Synthesis and Structure Activity Relationship of Pyridazine-Based Inhibitors for Elucidating the Mechanism of Amyloid Inhibition

Article abstract of DOI:10.1021/acs.chemrestox.8b00210

Conformational diseases, constituting a large number of diseases, have been connected with protein misfolding, leading to aggregation known as amyloid fibrils. Mainly due to the lack of detailed molecular mechanisms, there has not been an effective drug to combat amyloid-associated diseases. Recently, a small organic pyridazine-based molecule (RS-0406) has shown significant reductions in amyloid fibrils in both in vitro and in vivo animal studies. However, no information on molecular details of inhibition for the small molecule has been reported. In this work, we have decided to explore structure-activity relationship of pyridazine-based compounds to investigate structural parameters for amyloid inhibition. A number of closely related derivatives of RS-0406 were designed and synthesized to delineate the roles of structural properties, including bulkiness and halogen bonding, hydrogen-bonding ability, and the position of substituents on the flanking aromatic rings of the synthetic molecules. To examine the effectiveness of the synthesized compounds, amyloid fibril formation of hen egg white lysozyme was measured in the presence of each synthetic molecule. Our results indicated that in addition to the type of the aryl substituent, their positions on the ring were also important for their inhibitory roles in amyloid fibrils formation. Moreover, a fluorinated compound turned out to be a more effective kinetic inhibitor, displaying a delayed fibril nucleation than the original lead compound. Furthermore, biochemical structural analyses and molecular dynamics simulation revealed that the pyridazine-based compounds may mediate the inhibition of amyloid fibrils through stabilization of the protein monomer during partially unfolded state. The cytotoxicity assay revealed that the amounts of amyloid intermediates were reduced in the presence of the synthetic compounds. Eventually, IC₅₀ values were obtained for the synthetic compounds, and quantitative structure-activity relationship method was employed to suggest more effective amyloid inhibitors.

Full text of DOI:10.1021/acs.chemrestox.8b00210

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Article
Synthesis and Structure Activity Relationship of Pyridazine-Based
Inhibitors for Elucidating the Mechanism of Amyloid Inhibition
Hamid R. Kalhor, and Alireza Nazari Khodadadi
Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.8b00210 • Publication Date (Web): 24 Aug 2018
Downloaded from http://pubs.acs.org on August 25, 2018
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Synthesis and Structure Activity Relationship of PyridazineꢀBased Inhibitors for
Elucidating the Mechanism of Amyloid Inhibition
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Hamid Reza Kalhor*, Alireza Nazari Khodadadi
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Biochemistry Research Laboratory, Department of Chemistry,
Sharif University of Technology, Tehran, Iran
Corresponding Author:
* Email: kalhor@sharif.edu. Tel: +982166165315. Fax: +982166165315
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Abstract
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Conformational diseases, constituting a large number of diseases, have been connected with
protein misfolding, leading to aggregation known as amyloid fibrils. Mainly due to the lack of
detailed molecular mechanisms, there has not been an effective drug to combat amyloidꢀ
associated diseases. Recently, a small organic pyridazineꢀbased molecule (RSꢀ0406) has shown
significant reductions in amyloid fibrils in both in vitro and in vivo animal studies. However, no
information on molecular details of inhibition for the small molecule has been reported. In this
work, we have decided to explore Structure Activity Relationship (SAR) of pyridazineꢀbased
compounds to investigate structural parameters for amyloid inhibition. A number of closely
related derivatives of RSꢀ0406 were designed and synthesized to delineate the roles of structural
properties, including bulkiness and halogen bonding, hydrogen bonding ability, and the position
of substituents on the flanking aromatic rings of the synthetic molecules. To examine the
effectiveness of the synthesized compounds, amyloid fibril formation of Hen Egg White
Lysozyme (HEWL) was measured in the presence of each synthetic molecule. Our results
indicated that in addition to the type of the aryl substituent, their positions on the ring were also
important for their inhibitory roles in amyloid fibrils formation. Moreover, a fluorinated
compound turned out to be a more effective kinetic inhibitor, displaying a delayed fibril
nucleation than the original lead compound. Furthermore, biochemical structural analyses and
molecular dynamics simulation revealed that the pyridazineꢀbased compounds may mediate the
inhibition of amyloid fibrils through stabilization of the protein monomer during partially
unfolded state. The cytotoxicity assay revealed that the amounts of amyloid intermediates were
reduced in the presence of the synthetic compounds. Eventually, IC50 values were obtained for
the synthetic compounds, and QSAR method was employed to suggest more effective amyloid
inhibitors.
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Keywords: Protein misfolding, Pyridazine, Amyloid inhibitor, Kinetic inhibitors, Lysozyme,
QSAR
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INTRODUCTION
In order for a protein to gain its native function, it must undergo a folding process. To
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assist the folding process, a cell exploits a number of ways including molecular chaperons and
osmolytes1–3. However, in certain conditions, a protein (or a polypeptide) would not fold into its
native structure, leading to its misfolding4,5. Indeed, the misfolding process has been shown to be
accompanied by conformational change in proteins or polypeptides6. In humans, a number of
conformational diseases have been identified in which misfolded proteins or polypeptides are
accumulated as insoluble fibrils; these human debilitating diseases include Alzheimer’s,
Parkinson, type 2 diabetes, and mad cow7,8.Neurotoxicity has been shown to be the hallmark of
amyloid associated disesases9. Amyloid fibrils are unique insoluble, unbranched, and threadlike
fibrils with 70–120 Å diameters; since these fibrils are insoluble, it has been difficult to obtain
the atomistic details of their structures and interactions with other small molecules10,11. However,
using solid state NMR, double horseshoeꢀlike (Sꢀshape structure) cross–βꢀsheet structure in
amyloid fibrils has been reported12,13. The kinetics of amyloid formation can be divided into
three phases; the initial phase, called lag phase, is considered the rate limiting step in which
proteins become partially unfolded, leading to nucleus formation upon which other protein
monomers are assembled. In the second phase, known as elongation step, the heterogeneous
population of soluble oligomers are formed, producing protofibrils, and finally, in the last phase,
insoluble amyloid fibrils are formed in the process14,15. There exist a number of techniques that
can be exploited to detect different stages of amyloid formation including spectroscopic methods
to a much simpler approach as gel electrophoresis16,17. Proteins and polypeptides that have been
used widely as amyloid models include amyloid βꢀpeptide, amylin, insulin, transthyretin, human
lysozyme, and Hen Egg White lysozyme (HEWL)18–21. HEWL has been used extensively as an
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amyloid model mainly due to its well characterized misfolding pathways22. HEWL, which
mainly possesses alpha helices, can be easily converted to amyloid fibrils at low pH and high
temperature (57 °C) during several days. HEWL fibrillation has been highly used to investigate
the effects of inhibitors on the physical and chemical process of protein aggregation23. In
addition, it has been shown that HEWL aggregates can cause neurotoxicity similar to the
aggregation of amyloid beta peptide24.
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In the past few years, a number of studies have shown that both natural and synthetic
compounds can be exploited to inhibit or reduce amyloid formation25–29..In general, the inhibitors
fall into two categories: thermodynamic inhibitors which reduce the total amount of amyloid
fibrils, and kinetic inhibitors that could extend the time for nucleation of amyloid formation30,31
.
In recent reports in screening of molecular libraries, a pyridazineꢀbased belonging to small
organic molecule, coined RSꢀ0406, was identified as an effective amyloid inhibitor both in vitro
and animal studies32,33. This small organic molecule contains three aromatic rings, with
pyridazine as its central ring conjuring up a symmetrical structure with two hydroxyl moieties on
the outer rings. Although RSꢀ0406 is now in clinical trial phase II for further considerations,
there has not been any report illustrating experimental data on the molecular mechanisms or its
biochemical interactions in amyloid inhibition. It has been demonstrated that pyridazineꢀcore
compounds play special roles in a number of drugs that interact with proteins. Strong hydrogen
bonding ability, high dipolar moment, and higher solubility in water have made pyridazineꢀbased
compounds more intriguingly drugs, compared to other diazines34,35. In this work, several
symmetrical pyridazineꢀbased derivatives, analogous to RSꢀ0406, were synthesized, purified,
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and characterized by H and C NMR and HRMS analyses. The synthetic compounds were
examined for their effects on amyloid inhibition using ThioflavinꢀT (ThꢀT) binding, gel
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electrophoresis, and TEM. Furthermore, biochemical effects of the selected synthetic compounds
were examined by ANS binding and CD analyses. Moreover molecular dynamics were used to
look into the details of interactions between HEWL protein and synthetic compounds. The
results illustrated that in addition to the ability of hydrogenꢀbonding and hydrophobic
interactions, the position and the size of substituents play important roles in the inhibition of
amyloid formation. Furthermore, a fluorine atom in place of hydroxy group can be more
effective as a kinetic inhibitor (the ability to obstruct nucleation of amyloid fibrils). At the end,
using the IC50 measurements and the molecular structural descriptors of synthesized
compounds, QSAR model was generated; a compound containing carboxylic moiety at the para
position of aromatic ring was suggested to possess more inhibitory effect than the lead
compound. The predicted compound was synthesized, and when it was examined for its antiꢀ
amyloid activity, the compound was not soluble in the acidic medium, and its effect was
investigated in alkaline medium.
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Materials and Methods
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Synthesis of organic compounds:
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General information: 3ꢀ6ꢀdichloropyridazine was purchased from Sigma Aldrich. All of the
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other compounds were purchased from Merck, Germany. 1H and 13C NMR spectra were recorded
on a Bruker 500 instrument in DMSOꢀd6 solvent, With 1H at 500 MHz, and 13C at 125 MHz. 1H
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and C NMR chemical shifts were reported in ppm and were referenced to the residual solvent
signals (1H:(CD3)2SO at 2.50 ppm, CD3OD at 3.31 ppm, 13C: (CD3)2SO at 39.52 ppm).
General procedure for the synthesis of the pyridazineꢀbased compounds (Pyꢀ1 to Pyꢀ15)
Synthesis of all compounds was performed in the same setup while applying different methods
for isolation and purification. For setting up the reactions, the derivatives of aniline (2ꢀ3 eq) were
dissolved in 20 mL of 2ꢀethoxyethanol (2EE) and the solution was stirred at 60 °C for 30 min.
Subsequently, 3,6ꢀdichloropyridazine(1eq) was added, and the reaction was refluxed for 18ꢀ48 h.
N3, N6ꢀdi (3ꢀhydroxyphenyl) pyridazineꢀ3, 6ꢀdiamine (Pyꢀ1): The reaction was set up
according to the general procedure. 3ꢀaminophenol (1.10 g) and 3, 6ꢀdichloropyridazine (0.75 g)
were used to synthesize Pyꢀ1. After 24 h, the solvent was removed by vacuum evaporation. The
precipitate was washed with ethanol, dried in an oven, and subsequently purified by column
chromatography in (1:1 ethyl acetate/nꢀhexane) on silica. In a second procedure, the precipitate
after evaporation was recrystallized from ethanol, and a few drops of H2O (less than a milliliter)
was added to obtain a brown powder (1.24 g, yield 85%). 1HNMR (DMSOꢀd6, 500MHz): δ 6.59
(d, j=5, 15H), 6.95 (d, j=10, 13H), 7.00(s, 11H), 7.17 (t, j=10, 14H), 7.59 (s, 1H), 9.70 (s, OH),
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10.42 (s, 7H). 13C NMR (DMSOꢀd6, 125 MHz): δ108.99, 112.49, 127.79, 127.96, 130.39,
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139.62, 150.24, and 159.12. HRMS (ESI ), [M+H] calcd, 295.1189, found, 295.1189.
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N3, N6ꢀdi (4ꢀhydroxyphenyl) pyridazineꢀ3, 6ꢀdiamine (Pyꢀ2): The reaction was set up
according to the general procedure. 4ꢀaminophenol (1.10 g) and 3, 6ꢀdichloropyridazine (0.75 g)
were used to obtain a green powder (1.06 g, yield 72%). After 24 h and before the evaporation of
the solvent, ethanol was added dropꢀwise (the same amount as the volume of solvent) to the
mixture of reaction until the crude precipitate was formed. The crude solid was filtered, washed
with ethanol and was subsequently recrystallized from acetone. 1HNMR (DMSOꢀd6, 500MHz):
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+
δ 6.81(d, j=5, 11H, 15H), 7.30 (bs, 12H, 14H), 7.60 (s, 1H), 9.64 (s, 7H), 10.40 (s, 22H). HRMS (ESI ),
+
[M+H] calcd, 295.1189, found, 295.1189.
N3, N6ꢀdi (3ꢀmethoxyphenyl) pyridazineꢀ3, 6ꢀdiamine (Pyꢀ3): The reaction was set up
according to the general procedure. Mꢀanisidine (1.2 mL) and 3, 6ꢀdichloropyridazine (0.75 g)
were used to obtain a gray powder (1.25 g, yield 78%). After 28 h, the reaction was stopped, and
the solvent was removed by vacuum evaporation; the dried precipitate was stirred in basic water
(NaOH 1.25 M) containing a few drops of acetone for 1h. Subsequently, the solution was
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centrifuged at 400 rpm for 5 min. The precipitate was recrystallized from acetone. HNMR
(DMSOꢀd6, 500MHz): δ 3.75 (s, OCH3), 6.48(d, j=5, 15H) 7.12(s, 11H), 7.18(t, j=15, 13H),
7.26(d, j=10, 14H), 7.42 (s, 4H), 9.00 (s, 7H). 13C NMR (DMSOꢀd6, 125 MHz): δ 55.99, 104.67,
+
+
106.50, 111.33, 121.36, 130.47, 143.88, 153.33, and 160.85. HRMS (ESI ), [M+H] calcd,
323.1502, found, 323.1503.
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N3, N6ꢀdiphenylpyridazineꢀ3, 6ꢀdiamine (Pyꢀ4)36: Following the same procedure for the
synthesis of Pyꢀ3, aniline (1 mL ) and 3, 6ꢀdichloropyridazine (0.75 g) were used to obtain a
yellow powder (0.74 g, yield 57%). 1HNMR (DMSOꢀd6, 500MHz): δ 7.15(t, j=15, 13H), 7.40(t,
j=15, 12H, 14H), 7.57 (d, j=10, 11H, 15H), 7.78 (s, 4H), 10.72 (s, 7H).13C NMR (DMSOꢀd6, 125
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+
+
MHz): δ 121.897, 125.212, 127.696, 130.363, 138.978, 150.348. HRMS (ESI ), [M+H] calcd,
263.1291, found, 263.1291.
N3, N6ꢀdi (4ꢀbromophenyl) pyridazineꢀ3, 6ꢀdiamine (Pyꢀ5): Following the same procedure for
the synthesis of Pyꢀ3, in a similar scale, a gray powder (1.42 g, yield 68%) was obtained after 48
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h. HNMR (DMSOꢀd6, 500MHz): δ 7.14(s, 4H), 7.46(d, j= 10, 11H, 15H), 7.75 (d, j=10, 12H, 14H),
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9.21(s, 7H). C NMR (DMSOꢀd6, 125 MHz): δ 112.40, 120.61, 121.81, 132.45, 141.85, 153.03.
+
+
and 153.426. HRMS (ESI ), [M+H] calcd, 418.9501, found, 418.9499.
N3, N6ꢀdi (3,4ꢀdichlorophenyl) pyridazineꢀ3, 6ꢀdiamine (Pyꢀ6): Applying the same procedure
for the synthesis of Pyꢀ3; 3,4ꢀ dichloroaniline (2.00 g) and 3,6ꢀdichloropyridazine(0.75 g) were
used to obtain a brown powder (1.10 g, yield 55%) in 48 h. 1HNMR (DMSOꢀd6, 500MHz): δ
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7.15(s, 1H), 7.49(d, j=10, 15H), 7.62 (d, j=10, 14H), 8.25 (s, 11H), C NMR (DMSOꢀd6, 125
MHz): δ 118.806, 119.373, 121.803, 122.105, 131.470, 131.934, 142.647, and 153.200. HRMS
+
+
(ESI ), [M+H] calcd, 398.9732, found, 398.9737.
N3, N6ꢀdi (3ꢀfluorophenyl) pyridazineꢀ3, 6ꢀdiamine (Pyꢀ7). Synthesis of Pyꢀ7 was set up
according to the general procedure. 3ꢀfluoroaniline (1 mL) and 3, 6ꢀdichloropyridazine (0.75 g)
were used to give a yellow powder (0.44 g, yield 29%) in 48 h. The precipitate after vacuum
evaporation was stirred in 20 mL NaOH (1.25 M), without acetone. The precipitate was
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extracted using centrifugation (400 rpm for 5 min). Subsequently, the precipitate was dried and
recrystallized from acetic acid or purified by column chromatography in (3:1:0.1 ethyl acetate: nꢀ
hexane: acetic acid) on silica.1HNMR (DMSOꢀd6, 500MHz): δ 6.95(t, j=15, 14H) 7.34(d, j=5,
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15H), 7.43(q, j=25, 13H), 7.64(d, j=10, 11H), 7.80(s, 1H), 11.00(s, 7H). C NMR (DMSOꢀd6, 125
+
+
MHz): δ 117.60, 127.38, 131.87, 141.87, 150.40, 162.47, and 167.49. HRMS (ESI ), [M+H]
calcd, 299.1102, found, 299.1102.
N3, N6ꢀdi (3ꢀnitrophenyl) pyridazineꢀ3, 6ꢀdiamine (Pyꢀ8): Following the procedure for the
synthesis of Pyꢀ3, at the same scale, a dark brown powder (0.54 g, yield 30%) was obtained after
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48 h. HNMR (DMSOꢀd6, 500MHz): δ 7.23(s, 1H), 7.59 (t, j=15, 14H), 7.75 (d, j=5, 15H), 8.08 (d,
j=10, 13H), 8.86 (s, 11H), 9.66 (s, 7H). 13C NMR (DMSOꢀd6, 125 MHz): δ 112.300, 115.697,
+
+
121.954, 124.678, 121.053, 143.670, 149.358, and 153.426. HRMS (ESI ), [M+H] calcd,
353.0992, found, 353.0993.
N3, N6ꢀdi (3ꢀchlorophenyl) pyridazineꢀ3, 6ꢀdiamine (Pyꢀ9): Following the same procedure for
the synthesis of Pyꢀ3, at the same scale, a gray powder (0.33 g, yield 20%) was obtained after 48
1
h. HNMR (DMSOꢀd6, 500MHz): δ 6.91(d, j=10, 15H) 7.22(s, 1H), 7.29(t, j=15, 14H), 7.58(d,
j=10, 13H), 8.08(s, 11H), 9.63(s, 7H). 13C NMR (DMSOꢀd6, 125 MHz): δ 116.87, 117.67,
+
+
120.37, 121.65, 131.33, 134.04, 144.20, and 153.15. HRMS (ESI ), [M+H] calcd, 331.0511,
found, 331.0510.
N3, N6ꢀdi (4ꢀchlorophenyl) pyridazineꢀ3, 6ꢀdiamine (Pyꢀ10): Following the procedure for the
synthesis of Pyꢀ3, at the same scale, gray powder (0.74 g, yield 45%) was obtained after 48 h.
1HNMR (DMSOꢀd6, 500MHz): δ 7.11(s, 1H), 7.31(d, j=15, 11H, 15H), 7.79(d, j=15, 12H, 14H),
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9.14(s, 7H). C NMR (DMSOꢀd6, 125 MHz): δ 120.03, 121.38, 124.24, 129.53, 141.47, and
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+
+
153.04. HRMS (ESI ), [M+H] calcd, 331.0511, found, 331.0509.
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N3, N6ꢀdi (4ꢀmethyl benzoate) pyridazineꢀ3, 6ꢀdiamine (Pyꢀ11): Methyl 4ꢀaminobenzoate
was synthesized from 4ꢀaminobenzoic acid in the presence of methanol and thionylchloride37.
The reaction was set up according to the general procedure. Methyl 4ꢀaminobenzoate (1.5 g) and
3, 6ꢀdichloropyridazine (0.75 g) were used to obtain a light brown powder (0.47 g, yield 25%).
After 48 h, the reaction was stopped, and the solvent was removed by vacuum evaporation; the
viscous sediment was washed in methanol. The methanol was omitted with a syringe, and the
precipitate was washed several times with methanol; subsequently, the precipitate was dried and
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recrystallized from methanol containing a few drops of H2O. HNMR (DMSOꢀd6, 500MHz):
1HNMR (DMSOꢀd6, 500MHz): δ 3.83(s, 30H).7.68 (s, 1H), 7.78(d, j=5, 11H, 15H), 7.95(d,
j=10, 12H, 14H), 10.64(s, 7H). 13C NMR (DMSOꢀd6, 125 MHz): δ 52.00, 116.88, 120.87,
+
+
130.65, 146.16, 152.39, and 166.77. HRMS (ESI ), [M+H] calcd, 379.1400, found, 379.1400.
N3, N6ꢀdi (4ꢀnitrophenyl) pyridazineꢀ3, 6ꢀdiamine (Pyꢀ12): The reaction was set up according
to the general procedure. 4ꢀnitroaniline (1.5 g) and 3, 6ꢀdichloropyridazine (0.75 g) were used to
obtain an orange powder (0.53 g, yield 30%). After 18 h, the reaction was stopped and it was
allowed to cool off. Afterwards, using filter paper, the solvent was removed. The precipitate was
washed with ethanol and NaOH (1.25M); the precipitate was collected and dried in an oven, and
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it was recrystallized from methanol containing a few dross of NaOH (1.25M). . HNMR
(DMSOꢀd6, 500MHz): δ 7.33 (s, 1H), 7.96(d, j=10, 11H, 15H), 8.24 (d, j=10, 12H, 14H),
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10.02(s, 7H). C NMR (DMSOꢀd6, 125 MHz): δ 117.05, 117.44, 121.57, 139.73, 148.33, and
152.88.
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N3, N6ꢀdi (4ꢀfluorophenyl) pyridazineꢀ3, 6ꢀdiamine (Pyꢀ13): Following the procedure for
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48 h. HNMR (DMSOꢀd6, 500MHz): δ 7.11 (t, j=20 12H, 14H), 7.20 (s, 1H), 7.80(d, j=5, 11H,
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15H), 9.30(s, 7H).. C NMR (DMSOꢀd6, 125 MHz): δ 115.14, 119.14, 120.04, 138.22, 152.17,
+
+
and 156.16. HRMS (ESI ), [M+H] calcd, 299.1102, found, 299.1105.
N3, N6ꢀdi (3ꢀmethylphenyl) pyridazineꢀ3, 6ꢀdiamine (Pyꢀ14): Applying the same procedure
for synthesis of Pyꢀ3, at the same scale, a gray powder (0.56 g, yield 39%) was obtained after 48
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h. HNMR (DMSOꢀd6, 500MHz): δ 2.24(22H), 6.70(d, j=5, 13H), 7.09(s, 11H), 7.16(t, j=15,
13
14H), 7.52(d, j=10, 15H), 8.84(s, 1H), 9.63(s, 7H), C NMR (DMSOꢀd6, 125 MHz): δ 21.87,
+
+
115.22, 118.36, 120.60, 121.34, 128.97, 138.13, 142.12 and 152.67. HRMS (ESI ), [M+H]
calcd, 291.1604, found, 291.1601.
N3, N6ꢀdi (4ꢀbenzoic acid) pyridazineꢀ3, 6ꢀdiamine (Pyꢀ15): Pyꢀ11 (0.3 g) was dissolved in 5
mL DMSO; an aqueous solution of LiOH (0.65M) was added dropꢀwise as the same volume of
DMSO. After 24 h, the reaction was stopped by adding 20 mL of H2O and 5 mL of diethyl ether;
HCl were used to adjust pH of the aqueous phase (pH 2). Finally, the aqueous phase was
saturated with CHCl3. The brown solid precipitate was filtered (0.28 g, yield 93%). In a second
method which was similar to the synthesis of Pyꢀ12, with the only difference that NaOH was not
used for purification. 1HNMR (D2O, 500MHz): δ 6.80 (s, 1H), 7.25(d, 11H, 15H), 77.60(d, 12H,
14H), 13C NMR (D2O, 125 MHz): δ 117.11, 121.97, 128.91, 130.05, 143.00, 125.18, and
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175.35. HNMR (DMSOꢀd6, 500MHz): 7.69 (d, j=10, 11H, 15H), 7.84 (s, 1H), 7.96 (d, j=10,
13
12H, 14H), 11.00 (s, 7H), broad peak in 12.00 (carboxylic acid proton), C NMR (DMSOꢀd6,
+
+
125 MHz): 119.76, 125.75, 126.59, 131.20, 142.69, 150.13, 162.60. HRMS (ESI ), [M+H]
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calcd, 351.1087, found, 351.1088. IR (KBr) ν (cm−1), for Pyꢀ11(Ester): 3330 (ν, NH), 2950 (ν,
CꢀH), 1700 (ν, C꞊O), and for Pyꢀ15(Acid): 2600ꢀ3500(ν, OH), 1630 (ν, C꞊O).
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Preparation of stock solution from organic compounds
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All the synthetic compounds were dissolved in DMSO to the final concentration of 13900 µM
and then they were diluted with buffer to the specific final concentration.
Sample preparation for amyloid fibril formation:
Acidic condition: Solutions containing HEWL (349 µM) and the synthetic compound (349 µM)
were prepared in 100 mM of glycine buffer at pH 2.5. The solution was incubated at 57 °C for 10
days; samples were taken out every 24 h and stored at ꢀ20 °C for further analysis. DMSO (2.5 %
v/v) was used for dissolving the synthetic compounds in aqueous medium and thus a sample
containing 2.5% DMSO as a control was employed to determine whether DMSO itself had an
effect on the HEWL aggregation.
Basic condition: HEWL fibril formation in the basic medium was prepared as previously
reported38. Briefly, HEWL (349 ꢁM) was incubated for 14 h at 25ꢀ28ºC with 3 fold molar of the
synthetic compounds in phosphate buffer (10 mM pH 7.3). The incubated samples diluted 3 fold
with phosphate buffer (50 mM, pH 12.2) to induce the aggregation of HEWL. Then the solution
was incubated at 25ꢀ28 °C for 10 days.
Thioflavin T fluorescence Assay: For ThꢀT fluorescence intensity analysis; 15 µL from
aliquoted samples and 185 µL from stock solution of ThꢀT in sodium phosphate (NaH2PO4 50
mM, Na2HPO4 50 mM, ThꢀT 18µM) was mixed; subsequently, measurements were performed
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by exciting wavelength at 440 nm and emission wavelength at 485 nm using Synergy H4.To
check the error bar, tree independent experiment were used for each inhibitor39
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IC50 calculation: IC50 values were determined in two ways. In the first method, the ThꢀT
fluorescence assay was performed at 7 concentrations (1nM to 1mM) for each inhibitor and
2mg/mL HEWL in two or three independent experiments for each concentration of inhibitors.
The IC50 values were obtained from dose−response curves. In the second method the IC50
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values were determined with QSAR method40,41
.
Agarose gel electrophoresis: The procedure was performed as explained in details elsewhere39.
Briefly, 1% agarose gel using 0.1% SDS in buffer A (200mM Tris and 200mM glycine) was
prepared. The gel was run at 70 V for 2 h in buffer A containing 0.1 % SDS. The gel was stained
by Coomassie Brilliant Blue for 2 h and destined over nigh in destining solution.
Circular Dichroism (CD): HEWL samples were diluted in the final concentration 0.2mg/mL in
glycine buffer 100 mM and pH 2.5; then spectra were recorded from 190 to 260 nm (farꢀUV)
using CD spectrometer Aviv, USA modelꢀ125 and 1 mm path cell at room temperature. Spectra
for buffer without and with synthetic compounds at the same concentration were recorded.
ANS Fluorescence Assay: A stock solution of ANS was prepared at 0.01M by dissolving ANS
in absolute ethanol and filtered using a 0.2 µm cellulose filter. A final solution 100mM was
diluted by deionized water.). For ANS fluorescence intensity analysis; 10 µL from aliquoted
samples and 200 µL from 100mM stock solution was mixed, and subsequently, measurements
were obtained by excitation wavelength at 365 nm and emission spectra at 430 to 550 nm.
Transmission Electronic Microscopy (TEM): Samples were diluted 5 fold by absolute ethanol;
subsequently 10 µL of the diluted sample were placed on a copper 300 mesh grid, covered with
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carbonꢀcoated formvar film. After a few minutes, the grids were washed with deionized water
then stained with 1% (w/v) uranyl acetate, and the grid was incubated 30 min at room
temperature for drying. The prepared grids were viewed using Philips CM300 instrument at a
voltage of 80kV.
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MTT cell viability assay: The PC12 cell (as a model for neurosecretion cells), obtained from
Stem Cell Technology Center (Bonyakhteh, Tehran, Iran). The viability of PC12 cells was
determined using the MTT assay. The cells were cultured in DMEM (high glucose) medium,
supplemented with penicillinꢀstreptomycin (PenꢀStrep) 100U/mL and incubated at 37˚C in a 5%
CO2 humidified atmosphere. For cytotoxicity assay, HEWL native, synthetic compounds, and
aliquots incubated samples (fibrillation of HEWL in absence and presence of any synthetic
compound) were added to the cells (in a final concentration of 20 ꢁM) and incubated in the 48ꢀ
well plates for 24 h. Subsequently, 25 ꢁl of MTT (from stock 5 mg/mL in PBS) were added to
250 ꢁl of medium and incubated for 3 h. Finally, cells were treated with DMSO (10 min) and
absorbance was measured at 570 nm.
Docking and molecular dynamics simulation: The structure of HEWL protein was
downloaded from Protein Data Bank online server (PDB ID: 1AKI). The structures of the small
molecules were obtained from PRODRG 2.x online server. In order to assemble proteinꢀsmall
molecule complexes, the small molecules were docked to the protein using Auto Dock Tools
1.5.6 software. The resulting complexes as well as HEWL protein with no small molecule as a
control system, were subjected to molecular dynamics simulations using GROMACS package.
The OPLSaa force field was chosen; in each case, hydrogen atoms were added and titrable
residues were protonated corresponding to pH 2; the disulfide bonds of Cysteine residues were in
nonꢀreduced form. The protein (with or without small molecule) was centered in a cubic box
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with faces at least 1.0 nm apart from the closest atom of the protein. The resulting systems were
solvated by SPC216 water and neutralized by adding sufficient amount of ions; in addition,
further ions pairs (Na+ and Clꢀ) were added to reach the ionic concentration of 0.1 mol/L. Each
system was energy minimized by the steepest descent integrator and emtol value of 100.0
kJ/mol/nm. After minimization, 100 ps equilibration under NVT ensemble was carried out by
leapꢀfrog integrator, the temperature was set at 331 K. Furthermore, 100ps equilibration under
NPT ensemble was done using leapꢀfrog integrator, in which temperature and pressure were at
331K and 1.0 bar respectively. In each equilibration steps, position restraints on protein and the
small molecule atoms were applied. Then, the systems were simulated under NPT ensemble with
no position restraints and at the same temperature and pressure. The simulation time was 40ns in
each case. In all equilibration and MD simulation steps, integration time was assigned to be 2fs,
for equilibrations, velocities, coordinates, and energies were saved every 1.0ps and for MD
simulation, energies were saved every 10.0 ps. All bonds were constrained using Links
algorithm. Short range electrostatic and Van der Waals interactions were updated every 20fs
(10*2fs) with varlet cutoff scheme, the cutoff value was 1.0nm for equilibrations and 1.4nm for
MD simulation. Long range electrostatic interactions were calculated using PME method with
0.16 Fourier spacing value. Temperature couplings have been done by Vꢀrescale method with a
0.1ns time constant and pressure couplings have performed by ParrinelloꢀRahman method with
2ps time constant.
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QSAR method: The compounds of constituents of the database optimized in Gaussian03 and
Spartan. All of the molecular descriptors were obtained with PaDel program. The most effective
and optimal descriptors selected with Genetic Algorithm (GA) software, and finally chemoface
software was used to make a valid model and prediction of IC5040,41
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■
RESULTS
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Design of synthetic molecules
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To better investigate the mechanism of amyloid inhibition by pyridazineꢀbased inhibitors, a
number of derivatives of RSꢀ0406 were rationally designed and synthesized. To study the role of
hydrogenꢀbonding, the hydroxyl substituent on the lead compound (Pyꢀ1) was replaced with
methoxy (Pyꢀ3), fluorine atom (Pyꢀ7), nitro (Pyꢀ8), or no substitution (Pyꢀ4), (Table 1). To
compare the role of meta (C12) versus para (C13) position on aryl rings, compounds containing
hydroxyl moiety (Pyꢀ1/ Pyꢀ2), fluorine (Pyꢀ7/ Pyꢀ13), chlorine (Pyꢀ9/ Pyꢀ10), and nitro moiety
(Pyꢀ8/Pyꢀ12) were designed (Table 1). To probe the role of bulkiness and halogenꢀbonding, four
halides containing compounds: Pyꢀ13, Pyꢀ5, Pyꢀ10, and Pyꢀ6, were designed (Table 1). Because
of the symmetrical structure of the lead compound (RSꢀ0406), all other synthesized compounds
in this work were also kept symmetrical.
Synthesis of pyridizineꢀbased compounds
Pyꢀ1 was identified in screening of a large number of organic compounds, and so there has not
been any actual report on its synthesis except a patented document42. The synthesis of this
compound and their derivatives, as explained in details in the Materials and Methods section,
was performed without using any chemical catalyst or a base. Several solvents were examined
for the yield optimization of reactions including, DMF, DMSO, HMPA, and 2ꢀethoxy ethanol
(2EE); as a result, 2ꢀethoxy ethanol turned out as the best solvent. The synthesis of symmetrical
products indicated that the highest yields pertained to Pyꢀ1, Pyꢀ2 and Pyꢀ3, and the rate of
reaction for synthesis of Pyꢀ12 was the highest. After purification, a number of analytical
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techniques were employed for characterization of the products including 1H and C NMR, and
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HRMS, as shown in Fig. S1ꢀS15.
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In vitro biological activity of pyridazineꢀbased compounds in amyloid inhibition
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In order to study the biological role of the synthesized compounds, in vitro amyloid fibril
formation was performed in the presence of the synthetic compounds. In this assay, HEWL is
converted to amyloid fibril at low pH and high temperature. The amount of ThioflavinꢀT (ThꢀT)
fluorescence reflects the amounts of fibril produced during the course of reaction; that is, the
more protein fibrils are produced, the more ThꢀT molecules are bound to the fibrils, leading to
more fluorescence emission. As shown in Fig. 1A, the compound Pyꢀ1, containing hydroxyl
moiety at meta position (C12), displayed the most inhibitory effect thermodynamically; the next
best inhibitor was identified as Pyꢀ7 which contains fluorine in place of hydroxyl group. The
worst inhibitor (for the role of hydrogen bonding), turned out to be a compound Pyꢀ8. In general,
as the substituent on the aryl ring became bulkier, the amyloid inhibitory effect of the compound
became weaker (Fig. 1B); However, Pyꢀ5 (containing bromine), was more effective than Pyꢀ10
(containing chlorine), in inhibiting the amyloid formation. The compounds containing
substitution in meta (C12) versus para (C13) position on the aryl ring were investigated by
several functional groups such as hydroxyl, fluorine, chlorine, and a nitro moiety (Fig. 1C).
Interestingly, all of the compounds which possess a meta moiety (C12) displayed more
pronounced inhibitory effects than those containing a para moiety (C13) (Fig. 1C). The kinetic
studies of the synthesized compounds were also performed (Fig. S19). Interestingly, Pyꢀ7,
containing fluoroꢀmoiety, thermodynamically inhibited amyloid formation near 70 % and
delayed the aggregation process up to 125 h; in other words, Pyꢀ7 seems to be a more effective in
extending the lag time for nucleation of amyloid fibrils than Pyꢀ1(Fig. 1D).
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In order to actually detect the intermediates in the process of amyloid formation, gel
electrophoresis was performed; the gel electrophoresis can be very informative about the three
phases of amyloid fibril formation. Amyloid fibrils, due to their large size, are mostly retained in
agarose gel well during electrophoresis whereas heterogeneous population of soluble oligomers
are spread out as smears; the native protein (as a monomer) can be detected as a single band17.
When examining the amyloid fibrils intermediates, the amount of fibril in the absence of
inhibitors was higher than in the presence of inhibitors, confirming the ThꢀT binding assay (Fig.
S16).
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To better confirm amyloid inhibition mediated by the synthetic compounds: Pyꢀ1 (the most
effective inhibitor), Pyꢀ6 (the least effective inhibitor) and Pyꢀ7 were selected for TEM analysis.
In the absence of any synthetic compound, fibril formation was abundant; the sizes of fibrils
were estimated to be 20ꢀ30 nm; however, in the presence of Pyꢀ1 the fibrils were immensely
reduced (Fig. 2A and 3C). In the presence of Pyꢀ6, the amount of fibril was not reduced
significantly compared to the control (Fig. 2B). Interestingly, in the presence of Pyꢀ7, not only
the amount of amyloid fibrils was reduced but also the presence of substantial low molecular
weight was detected; these particles may represent either nucleus or oligomers that did not
convert to amyloid fibril (Fig. 2D).
Protein structural analysis
The effects of synthesized compounds on the secondary structure of HEWL was examined using
Circular Dichromism (CD). As seen in Fig. 3A, the secondary structure of native HEWL in the
absence of any synthetic compound (control) were detected as mostly alpha helices, at the
beginning of amyloid process. However, at the late stage of amyloid formation, a remarkable
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conformational change, from alpha helix (at 208 and 222 nm) to the beta sheet (217 nm) was
observed. In the presence of Pyꢀ1, the conformational change to beta sheet was not seen as it was
observed in the control, displaying higher alpha helices at the late stage of amyloid formation
(Fig. 3B). However, the high content of alpha helix was not observed with Pyꢀ6 whereas in the
presence of Pyꢀ2 the content of alpha helices of the protein was nearly conserved, but not as
efficient as in the presence of Pyꢀ1, during the late stage of amyloid formation. The CD analysis
confirmed the importance of position of substituent for the inhibitory ability of Pyꢀ1 and Pyꢀ2 in
amyloid fibril formation (Fig. 3C and Fig. 3D). Additionally, few signals were also observed in
the presence of synthetic compounds between 190ꢀ200 nm, demonstrating significant changes in
secondary structure of the protein during amyloid fibrillation; these secondary structural changes
have been reported in the previous studies43.
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To investigate the hydrophobic exposure of protein core during amyloid formation, ANS
binding of HEWL was measured in the absence and presence of synthetic compounds.
Generally, as proteins become unfolded, during the misfolding process, more hydrophobic core
of the proteins is exposed; therefore, more ANS binding can occur, leading to the emission of
more fluorescence intensity. The ANS binding experiments indicated that as amyloid formation
progressed, in the absence of any synthetic compound, more ANS fluorescence was observed
due to increased binding of ANS to the hydrophobic regions of HEWL (Fig. S17A). In the
presence of synthetic compounds, a reduction of ANS fluorescence was detected for Pyꢀ1, Pyꢀ2,
and Pyꢀ4. However, ANS fluorescence in the presence of Pyꢀ6 displayed little reduction in the
fluorescence emissions (Fig. S17B). These results not only confirmed the ThꢀT binding assay,
but they also indicated that the protein structure in the presence of synthetic compounds was less
unfolded and exposed to the environment, giving rise to less amyloid fibril.
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To gain more insight into the effects of the synthesized compounds on cytotoxicity of amyloid
intermediates, the viability of PC12 cell line (a neuron model cell line) was examined using MTT
assay. The amyloid formation assay was performed in the presence of various synthetic
compounds, and subsequently the mixtures, along with control sample lacking any synthetic
compound, were incubated with PC12 cells. As shown in Fig. 4A, while no cytotoxicity was
observed for HEWL native, HEWL amyloid fibrils reduced the percentage of cell viability,
indicating that amyloid intermediates were toxic to cells. The HEWL samples that were
incubated in the presence of Pyꢀ1, Pyꢀ2, and Pyꢀ7 displayed higher cell viability than other
compounds, corroborating the ThꢀT binding assay. Morphological changes of PC12 cells also
confirmed the protective effect of pyridazineꢀbased compounds on cytotoxicity of aggregated
HEWL (Fig. 4B).
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Docking and MD analysis of synthetic compounds
In order to better investigate the details of molecular interactions of the synthetic compounds and
HEWL, molecular dynamic simulation was performed. Initially, Pyꢀ1, Pyꢀ7 and Pyꢀ4 were
docked onto HEWL, and simulations were carried out at high temperature and low pH
(resembling the experimental amyloidogenic conditions). RMSD results showed that in the
presence of Pyꢀ1, HEWL became more stable as compared to other two compounds (Fig. 6A and
6B). Interestingly, both Pyꢀ1 and Pyꢀ7 displayed significant noncovalent interactions with amino
acid residues that are located in the amyloidogenic region of HEWL, including hydrophobic
cluster 57ꢀ107 with Trp61 residue44,45. The types of interactions that Pyꢀ1 and Pyꢀ7 formed with
HEWL included hydrophobic, hydrogenꢀbonding and van der Waals interactions (Fig. 6C and
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Fig. 6D). The interactions of docked Pyꢀ4 lacking hydroxyl group was performed for comparison
with the control, but RMSD for Pyꢀ4 showed not much difference with the control (Fig. 6B).
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Applying QSAR method for obtaining more effective inhibitors
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Since some of the synthetic compounds have shown antiꢀamyloid activities close to the original
lead compound, one meaningful approach would be exploiting the pyridazineꢀbased amyloid
inhibitors to perform QSAR. IC50 inhibitory effects of the synthetic compounds were measured
(Table 2). A best QSAR model was created using IC50 values (R2= 0.966, RMSE=0.146). Pyꢀ15
containing carboxylic group was predicted to display more potent IC50 (smaller IC50) than the
original compound (Table 2). Therefore, Pyꢀ15 was synthesized, purified and confirmed as
described in the Materials and Methods section and in Fig. S15. When amyloid assay was
performed, in theꢀpresence of Pyꢀ15 the compound was precipitated out due to highly acidic
condition of the assay.
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DISCUSSION
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Due to the physicochemical properties of pyridazine, pyridazineꢀbased compounds have
been used widely in drugꢀprotein interactions35,46. However, limited pyridazineꢀbased
compounds have been introduced as amyloid inhibitors. RSꢀ0406 has recently been reported as
an important amyloid inhibitor32,33; the mechanism of the amyloid inhibition has been largely
missing. In this study, a number of related compounds were synthesized in order to gain more
mechanistic information on the inhibition of amyloid by pyridazineꢀbased compounds and also to
make a data set of RSꢀ0406 analogs for finding a better amyloid inhibitor. The pyridazineꢀbased
compounds were synthesized using amination reactions without using any catalyst or base (Table
1). It is worth mentioning that the electrophile (3, 6ꢀdichloropyridazine) must be added after
dissolving the nucleophile (aniline derivatives); otherwise, there could be little products mainly
due to high dipole momentum (3.9 D) of pyridazine which is generated by two nitrogen atoms
adjacently positions in its ring47. Additionally, the solvent (2EE) used in the reaction, also
possesses a high dipole moment (2.08 D); if both solvent and reactants were added
simultaneously, the nucleophilic substitution reaction would most likely be hindered.
Nucleophilic substitution at carbons of the pyridazine ring occurs by additionꢀelimination (SNAr)
mechanism. Both the leaving group (chlorine atom) and the nucleophile (ArꢀNH2) haveꢀ
determinant roles in ꢀthe reaction mechanism. Since, in this work, no chemical catalyst or a base
was used to synthesize novel compounds, rate and the yield of reaction was mainly dependent on
the leaving group, ArꢀNH2 substituent, temperature, and solvent. All of the compounds were
synthesized in the same solvent and temperature; therefore, substituent of ArꢀNH2 played a key
role in the synthesis of pyridazineꢀbased compounds. A fourꢀstep mechanism has been proposed
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for the synthesis of Pyꢀ1 to Pyꢀ15, indicating the rate determining step as disruption of
pyridazine aromaticity (Fig. S20). The synthesis of symmetrical products indicated the highest
yields pertained to Pyꢀ1, Pyꢀ2, and Pyꢀ3 which contain the electronꢀdonor moiety as an activator
in the first step of reaction. Interestingly, the synthesis of Pyꢀ12 performed in 18 h and the
product was precipitated out in the mixture of reaction. Further looking into the effects of
substituent of ArꢀNH2, Mulliken atomic charge on chlorine (leaving group) was calculated (Fig.
S20). In fact, a nitro group, in para position to ꢀNHꢀ (Py12), was partially blocked from electron
donation from ꢀNHꢀ to pyridazine ring, resulting in a faster reaction during the slowest step of
the reaction (third step).
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The results demonstrated that several structural parameters including hydrogen bonding,
position of the aromatic substituent, and bulkiness (halogenꢀbonding ability) most likely are
involved in inhibition of amyloid by the pyridazineꢀbased compounds. Most notable factor,
based on our results, is the position of the substituent on the outer aromatic rings of the synthetic
compounds (Fig. 1C). To better understand the position effect, the optimized structures of
synthetic compounds were compared using Gaussian48. It was revealed that the paraꢀsubstituted
compounds of the library were all entirely flat as compared to their meta counterparts (Fig. 5).
Therefore, it could be reasoned that the lesser inhibitory effect of para substituted compounds
might be related to their inability to finely tune to interact with the protein during unfolding
process. Indeed, Pyꢀ15 which has para carboxyl substitution was predicted to have a better
inhibitory effect than its meta counterpart. However, its optimized structure revealed a lack of
flat structure, similar to the other meta substituent compounds, as opposed to its flat meta
counterpart (Fig. 5).
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The cytotoxicity effect of protein aggregate in the presence of the synthetic compounds
indicated that those compounds that displayed less ThꢀT binding assay also caused less toxicity
at the cellular level (Fig. 4). Interestingly, Pyꢀ7 displayed more cell viability than original lead
compound (Pyꢀ1). This difference might be due to more toxicity of oligomers than the amyloid
fibrils that have been reported previously49 and perhaps Pyꢀ7 prevented oligomer formation more
effectively as supported by its longer lag time in ThꢀT binding assay and gel electrophoresis. It
must be noted that toxicity and the amount of amyloid fibrils formed are not always correlative
where an inhibitor might increase fibril formation but reduce toxicity50.
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In the absence of any crystal structure of the amyloid forming protein (HEWL) in the
presence of the pyridazineꢀbased inhibitors, it can be suggested that a hydroxyl group on the aryl
ring, as in Pyꢀ1, can be very effective to inhibit amyloid formation. However, when the hydroxyl
group is in para position, the inhibitory effect is reduced. Since antiꢀamyloid properties of several
compounds have been linked to their antioxidant effects, Bond Dissociation Energy (BDE) has
been used as a parameter to compare antioxidant potency51–53. Interestingly, when BDE was
calculated for both Pyꢀ1 and Pyꢀ2, the meta substituted compound (Pyꢀ1) displayed a lower BDE
(Fig. S18), therefore, Pyꢀ1 can potentially act as an antioxidant more effectively than Pyꢀ2.
Interestingly, when the hydroxyl groups were completely removed (as in Pyꢀ4), amyloid
inhibition was significantly reduced; however, the amyloid formation was not completely
prevented most likely due to the presence of pyridazine ring and hydrogenꢀbonding between the
amino group of the inhibitor and the protein (Fig. 6B). In a very recent study, a molecular
dynamics studies using Pyꢀ4 and Pyꢀ1 against Abeta peptide oligomer indicated the role of the
hydrogenꢀbonding in pyridazineꢀbased inhibitors54.
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Compound Pyꢀ8 containing two nitro groups also displayed much less inhibiting effect
than all the synthetic compounds that were studied for their hydrogen bonding abilities (Fig. 1A).
Although the nitro moiety is capable of making hydrogenꢀbonding, it could also reduce the
solubility of organic compounds in aqueous solution. Indeed, indoleꢀbased compounds
containing nitro groups have shown much less inhibitory effects55. Noncovalent proteinꢀligand
interactions have been shown to be influenced by halogens through halogen bonding and
hydrogenꢀbonding56. A halogenꢀbond is defined as a noncovalent bond between a halogen atom,
(Cl, Br, and I substitution of ligand) as a Lewis acid, and an electron donor, (Nitrogen and
Oxygen in backbone of protein) as a Lewis base. However, fluorine cannot form a halogen bond
due to its high electronegativity and inability to interact with nucleophiles56–58. Realizing that in
the present work Pyꢀ5, containing bromoꢀmoiety, turned out to be more effective amyloid
inhibitor than Pyꢀ9 and Pyꢀ10, this effect may be explained mainly by more halogen bonding
ability of bromine than chlorine atom since halogen bonding is enhanced with the increasing in
size of halogen atom. However, Pyꢀ6, containing two chloro moieties, displayed less inhibitory
effect than Pyꢀ9 and Pyꢀ10 which both contain one chloro moiety. Therefore, a bulkier ligand
can have weaker interactions with proteins due to its steric effect. In addition, the compound
containing fluoro (Pyꢀ7) can exert a similar effect as the hydroxyl group in Pyꢀ1 because organic
fluorine substitution is small and capable of making hydrogenꢀbonding59. In molecular dynamic
simulation, RMSD values indicate stabilization of protein ligand complex. The RMSD trajectory
of HEWL/Pyꢀ1 complex demonstrated that with the progress in simulation the complex became
more stable than complexes of HEWL/ Pyꢀ4 and HEWL/Pyꢀ7, confirming the effectiveness of
interactions between the Pyꢀ1 and ꢀHEWL. More interestingly, when the interactions of small
synthetic compounds during simulation were analyzed, both Pyꢀ1 and Pyꢀ7 demonstrated
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noncovalent interactions such as hydrogen bonding and πꢀπ interaction (parallel and vertical)
with residues in the amyloidogenic region of HEWL.
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To generate a reasonable QSAR model, the pyridazineꢀbased compounds that were
synthesized in this study were employed in a library in such a way that most of the compounds in
the library would have a similar pharmacophore, making the prediction more reliable60. In
addition, the IC50 values were measured for most of the pyridazineꢀbased compounds (Pyꢀ1 to
Pyꢀ11). Using the measured IC50 values, a model was built; subsequently, among a number of
suggested pyridazineꢀbased compounds, the model predicted that Pyꢀ15 as more effective than
the original lead compound (Table 2). Interestingly, the predicted compound (Pyꢀ15) which
possesses a para carboxy moiety, were predicted to have a lower IC50 than its meta counterpart
(Pyꢀ16). However, when the two compounds were compared structurally, Pyꢀ15 displayed a nonꢀ
planer structure similar to the other meta substituted compounds as opposed to its meta
counterpart (Fig. 5). We have also examined the effect of Pyꢀ15 as an amyloid inhibitor under
nonꢀacidic condition due to low solubility of the compound in low acidic medium. The results
did not match the QSAR prediction most likely due to a different condition (low pH) that the
model was built upon.
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Supporting Information Available: Figures S1ꢀS15 includes NMR spectra analysis for
synthetic compounds (Pyꢀ1ꢀ Pyꢀ15). Fig. S16 shows gel electrophoresis of aggregation assay in
the absence and presence of inhibitors. Fig. S17 ANS binding assay HEWL in absence and
presence of inhibitor. Fig. S18 demonstrates BDE of hydroxyl moiety on Pyꢀ1 and Pyꢀ2, and Fig.
S19 depicts kinetic studies of pyridazineꢀbased compounds. Fig. S20 Proposed mechanism for
the syntactic compounds.
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Funding Sources: This work was partially supported by Sharif University of Technology grant
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