Azo-dyes based small bifunctional molecules for metal chelation and controlling amyloid formation

Article abstract of DOI:10.1016/j.ica.2017.11.029

Chemical tools are needed to discover new effective drugs for tackling multifaceted complex neurodegenerative diseases like Alzheimer's disease (AD). Multifunctional nature of two compounds, 5-((4-nitrophenyl)diazenyl)quinolin-8-ol (HL1) and 4-((4-nitrophenyl)diazenyl)benzene-1,3-diol (HL2) is reported w.r.t. their ability to bind Cu²⁺ ions and amyloid aggregates related to AD. HL1 and HL2 have half congo-red type azo-stilbene structural framework incorporated with metal chelating groups, designed to chelate metal ions from metal-amyloid species. Metal binding studies of HL1 and HL2 are established by the methods of Job's Plot, UV-vis spectra with metal ions and stability constant determination. In addition, their metal complexes are isolated, purity checked by elemental analysis, spectroscopically characterized and their structural analyses were obtained from DFT based calculations including binding energy determination. Chicken egg white Lysozyme (CEWL) was used as a model peptide for fibrillation studies. HL1 is found as an excellent colorimetric sensor for amyloid fibrils. Inhibitory effect of HL1 and HL2 and their isolated metal complexes L1-Cu and L2-Cu on CEWL fibrillation was studied using ThT and ANS fluorescence assay along with TEM imaging. In addition, the cell toxicity studies on these compounds suggest that although azo dyes may be non-toxic but having a nitro-substitution lead to significant cell toxicity. Overall, these results suggest that this new class of multifunctional small molecules can interact with amyloids as well as metal ions and could be potential anti-aggregation metal chelating agents.

Full text of DOI:10.1016/j.ica.2017.11.029

Accepted Manuscript
Azo-dyes based small bifunctional molecules for metal chelation and controlling
amyloid formation
Monika Rana, Hong-Jun Cho, Tapta Kanchan Roy, Liviu M. Mirica, Anuj K.
Sharma
PII:
S0020-1693(17)31443-3
https://doi.org/10.1016/j.ica.2017.11.029
ICA 17999
DOI:
Reference:
Inorganica Chimica Acta
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16 September 2017
6 November 2017
17 November 2017
Please cite this article as: M. Rana, H-J. Cho, T.K. Roy, L.M. Mirica, A.K. Sharma, Azo-dyes based small
bifunctional molecules for metal chelation and controlling amyloid formation, Inorganica Chimica Acta (2017),
doi: https://doi.org/10.1016/j.ica.2017.11.029
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Azo-dyes based small bifunctional molecules for metal chelation and
controlling amyloid formation
Monika Rana a, Hong-Jun Cho b, Tapta Kanchan Roy c, Liviu M. Mirica b, Anuj K. Sharma a,*
aDepartment of Chemistry, Central University of Rajasthan, NH-8, Bandarsindri, Ajmer, Rajasthan, 305817, India, E-mail: anuj.sharma@curaj.ac.in;
aks.iitk@gmail.com
bDepartment of Chemistry, Washington University, One Brookings Drive, St. Louis, Missouri 63130-4899.
cDepartment of Chemistry and Chemical Sciences, Central University of Jammu, Jammu, India, 180011.
Abstract
Chemical tools are needed to discover new effective drugs for tackling multifaceted complex
neurodegenerative diseases like Alzheimer’s disease (AD). Multifunctional nature of two compounds, 5-
((4-nitrophenyl)diazenyl)quinolin-8-ol (HL1) and 4-((4-nitrophenyl)diazenyl)benzene-1,3-diol (HL2) is
reported w.r.t. their ability to bind Cu2+ ions and amyloid aggregates related to AD. HL1 and HL2 have
half congo-red type azo-stilbene structural framework incorporated with metal chelating groups, designed
to chelate metal ions from metal-amyloid species. Metal binding studies of HL1 and HL2 are established
by the methods of Job’s Plot, UV-vis spectra with metal ions and stability constant determination. In
addition, their metal complexes are isolated, purity checked by elemental analysis, spectroscopically
characterized and their structural analyses were obtained from DFT based calculations including binding
energy determination. Chicken egg white Lysozyme (CEWL) was used as a model peptide for fibrillation
studies. HL1 is found as an excellent colorimetric sensor for amyloid fibrils. Inhibitory effect of HL1 and
HL2 and their isolated metal complexes L1-Cu and L2-Cu on CEWL fibrillation was studied using ThT
and ANS fluorescence assay along with TEM imaging. In addition, the cell toxicity studies on these
compounds suggest that although azo dyes may be non-toxic but having a nitro-substitution lead to
significant cell toxicity. Overall, these results suggest that this new class of multifunctional small
molecules can interact with amyloids as well as metal ions and could be potential anti-aggregation metal
chelating agents.
the site of binding and selectively chelate metal ions and control
their redox properties; (iii) ability to act as antioxidants; (iv)
acetylcholinesterase inhibition; (v) anti-inflammatory activity,
etc. [2c, 6b, 7a, 9]. These MFCs are expected to be more efficient
and potential neuroprotective disease-modifying drugs [10].
However, one limiting factor with these molecules is entering in
human brain crossing through blood brain barrier (BBB) [9, 11].
Molecules with a good balance of hydrophobic/hydrophilic
properties are suggested to have better chance of clearing BBB.
A number of bifunctional chelators have recently been
reported from various groups (Fig. 1) [2c, 3c, 7a, 9]. 8-hydroxy-
quinoline, clioquinol, ThT and stilbene frameworks have been
utilized for the construction of novel metal chelators that can
have amyloid interaction [2c, 3c, 6b, 7a, 9, 12]. A range of
benzothiazole and stilbene compounds bind with some extent of
selectivity to amyloid aggregates [6b, 13]. Despite these reports,
this field of multifunctional chelator design is still new and
numerous opportunities exist to improve the design of new such
small molecules [3c, 8].
Congo-Red (CR), an azo-dye, is currently treated as the gold
standard for amyloid detection and aimed for detecting amyloids
in early stages of AD [14]. We have started to design and
construct several new aza- and azo-stilbene derivatives and
developing these molecules into metal chelators to form
multifunctional compounds (Fig. 1). The use of azo-stilbene
framework has perhaps also originated from resveratrol and
subsequent modifications led to aza-stilbene type framework
1. Introduction
Aetiology of Alzheimer’s disease (AD) is still poorly understood
[1]. AD is recognized as a multi-faceted disorder in which the
patient brain shows protein misfolding and aggregation [2]. It has
been established that there are several factors responsible for the
neurodegeneration among which the role of metal ions have
caught especial attention [1b, 2b, 3]. Studying the role of metal
ions is also a challenging research topic [4]. Oxidative stress and
free radical formation and role of metal ions like Cu and Fe in
these processes is strongly indicated in recent research [4-5].
Therefore, inorganic chemistry of biological systems with special
emphasis on metal ion dyshomeostasis in neurodegenerative
diseases has been stimulated greatly in recent years [3a, 3c, 6].
Individuals with neurodegenerative diseases can be benefited
by developing multifunctional drugs which can target multiple
problems [7]. Understanding of some of the common structural
and pathogenic features of these diverse protein aggregation
processes may provide more effective therapeutic approaches.
Chemical tools and novel chemical agents are high in demand
that can deplete the monomeric precursor proteins, inhibiting
their aggregation, enhancing clearance and block the common
aggregation-induced pathways leading to cellular toxicity. Use of
smart metal chelators for developing effective AD therapeutics
has received special attention in medicinal research [1b, 3c, 8].
These so called multifunctional compounds (MFCs) comprises
of two or more following properties: (i) reducing the aggregation
of peptides into amyloid plaques and oligomers, (ii) recognizing

modified into metal chelators [15]. CR has been indicated to be
useful for early stage neurotoxic oligomers and detection of early
symptoms of AD [16]. However, to the best of our knowledge,
multifunctional compounds based on azo-dyes or CR has not
been used in the context of metal chelation from metal-amyloid
species. Metal chelating properties in combination with these
amyloid binding features are expected to lead to interesting new
multifunctional compounds.
In this report, we used two simple bi-dentate metal
chelators HL1 and HL2 (Fig. 1), their synthesis, isolation of their
Cu complexes and amyloid interaction properties are
investigated. Interestingly, these compounds exhibit amyloid-
metal dual affinity as well as inhibitory effect on chicken egg
white (CEWL) fibrilization.
solution of 1:1 concentrated HCl (5 mL) and distilled water (5
mL). It was cooled down to 0-5 °C using an ice-bath and a cold
aqueous solution of NaNO2 (1 g, 14.48 mmol) was added to it
drop by drop with constant stirring. The resulting solution was
stirred for 30 minutes and colour changed from dark brown to
violet, probably due to formation of intermediate diazonium
chloride salt. A solution of 8-hydroxyquinoline (1.05 g, 7.24
mmol) in the mixture of methanol (2 mL) and NaOH (10% w/v, 8
mL) was added to above diazonium salt solution while
maintaining the temperature at 0-5 °C. The resulting dark violet
solution was stirred for 1h and then acidified with few drops of
conc. HCl to obtain a red colour precipitate. It was filtered and
dried over high vacuum (1.61 g, yield 75%). 1H NMR (500 MHz,
DMSO-d6/CDCl3): δ 9.37 (br, s, 1H), 8.98 (d, 1H), 8.60-8.00 (m,
5H), 7.90-7.65 (m, 1H), 7.50-7.10 (m, 1H). FT-IR (KBr): 3448,
3199, 1560, 1385, 1265. UV-vis, MeOH, λmax, nm, (ε, M-1 cm-1):
418 (14,000). Melting Point: 275-278°C. HRMS: Calcd for
[M+H]+, 295.0831; Found, 295.0842.
2.2 Synthesis of 4-((4-nitrophenyl)diazenyl)benzene-1,3-diol
(HL2)
To a flask containing 4-nitroaniline (1 g, 7.24 mmol) was
added solution of 1:1 concentrated HCl (5 mL) and distilled water
(5 mL). It was cooled down to 0-5°C using an ice-bath and a cold
aqueous solution of NaNO2 (1g, 14.48 mmol) was added to it
drop by drop with constant stirring. The resulting solution was
stirred for 1h, colour changed from dark brown to violet,
probably due to formation of intermediate diazonium chloride
salt. A solution of resorcinol (0.7 g, 7.17 mmol) in the mixture of
methanol (2 mL) and NaOH (10% w/v, 8 ml) was added to the
above diazonium salt solution while maintaining the temperature
at 0-5 °C. The resulting dark violet solution was stirred for 1h. A
few drops of conc. HCl were added which led to the formation of
a red colour precipitate. Precipitate was filtered off, dried on high
vacuum (1.21 g, yield 65%). 1H NMR (500 MHz, CDCl3)
δ(ppm): 8.35 (d, J = 8.6 Hz, 2H), 7.89 (d, J = 8.6 Hz, 2H), 7.74
(d, J = 8.8 Hz, 1H), 6.62 (d, J = 7.5 Hz, 1H), 6.43 (s, 1H). 13C
NMR (125 MHz, CDCl3) δ(ppm): 163.4, 158.9, 153.5, 147.5,
136.4, 133.8, 125.1, 121.6, 110.8, 103.9. FT-IR (KBr): 3442,
3168, 1594, 1384, 1209. UV-vis, CH3OH, λmax, nm (ε, M-1 cm-1):
442 (19500). Melting Point: 175-180°C. HRMS: Calcd for
[M+H]+, 260.0671; Found, 260.0663.
Fig. 1 (Top) Molecular structure of resveratrol, Thioflavin-T and
Clioquinol; (Middle) Structures of selective bifunctional chelators
recently reported; and (Bottom) Structure of HL1 and HL2,
multifunctional molecules studied in this work.
2. Experimental
2.1. General
All reagents were purchased from commercial sources and
used as received unless stated otherwise. Solvents were purified
by distillation prior to use. 4-(phenyldiazenyl)phenol was
obtained from Sigma Aldrich. 4-(phenyldiazenyl)benzene-1,3-
diol and 5-(phenyldiazenyl)quinolin-8-ol were prepared
following a literature procedure [17]. All aqueous solutions and
buffers were prepared using commercially available distilled
1
water. H and 13C NMR spectra were recorded on a BRUKER-
500 spectrometer (500 MHz). Chemical shifts (δ) are reported in
ppm. The UV-visible spectra were recorded on a Carry-100 UV-
Visible Spectrophotometer and data are reported as λmax (nm)
absorbance. FT-IR spectra were recorded on PerkinElmer FT-IR
Spectrum-Two model using KBr. The melting/decomposition
points were determined in house by placing a finely powdered
sample in a glass capillary and heating by using BUCHI digital
melting point apparatus. High-resolution mass spectra were
recorded on XEVO G2-S QT of Waters Corporation, USA at
MNIT, Jaipur.
2.3. Synthesis of (L1)2Cu
.
A solution of Cu(ClO4)2 6H2O (0.125 g, 0.34 mmol) in
methanol (10 mL) was added to a stirring solution of HL1 (0.2g,
0.68 mmol) in 30 mL of methanol and Et3N (0.14 g, 1.35 mmol).
The resulting reaction mixture was stirred at room temperature
for 2h. The precipitate formed was filtered and washed twice with
Et2O and CH3CN. It was dried under vacuum (0.135 g, yield
60%). UV-vis, DMF, λmax, nm (ε, M-1 cm-1): 435 (9,100), 500 (sh,
8,100). Anal. Calcd for C30H18N8O6Cu: C, 55.43; H, 2.79; N,
17.24. Found: C, 55.16; H, 2.34; N, 17.22.
2.2. Synthesis of ((4-nitrophenyl)diazenyl)quinolin-8-ol (HL1)
A slightly modified method than reported was used [18]. To a
flask containing 4-nitroaniline (1 g, 7.24 mmol) was added a
2.4. Synthesis of (L2)2Cu

.
complex. Titrations were carried out at 25 ºC using a Eutech pH
meter 700 with pH glass-electrode (ECFC7252101B) with a
ground-joint diaphragm. Electrode has a porous HDPE pin type
liquid junction and Ag/AgCl type internal reference. It can
measure pH range from 0-13 in the 0-80 ºC temperature range. It
has a stainless steel 304 temperature probe (PH5TEMB01P).
Before experiment, the standardization was done with aqueous
buffer solutions at pH 4.00 and 7.00. Due to the limited solubility
of HL2 in water, MeOH stock solutions (1 mM) were used and
titrations were performed in MeOH-H2O (20% v/v) mixture
containing 0.1 M NaCl. For titration, pH of solution was raised
by adding 0.1M NaOH solution using a Hamilton microsyringe
(10 µL). To compensate for the expected methanol-water liquid
junction potential, a correction of 0.015 pH units was done by
subtracting from the measured pH readings [19]. For the acidity
constants determination, a solution of HL2 (50 µM) was titrated
with 0.1M NaOH at room temperature. At least twenty UV-vis
spectra were collected in pH 3-12 range. Similarly, stability
constant was determined by titrating solution of HL2 (20 µM)
A solution of Cu(ClO4)2 6H2O (0.14 g, 0.38 mmol) in CH3OH
(10 mL) was added to a stirring solution of HL2 (0.2 g, 0.77
mmol) in (10 mL) CH3OH and Et3N (0.156 g, 1.54 mmol). The
resulting reaction mixture was stirred at room temperature for 2h.
The dark brown precipitate formed was filtered and washed thrice
with Et2O. It was dried under vacuum (0.2 g, yield 89%). UV-vis,
CH3CN, λmax, nm (ε, M-1 cm-1): 435 (13,850). ESI-MS: Calcd for
[M+H]+, 580.04; Found, 580.31. Anal. Calcd for C24H16N6O8Cu:
C, 49.70; H, 2.76; N, 14.49. Found: C, 49.39; H, 2.76; N, 14.78.
2.5. CEWL fibrillation
Chicken Egg White Lysozyme (CEWL) protein was obtained
from Sigma and stored at -80 °C. Small aliquots were carefully
weighed out at room temperature in microcentrifuge tubes.
CEWL aliquot was dissolved in DMSO and diluted in phosphate
buffer saline (PBS, pH 7.4) TO reach a final concentration of 20
µM. Final DMSO amount was maintained <2% of total volume
throughout all assays. These solutions were incubated in absence
and presence of inhibitors for 48 h at 60 °C with continuous
agitation of 1200 rpm. The extent of fibrils formed after this time
was checked by ThT fluorescence and TEM. It was observed that
there is no fibrillation of CEWL at room temperature (25 °C)
even after 7 days.
.
and 0.5 equivalent amount of CuCl2 2H2O (10 µM) with small
aliquots of 0.1 M NaOH at room temperature. At least twenty
UV-vis spectra were collected in pH 3-12 range. The acidity and
stability constants were calculated using the HYPSPEC computer
program [20]. Speciation plots of the compounds and their metal
complexes were calculated using the program HySS2009
(Protonic Software, UK) [21].
2.6. Fluorescence measurements
All fluorescence measurements were performed using Perkin
Elmer LS-55 Fluorescence Spectrometer unless stated otherwise.
2.10. Computational Details
All the theoretical calculations were performed using density
functional theory based B3LYP [22] level of theory in conjuction
with 6-31+G** basis set [23] for all except metal atom where we
used Def2-DZVP with effective core potential [24]. Full
geometry optimizations for all complexes considered here have
been performed followed by harmonic vibrational frequencies to
validate each structure as true minima. All the theoretical
calculations have been performed using the Gaussian09 program
package (reference R1, ESI). Further validation for the accuracy
of the level of theory used here were tested. First, to check the
accuracy of the Def2-DZVP basis used for metal atom, we used
6-31+G** basis for all the atoms. Next, we also tested the solvent
effects on the structural parameters by means of the Polarizable
Continuum Model (PCM) using the integral equation formalism
variant (IEFPCM)[25] where CH3CN was used as solvent at the
B3LYP/6-31+G**/Def2-DZVP level of theory.
2.7. Thioflavin-T Assay
For monitoring the progress of aggregation, Thioflavin-T
(ThT) fluorescence assays were perfomed as follows: 1 mM stock
solutions of ThT was prepared in DMSO and diluted in PBS as
needed. For inhibition of CEWL fibrillation by inhibitors used in
these studies, inhibitors (20 µM) and CEWL solutions (20 µM in
PBS, pH 7.4) were incubated for 48-72 h at 60°C with constant
agitation. For monitoring the extent of aggregation, samples were
diluted to 2.5 µM CEWL in PBS containing ThT (10 µM).
Fluorescence spectra were recorded for each sample using λex
435 nm and measuring the fluorescence intensity at 485 nm.
=
2.8. 8-Anilino-1-naphthalenesulfonic acid ammonium salt (ANS)
assays
5 mM stock solution of ANS was prepared in PBS having 1%
DMSO. Then, it was diluted to 50 µM in PBS for recording UV-
vis spectra. Emission spectra were recorded at λex = 380 nm. For
monitoring the extent of aggregation, samples were diluted to 2.5
µM CEWL in PBS containing ANS (10 µM). Fluorescence
spectra were recorded for each sample using λex = 380 nm
measuring the fluorescence intensity at 480 nm.
2.11. Transmission Electron Microscopy (TEM)
Glow-discharged grids (Formar/Carbon 300-mesh, Electron
Microscopy Sciences) were treated with preformed CEWL
aggregates in absence and presence of compounds for 30 min at
room temperature. Excess solution was removed using filter
paper and grids were rinsed with distilled H2O. Grids were
stained with ammoniumm molybdate (1% w/v, H2O) for 1 min,
blotted with filter paper, and dried overnight at room temperature.
Images were captured using a FEI G2 Spirit Twin microscope
(200 kV). TEM analysis was performed at the Material Research
2.9. Acidity and stability constant determination.
UV-vis pH titrations were employed for the determination of
acidity constants of HL2 and stability constants of its Cu

Centre at MNIT, Jaipur.
2.12. Cell toxicity assays
Mouse neuroblastoma Neuro2A (N2A) cell lines were
purchased from the American Type Culture Collection (ATCC).
Cells were grown in Dulbecco's Modified Eagle's medium
(DMEM)/10% FBS, which is the regular growth media for N2A
cells. N2A cells were plated to each well of a 96 well plate
(1x104/well) with DMEM/10% FBS. The media was changed to
serum-free medium containing N2 supplement. After 1 h, the
compounds were added into each well (final volume: 100 µL)
with different concentrations (2, 5, 10 µM), followed by
incubation for 24 h at 37 °C. Due to the poor solubility of
compounds in water or media, their stock solutions were prepared
in DMSO and diluted in media. The final DMSO concentration in
this way was less than 1%. Our control experiments suggest that
there is no toxicity caused by 2% DMSO. At last, each well was
treated with 10 μL of Alamar blue reagent and the cells were
incubated for 1 h. The fluorescence (excitation: 560 nm,
emission: 590 nm) was measured using a SpectraMax M2e plate
reader (Molecular Devices, USA).
Fig. 2 Design concept of multifunctional chelators HL1 and HL2
used in current studies.
3.2. Synthesis, characterization and drug-likeness of HL1 and
HL2
Synthesis of both compounds HL1 and HL2 is described above in
experimental section. Standard synthetic methods of azo-coupling
were followed to obtain these compounds in good yields (Scheme
1). During the reactions, a distinct colour change due to azo dye
formation suggested product formation.
Scheme 1. Synthesis of HL1, HL2 and their Cu complexes
3. Results and discussion
3.1. Design Concept for HL1 and HL2
Our general strategy to develop novel multifunctional
compounds is to synthesize small molecule metal chelators
functionalized with amyloid recognition moieties. In the current
studies, we choose molecular frameworks that are expected to
bind amyloid aggregates. Stilbene and fluorescent dye thioflavin-
T (ThT) derivatives and few other molecular frameworks have
been utilized previously for this purpose [2c, 6b, 7a, 15a].
Iodinated 2-(2-hydroxyphenyl)benzothiazole and a few related
compounds were also shown to be potential metal chelating
agents for AD by Rodríguez-Rodríguez et al [13]. A generic
scheme of our approach is shown in Fig. 2. The design of these
molecules is partly inspired by the properties of CR known to
stain ex-vivo amyloid plaques 14, 16, 26]. Half CR type
molecules, resveratrol-stilbene framework have been adopted
since these are known for their amyloid binding affinities [15b,
15c]. In addition, metal chelation properties of 8-
hydroxyquinoline (HQ) or clioquinol (CQ) were incorporated
(Fig. 1 and 2) [27]. In HL2, metal chelation part was developed
by using resorcinol as starting material, thus generating the azo-
stilbene character (Fig. 1). To the best of our knowledge, this
design has never been investigated in the context of bioinorganic
chemistry in general or multifunctional metal chelators for
controlling metal induced abnormalities in particlular.
1
Intense coloured products were isolated and characterized by H,
13C NMR, HRMS methods. 1H NMR chemical shifts were
broadened in some cases, probably due to some cis-trans
isomerisation about azo-group. UV-vis spectrum of HL1 shows
an absorption band at 418 nm in MeOH (Fig. S1, ESI). However,
HL1 exhibit an additional absorption band at 630 nm in DMF,
probably due to a change in conformation (Fig. S1, ESI). Copper
complex of HL1 in DMF exhibit two absorption bands at 435 nm
and a shoulder at 500 nm, respectively (Fig. S2, ESI). When
EDTA is added to a solution of (L1)2Cu in DMF, both the
absorption bands at 418 nm and 630 nm reappeared
corresponding to the HL1 (Fig. S3, ESI). Although it suggests a
reversible process, it was observed that EDTA could generate the
free ligand only partially even after adding excess and keeping
for days. It could be due to the chelating ability of HL1 for Cu is
similar to EDTA and equilibrium develops after certain time.
HL2 show a UV absorption bands at 410 nm assignable to n→*
transitions (Fig. S3, ESI). A shift in this absorption band was

observed for the copper complex of HL2 which exhibit
absorption band at 435 nm in CH3CN (Fig. S4, ESI). HL2 and
(L2)2Cu spectra were recorded in DMF also. In DMF, HL2 and
(L2)2Cu both show two absorption bands at 445 nm and 545 nm.
(Fig. S5, ESI).
other phenol group also get deprotonated but at very high pH
(~11) and spectra again changes but data is not shown here.
0.8
0.6
0.4
0.2
0.0
An important qualification for small molecules to fit for
any application in brain related diseases is its ability to cross
through blood brain barrier (BBB). The BBB permeability of a
metal-binding ligand is affected by many properties such as
molecular size, charge and lipophilicity [1b, 3c]. According to
Lipinski rules [11] for bioavailability, at least three out of the five
following rules for molecules are to be obeyed: molecular weight
<500, calculated logarithm of octanol–water partition coefficient
(clogP) <5, the number of hydrogen-bond donors (HBD) <5, the
number of hydrogen-bond acceptors (HBA) <10. Clearly, highly
polar compounds would have difficulty at the stage of passing
through the BBB. In order to evaluate theoretically whether or
not a molecule could pass through the BBB by passive diffusion,
the logBB parameter can be predicted using the relation, log BB
300
400
500
600
700
Wavelength(nm)
HL2- Speciation
LH
100
80
60
40
20
0
=
−0.0148 TPSA + 0.152 clogP + 0.139 [15a]. This
LH
L
2
phenomenological expression has shown good predictive ability
for a large number of molecules. Freely available software from
the http://www.molinspiration.com/cgi-bin/properties was used to
calculate above properties. Although, most of the parameters for
two compounds investigated here are in the range of Lipinski
parameters, except that TPSA (polar surface area) which is on
slightly higher side due to the hydrophobic nature of HL1 and
HL2 (Table 1).
Table 1. Drug-like parameters for compounds HL1-HL2.
Compounds
HL1
cLogP TPSA
M.Wt. HBD HBA logBB
3
5
7
9
11
13
4.08
3.32
5
103.67
111.01
90
294.27
259.22
500
1
2
5
7
-0.775
pH
HL2
Lipinski rulesa
7
-0.999
Fig. 3 Variable pH-UV spectra of HL2 (50 µM, 25 °C, I = 0.1 M
10
>0.3 to 1.0
aMW: molecular weight; clogP: calculated octanol-water partition
coefficient; HBD: hydrogen-bond donor atoms; HBA: hydrogen-
bond acceptor atoms; PSA: polar surface area. (Lipinski, C. A.;
Lombardo, F.; Dominy, B. W.; Feeney, P. J. Adv. Drug Delivery
Rev. 1997, 23, 3-25; Clark, D. E.; Pickett, S. D. Drug Disc.
Today 2000, 5, 49-58).
NaCl) and the species distribution plot cacuclated by HYSS.
3.4. Metal chelating properties of HL1 and HL2 towards Cu2+
Metal binding ability for both HL1 and HL2 were studied in
solution using methanol as solvent. The binding stoichiometry of
HL1 and HL2 with Cu2+ was determined by Job’s Plot method. A
notable shift was observed in the spectra of HL1 and HL2 when
their solution was mixed with Cu2+ ions (Fig. S6 and S7, ESI).
For both the ligands, a stoichiometric break at 0.4 mole fraction
of the metal ion, suggests the formation of a mixture of 1:1 and
1:2 metal-ligand species (Fig. 4). Given that these are bidentate
ligands and are expected to form (L)2Cu complexes, a 1:1
stoichiometry suggest the presence of some solvento complexes
in solution. In addition, Cu2+ complexes of HL1 or HL2 were
3.3. Protonation constants for HL1 and HL2
As both HL1 and HL2 contain phenoxo/hydroxo and pyridine
functional groups which can undergo protonation, we determined
their acidity constants (pKa) by spectrophotometric-
potentiometric titrations [2c, 28]. HL1 shows a limited solubility
in aqueous media and satisfactory data could not be obtained for
pKa determination. We tried to use more soluble analogues i.e.,
(phenyldiazenyl)quinolin-8-ol and 8-hydroxyquinoline (HQ) as a
model for this ligand but these ligands did not show any
appreciable change in spectra to be used for pKa determination.
For HL2, UV-vis titrations in the pH range 3-12 undergo several
spectral shifts (Fig. 3). First at pH 3, a peak at 410 nm is
dominant and as the pH rises, this peak shifts to a new peak at
450 nm. The best fit to this data could be obtained with pKa
values of 6.117 and 9.951. These pKa values may be assigned to
the deprotonation of nitrogen atoms of azo group, while the
second one is deprotonation of one of the phenol groups. The
.
synthesized by reacting CuClO4 6H2O with the corresponding
ligand in MeOH in presence of a slight excess amount of Et3N.
Their UV-vis spectra of these isolated complexes show a shift
from parent ligand spectra suggesting the formation of Cu2+
complexes (Fig. S2 and S4, ESI).

0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
-0.1
L2-Cu
0.4
0.3
0.2
0.1
0.0
L1-Cu
0.0
0.2
0.4
0.6
0.8
1.0
0.0
0.2
0.4
0.6
0.8
1.0
Mole Fraction of Cu
Mole fraction of Cu
Fig. 4 Job’s plot for HL1 and HL2 with Cu2+ in MeOH.
3.5. Theoretical investigations on equilibrium structures and
binding energies of Cu complexes
To assess the structures of metal complexes, the calculated
equilibrium geometries of most probable Cu2+ complexes were
studied using first-principles based quantum mechanical
calculations. The structural analysis of equilibrium geometries of
most probable Cu2+ complexes was performed. Both the Cu2+
complexes of HL1 and HL2 were optimized to get their
geometries and other metric parameters. As these Cu2+ complexes
are bidentate ligands, a 2:1 ligand to metal stoichiometry is
expected which is also suggested by Job’s plot. A systematic
analysis of the optimized geometrical data such as bond lengths,
bond angles and dihedral angles can provide useful information
about the metal chelation. For (L1)2Cu complex, a perfect square
planar geometry was observed with dihedral angle 180.0 around
the Cu2+ (Fig. 5). The Cu-N and Cu-O equilibrium bond lengths
were found 1.9962 Å and 1.9501 Å, respectively. These bond
lengths are in good agreement with reported literatures for Cu-
O(phenolate), Cu-N(amine/pyridine) [7a, 29]. In (L2)2Cu, due to
the presence of azo group, there is a possiblity that either of the
nitrogens can coordinate with Cu2+ to form Cu-N bond which
afford five or six-membered ring (Scheme S1, ESI). In the
optimized structures, both the possibilities were analyzed.
Structural parameters showed that 6-membered ring is less
distorted from planarity than 5-membered ring. Consequently, the
Cu-O and Cu-N bond distances for 6-membered ring are closer
than the five-membered ring to the corresponding values of L2.
While the two 5-membered rings of the corresponding complex is
almost planar with a dihedral angle very close to 0, the
corresponding 6-membered rings are non-planar with a dihedral
angle ~11 to reduce the ring strain. As a result it was observed
that 6-membered ring was more stable than the 5-membered ring
by 14.42 kcal/mol [30].
Fig. 5 DFT optimized structures of (L1)2Cu and (L2)2Cu
complexes (both 5-membered ring and 6-mebered chelating
ring are shown for (L2)2Cu). Coordinating atoms are name as
N1 and O1 in (L1)2Cu; and N2/N3 and O1 in (L2)2Cu,
respectively.
Table 2. DFT-optimized bond lengths and bond angles data for
(L1)2Cu and (L2)2Cu. Bond lengths are in Å and angles are in
degree.
(L1)2Cu
(L2)2Cu
5-membered
Cu – O(1)
1.9501
1.9962
1.9501
Cu – O(1)
Cu – N(1)
Cu – N(3)
1.895
2.175
2.175
Cu – N(1)
Cu – O(2)
Cu – N(2)
O(1)–Cu–N(1)
O(1)–Cu–O(2)
N(1)–Cu–N(2)
O(1)–Cu–N(2)
Dihedral angle
sq. pl.
1.9962
83.60
179.99
199.99
96.40
Cu – O(2)
1.895
82.44
147.81
111.72
129.02
98.08
O(1)–Cu–N(1)
O(1)–Cu–O(2)
O(1)–Cu–N(3)
N(1)–Cu–N(3)
179.99
O(1)–Cu–N(1) – N(3)
O(2)–Cu–O(1) – N(3)
119.35
Selected reported values of Cu-N and Cu-O bond lengths are as follows:
Cu-N
1.980 and 1.965 [ref7a] ; 2.002 and 2.042 [ref28]; 2.004 and
2.024 [ref13]; 1.980-2.024 [ref31]
Cu-
O(phenolate)
1.906 [ref28]; 1.917 and 1.904 [ref13]; 1.876 and
1.873[ref7a]; 1.961[ref31]
In order to further check the accuracy of the Def2-DZVP basis
used for metal, one selected complex (L2)2Cu was further
analysed by using 6-31+G** basis for all the atoms. Next, solvent
effects on the structural parameters was also tested by means of
the Polarizable Continuum Model (PCM) using the integral
equation formalism variant (IEFPCM) [25] where CH3CN was
used as solvent at the B3LYP/6-31+G**/Def2-DZVP level of
theory. It is found that there are no noticeable changes in the
optimized structures at different levels. All the parameters (see
Table S1, ESI) found at different methods were very much
similar to each other which shows that B3LYP/6-31+G**/Def2-
DZVP level of theory is adequate for the study of these
complexes (Table S1, ESI).
To further assess the ability of metal chelation we explored the
binding energies for each complex. The binding energy between
the BS and each metal in vacuo is defined by the formula: ΔE
(binding energy) = E (metal-binding complex in vacuo) − [E
(Ligand in vacuo) + E (free metal ion in vacuo)]. The calculation
of the interaction energies were considered using rigid
geometries, such that when the metal-ligand bonds are broken,
the ligands retain the same geometries they had in the equilibrium
structure. It is found that the binding energies for the L1-Cu

Stability Constant LCu-speciation
(L) Cu
complex, L2-Cu complex with 5-membered ring and L2-Cu
complex with 6-membered are 619.75, 619.01 and 629.20
kcal/mol, respectively. That shows that all ligands bind with Cu2+
strongly and approximately equal strength whereas 6-membered
L2-Cu complex binds marginally better. It further supports our
previous observation regarding the more stability of 6-membered
L2-Cu complex over 5-membered one. Finally, the overall
equilibrium structural parameters and binding energies for all the
cases obtained suggest that these ligands are very much capable
of binding to Cu2+ ions and can potentially be used for copper
chelation therapy.
100
80
60
40
20
0
2
free Cu
3.6. UV-vis determination of stability constants
pH-spectrophotometric titrations were also performed to
determine the stability constants and solution speciation of Cu2+
with HL2 system (Fig. 6). The pKa values of the HL2 were
included in the calculations of stability constants. Two models
were tried to fit the observed data: first the formation of both 1:1
and 1:2 metal to ligand species and second, only formation of 1:2
metal to ligand species were taken in account. While first model
did not give any reasonble fitting of the data, second model fitted
the data easily. The best fit to the data shows that HL2 binds Cu2+
with a stability constant of 16.88(1) for the reaction 2L2+Cu =
(L2)2Cu. For the sake of simplicity, in the data fitting model HL
is represented to have only one phenol group which can
coordinate with metal ion. The speciation diagram for the Cu-
HL2 system is shown in Fig. 6. In addition, the concentration of
free Cu2+ (pCu = -log[Cufree]) can be determined at specific pH
values and total ion concentrations, pCu values of 9.46 at pH 7.4.
Only a 0.003% of total Cu was remaining free at pH 7.4 as
observed in the fitted data. These pCu values are similar
compared to other bidentate lignads reported in the context of
metal chelation from amyloids [7a, 9]. Unfortunately, the
spectrophotometric titration of HL1 in presence of Cu2+ did not
give satisfactory spectral data to determine stability constants due
to precipitation of (L1)2Cu. Use of 8-hydroxyquinoline as a
model study also did not help as this system exhibit very little
change in spectra. The use of higher concentrations lead to the
precipitation of the Cu complexes.
2
6
10
pH
Fig. 6 Variable pH UV spectra of the HL2-Cu2+ system ([HL2] = 20 µM;
[Cu2+] = 10 µM, 25 °C, I = 0.1 M NaCl) and the species distribution plot
cacuclated by HYSS.
3.7. HL1 as amyloid sensor
Azo dyes are known for their staining of fibres. Due to the azo
dye function associated with these molecules, we were curious to
see if HL1 and HL2 can stain amyloid fibrils and give some
further evidence that these MFCs can bind to amyloid fibrils.
Staining assays were performed with both in solution and isolated
amyloid fibrils (centrifuged pellet). Preformed fibrils (20 µM)
were taken in a clean glass vial and HL1 and HL2 (1:1
DMSO:EtOH) were added to it. Addition of HL1 leads to a
sudden colour change to violet but addition of HL2 did not cause
any significant colour change (Fig. 7-label 1-4). In addition, when
HL1 was added in an isolated pellet of CEWL fibrils, it resulted
into a similar colour change (Fig. 7-label 5-6). The purple colour
did not fade away even after several washes with PBS buffer. It
clearly indicates the tight binding affinity of HL1 for amyloid
fibrils. Although it was expected to see some colour shift but
more interesting fact was to see such a drastic colour change
which persists for months without fading. This is encouraging for
investigating further and using this class of compounds for
histological applications in imaging of amyloid species. It will
help to find out molecular motifs that can be used in future
studies for the development of novel amyloid-binding
compounds.
0.5
0.4
0.3
0.2
0.1
0.0
300
400
500
600
700
Wavelength(nm)

Metal binding properties of HL1 and HL2 are evident from the
above discussion. The inhibitory effect of HL1, HL2 and their
isolated Cu complexes on CEWL aggregation was also
investigated. For these studies, equivalent amounts of HL1, HL2
and their isolated Cu complexes were added to CEWL solutions
(20 µM in PBS) before incubation for 48h at 60°C. We were
curious to see if metal complexes can inhibit aggregation or cause
disaggregation of amyloid plaques. Metal complexes as inhibitors
of aggregation is an important aspect but neglected so far and
needs more attention. Inhibitory effect of Cu complexes is needed
to be evaluated since HL1 and HL2 are good metal chelating
agents and expected to chelate metals from amyloids plaques
when applied in vivo. ThT and ANS fluorescence assays along
with TEM imaging were performed to assess the extent and
nature of aggregates formed.
Fig. 7 Amyloid staining experiment: Vial 1: HL1 with CEWL fibrils; vial
2: HL1 only; vial 3: HL2+CEWL fibrils; vial 4: HL2 only; (on the glass
slide) pellets of amyloid fibrils with HL1 (5); and without HL1 (6),
respectively.
In order to further understand the observed colour change of
HL1 with amyloids, its spectra were recorded in absence and
presence of fibrils. For this, stock solution of 2mM of HL1 in 1:1
DMSO and EtOH was prepared due to its limited solubility in
aqueous media. Varying amounts of HL1 (0.2-0.7 mM) were
added successively to the solution containing CEWL fibrils (20
µM) and their spectra were recorded. Spectra with 0.5 and 0.7 mM
solution of HL1 in absence and presence of fibrils are shown in
Fig. 8. In the diluted solutions, presence of fibrils leads to only
small change in spectra. However, in case of 0.7 mM HL1
concentration a significant spectral shift was obtained due to the
presence of amyloid fibrils and a new absorption band at ~580 nm
appeared in the spectra. It can be understood that the conformation
of HL1 bound to CEWL fibrils exhibit an absorption band at ~580
nm. It is interesting to note that absorption spectra of HL1 exhibit a
broad absorption band at 630 nm when spectra were recorded in
medium containing DMF (Fig S1, ESI), probably due to the similar
reason. As HL1 binds to the β-sheets of amyloid fibrils, the
conformation associated with purple band becomes more stable.
However, more experimental evidences are needed to support this
hypothesis.
3.9. ThT fluorescence assay to study inhibition
ThT fluorescence assay were used to quantify the extent of fibril
formed when CEWL was incubated in fibrillation conditions with
and without inhibitors. HL1, HL2 and their copper complexes
were tested for the inhibition of CEWL fibrillation. Interestingly,
HL1, HL2 and their copper complexes significantly reduce the
ThT emission intensity indicating the inhibition of CEWL
fibrillation (Fig. 9. HL1 and HL2 inhibit fibrillation by 76% and
92% respectively. (L1)2Cu and (L2)2Cu complexes inhibit
fibrillation by 44% and 62%, respectively. The reduction in the
extent of inhibition by non-planar (L)2Cu complexes compared to
parent ligands is understandable due to poor intercalation
properties which depends on molecular planarity. Interestingly,
complexes of HL1 and HL2 are also good inhibitors of amyloid
aggregation.. In addition, centrifugation was performed for these
samples and both the residue (bottom part) and supernatants were
separately analysed by ThT fluorescence assay. It was interesting
that both the supernatant and residues show very less ThT
fluorescence and suggests very low fibrillation in presence of
inhibitors compared to those in absence of inhibitors (Fig. S8 and
S9, ESI). These studies clearly show that the ligands and their
metal complexes investigated here have an inhibitory effect on
CEWL fibrillation
1.0
Fibrils
Fibrils+500M L1
0.8
0.6
0.4
0.2
0.0
L1 500M
Fibrils+700M L1
L1 700M
Fibrils
HL1
HL2
L1-Cu
800
L2-Cu
600
400
200
0
300
400
500
600
700
460
480
500
520
540
560
580
600
Wavelength(nm)
Wavelength(nm)
Fig. 8 Comparison of UV-vis spectra of HL1 (500 µM and 700 µM) in
absence and presence of amyloid fibrils (20 µM).
3.8. Inhibition of CEWL fibrillation by HL1, HL2 and their Cu
complexes

120
100
80
60
40
20
0
120
100
80
60
40
20
0
Lysozyme
HL1
HL2
L1-Cu
L2-Cu
Lysozyme
HL1
HL2
L1-Cu
L2-Cu
Fig. 10 Normalized ANS fluorescence intensities when mixed with
CEWL aggregates formed in fibrillation conditions [60 C, 1200 rpm,
48h] with various additives as indicated.
Fig. 9 (Top) ThT fluorescence spectra when mixed with CEWL
aggregates formed in fibrillation conditions [60 C, 1200 rpm, 48h] with
various additives as indicated. (Bottom): Normalized ThT fluorescence
when mixed with CEWL aggregates formed in fibrillation conditions [60
C, 300 rpm, 48h] with various additives as indicated.
3.11 TEM Analysis
All the samples studied above by ThT and ANS fluorescence
assays were also analysed by TEM in order to assess the extent of
aggregation and morphology of different aggregates formed. In
general, a big surface of TEM grids were analysed and many
images were taken. The images shown in manuscript are the most
representative of the entire sample. CEWL was observed to form
large amyloids in fibrillation conditions (Fig. 11, panel A). In
presence of either HL1 (Fig. 11, panel B) or HL2 (Fig. 11, panel
C) CEWL show much less aggregates. HL2 was observed to be a
better inhibitor of fibrillation process than HL1 as observed by
very few aggregates in panel C of Fig. 11. This is also consistent
with the results of ThT fluorescence assay. In addition, the
samples containing the aggregate formed by CEWL in presence
of Cu complexes of HL1 and HL2 were also analysed.
Interestingly, while (L1)2Cu show reduced amyloid aggregates,
(L2)2Cu forms amorphous aggregates. Since, amorphous
aggregates are known to reduce ThT fluorescence [4], these
results are consistent with ThT and ANS fluorescence assay.
Overall, both HL1 and HL2 and their Cu complexes are good
inhibitors of amyloid formation.
3.10. ANS fluorescence assay to study inhibition
1-Anilinonaphthalene-8-sulphonate (ANS) fluorescence assays
were also performed to study the inhibition in order to obtain
more information about the nature of aggregates formed. ANS
and Bis-ANS have been widely used non-covalent fluorescent
probes for studying protein fibrillation. ANS contains aniline and
naphthelene as hydrophobic moieties whereas sulphonate group
as polar part. It interacts through its sulphonate part with the
positively charged amino acids such as lysine, arginine and
histidine. The aromatic hydrophobic part coordinates with the
nonpolar entities of proteins. The reported excitation wavelength
of ANS is 350-380 nm and the emission maximum of ANS in
water is 500 nm. It is known that ANS is virtually non-
fluorescent in aqueous medium but becomes highly fluorescent in
nonpolar organic solvents due to its binding to hydrophobic
regions of proteins resulting into ~100 fold increase in its
fluorescence quantum yield and a blue shift of emission
wavelength (from 530 nm to 475 nm) [32]. Also, it is reported
that when ANS is bound to protein oligomers, its emission
maximum is blue-shifted to 440 nm [33]. We recorded the UV-
vis spectra of ANS in PBS and first absorption band was
observed at 265 nm and another broad absorption band between
300-400 nm (Fig. S10, ESI). ANS exhibit an emission band at
480 nm when excited at 380 nm. A several fold increase in
fluorescence intensity of ANS at 480 nm was observed in
presence of CEWL fibrils. Monomeric CEWL did not cause any
such increase (Fig. S11, ESI). Increase in fluorescence intensity
at 480 nm was used to quantify the CEWL fibrils formed either in
presence or absence of inhibitors. It was interesting to observe
that HL1 and HL2 caused >90% inhibition to fibrillation (Fig.
10). These results are consistent with the ThT data obtained
above. (L1)2Cu and (L2)2Cu show >70% and >80% inhibition of
CEWL fibrillation, respectively. Interestingly, a slight blue shift
in ANS spectra was observed for the sample containing (L1)2Cu
complex (Fig. S12, ESI).
Fig. 11 TEM images of CEWL species from inhibition experiments
([CEWL] = 20 μM, [compound] = 20 μM, 48 h, 60 °C). All scale bars
represent 500 nm. Panels: A. CEWL; B. CEWL + HL1; C. CEWL +
HL2; D. CEWL + (L1)2Cu; E. CEWL + (L2)2Cu.

3.12. Cell toxicity Studies
Cu as 6-membered chelate ring in case of L2-Cu complex binds
marginally better than the 5-membered one. HL1 stains CEWL
aggregates very well and suggest a strong binding nature of such
molecules towards amyloid plaques. The inhibitory effect of
HL1, HL2 and their Cu complexes on CEWL fibril formation
was studied. Interestingly, HL1 and HL2 and their Cu complexes
are excellent anti-amyloidogenic agents as established by ThT
and ANS fluorescence assays. Cellular studies suggest that
although this molecular design could be non-toxic, nitro group
substitution results into decreased cell survival. Non-nitro
analogues were less toxic. These studies will be helpful for the
design of future anti-aggregation multifunctional metal chelators.
Cell toxicity studies were also performed with Neuro2A (N2A)
cells using an Alamar Blue reagent to check the toxic effects of
HL1 and HL2 as well as their non-nitro analogues. The objectives
with cellular studies were to check if, in general, our molecular
design is toxic to cells or not and also to see if there is any effect
of azo- and nitro- groups on cellular toxicity. The cell toxicity
data is given in Table S2, ESI and shown in Fig. 12. First, we
investigated the cellular toxicity of 4-(phenyldiazenyl)phenol at
various concentrations ranging from 2 µM to 10 µM and
observed a >90% cell survival (Fig. 12, A1-A3). This suggests
that 4-(phenyldiazenyl)phenol as such is non-toxic molecular
framework. However, while 5-(phenyldiazenyl)quinolin-8-ol (the
non-nitro analogue of HL1) was found to exhibit a ~90% cell
survival at 2 µM concentration, higher concentrations lead to
significant cell toxicity (Fig. 12, B1-B3). HL1 was found to
exhibit an even poor cell survival in the range of 20-30% only at
the different concentrations tested (Fig. 12, C1-C3). This study
clearly suggests that having a NO2 group substitution is toxic to
cells. The same pattern was also observed for the HL2 and its
non-nitro analogue (Fig. 12, D1-D3 & E1-E3). Interestingly, 4-
(phenyldiazenyl)benzene-1,3-diol (the non-nitro analogue of
HL2) was found to be non-toxic with a >90% cell survival at
different concentrations. HL2 lead to only ~45% cell survival at
various concentrations (Fig. 12, E1-E3).
Acknowledgement
AKS acknowledge Science and Engineering Research
Board for financial support (grant ref.: EMR/2016/001452) and
DST-INSPIRE research grant (IFA-13, CH-97). MR
acknowledges Central University of Rajasthan for fellowship.
AKS is thankful to Dr. Jaykant Yadav for helping with amyloid
preparation. TKR thanks to Prof. R. T. Pardasani, and CMSD,
University of Hyderabad for computational support under DST-
FIST grant. L.M.M. acknowledges research funding from NIH
(R01GM114588) and the Alzheimer’s Association (NIRG 12-
259199). AKS is DST-INSPIRE faculty awardee. The paper is
dedicated to the memories of Dr. Sunil G. Naik.
Supporting Information
UV-vis spectra of HL1 and HL2 and their copper complexes,
Job’s plot spectra, ANS spectra, Table of comparison of
theoretical and experimental structural parameters, Structures
depicting 5- and 6-membered ring drawn for (L2)2Cu complex,
Table of Cell toxicity data.
140
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The highlights of this manuscript are following:
1. Metal chelation therapy and inorganic
chemistry of Alzheimer’s disease has
garnered special attention in recent years.
We report development of two new
multifunctional chelators HL1 and HL2
having an amyloid binding framework of
Congo-Red inspired azo-stilbene.
2. The binding to amyloids of HL1/HL2
framework was established by direct
staining
of
amyloids
and
spectroscopically.
3. Metal binding properties of HL1 and HL2
are investigated by Job’s plot methods for
stoichiometry determination, stability
constants determination using pH-
spectrophotometry, and DFT studies for
Cu2+ binding are described in details.
4. Inhibitory effects of HL1 and HL2 and
their isolated metal complexes L1-Cu and
L2-Cu on CEWL fibrillation were also
studied using ThT, ANS assay and by
TEM. Last, preliminary cell toxicity
studies were also performed.
Congo-red inspired multifunctional
compounds for metal chelation, amyloid
staining and inhibition of chicken egg
white lysozyme fibrillation