Synthesis and characterization of 2-benzylidene-1,3-indandione derivatives as in vitro quantification of amyloid fibrils

Article abstract of DOI:10.1007/s13738-019-01776-2

Timely detection of amyloid aggregations has a critical role in the treatment of degenerative nervous system disorders such as Alzheimer’s, Parkinson’s disease and systemic amyloidosis. Thioflavin T (ThT) is a dye considered for the detection of amyloids.

Full text of DOI:10.1007/s13738-019-01776-2

Journal of the Iranian Chemical Society
https://doi.org/10.1007/s13738-019-01776-2
ORIGINAL PAPER
Synthesis and characterization of 2‑benzylidene‑1,3‑indandione
derivatives as in vitro quantifcation of amyloid fbrils
Hadi Adibi1 · Maryam Mehrabi2 · Kazhal Amiri3 · Saeed Balalaie4,5 · Reza Khodarahmi5,6
Received: 19 February 2019 / Accepted: 30 August 2019
© Iranian Chemical Society 2019
Abstract
Timely detection of amyloid aggregations has a critical role in the treatment of degenerative nervous system disorders such
as Alzheimer’s, Parkinson’s disease and systemic amyloidosis. Thiofavin T (ThT) is a dye considered for the detection of
amyloids. However, ThT cannot cross the blood–barrier barrier due to positive charge and low lipophilicity. In the present
study, a variety of 2-benzylidene-1,3-indandione derivatives 5a–5j were synthesized as neutral fuorescence probe candi-
dates for identifcation of amyloid. Among these compounds, only compound 2-(2-hydroxybenzylidene)-1,3-indandione 5a
was selected for further examination, because its fuorescence intensity showed the signifcant increasing upon interaction
with amyloid aggregates. The compound 5a was compared to standard and conventional probe such as ThT. The compound
5a was excited at its specifed wavelength, and the fuorescence emission signal was recorded in the presence of diferent
concentrations of the protein/peptide (amyloid aggregated and native protein). According to the obtained results, it can be
seen that 2-(2-hydroxybenzylidene)-1,3-indandione 5a selectively and specifcally bind to amyloid fbrils, such as ThT. Due
to the neutrality of electrical charge and high lipophilicity coefcient of the synthesized compound 5a, it is possible for it to
cross from the blood–brain barrier. Our results show that this synthetic derivative can be considered as a suitable probe to
detect in vitro amyloid aggregations.
Keywords Amyloid aggregation · Alzheimer’s disease (AD) · Indanone derivatives · Thiofavin T (ThT) · β-Amyloid
plaques
Introduction
Fibrillar protein aggregates are a major cause of amyloidosis, a
group of diseases created by self-aggregation of some normal
* Reza Khodarahmi
proteins into harmful flamentous deposits (amyloid fbrils).
Amyloid fbrils are correlated with the most neurodegenera-
tive disorder, such as Alzheimer’s, Parkinson’s, and systemic
amyloidosis [1]. For example, Alzheimer’s disease (AD) is a
type of degenerative nervous system disorders that gradually
declines the mental abilities of the patient [2, 3]. Amyloid-
beta (Aβ) and Tau are the two major proteins extensively
involved in the AD pathology. Both proteins convert from
innocuous soluble forms to cytotoxic oligomers [4] and fb-
ers (with cross-β-sheet structure) [5]. There are great interests
about the pathological process of Tau aggregation, because
Tau deposition more occur in the temporal lobe of the brain
and it is a better predictor of AD than Aβ deposition that hap-
pen in any region of the brain [6]. This intrinsically disordered
protein consists of three or four repeated regions with similar
amino acid sequences. Recently, studies have demonstrated
rkhodarahmi@kums.ac.ir; rkhodarahmi@mbrc.ac.ir
1
Pharmaceutical Sciences Research Center, Health Institute,
Kermanshah University of Medical Sciences, Kermanshah,
Iran
2
Department of Biology, Faculty of Sciences, Razi University,
Kermanshah, Iran
3
Student Research Committee, Kermanshah University
of Medical Sciences, Kermanshah, Iran
4
Peptide Chemistry Research Center, K. N. Toosi University
of Technology, P.O. Box 15875-4416, Tehran, Iran
5
Medical Biology Research Center, Health Technology
Institute, Kermanshah University of Medical Sciences,
Kermanshah, Iran
6
Pharmacognosy and Biotechnology Department, Faculty
of Pharmacy, Kermanshah University of Medical Sciences,
Kermanshah, Iran
Vol.:(0123456789)
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Journal of the Iranian Chemical Society
that only a small part of the Tau molecule is contributed in
the aggregation process [5]. In Tau polymerization, the for-
mation of β-sheet structure is attributed to two hydrophobic
short hexapeptide motifs in the second and third repeat of Tau,
PHF6*275VQIINK280(in R2) and PHF6306VQIVYK311(inR3),
and it has been verifed that the interaction between these two
regions causes to the unique PHF structure [7]. In our pre-
vious study, we synthesized a modifed PHF6 heptapeptide
(FVQIVYH) and amyloid fbril formation by tau protein and
the modifed PHF6 heptapeptide (FVQIVYH) were verifed
through some specifc methods of amyloid detection (ThT
fuorescence, XRD, far UV-CD and AFM). This modifed
PHF6 heptapeptide (FVQIVYH) is known as FV peptide [8].
The greater we understand amyloidosis molecular details,
the greater we can control amyloid formation and conse-
quently, neurodegenerative diseases. This requires a reliable
probe for identifying amyloids. Detection of amyloid for-
mation is carried out in a variety of ways, including iodine
colorimetric method, crystalline violet staining, optical dis-
persion, Congo red (CR), transmission electron microscopy
(TEM), atomic force microscopy (AFM), thiofavin S (ThS),
and thiofavin T (ThT) [9]. ThT, in the form of dimer, hex-
amer and micelle, has the ability to interact with amyloid. Its
structure is two-ring of benzothiazole and benzylamine; these
two rings have the ability to rotate freely around their shared
carbon–carbon bond in aqueous media that rapidly quenches
excited states generated by photon excitation. This rotation is
limited when ThT bind to amyloid fbrils, so it exhibits fuo-
rescence properties [10–12]. ThT is not able to pass through
the cell membrane due to the ThT positive charge, so it is not
suitable for the study of amyloid aggregation in the cell [13].
In addition, compounds without a net positive charge can
mainly cross the blood–brain barrier (BBB) [14].
(Darmstadt, Germany). All solutions were prepared by steri-
lizing double distilled water. The structure of compounds was
characterized by IR, 1H-NMR spectra and mass spectrometry
(MS). IR spectrum was recorded on a Shimadzu 470 spectro-
photometer (KBr disk). 1H-NMR spectrum was recorded on a
Bruker FT-250 NMR spectrophotometer using DMSO-d6 as
solvent and tetramethylsilane (TMS) as an internal standard.
The MS analysis was performed using a MS system comprising
a Finnegan MAT Spectra System P4000 pump coupled with
a UV6000LP diode array detector and a Finnigan AQA mass
spectrometer. A Cary 100 Bio (Varian) spectrophotometer was
used for protein determination. All fuorescence measurements
were performed in the ratio mode using a 1-cm cell in a Cary
Eclipse (Varian) fuorescence spectrophotometer, equipped
with a 150 W xenon lamp and a thermostatic cell holder, at
room temperature or as stated. Appropriate vehicle controls
were run in all experiments. All the reported results are aver-
ages of 2–3 separate experiments whenever the coefcients of
variation were less than 5%. Other devices used in this study
were pH/EC Meter (Wm-50EG) (Dkktoad) and Heater/Stirrer
RH basic 2 (IKA®).
Synthesis of 2‑benzylidene‑1,3‑indandione deriva‑
tives 5a–5j
In a round-bottomed fask (25 ml), 1,3-indandione (1 mmol,
0.146 g), benzaldehyde derivatives (1 mmol), pyridine
(100 μl) and ethanol (5 ml) were stirred under refux condi-
tions for 2–3 h. After the reaction was completed (TLC analy-
sis), the reaction mixture was allowed to stand at room tem-
perature for the next 12 h and then the cold distilled water was
added to the solution and the precipitate was fltered, washed
with n-hexane and distilled water, crystallized with appropri-
ate solvent and dried to aford the corresponding; compounds
5a–5j were obtained in % 92–99 yields (Scheme 1; Table 1)
[15, 16]. Structural assignments of the products are based on
their IR, 1H-NMR, MS and melting point.
In the current study, with the purpose of produc-
ing such compounds, a variety of 2-benzylidene-1,3-in-
dandione derivatives were synthesized as markers for
identification of amyloid (Scheme 1). Among them,
2-(2-hydroxybenzylidene)-1,3-indandione 5a has been found
neutral ThT analog fuorescence probe, and it employed to
determine quantitatively the amyloid fbrils comparison with
conventional methods. The resulting data may be useful in
providing mechanistic insights to develop potential diag-
nostic, curative, and/or preventive strategies in vivo against
amyloid-related neurodegenerative disorders.
2‑(2‑Hydroxybenzylidene)‑1H‑indene‑1,3(2H)‑dione
(compound 5a)
Materials and methods
Chemicals and equipments
Acetone, bovine serum albumin (BSA), Congo red (CR) and
ThT were provided by Sigma (St. Louis, MO, USA.). The
chemical compounds were purchased from Merck chemicals
M.W. =250. 2; m.p. =196 °C; color: Orange; FT-IR (KBr,
cm−1)∶ ̄ꢀ 3147 (stretch, CHArom), 2939 (stretch, CHAliphatic),
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Journal of the Iranian Chemical Society
Scheme 1 The synthesis of 2-benzylidene-1,3-indandione compounds: reaction conditions and the mechanism
Table 1 The synthesized compounds
Compound
Name
Yield (%)
Excitation wavelength (nm)
5a
5b
5c
5d
5e
5f
2-(2-Hydroxybenzylidene)-1H-indene-1,3(2H)-dione
2-(3-Hydroxybenzylidene)-1H-indene-1,3(2H)-dione
2-(4-Hydroxybenzylidene)-1H-indene-1,3(2H)-dione
2-(2,3-Dihydroxybenzaldehyde)-1H-indene-1,3(2H)-dione
2-(2,4-Dihydroxybenzaldehyde)-1H-indene-1,3(2H)-dione
2-(2,5-Dihydroxybenzaldehyde)-1H-indene-1,3(2H)-dione
2-(3,4-Dihydroxybenzaldehyde)-1H-indene-1,3(2H)-dione
2-(2-Hydroxy-3-methoxybenzaldehyde)-1H-indene-1,3(2H)-dione
2-(4-Hydroxy-3-methoxybenzaldehyde)-1H-indene-1,3(2H)-dione
2-(4-Isopropylbenzaldehyde)-1H-indene-1,3(2H)-dione
98
98
97
97
99
94
94
96
95
92
320
390
400
400
300
400
400
360
420
370
5 g
5 h
5i
5j
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Journal of the Iranian Chemical Society
1712 (stretch, C=O), 1674 (stretch, C=CVinyl ), 1570
(stretch, C=CArom), 1462 (stretch, C=CArom), 1276 (stretch,
OHphenol), 1215 (stretch, C–O), 1157 (stretch, O–C, 732
M.W. =250.2; m.p. =215 °C; color: Yellow; FT–IR (KBr,
cm−1): ̄ꢀ , 3398 (stretch, OH), 3190 (stretch, CHArom ),
2954 (stretch, CHAliphatic ), 1708 (stretch, C=O), 1666
(stretch, C=CVinyl ), 1566 (stretch, C=CArom ), 1477
(stretch, C=CArom), 1257 (stretch, OHphenol), 1207 (stretch,
1
(OOP, C–H); H–NMR (300 MHz, DMSO-d6): δ 10.83
(s, 1H, HPhenol), 8.86 (d, J=7.9 Hz, H4), 8.31 (s, 1H, Hvinyl),
1
7.96 (d, J= 6.35 Hz,1H, H ), 7.94 (m, 2H, H ꢀ), 7.91 (d,
C–O), 1087 (stretch, C–O), 732 (OOP, C–H); H–NMR
ꢀ
6ꢀ
J=6.35 Hz, 1H, H ), 7.44 (t, J=7.45 Hz, 1H,4H,5 ), 6.98 (d,
(300 MHz, DMSO-d6): δ 10.91 (s, 1H, OHPhenol),
8.52–8.55 (d, 2H, J = 8.7 Hz, H2ꢀ,6ꢀ ), 7.88–7.95 (m, 4H,
H4,5,6,7), 6.93–6.96 (d, 2H, J = 8.4 Hz, H3ꢀ,5ꢀ), 6.2 (s, 1H,
HVinyl); Mass (m/z, %): 250 (M, 5.35), 233 (M-17, 18),
194 (M-56, 8.8), 165 (M-85, 7.57), 139 (M-111, 8.14),
118 (M-132, 8.51), 104 (M-146, 57), 89 (M-161, 4.44),
76 (base peak, 100), 63 (M-187, 5.52), 50 (M-200, 1.65).
3ꢀ
6
J=8.2, 1H, H7), 6.92 (t, J=7.55, 1H, H5); Mass (m/z, %):
252 (M, 122), 233 (M-11, 51.18), 221 (M-21, 5.55), 225
(M-45, 23.31), 114 (M-56, 1.25), 165 (M-85, 25.51), 145
(M-125, 81.14), 125 (M-145, 4.24), 81 (M-161, 51.18), 16
(M-114,4.21), 63 (M-181, 1.11), 52 (M-222, 1.11).
2‑(3‑Hydroxybenzylidene)‑1H‑indene‑1,3(2H)‑dione
(compound 5b)
2‑(2,3‑Dihydroxybenzaldehyde)‑1H‑in‑
dene‑1,3(2H)‑dione (compound 5d)
M.W.=250. 2; m.p.=221 °C; color: Lemon; FT–IR (KBr,
cm−1): ̄ꢀ 3741(overtone, C=O), 3240 (stretch, OH), 3024
(stretch, CHArom), 2924 (stretch, CHAliphatic), 1720 (stretch,
C=O), 1666 (stretch, C=CVinyl), 1585 (stretch, C=CArom),
1454 (stretch, C=CArom), 1230 (stretch, C–O), 1180 (stretch,
M.W. = 266.2; m.p. = 179 °C; color: Yellow; FT–IR
(KBr, cm−1): ̄ꢀ 3738(overtone, C=O), 3444 (stretch, OH),
3421 (stretch, OH), 3089 (stretch, CHArom), 2924 (stretch,
CHAliphatic), 1708 (stretch, C=O), 1674 (stretch, C=CVinyl),
1570 (stretch, C=CArom), 1473 (stretch, C=CArom), 1273
(stretch, OHphenol), 1215 (stretch, C–O), 1153 (stretch,
O–C), 732 (OOP, C–H); 1H–NMR (300 MHz, DMSO-d6):
δ 10.19 (s, 1H, HPhenol), 8.97–8.98 (d, J =7.1 Hz, H4), 8.58
(s, 1H, Hvinyl), 7.95–7.96 (d, J = 5.05 Hz, 1H, H6ꢀ), 7. 4 (m,
2H, H4ꢀ5ꢀ), 7.39–7.41 (t, J = 11.45 Hz, 1H, H ), 6.94–6.96
(d, J = 7.95 Hz, 1H, H7), 6.74–6.77 (t, J = 165.75 Hz, 1H,
H5); Mass (m/z, %): 266 (M, 14), 249 (M-17, 14), 222
(M-44, 11.1), 181 (M-85, 16.2), 165 (M-101, 25.1), 152
(M-114, 28.8), 134 (M-132, 15.5), 104 (M-162, 60.7), 93
(M-173, 29.6), 76 (base peak, 100), 63 (M-203, 44.4), 50
(M-216, 85.1).
1
O–C, 1157 (stretch, O–C), 740 (OOP, C–H); H-NMR
(300 MHz, CHCl3): δ 8.56 (s, 1H, H2′), 8.26 (d, J=7.5 Hz,
2H, H4′), 8.04 (d, J=4.45 Hz, 2H, H4,7), 7.85 (d, J=8.55 Hz,
2H, H5,6), 7.81 (s, 1H, Hvinyl), 7.53 (d, J=7.9 Hz, 1H, H6′),
7.47 (t, J=7.81 Hz, 1H, H5′); Mass (m/z, %): 250 (M, 37.50),
233 (M-17, 8.34), 165 (M-85, 80), 139 (M-111, 20), 118
(M-132, 3.33), 104 (M-146, 5.55), 89 (M-161, 4.47), 76 (base
peak, 100), 63 (M-187, 6.49), 50 (M-200,3.57).
2‑(4‑Hydroxybenzylidene)‑1H‑indene‑1,3(2H)‑dione
(compound 5c)
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Journal of the Iranian Chemical Society
M.W. =266.2; m.p. =185 °C; color: Brown; FT–IR (KBr,
cm−1): ̄ꢀ 3738(overtone, C=O), 3414 (stretch, OH), 3109
(stretch, CHArom), 2912 (stretch, CHAliphatic), 1716 (stretch,
C=O), 1685 (stretch, C=CVinyl), 1566 (stretch, C=CArom),
1458 (stretch, C=CArom), 1246 (stretch, OHphenol), 1219
(stretch, C–O), 1157 (stretch, O–C), 740 (OOP, C–H);
1H–NMR (300 MHz, DMSO-d6): δ 9.53–9.55 (d,
J = 7.15 Hz, 1H, H4), 8.91 (s, 1H, Hvinyl), 8.06–8.09 (t,
J = 15.15 Hz, 1H, H6ꢀ ), 8.03–8.04 (d, J = 6.05 Hz, 2H,
H3ꢀ,4ꢀ), 7.96–7.99 (t, J = 12. 5 Hz, 1H, H ), 6.85–6.86 (d,
J=8. 1 Hz, 1H, H7); Mass (m/z, %): 274 6(M+8,14.8), 266
(M, 3.7), 246 (M-20, 22.2), 189 (M-77, 62.9), 163 (M-103,
18.5), 139 (M-127, 12.5), 114 (M-152, 29.6), 103 (M-162.
26.6), 93 (M-173, 46.6), 76 (M-190, 93.3), 63 (M-203,
44.4), 55 (M-211, 54.8), 43 (M-223, 100).
2‑(2,4‑Dihydroxybenzaldehyde)‑1H‑in‑
dene‑1,3(2H)‑dione (compound 5e)
M.W. = 266.2; m.p. = 232.4 °C; color: Brown; FT–IR
(KBr, cm−1): ̄ꢀ 3749(overtone, C=O), 3248 (stretch, OH),
3097 (stretch, CHArom), 2927 (stretch, CHAliphatic), 1712
(stretch, C=O), 1658 (stretch, C=CVinyl ), 1554 (stretch,
C=CArom ), 1469 (stretch, C=CArom ), 1300 (stretch,
OHphenol ), 1215 (stretch, C–O), 1161 (stretch, O–C),
2‑(3,4‑Dihydroxybenzaldehyde)‑1H‑in‑
dene‑1,3(2H)‑dione (compound 5 g)
1
729 (OOP, C–H); H–NMR (300 MHz, DMSO-d6): δ
10.95 (s, 1H, HPhenol), 10.80 (s, 1H, HPhenol), 8.95–8.96
(d, J = 7. 95 Hz, 1H, H4), 8.28 (s, 1H, Hvinyl), 8.17–8.19
(d, J = 8.2 Hz, 1H, H ), 7.80 (s, 1H, H ), 7.68–7.71 (t,
ꢀ
J = 15.65 Hz, 1H, H6)6,ꢀ7.51–7.54 (d, J =38.2 Hz, 1H, H6′),
6.65–6.67 (d, J = 7.7 Hz, 1H, H7), 6.40–6.43 (t, J = 15.75,
1H, H5); Mass (m/z, %): 266 (M, 11.8), 246 (M-20, 68.1),
218 (M-48, 25.1), 189 (M-77, 95.5), 163 (M-103, 25.9),
152 (M-114, 8.8), 139 (M-127, 10.3), 113 (M-153, 34.8),
101 (M-165, 9.6), 88 (M-178, 31.1), 63 (M-203, 45.1), 50
(M-216, 63.7).
M.W. = 266.2; m.p. = 250 °C; color: Light Green;
FT–IR (KBr, cm−1): ̄ꢀ 3749 (overtone, C=O), 3456
(stretch, OH), 3240 (stretch, OH), 3097 (stretch, CHArom),
2924 (stretch, CHAliphatic ), 1670 (stretch, C=CVinyl ),
1562 (stretch, C=CArom), 1450 (stretch, C=CArom), 1292
(stretch, OHphenol), 1246 (stretch, OHphenol), 1188 (stretch,
2‑(2,5‑Dihydroxybenzaldehyde)‑1H‑in‑
dene‑1,3(2H)‑dione (compound 5f)
1
C–O), 1161 (stretch, O–C), 732 (OOP, C–H); H–NMR
(300 MHz, DMSO-d6): δ 10.51 (s, 1H, HPhenol), 8.35 (s,
1H, Hvinyl), 7.96 (d, J = 6.35 Hz, 1H, H ), 7.84–7.86 (d,
ꢀ
J=8.42 Hz, 2H, H2ꢀ,5ꢀ), 7.66–7.71 (t, J=614.1 Hz, 1H, H ),
6
6.90–6.93 (d, J = 8.4 Hz, 1H, H7); Mass (m/z, %): 266
(M+, 14.8), 249 (M-17, 7.4), 233 (M-33,8), 189 (M-77,
5.9), 176 (M-90, 12.5), 163 (M-103, 22.2), 152 (M-114,
19.2), 134 (M-132, 14. 8), 121 (M-145, 17), 104 (M-162,
62.9), 93 (M-173, 26.6), 76 (base peak, 100), 63 (M-203,
33.3), 50 (M-216, 74.8), 41 (M-125, 41.4).
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Journal of the Iranian Chemical Society
2924 (stretch, CHAliphatic ), 1716 (stretch, C=O), 1681
(stretch, C=CVinyl), 1577 (stretch, C=CArom), 1512 (stretch,
C=CArom), 1257 (stretch, OHphenol), 1207 (stretch, C–O),
1157 (stretch, O–CH3), 1130 (stretch, O–C), 736 (OOP,
2‑(2‑Hydroxy‑3‑methoxybenzaldehyde)‑1H‑in‑
dene‑1,3(2H)‑dione (compound 5 h)
1
C–H); H–NMR (300 MHz, DMSO-d6): δ 10.59 (s, 1H,
H
Phenol), 8.70 (s, 1H, Hvinyl), 7.96–7.97 (d, J= 3.65 Hz, 1H,
H ), 7.95 (m, 2H, H ), 7.91–7.92 (t, J=4.2 Hz, 1H, H ),
ꢀ
2ꢀ,5ꢀ
6.693–6.95 (d, J = 8.3 Hz, 1H, H7), 3.92 (s, 1H, HMethoxy6);
Mass (m/z, %): 280 (M+, 100), 263 (M-17, 17.7), 249
(M-31, 18.5), 237 (M-43, 20.7), 209 (M-71, 13.3), 181
(M-99, 16.2), 165 (M-115, 11.8), 152 (M-128, 46.6), 133
(M-147, 8.8), 104 (M-176, 25.1), 76 (M-204, 39.2), 63
(M-217, 8), 51 (M-229, 14).
2‑(4‑Isopropylbenzaldehyde)‑1H‑in‑
dene‑1,3(2H)‑dione (compound 5j)
M.W. =280.2; m.p. =225 °C; color: Light Orange; FT–IR
(KBr, cm−1): ̄ꢀ 3738(overtone, C=O), 3410 (stretch, OH),
3093 (stretch, CHArom), 2924 (stretch, CHAliphatic), 1712
(stretch, C=O), 1674 (stretch, C=CVinyl ), 1577 (stretch,
C=CArom ), 1477 (stretch, C=CArom ), 1265 (stretch,
OHphenol), 1215 (stretch, C–O), 1157 (stretch, O–CH3), 736
1
(OOP, C–H); H–NMR (300 MHz, DMSO-d6): δ 10.05
(s, 1H, HPhenol), 8.45–8.47 (d, J = 8.15 Hz, 1H, H4), 8.33
(s, 1H, Hvinyl), 7.93–7.94 (d, J = 2.75 Hz, 1H, H6ꢀ ), 7.92
(d, J = 7.95 Hz, 2H, H4ꢀ,5ꢀ ), 7.90 (t, J = 2.9 Hz, 1H, H6),
7.18–7.20 (d, J = 8 Hz, 1H, H7), 6.86–6.89 (t, J = 16.1 Hz,
1H, H5), 3.85 (s, 1H, HMethoxy ); Mass (m/z, %): 280
(M+,11), 266 (M-14,5), 249 (M-31, 4.4), 235 (M-45,
12.5), 206 (M-74, 8), 181 (M-99, 9.6), 165 (M-115, 16.2),
152 (M-128, 38.5), 126 (M-154, 14.8), 104 (M-176, 51.8),
93 (M-187, 21.4), 76 (base peak, 100), 63 (M-217, 26.6),
50 (M-230, 54.8), 41 (M-239, 54.8).
M.W.=276.3; m.p.=230 °C; color: Lemon; FT–IR (KBr,
cm−1): ̄ꢀ 3506 (overtone, C=O), 3441 (stretch, OH),
3059 (stretch, CHArom), 2958 (CHAliphatic), 2924 (stretch,
CHAliphatic), 1701 (stretch, C=O), 1658 (stretch, C=CVinyl),
1589 (stretch, C=CArom), 1462 (stretch, C=CArom), 1257
(stretch, OHphenol), 1192 (stretch, C–O), 1157 (stretch,
O–CH3), 736 (OOP, C–H); 1H–NMR (300 MHz, DMSO-
d6): δ 8.64–8.66 (d, J=7.65 Hz, 1H, H4); Mass (m/z, %):
276 (M+, 55.5), 261 (M-15, 61.4), 246 (M-30, 9.6), 233
(M-43, 100), 215 (M-61, 16.2), 202 (M-74, 17.7), 189
(M-87, 9.6), 176 (M-100, 6.6), 128 (M-148, 11.8), 115
(M-161, 13.3), 104 (M-172, 13.3), 91 (M-185, 8), 76
(M-200, 22.9), 50 (M-226, 9.6).
2‑(4‑Hydroxy‑3‑methoxybenzaldehyde)‑1H‑in‑
dene‑1,3(2H)‑dione (compound 5i)
Formation of the FV peptide fbrils
To prepare 400 μM of the FV peptide fbrils, 2 mg of the FV
peptide was dissolved in 5 ml of 50 mM phosphate bufer
with pH 7, and it was well adjusted to make a clear solu-
tion, and the peptide was completely dissolved. Then it was
placed at room temperature for 30–40 min to form amyloid.
After this time, the solution was turbid, and amyloid was
formed [17].
M.W. = 280.2; m.p. = 198.8 °C; color: Yellow; FT–IR
(KBr, cm−1); ̄ꢀ 3749(overtone, C=O), 3414 (stretch, OH),
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Fig. 1 Absorption spectrum of 5a in the presence of FV peptide:
compound 5a alone (line), compound 5a with peptide FV (.-.-.),
subtraction of 5a+peptide absorption spectrum from peptide (thick
dashed line), and peptide alone (thin dashed line). The concentration
of 5a was 22 μM. Peptide FV concentration was 160 μM
Fig. 3 CR absorption spectrum in the presence of FV peptide: CR
alone (line), CR with peptide FV (dashed line). The concentration of
CR was 10 μM
Lysozyme formation
Four milligrams of lysozyme was weighted, and it was com-
pletely dissolved in 1 ml of 50 mM glycine with the pH 2.5.
The pH of the solution was adjusted with concentrated HCl
(4 M). Tube lid was covered by nylon, and the prepared solu-
tion in bufer was kept in 57 °C water bath for 72 h to aggre-
gate lysozyme. (Increasing temperature could accelerate the
process of aggregation and amyloidization.) ThT emission
was measured alone and with lysozyme, at 24-h intervals, to
ensure about progressing of lysozyme aggregation. Increas-
ing ThT emission at 450 nm indicated that lysozyme was
amyloidized, and increasing emission in any 24-h indicated
the correct process.
Fig. 4 ThT absorption spectrum in the presence of FV peptide: ThT
alone (line), ThT with peptide FV (dashed line). The concentration of
ThT was 12 μM
Preparation of beta‑lactoglobulin (BLG)
0.0046 g of BLG was weighted to make a solution with a
concentration of 0.5 mM, and it was dissolved in 0.5 ml of
50 mM phosphate bufer.
Fig. 2 Absorption spectrum of 5a in the presence of FV peptide:
compound 5a alone (line), compound 5a with peptide FV (.-.-.),
subtraction of 5a+peptide absorption spectrum from peptide (thick
dashed line), and peptide alone (thin dashed line). The concentration
of 5a was 10 μM. Peptide FV concentration was 160 μM
Fig. 5 Fluorescence emission spectrum of 5a in the presence of pep-
tide FV: 5a alone with the concentration of 20 μM (line), 5a with
peptide FV with the concentration of 160 μM (dashed line)
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Journal of the Iranian Chemical Society
measurements were done at room temperature. All measure-
ments were applied at maximum excitation for each dye.
Results and discussion
A variety of 2-benzylidene-1,3-indandione derivatives
5a–5j were synthesized (Table 1) and characterized with
1H-NMR, FT-IR and mass spectra. For the synthetic com-
pounds, maximum wavelengths were determined by tracking
absorption spectra of the compounds, alone and in the pres-
ence of the FV peptide (all data not shown) (Figs. 1, 2), and
the observed redshifts compared with CR (Fig. 3) and ThT
(Fig. 4). By this way, excitation wavelengths were identifed
for fuorescence studies. Thereafter, formation of amyloid
fbrils by the FV peptide was verifed using ThT fuorescence
spectroscopy as well as Congo red binding assay. The bind-
ing of all compounds to FV peptide (amyloid fbrils) shows
diferent behavior for diferent compounds, unlike the ThT
and compound 5a; the fuorescence intensity of the other
compounds did not show the signifcant increasing upon
interaction with the FV peptide. According to these results,
we excluded the rest of the compounds (except compound
5a) from subsequent experiments. It is widely believed
that the dramatic increase in ThT fuorescence results from
the selective immobilization of ThT conformers [18, 19].
In solution, a low energy barrier allows the benzylamine
and benzathiole rings of ThT to rotate freely about their
shared carbon–carbon bond. This rotation rapidly quenches
excited states generated by photon excitation, causing low
fuorescence emission for free ThT. In contrast, rotational
immobilization of ThT preserves the excited state, result-
ing in a high quantum yield of fuorescence [20]. In this
way, there is a similar structure for Compound 5a in which
phenol ring freely rotates around the single carbon–carbon
bond and quench the fuorescence emission of it, in solu-
tion. Amyloid fbrils are likely to present a binding site that
Fig. 6 The common structure of amyloid fbrils and a structural
rationale for fbril–dye interactions. Top cross-β structure of amyloid
fbrils, formed from layers of laminated β-sheets. Bottom “Channel”
model of dye binding to fbril-like β-sheets. Dye is proposed to bind
along surface side-chain grooves running parallel to the long axis of
the β-sheet
Spectroscopic studies
Fluorescence excitation and emission spectra were done by
Cary Eclipse fuorescence spectrophotometer (Varian, Aus-
tralia). The slit width of fuorescence excitation and emis-
sion spectra was set at 5 and 10 nm. Correction of obtained
spectra was performed by Cary Eclipse fuorescence spec-
trophotometer for the dependence of wavelength to intensity
of excitation source. The main purpose of this part was to
study the fuorescence reaction of each dye in the presence
of native and aggregated proteins. Spectroscopic measure-
ments were performed in standard quartz cells (1*1 cm). All
Fig. 7 Fluorescence emission
spectra of 5a and ThT in the
presence of diferent concen-
trations of the FV peptide (0,
5, 10, 30, 50, 80 μM). The
concentration of the compound
5a was 20 μM
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Fig. 8 Fluorescence emission
spectra of 5a and ThT in the
presence of diferent concentra-
tions of lysozyme (0, 5, 10, 30,
50, 80 μM). The concentration
of the compound 5a was 20 μM
Fig. 11 Evaluation of fuorescence emission for the compound 5a
after binding to native lysozyme at indicated concentration: the com-
pound 5a (∆), standard compound ThT (〇). The concentration of 5a
and ThT were 20 μM
Fig. 9 Evaluation of fuorescence emission for synthesized compound
5a after binding to native BSA at indicated concentration: The com-
pound 5a (∆), Standard compound ThT (〇). The concentration of 5a
and ThT were 20 μM
Fig. 12 Evaluation of fuorescence emission for the compound
5a alone (line), with native lysozyme (dashed line), and amyloid
lysozyme (.-.-.-). The concentration of 5a and lysozyme were 20 μM
Fig. 10 Evaluation of fuorescence emission for the compound 5a
after binding to native BLG at indicated concentration: the compound
5a (∆), standard compound ThT (〇).The concentration of 5a and
ThT were 20 μM
sterically “locks” the bound compound 5a and prevents its
rotation, thus leading to an enhancement of its fuorescence.
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Journal of the Iranian Chemical Society
Acknowledgements We gratefully acknowledge Vice Chancellery for
Research and Technology, Kermanshah University of Medical Sciences
for fnancial support (Grant No. 93209). This article resulted from the
Pharm.D thesis of Kazhal Amiri, major of Pharmacy, Kermanshah
University of Medical Sciences, Kermanshah, Iran.
Binding of the compound 5a to the FV peptide was exam-
ined by observing fuorescence emission spectrum between
300 and 600 nm using specific excitation wavelengths
(320 nm) and at a fxed concentration (20 µM) of compound
5a (Fig. 5). As indicated in Fig. 5, the fuorescence intensi-
ties (emission at 440 nm) increased signifcantly upon inter-
action with amyloid aggregates.
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Conclusion
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According to the results of this study, we have proved that
our synthetic derivatives have the ability to compete with
standard samples such as ThT. Due to neutrality nature of
our synthetic compound 5a and its high lipophilicity, it can
be considered as a more potent probe than standard sample,
ThT, for crossing blood–brain barrier.
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