Synthesis of benzylidene-benzofuranone derivatives as probes for detection of amyloid fibrils in cells

Abstract Aggregated protein is the common cause of a wide variety of neurodegenerative diseases, such as Alzheimer’s disease (AD), Parkinson’s disease, etc. It is proven that protein aggregation like amyloid β (Aβ) is one of the critical factors causing AD and, its diagnosis in the early stages of the disease is important for the treatment or prevention of AD. To have a better understanding of protein aggregation and its pathologies, there is a huge need to design and develop new and more trustworthy probe molecules for in vitro amyloid quantification and in vivo amyloid imaging. In this study, 17 new biomarker compounds, have been synthesized from benzofuranone derivatives, to detect and identify amyloid in vitro (dye-binding assay) as well as in the cell by staining method. According to the results, some of these synthetic derivatives can be considered suitable identifiers and quantifiers to detect amyloid fibrils in vitro. Compared to thioflavin T, 4 probes out of 17 probes have shown good results in selectivity and detectability of Aβ depositions, and their binding properties were also confirmed with in silico analysis. The drug-likeness prediction results for selected compounds by the Swiss ADME server show a satisfactory percentage of blood-brain barrier (BBB) permeability and gastrointestinal (GI) absorption. Among all of them, compound 10 was able to show better binding properties than others, and in vivo study showed that this compound was capable of detecting intracellular amyloid. Communicated by Ramaswamy H. Sarma


Introduction
In general, protein aggregation is a biological process named amyloidosis, which is the main cause of many neurodegenerative diseases, such as Alzheimer's disease (AD), Parkinson's disease, and prion disease, and so on.Amyloidosis is the deposition of protein peptides/proteins in different parts of the body and organs like the heart, kidney, brain, etc. (Khanam et al., 2016;Lee et al., 2005;Obici et al., 2005).Among these biological disorders, AD has great importance because of its devastating effects on patients' lives and their families (Manzini & Vale, 2020).AD is a common form of dementia, and a progressive decline in cognition, which gradually leads to forgetfulness, mood and behavioral change, impairs daily activities, and finally brings the death to the patient (Alzheimer's Association, 2018).That is why scientists and physicians have paid special attention to this disease.There are two hypotheses for AD, one is Amyloid b (Ab) peptide aggregation outside the neurons producing fibrils or plaques, and the other is neurofibrillary tangles formation (NFTs) caused by twisted tau proteins inside the neurons (Breijyeh & Karaman, 2020;O'brien & Wong, 2011;Rahman et al., 2020).The Ab plaques are made up of peptides that have mainly 40 and 42 amino acids, which block cell signaling (Ayodele et al., 2021;Mendez, 2017).On the other hand, NFTs impair the passway of nutrients inside the neurons.These two together cause cells' destruction and shrinkage of the brain, causing the memory to be lost and slowing down the function of vital organs such as the heart, eventually causing death (Cermakova et al., 2015).Today it is estimated that every 3 seconds, somebody is diagnosed with AD, mostly at ages 65 and older (Morris et al., 1996;Roy et al., 2021).
To have a better understanding of amyloid fibrils formation and its pathology, there is a huge need to develop and synthesize new probe molecules to detect amyloid aggregations.The process of dementia in a patient with AD could not be reversed, once the neuron cells are dead, therefore developing new probe molecules to detect Ab depositions have a great interest to scientists and researchers.These synthesized biomarkers may help the diagnosis of AD in the early stages, which means physicians could start treatment sooner and more effectively so that progression and symptoms of the disease could be delayed or stopped before damage to the brain is completely done.The best way to identify Ab is the staining method using fluorophores/probes.A reliable probe should have three important features, the first selectivity only to Ab fibrils, the second sensitivity to low concentrations of Ab fibrils, and the third the lack of cytotoxicity.Thioflavin T (ThT), as a classical biomarker has serious drawbacks in identifying Ab, especially for in vivo staining, including low lipophilicity, due to its permanent positive charge, which means this molecule cannot easily passes the cell membranes and blood-brain barrier (BBB), the other issue is that at low concentrations of Ab fibrils, its fluorescence intensity is weak.Apart from ThT, Congo Red (CR) was one of the first known probes to detect amyloid fibrils, but it also has major drawbacks, including staining instability, the lack of selectivity, and sensitivity to Ab (Kumar & Pandey, 2013;Ono et al., 2006).In previous studies, various probes have been designed and synthesized to detect amyloid fibrils.However, a probe that can effectively detect amyloid fibrils at low concentrations within the cell has not yet been identified.In this study, following our studies (Abbasbeigi et al., 2019;Ghadami et al., 2013), 17 new compounds were synthesized, and 4 of them had the best results as they could properly detect the amyloid depositions in vitro, and more importantly one of them could detect amyloid inside the cell.

Reagents
In this study, all the applied materials and reagents were provided by Sigma Aldrich and Merck companies.For thinlayer chromatography (TLC), "Merck silica gel 60 F254" sheets were used.Distilled water was used for all of the experiments.Shimadzu 470 spectrophotometer device was used for FT-IR spectra.A Bruker 500 was used for NMR spectra and chemical shifts were expressed in ppm by using DMSOd 6 as solvent and coupling constants (J), reported in Hertz (Hz).Multiplicities of the peaks have been abbreviated as singlet (s), broad singlet (bs), doublet (d), triplet (t), and quartet (q).Melting points were measured by an Electrothermal apparatus IA900 series.

Chemistry
First lab works have been done by synthesizing 17 different probes from the two compounds 6-hydroxycoumaranone and 7-methoxycoumaranone, reacted with benzaldehyde derivatives to form probe compounds such as (Z)-2-(2-fluorobenzylidene)-6hydroxybenzofuran-3(2H)-one or (Z)-2-(2-fluorobenzylidene)-7methoxybenzofuran-3(2H)-one, afterward, in the biochemical laboratory they are examined and tested to find out which ones are better detector of Ab plaques.The methods and reactions for all of the 17 probe compounds are the same but the substituents and their positions are different.It was started with the primary compound 6-hydroxycoumaranone.So first 0.15 g of 6hydroxycoumaranone was weighed and put in an Erlenmeyer, next 1 mmole of 2-flourobenzaldehyde (0.124 g) and piperazine (0.043 g) as basic catalyst was weighed and added, afterward 10 mL ethanol was added and stirred under reflux at 24-48 hours.Synthesized products were crystallized in the appropriate solvent (EtOH) and examined to identify their structure by 1 H and 13 C NMR, FTIR, and MS analyses (Supplementary Figure 1).The physical analysis and the spectral data of the synthesized 6-hydroxy-3-benzofuranone based compounds and 7-methoxy-3-benzofuranone based compounds are presented in supplementary file.

Preparation of BSA amorphous
For this study, the BSA was purchased from Sigma-Aldrich Company (St. Louis, Mo, USA).To prepare the BSA stock solution, since BSA protein was solid powder, therefore, a tiny amount was taken and dissolved in 2 mL PBS with pH ¼ 7.4 in an Eppendorf, next with help of a UV/Vis spectrophotometer its concentration was obtained and diluted to get the wanted concentration.To prepare the amorphous aggregation, 2 mL of 2 mg/mL BSA protein was taken and adjusted to pH 5.7 then incubated at 70 � C for 2 hours.

Preparation of amyloid fibrils of human insulin and amyloid b
Recombinant human insulin was purchased from Sigma-Aldrich Company (St. Louis, Mo, USA). 2 mL of 2 mg/mL solution of human insulin was adjusted at pH 5.4 and then incubated at 60 � C for 3 hours to have amyloid fibrils.Human b-Amyloid (1-42) protein fragment was purchased from Abcam Company (Cambridge, UK).To prepare Ab fibrils solutions, the powder of Ab peptide was dissolved in HFIP and was mixed gently, then, HFIP was evaporated, after that DMSO was added and the mixture was sonicated for 5 minutes (Stine et al., 2010).To prepare Ab fibril, the solution of Ab monomers was prepared in phosphate buffer and then 10 mM HCl was added to have the peptide at a final concentration of 100 lM, and then the peptide was incubated for 24 h at 37 � C (Stine et al., 2010).The next step was confirmation, and observation of Ab fibrils by scanning electron microscopy method (SEM).

Preparation of stock and working solutions
Stock solutions of probe compounds (20 mM) were prepared in phosphate buffer, and dimethyl sulfoxide (DMSO) less than 3% v/v was used as the environment and solvent for them.To provide working solutions, probe compounds were dissolved in 20 mM sodium phosphate buffer pH 7.4.The mixture solutions of the protein-probe compound were made by mixing the different concentrations of aggregated or native proteins with an aliquot of different concentrations of the compounds.

Scanning electron microscope (SEM) imaging
TESCAN MIRA3, a scanning electron microscope were used to analyze all the samples.A small amount of samples were aliquoted on SEM stubs and then dried at room temperature.
We used a sputter coater to coat all the samples with 10 nm gold.

UV/Vis and Fluorescence spectroscopic measurements
The UV-Vis spectroscopy (Shimadzu UV-1800, Shimadzu, Japan) was applied to measure the absorbance of the compounds to determine the spectra properties, which later, were used as excitation wavelengths for fluorescence spectroscopy of the synthesized probe molecules.The absorbance wavelengths of compounds were observed in the range of 200 -800 nm.All the compounds had the same figures (k max ¼ 380 nm), Supplementary Figure 2.For fluorescence measurement, and binding assay the Shimadzu fluorescence-spectrophotometer was used.The fluorescent spectra of each dye, binding to proteins were measured separately, in the presence of a native, amorphous BSA, amyloid of human insulin, and Ab solutions.In addition, the spectra of proteins alone and the compounds alone with similar concentration were recorded and were used as a baseline for fluorescence analysis of mixed samples.For accuracy and precision, all the dye ligand-binding assay tests were done three times.In the present study, all experiments were performed three times and the values were reported as the mean value ± SEM.

Docking of synthesized probes
For docking or ligand binding investigation of 17 synthesized biomarkers.The Auto Dock 2.4 software has been used (Morris et al., 1996).To draw the molecular structures of each compound, the software ChemDraw was used (Esteb et al., 2010), and their 3-D structures were plotted in Chem Office version 14.The GroMACs software has been used for the energy minimization (Hanwell et al., 2012).The 3D structure of Ab fibrils with1-42 sequence amino acids obtained from protein data bank (RCSB) (PDB ID: 2beg).Ab is considered as a receptor and synthesized probe molecules as ligands.The protein was energy minimized in water by GroMACs software and then was transferred to Auto-Dock tools, and added Gasteiger charges to prepare ligands and Kollman charges (Morris et al., 1996).For docking analyzes, all the volume of the protein was taken to monitor every single possibilities of receptor binding ligands.To have energetic maps of the software, Auto grid-4 was used, although, some of them have been done manually (Morris et al., 1998), and (Auto-Dock Tools) Lamarckian genetic algorithm distinguished specifically the most stable protein binding ligand complexes' structures (Morris et al., 1998).LigPlot plus (Laskowski & Swindells, 2011) was used for visualizing a 2D (two-dimensional) diagram form of receptor-ligand interaction.

In vitro cytotoxicity assay
For the examination of the compounds' effects as amyloid probes, the Sh-SY5Y cell line (neuroblastoma cell line) was chosen for the MTT assay.96 wells of cultured plate were used, and 20 � 10 3 cells were seeded in these plates, afterwards, they were incubated for 24 h with each of the synthetic compounds with 0-400 mM concentration.The medium was refilled at the scheduled time gap with 0.5 mg/mL MTT solution.DMSO was added to solubilize crystals of formazan, and the samples' absorbance was measured at 570 nm by using an Eliza Micro Plate reader (BioTek Instruments, USA), and the viability percentage of the cell was computed as each absorbance of the sample divided to the control absorbance.All the MTT assays were carried out three times, and also the IC 50 values were obtained as the concentration of compounds that kill 50% of the cells.IC 50 value obtained via plotting log10 percentage of proliferation versus concentration of probes by using Prism software.In the present study, all experiments were performed three times and the values were reported as the mean value ± SEM.

Staining with amyloid-diagnostic dye
SH-SY5Y cells were plated and transfected with 2 mM BSA or Ab 42 fibrils (the Ab 42 fibril formation was described previously).BSA or Ab 42 fibrils were transfected to the cells using the PULSin protein delivery reagent (Polyplus-transfection), in the presence of 100 ml of 20 mM Hepes Buffer and 4 ml of PLUSin reagent in cell culture medium without FBS.The cells were washed with PBS, after transfection, were fixed in 2% (w/v) buffered paraformaldehyde at 20 � C for 10 min, and permeabilized for 5 min with a 0.5% (v/v) Triton X-100 solution.The presence of amyloid structures was then monitored by using the amyloid-diagnostic dyes, for 1 h at 37 � C. To determine the presence of Ab 42 fibrils in the cell, a specific Ab 42 fibril primary antibody (6E10, 2 lg/mL per sample) and rabbit anti-mouse IgG (H þ L) secondary antibody Alexa FluorV R 488 conjugate (Product#A27023, Thermofisher) was used in phosphate-buffered saline (PBS) containing 0.2% BSA for 45 minutes at a concentration of 1 mg/mL at room temperature.The cell membrane was stained with Wheat Germ Agglutinin, Alexa Fluor V R 594 Conjugate (WGA).Fluorescence emission was detected after excitation at 488 nm and 594 nm for coverslips stained with Alexa Fluor V R 488, and WGA, respectively, and 380 nm for those labeled with compound by the confocal laser scanning microscope (Zeiss, Germany).

Results
Alzheimer's disease is the predominant disease of the nervous system and the fourth leading cause of death after heart disease, cancer, and stroke in advanced societies.Alzheimer's is a progressive disease characterized by specific behavioral symptoms, mainly forgetfulness and memory loss.Clinical features of Alzheimer's disease include decreased cognitive function, impaired normal daily activities, and changes in behavioral practices (Burns & Iliffe, 2009).Amyloid fibrils are formed from a process called amyloidosis, which is explained as the aggregation of protein.It is suggested that amyloid fibrils are the common pathological feature of many neurodegenerative disorders, so developing molecules to be used as markers to detect amyloid deposits has been a goal of researchers for many years.Despite all the optimism surrounding a large pipeline of drugs in clinical trials, so far, Alzheimer's treatments have been filled with disappointment as many promising drugs have failed to show significant improvement in slowing down the progression of the disease, therefore synthesizing compounds may help to have a better understanding and lead to early diagnose stages of neurodegenerative disease caused by amyloid fibrils.In this study, synthesizing and in vitro and in vivo investigations of these kinds of new fluorophores have been tested.To evaluate the ability of probe to see if they could identify amyloid fibrils, Seventeen dyes have been synthesized (Scheme 1) and five protein solutions were used, including native and amyloid human insulin, native and amorphous BSA, and finally human Ab fibrils.To determine if the probes only detect the amyloid, each compound was mixed with different protein solutions including native, amyloid fibril and amorphous, and the results of the fluorescence binding were recorded.The native BSA and native human insulin could form amorphous or amyloid fibrils aggregation according to different protocol conditions (R. Li et al., 2016;Militello et al., 2004).In this research, to convert native BSA to amorphous structure, 2 mL of 2 mg/mL native BSA with pH 5.7 was incubated at 70 � C for 2 hours and the amorphous structure was confirmed by SEM (Figure 1A). 2 mL of 2 mg/mL recombinant human insulin with pH 5.4, was incubated at 60 � C for 3 hours to have amyloid fibril, and the formation of insulin fibrils was monitored with the help of ThT assay (data not shown), and SEM microscopy (Figure 1B).Four compounds (6, 7, 9 and 10) were selected from seventeen molecules and different concentrations of them and ThT (as a standard dye) were mixed with insulin amyloid fibrils at concentrations of 0.02, 0.05 and 0.1 mg/mL separately.Synthesized compounds and standard ThT have different concentrations from very diluted to very concentrated solutions.Figure 3 shows that as the concentration of probes (x-axis) increases, the fluorescence intensity for insulin amyloid fibrils increases (y-axis).Experiments were performed with different concentrations of insulin amyloid fibrils (0.02, 0.05 and 0.1 mg/mL) to observe the accuracy and repeatability of the results.The concentration of the compound varies from 0 to 250 lM.For compound 6, there is a peak of intensity around 100 mM, but then, as the concentration of the probe increases, the fluorescence intensity becomes constant.For compound 7, its peak intensity is about 70 lM, but with increasing the dye concentration, quenching occurs, and the intensity decreases.The peak intensity of compound 9 is about 100 lM, and after that, its fluorescence intensity (FI) becomes constant.Although the fluorescence intensity of this compound is well-differentiated by varying the concentration of amyloid mass, its fluorescence intensity is not comparable to that of compound 10.For compound 10, its peak intensity is about 40 lM.Among the synthesized probes, compound 7 has the least fluorescence intensity.The fluorescence intensity of compound 7 with a peak is about 32,000, but compound 10 has the highest intensity, close to 80,000.However, by increasing the concentration of compound 10, quenching happened, the same as ThT, but there is a big difference, for ThT maximum peak intensity is about 100 mM (FI: 14,000), while for compound 10 is at 40 mM.
Comparisons of fluorescence intensities and their dissociation constants were examined using a Scatchard plot (Supplementary Figure 3) and their order is ThT > compound  increasing the amyloid concentration, its intensity increases significantly.Therefore, it was decided to observe the binding behavior of desired compounds with amyloid-b (Ab) protein and compare it with ThT in situ as well as in binding assay.For this purpose, the dye-binding assay of Ab fibril in the presence of different concentrations of the desired compounds was used.First of all, the formation of amyloid fibrils of Ab was observed by SEM (Figure 4A). Figure 4B shows a comparison of ThT and probes binding assay, as we mentioned earlier, the binding assay was carried out on human Ab at a concentration of 0.1 mg/mL, mixed with different concentrations of four selected probes to evaluate and compare their fluorescence intensities, to see if they have the same performance as they did on insulin amyloid fibrils.Figure 4B is the evidence showing the behavior of synthesized probes attached to Ab, which is almost identical to previous experiments on human insulin amyloid.Binding assay of compounds 6, 7, 9 and 10 provide information that compound 10 has the highest fluorescence intensity compared to the others and its maximum fluorescence intensity occurs at a concentrations of about 40 lM and then quenched.Figure 5 illustrates the two-dimensional aspects of molecular docking for the four selected compounds 6, 7, 9, 10 and ThT to Ab amyloid fibrils.As far as we know, the exact fibril binding mechanism of amyloid fluorophores like ThT is still unknown, but it has been confirmed that ThT as a standard probe binds to Ab fibrils.Some studies have suggested that molecular binding of ThT to Ab fibrils happens along their fibril axis (Ghadami et al., 2013;Jin et al., 2003;Krebs et al., 2005).Figure 5 illustrates Ab fibrils in which the b-sheets are arranged in a way that they bound and set next to each other tidily, forming a U-shaped chain and channel.It is confirmed that probe compounds that are analog to ThT could penetrate this channel and bind to the b-sheets of amyloid fibril (Ghadami et al., 2013;Jin et al., 2003;Krebs et al., 2005).Docking studies have shown that probes similar to ThT insert between amyloid b-sheets.Ligaplot plus software (Laskowski & Swindells, 2011) was used to compare the binding site of compounds/ThT to amyloid beta.The results showed that amino acids and binding forces between ligands and amyloid beta are different from thioflavin T. The in silico results may be a justification for the difference in the dissociation constant of compounds and thioflavin T (dyebinding assay).These data support our experimental, and it confirms that compounds 6, 7, 9 and 10 are possible appropriate alternatives to ThT which are worth to further experiments and in vivo studies.Since compounds without a permanent positive charge are mainly capable of crossing the blood-brain barrier (BBB) (Banks, 2009;Lipinski et al., 1997), in this study, we employed benzylidene-benzofuranone derivatives as fluorescent probes to quantitatively determine the amyloid fibrils made of Ab.However, to clarify the subject, pharmacokinetic parameters were provided as important filters in drug discovery.The drug-likeness prediction results for synthesized compounds by Swiss ADME (Daina et al., 2017) are given in Table 1, indicating that all selected compounds have no violations due to the Lipinski's rule of five (Lipinski et al., 1997).Predicted water solubility parameter indicated that those compounds possess permissible value.Also, it shows a satisfactory percentage of bloodbrain barrier (BBB) permeability and gastrointestinal (GI) absorption for the selected compounds.More experiments and in vivo studies are needed to determine these compounds' complete properties.
In order to find out the toxicity of synthesized biomarkers, a cell viability assay was applied with different concentrations of probe compounds.As it is known, IC 50 is a concentration of a drug or substance at which 50% of the cells will be inhibited from biological functions, in other words, below the IC 50 , the substance has cell toxicity.As could be seen (Figure 6A), all the 17 synthesized compounds have no toxicity.Many studies express that most of the dyes as biological markers have toxicity at high concentrations, which synthesized compounds of this study may be included at higher concentrations (Bieschke, 2013;  Sipe & Cohen, 2000).Compound 10, which had the best performance in vitro, was used for in vivo study.Cells containing Ab fibril were exposed to this probe, then the ability of compound to stain the amyloid-containing cells was evaluated in comparison with the control cell using a confocal microscopy.After 1 hour of incubation of cells containing Ab with the desired compound, the cells were examined by a microscope.As can be seen from Figure 6D, the desired compound was able to stain amyloid intracellularly, and instead, no staining was seen in the negative control, which contained only albumin protein (Figure 6B).In addition, a primary anti-amyloid antibody was used to detect the presence of amyloid inside the cell (Figure 6C) by employing a fluorescent dye-attached secondary antibody (see Materials and Methods for more information).

Discussion
Thousands of scientists and researchers have long been working on new ways to identify and quantify AD by synthesizing molecules as detectors.For this purpose, scientists have been conducting research on chemical probes and markers to identify and quantify key AD-causing elements, mainly amyloid b and tau proteins (Fantini et al., 2020).The main pathological causes of Alzheimer's disease, which are the deposition of Ab and tau proteins in the brain, must be identified in the early stages of the disease, so it is necessary to design and develop detectors with appropriate, specific, and selective binding to these aggregated proteins.These detections are critical in the early diagnosis of AD, as these efforts will help scientists and physicians detect the symptoms of neurological disease earlier, so they can stop or delay symptoms in the early stages before injury occurs.Then physicians can start providing treatments sooner and, more importantly allow them to try new medicines, because dementia after neuron death cannot be reversed.Alzheimer's disease is the most common cause of dementia due to the aggregation of amyloid fibrils in the brain.There is a strong need to design and develop new and more reliable probes for in vitro quantification of amyloid fibrils and in vitro amyloid imaging.Therefore, it is necessary to find sensitive compounds to detect amyloid fibrils at low concentration, especially inside the cell.For these reasons, new compounds have been synthesized.In this article, the identification and quantification of amyloid fibrils in vitro and in vivo by newly  synthesized compounds are tested and discussed.After the synthesis of the desired compounds, their structure was examined using 1 H NMR, 13 C NMR, FTIR and MS techniques (Supplementary Figure 1).The absorption spectroscopy and fluorescence properties of the compounds were determined in the presence and absence of amyloid, and their excitation and emission wavelengths were determined (Supplementary Figure 2).Each compound was excited at its specific wavelength and the emission results of the compounds in different protein solutions, including native BSA, amorphous BSA, native recombinant human insulin, and insulin amyloid fibril were recorded and compared to standard ThT.To study the abilities of 17 synthesized compounds to bind only to amyloid, not native protein or amorphous aggregation, initially, it was needed to provide amyloid of insulin and amorphous aggregates of BSA (Figure 1), subsequently, the binding of the synthesized fluorophores to them was evaluated (Figure 2).More than 90% of BSA is an alpha helix, and it is proven that BSA has the capability to form both amorphous and amyloid forms (R. Li et al., 2016).In this study, the amorphous structure of BSA was used.To obtain human insulin fibrils by the oligomerization process, protein samples were exposed to acidic pH and thermal conditions (Hua & Weiss, 2004;Wang, 2005).The morphology of amyloid fibrils of insulin was observed by the scanning electron microscopy (SEM) method.Insulin is being widely used as a model protein by researchers because it is highly susceptible to the formation of amyloid fibrils in vitro under certain experimental conditions (Y.Li et al., 2012;Nilsson, 2016;Schlein, 2017).In addition, Insulin has specific roles in the body and brain and dysregulation of these functions may contribute to the expression of late-life neurodegenerative disease (Craft & Watson, 2004;Hua & Weiss, 2004;Nilsson, 2016).As we mentioned, four different protein states, including native BSA, amorphous BSA, native insulin and insulin amyloid fibrils were considered and they were reacted one by one with 17 synthesized compounds and their function was evaluated in terms of binding.Finally, those compounds that can bind to amyloid and have a high function in differentiating between amyloid and other protein states were selected for further experiments.As shown in Figure 2, only compounds 6, 7, 9 and 10 were able to distinguish amyloid structures from the amorphous aggregation and native protein.Then, the binding properties of the desired compounds at different concentrations in the presence of different concentrations of insulin amyloid fibrils was investigated.In addition, by increasing the mass of amyloid fibrils, the fluorescence intensity of the desired compounds has increased (Figure 3).Binding properties appear to vary depending on the concentration of amyloid or the synthesized compounds.For this reason, different concentrations (0, 0.02, 0.05 and 0.1 mg/mL) of insulin amyloid fibrils were prepared and mixed separately with each of the four synthesized compounds in concentrations from 0 to 250 mM, and then their fluorescence intensities were obtained and compared with ThT as a standard probe, which is well-known in vitro amyloid detector and is a suitable standard for evaluating the performance of synthesized probes.Compounds 6, 7 and 9 show approximately the same behavior, although all of these probes showed higher dye/amyloid fluorescence intensities than ThT (Figure 3).Moreover, with increasing the concentration of the probe in the presence of an enhanced concentration of insulin amyloid, the intensity of fluorescence increases.On the other hand, compound 10 showed much higher fluorescence intensity (almost twice) under the same experimental conditions (Figure 3), which indicates a stronger binding of compound 10 to amyloid fibril.However, to show the binding strength of the compounds to the amyloid fibrils, linear regression and Scatchard analysis were used and the dissociation constants (K d ) of the compounds are as follows: ThT > compound 6 > compound 7 > Compound 9 > Compound 10 (see the Supplementary Figure 3 for more details).For compound 10, after a while, its fluorescence intensity decreases with increasing the concentration of the compounds and shows that the extra probes quench the fluorescence.The same behavior is seen in ThT, but the main difference is that compound 10 reaches its maximum fluorescence at a very low concentration of about 40 lM, while thioflavin reaches its maximum fluorescence intensity at 120 lM.Also, the binding assay of selected compounds with Ab was performed to compare with insulin amyloid fibrils.Recombinant human Ab 1-42 at 0.1 mg/mL concentration was mixed with different concentrations of four probes to evaluate their fluorescence intensities.The main aim of this experiment was to see if they have the same performance as they did on insulin amyloid fibrils.Figure 4 illustrates the behavior of desired compounds bounding to Ab that are almost the same as the previous experiments which were on human insulin amyloid fibrils.Detection of amyloid fibrils at very low concentrations is important in the diagnosis of diseases such as Alzheimer's, because several studies have shown that the concentration of amyloid in a patient's body is in the nanogram range (Lue et al., 2017;Small, 2000) and is much lower in the blood because Ab cannot simply cross the bloodbrain barrier (BBB) (Risacher et al., 2019).In addition, some studies have shown that synthesized compounds at high concentrations are toxic (Benderdour et al., 1998;Christie & Costa, 1984), although the compounds in this study did not have toxicity up to the concentrations used in the experiment (Figure 6A).Another important point is that ThT has a positive charge that cannot easily cross the BBB, and in fact the possibility of using ThT or similar compounds with the charge to diagnose amyloid in body or in vivo seems very challenging and difficult.However, the synthesized compounds in this study have no charge and have a much stronger fluorescence intensity than ThT.The exact binding mechanism of amyloid fluorophores like ThT is still unknown, but it has been confirmed that ThT as a standard probe binds to Ab amyloid (LeVine, 1995).Some studies have suggested that the molecular binding of ThT to Ab fibrils happens along their fibril axis (Biancalana & Koide, 2010; Khurana et al., 2005;Krebs et al., 2005;LeVine, 1995).Figure 5 illustrates Ab fibrils (PDB ID: 2beg), and it could be seen that b-sheets are arranged in a way they bound and set next to each other tidily, like a chain and formed U shape channel.It is confirmed that compounds that are analog to ThT could penetrate this channel and bind to the b-sheets of amyloid fibrils (Biancalana & Koide, 2010;Khurana et al., 2005;Krebs et al., 2005;LeVine, 1995).The fluorescence of thioflavin T bound to amyloid fibrils is strongly influenced by its structural and electronic properties (Wolfe et al., 2010).The ThT rings can be considered as two rigid planes separated by a single bond around which rotation can occur (u), causing them to be non-coplanar (u ¼ 0 � ).The N-methyl group of the benzothiazole ring gives a steric clash that results in an optimal angle of u ¼ 37 � .By absorbing a photon, ThT enters the electronically excited state, where the angle between these two planes in this excited state is 90 degrees.In the excited state, a phenomenon known as twisted intramolecular charge transfer (TICT) occurs, where the charge on the benzothiazole ring can remain localized or delocalized over the combined ring system.In the second case, angles u > �60 � are formed, and the ThT quantum yield approaches zero (Stsiapura et al., 2007).Therefore, ThT is weakly fluorescent in free solution because it goes into a state with an angle of u > 60 � after photon absorption.If the conditions are such that rotation around u is limited by protein binding or solvent viscosity, then the time required to rotate up to 60 � is greater than the lifetime of the excited state and radiative decay occurs (Harel et al., 2008;Stsiapura et al., 2007;2008;Stsiapura et al., 2008, J Phys Chem B;Harel et al., 2008, J Am Chem Scheme 2. The pathology of Alzheimer's disease and the role of the probes to detect amyloid.Soc).Interestingly, a study of the crystal structure of the non-amyloid protein with ThT is in stark contrast to these properties as the density of the dimethylaminobenzene ring suggests a disordered range of u angles, all of which are > 60 � .Moreover, rational design of ThT derivatives or other compounds may allow greater selectivity and/or sensitivity to amyloid states.Figure 5 shows the molecular docking comparison of standard ThT and synthesized compounds to b-sheets of Ab and the similarity of their performance.Since synthesized probe compounds and ThT are phenolic compounds when they are sited between b-sheets their resonance rotation would be restricted and suppressed, as a result their fluorescence intensities would increase, and that is the advantage of these fluorophores (Biancalana & Koide, 2010;Khurana et al., 2005).
It is known that Ab fibrils are one of the major cause of Alzheimer's disease.Scheme 2 shows the pathology of Alzheimer's disease in an Alzheimer patients compared to a healthy person, as well as the possible role of synthesized compounds in the early detection of the disease.Many compounds have been synthesized for the diagnosis of amyloid fibrils in vitro, but there are fewer known compounds for intracellular detection.In this study, compound 10, which had the best binding properties in vitro, was used for in vivo study, and the results showed that this compound could detect intracellular amyloid (Figure 6B).Moreover, further studies are needed to draw accurate conclusions about this compound, but it could be a good candidate for intracellular amyloid diagnosis.

Conclusion
Developing molecules to be used as markers to detect amyloid deposits has been a goal of researchers for many years.Despite all the optimism surrounding a large pipeline of drugs in clinical trials, so far, treatments of AD have been filled with disappointment as many promising drugs have failed to show significant improvement in slowing down the progression of the disease, therefore synthesizing probe compounds may help to have a better understanding and lead to early diagnose stages of neurodegenerative disease.These chemical probes must detect amyloid fibrils in vitro and stain amyloid fibrils in vivo, which may help us in early stages of AD diagnosis.The Ab plaque deposition has been studied by various strategies as well as dye-binding assay, for example Congo red dye, that is not specific and sensitive or Thioflavin T as the most common dye in quantifying Ab deposition in vitro, which has drawbacks and cannot be used in vivo, unless become modified, because it has a poor florescence emission at low concentrations of Ab deposition, and has a positive charge, means low lipophilicity, and it is not able to pass the BBB.In this study, various benzylidene-benzofuranone derivatives were synthesized and it was shown that 4 compounds out of 17 compounds distinguished amyloid from amorphous and native proteins and had a stronger signal than thioflavin T.Among all of synthesized probes, compound 10 was able to show better binding properties than others, and in vivo study showed that this compound was capable to detect intracellular amyloid.Proving the ability of these compounds to identify specific amyloid fibrils, quantitatively and qualitatively in the brain of patients with degenerative nervous system diseases requires further studies and experiments.
Scheme 1. Synthesis of compounds.A) reagents and conditions, B) The chemical structures of 6-hydroxy-and 7 methoxy benzofuranone derivatives reacted with different benzaldehydes bearing halogens.

Figure 1 .
Figure 1.Amyloid and amorphous aggregation.(A) SEM image at the end of aggregation of BSA (2h) at pH 5.7, 70 � C was measured and showed amorphous aggregation at mentioned condition.(B) SEM image at the end of aggregation of human insulin (3 h) at pH 5.4, 60 � C was measured and showed the fibril formation at mentioned condition.

Figure 2 .
Figure 2. Fluorescence emissions of synthesized compounds (1-17) after binding to different proteins.Fluorescence spectrometry analysis of probe compounds and their performance on different proteins, when the concentrations of proteins are being increased from 0, 0.02, 0.05, 0.1, and 0.2 mg/mL.20 mM of compounds were mixed with each one of 4 protein solutions, these proteins are displayed as: insulin amyloid fibrils (dark purple), BSA amorphous (dark blue), native insulin (light purple), and BSA native (light blue).Data are presented as mean ± SEM (N ¼ 3).

Figure 3 .
Figure 3.The dye-binding fluorescence of insulin amyloid fibrils.The Fluorescence intensity changes under the effect of various concentrations of amyloid mass and probes.The insulin amyloid fibrils at three different concentrations of 0.02 (close circle), 0.05 (close square), and 0.1 (close triangle) mg/mL were treated with different concentrations of dyes.Data are presented as mean ± SEM (N ¼ 3).

Figure 4 .
Figure 4.The binding assay of probes to Ab fibrils.A) SEM image of Ab fibril formation which is explained at mentioned conditions in the material and method section.B) The dye-binding assay of desired compounds exposed to human Ab plaques (0.1 mg/mL).Data are presented as mean ± SEM (N ¼ 3).

Figure 5 .
Figure5.The molecular docking of the 4 selected compounds and ThT to Ab fibrils (PDB ID: 2beg) which shows that they bind to the same site.The Ab and the compounds were energy minimized by Gromacs and then the PDB files were moved to Autodock and possible sites were calculated, then the files were transferred to LigPlot plus software to create two-dimensional schematic representation of hydrogen bonding and hydrophobic interactions of Ab and compounds.Hydrogenbond and hydrophobic contacts are illustrated as green and red dashed lines respectively.The three-dimensional figure was created using by PyMOL software (http://pymol.org).

Figure 6 .
Figure 6.Cellular studies of compound cytotoxicity and amyloid fibril detection.A) The cytotoxicity of synthesized probe compounds to Sh-SY5Y cells.B) Representative confocal microscopy images of Sh-SY5Y cells transfected with vehicle, BSA (C), or Ab 42 fibrils (B, D).Cells were then stained with primary and secondary antibodies to show the presence of Ab 42 fibrils inside the cells (B).Cells were stained with compound 10 (B, D). Green and red fluorescence: amyloid-bound diagnostic dye and WGA-stained cell membrane, respectively.

Table 1 .
Pharmacokinetic properties of synthesized compounds.