employed in the last years to identify inhibitors of rPrP
aggregation in vitro (Ferreira et al. 2014, 2018; Vieira et al.
2014; Schmitz et al. 2016; Hyeon et al. 2017).
1
0a and 10b, the corresponding amines, i.e., ethylenedia-
mine or piperazine, were slowly added to a solution of the
acyl-chloride 14 in dichloromethane until the complete
consumption of 14 was observed. In the described condi-
tions, only trace amounts of the undesired products were
detected by TLC monitoring. The acyl-chloride 14 was
obtained from the corresponding benzoic acid 15 by clas-
sical functional group interconversion (Ahmad et al. 2010),
as detailed in Scheme 2.
All benzamide derivatives 10a, 10b, 13a, and 13b were
As all trimethoxy-benzamide derivatives were predicted
to bind to PrP globular domain, we investigated whether
1
13
characterized by H and C NMR. Moreover, the designed
bis-trimethoxy-benzamide analogs 10a and 10b were ana-
lyzed by HRMS and showed spectral data consistent with
the assigned structures.
they could inhibit rPrP conversion in the RT-QuIC assay.
We performed the reactions using hamster rPrP9
0–231
as
substrate and brain homogenate from a patient with sCJD as
Sc
a source of PrP . At all concentrations tested (50, 100, and
Interaction between PrP and trimethoxy-benzamide
derivatives assessed by molecular docking
200 μM), the analogs neither inhibited the conversion nor
delayed the process, except for 8a, which shortened the lag
phase (Figs. 4 and S23). These findings indicate that the
C
In order to investigate the binding mode of the trimethoxy-
benzamide derivatives, we docked them into mouse PrP
globular domain (PDB: 1AG2) using AutoDock software.
Initially, we performed a blind docking, which reinforced
that their most probable site of interaction within PrP
globular domain was the hotspot region, previously
described (Langella et al. 2004; Kuwata et al. 2007). In
order to further explore the specific interaction of these
compounds with the PrP hotspot site, we firstly docked
GN8 (4) into this pocket and obtained interactions and
spatial orientation in accordance with the results found by
Kuwata’s group (Kuwata et al. 2007; Yamamoto and
Kuwata 2009; Ishikawa et al. 2009) (Fig. S21). The vali-
dated docking methodology was then performed with the
trimethoxy-benzamide analogs. All these compounds
exhibited similar orientation and hydrogen bonding with
Lys194 (ranging from 2.6 to 3.3 Å) and Asn159 (ranging
from 2.6 to 3.5 Å), as well as hydrophobic interactions with
Leu125, Leu130, Gln160, Val161, Tyr162, Ile182, Thr183,
and Gln186 (Figs. 3 and S22). Their binding energy to PrP
ranged from –6.35 to –7.88 kcal/mol, which was similar to
the value found for GN8 binding (–8.83 kcal/mol) (Figs. 3
and S21). In summary, these results suggested that these
molecules might act as PrP ligands.
putative interaction of the designed compounds with PrP ,
as predicted by molecular docking, was not able to prevent
monomeric rPrP conversion into rPrP aggregates in vitro.
However, the reduced lag phase observed in the presence of
8a suggests that this derivative might interact with PrP
globular domain.
In order to exclude the possibility that the compounds
could be quenching ThT fluorescence rather than inhibiting
rPrP fibrillation, the analogs were added to the RT-QuIC
reaction media after the conversion reached the plateau
(17 h). No decrease in ThT fluorescence was observed (Fig.
S24).
Although no inhibitory effect in the prion conversion
assay was observed, it is relevant to highlight that we were
able to identify a new chemical entity (NCE) bearing a
novel 4-amino-quinoline trimethoxy-benzamide structural
pattern, i.e., 8a, which was able to modulate rPrP aggre-
gation in vitro. Based on these data, the 4-amino-quinoline
heterocyclic moiety was considered as preferential for the
C
proposed interaction with PrP ; thus 8a represents a useful
starting point for future structural optimization in the design
of novel anti-prion drug candidates.
Interaction between rPrP and trimethoxy-
benzamide derivatives assessed by STD-NMR and
ITC
Modulation of rPrP aggregation assessed by RT-
QuIC
Considering the results from the RT-QuIC assay, we next
performed a saturation transfer difference nuclear magnetic
resonance (STD-NMR) experiment to investigate the direct
interaction of compound 8a and its conformationally
The real-time quaking induced conversion (RT-QuIC) is a
cell-free diagnostic tool widely used to detect and amplify
minute amounts of PrPSc from different biological samples,
such as brain, cerebrospinal fluid, blood, nasal brushing,
saliva, urine, and feces (Wilham et al. 2010; Atarashi et al.
9
0–231
restricted analog 8b with mouse rPrP
.
STD-NMR signals were observed for nearly all hydro-
2
011; Orru et al. 2017). In this assay, soluble, monomeric
gens of 8a (hydrogens 2 and/or 5, 6, 8, 3′, 6′, and methoxyl

Medicinal Chemistry Research
Fig. 3 Binding mode analysis of
the trimethoxy-benzamide
analogs with mouse PrP globular
domain (PDB: 1AG2). Residues
involved on the interactions are
highlighted in dark gray, and
hydrogen bonds are colored in
yellow dashed lines. The
binding energy values were the
following: –7.00 kcal/mol for
8
–
–
–
–
a, –7.66 kcal/mol for 8b,
6.78 kcal/mol for 9a,
7.88 kcal/mol for 9b,
6.35 kcal/mol for 10a, and
6.90 kcal/mol for 10b
groups) and 8b (hydrogens 2, 3, 5, 8, 3′, 6′, and methoxyl
We also analyzed the interaction of 8a and 8b with
90–231
90–231
groups) in the presence of rPrP
, suggesting that both
rPrP
using single-injection isothermal titration calori-
ligands bind directly to rPrP9
0–231
via hydrophobic interac-
metry (ITC). Addition of either 8a or 8b to rPrP
90–231
pro-
tions (Fig. 5). The signals highlighted at approximately
.6 ppm (Fig. 5c), 7.3 ppm, and 8.5 ppm (Fig. 5b) corre-
spond to protein resonances that were selectively saturated,
duced an endothermic heat of reaction (Fig. 6), which
indicated that both compounds display mainly hydrophobic
6
9
0–231
interactions with rPrP
. This is in accordance with the
despite the spinlock filter used during the experiment.
results from the STD-NMR analysis.
Comparison of the STD-NMR spectra obtained for 8a
and 8b in complex with rPrP9
0–231
suggests that the binding
Cellular efficacy/toxicity evaluation of trimethoxy-
epitope for both ligands are similar, reinforcing the rele-
vance of the 4-amino-quinoline and trimethoxy-benzamide
moieties for this complementary molecular recognition.
Interestingly, 8b showed higher intensity STD-NMR sig-
nals than 8a, suggesting that 8b has a tighter interaction
benzamide derivatives
We next investigated whether the analogs 8a and 8b could
Res
Sc
change PrP
levels in PrP -infected murine neuro-
blastoma (ScN2a) cells. This method has been extensively
used to assess the anti-prion activity of molecules, as it
employs a robust and relatively economical model and
9
0–231
with rPrP
. In contrast, 8b yielded no significant change
in the RT-QuIC assay.

Medicinal Chemistry Research
Fig. 4 RT-QuIC kinetics using
9
0–231
hamster rPrP
at 6 μM as the
substrate and sCJD agent-
infected human brain
homogenate as the seed. The
reactions were performed in the
absence (solid black line) or in
the presence of the compounds
at 50 μM (dotted gray line),
1
2
00 μM (dashed gray line), and
00 μM (solid gray line).
Normal brain homogenate
NBH) was used as a negative
(
control of seeding (white
circles). Each curve represents
the average ThT fluorescence of
four replicates. The individual
replicates are plotted in Fig. S23
allows the evaluation of prion strain-related effects
10 μM (Fig. S25). These results suggested that the 7-chloro-
4-amino-quinoline and 4-amino-quinazoline portions are
toxicophoric in the cell lines used, while the trimethoxy-
benzamide region is not.
(
(
Kocisko et al. 2003). We treated both RML-strain ScN2a
ScN2a-RML) and 22L-strain ScN2a (ScN2a-22L) cell
lines with 8a and 8b at concentrations ranging from 0.1 to
0 μM. In ScN2a-RML cells, 8b induced a significant
1
Although the analogs 8a, 8b, 9a, and 9b were toxic to
ScN2a cells, they were not equally toxic to other mamma-
lian cell lines, including human cell lines (data not shown).
These findings indicated that the 7-chloro-4-amino-quino-
line and 4-amino-quinazoline rings are not universally
toxicophoric, thus these compounds might even be tested in
Res
decrease in PrP content at 1 and 10 μM, while 8a pro-
moted a similar outcome only at 10 μM (Fig. 7). In ScN2a-
2
2L cells, both 8a and 8b prompted a significant decrease in
Res
PrP content only at 10 μM (Fig. 7).
Simultaneously, we examined whether the compounds
were affecting cell viability. Both 8a and 8b significantly
reduced ScN2a-RML and ScN2a-22L cell viability at
Sc
other PrP -infected cell lines.
1
0 μM, while 8b also significantly decreased ScN2a-RML
Res
cell viability at 1 μM (Fig. 7). The PrP content reduction
profiles were reasonably correlated with the cell viability
Conclusion
reduction profiles (Fig. 7). These results indicated that these
This study has described the synthesis and biological testing
of a novel series of trimethoxy-benzamide derivatives (8a,
8b, 9a, 9b, 10a, and 10b) designed as anti-prion com-
pounds. Molecular docking studies predicted that all the
compounds were expected to bind to a hotspot region in PrP
globular domain with very similar spatial orientation and
interaction mode. The analogs were then further evaluated
Res
compounds were decreasing the final PrP
levels by
inducing cell death rather than by effectively inhibiting
Res
Res
PrP formation and/or promoting PrP clearance.
The analogs 9a and 9b exhibited similar profiles to those
displayed by 8a and 8b, while 10b was neither effective in
Res
decreasing PrP content nor significantly cytotoxic (Fig.
S25). The derivative 10a was also non-toxic and promoted
moderate reduction in PrP levels at the concentration of
by RT-QuIC and, although none of them prevented the
Res
90–231
conversion of monomeric rPrP
into rPrP fibrils, the

Medicinal Chemistry Research
Fig. 5 STD-NMR spectra of 8a and 8b in complex with mouse
NMR spectrum was collected in a different on-resonance frequency:
9
0–231
rPrP
. a Chemical structures of 8a and 8b, with the corresponding
–0.11, 0.21, 0.38, 7.28, 8.45 ppm, as indicated. c STD-NMR spectra of
1
hydrogens showing STD-NMR signals highlighted in red. For 8a,
1 mM 8b in the presence of 10 μM rPrP in 100% D2O. H reference
hydrogens 2 and 5 are highlighted in blue, since they could not be
spectrum is depicted on top of the figure. Each STD-NMR spectrum
was collected in a different on-resonance frequency: –0.19, 0.12, 0.76,
6.52, 6.62 ppm, as indicated
discriminated by their STD-NMR signals. b STD-NMR spectra of
1
1
mM 8a in the presence of 10 μM rPrP in 100% D2O. H off-reso-
nance, reference spectrum is depicted on top of the figure. Each STD-
Fig. 6 Single-injection ITC
binding isotherm of 8a and 8b to
9
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mouse rPrP
. The curves
shown were normalized by
subtracting the heat of dilution
of the compounds in buffer from
the heat of dilution of the
compounds in rPrP solution. The
experiment was carried out at
2
5 °C with protein:ligand molar
ratio of 1:10

Medicinal Chemistry Research
Products, Federal University of Rio de Janeiro (IPPN-UFRJ, BR) for
NMR analysis. This study was financed in part by the Coordenação de
Aperfeiçoamento de Pessoal de Nível Superior - Brazil (CAPES) -
Finance Code 001.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
Publisher’s note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
Fig. 7 Effect of 8a and 8b on PrPRes content and cell viability in PrPSc-
infected murine neuroblastoma (ScN2a) cells. ScN2a cells infected
with RML strain (left) or 22L-strain (right) were treated with 8a
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Persistent Viral Diseases from Rocky Mountain Laboratories of the
National Institutes of Health (USA); and the funding agencies INCT-
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