Toxic Tau Oligomers Modulated by Novel Curcumin Derivatives

The pathological aggregation and accumulation of tau, a microtubule-associated protein, is a common feature amongst more than 18 different neurodegenerative diseases that are collectively known as tauopathies. Recently, it has been demonstrated that the soluble and hydrophobic tau oligomers are highly toxic in vitro due to their capacity towards seeding tau misfolding, thereby propagating the tau pathology seen across different neurodegenerative diseases. Modulating the aggregation state of tau oligomers through the use of small molecules could be a useful therapeutic strategy to target their toxicity, regardless of other factors involved in their formation. In this study, we screened and tested a small library of newly synthesized curcumin derivatives against preformed recombinant tau oligomers. Our results show that the curcumin derivatives affect and modulate the tau oligomer aggregation pathways, converting to a more aggregated non-toxic state as assessed in the human neuroblastoma SH-SY5Y cell line and primary cortical neuron cultures. These results provide insight into tau aggregation and may become a basis for the discovery of new therapeutic agents, as well as advance the diagnostic field for the detection of toxic tau oligomers.

. Biochemical analysis of oligomeric tau treated with Hemi-curcuminoid (HemiC) derivatives and untreated control. (A) Western blot analysis of tau oligomers alone and pretreated with curcumin and Hemicurcuminoid analogs probed with T22, shows that some of the compounds can alter the aggregation states of preformed tau oligomers. (B) Dot Blots analysis of oligomeric tau alone and in the presence of HemiC, probed with the Tau Oligomer Monoclonal Antibody (TOMA1), T22 and Tau5, shows that some of the HemiC compounds are able to decrease tau oligomer levels as compared to the untreated control. (C) ELISA analysis of oligomeric tau with and without HemiC analogs shows that some HemiC affect tau aggregation pathways reducing tau oligomer levels as compared to the untreated control while there is no change in total tau protein as assessed by Tau 5 immunoreactivity. Data were compared by one-way analysis of variance (ANOVA), followed by Dunnett's multiple comparison test: *p<0.05; **p<0.01; ****p<0.0001. Bars and error bars represent the mean and standard deviation. Figure S2. Biochemical analysis of oligomeric tau treated with Curcumin-like (CL) derivatives and untreated control. (A) Western blot analysis of tau oligomers alone and those pretreated with curcumin and CL analogs, probed with T22, shows that the compounds are able to alter the aggregation states of preformed tau oligomers. (B) Dot blot analysis probed with anti-oligomeric monoclonal and polyclonal tau antibodies, respectively TOMA1 and T22, and total tau antibody, Tau 5. (C) ELISA analysis of oligomeric tau shows a significant decrease in the tau oligomer levels in the presence of the CL compounds as compared to the untreated control, TauO. Data were compared by one-way analysis of variance (ANOVA), followed by Dunnett's multiple comparison test: **p<0.01; ***p<0.001; ****p<0.0001. Bars and error bars represent the mean and standard deviation. Figure S3. Biochemical analysis of oligomeric tau with and without Heterocyclic curcumin (CH) derivatives treatment. (A) Western blot analysis of tau oligomers alone and pretreated with curcumin and Heterocyclic curcumin analogs probed with T22, shows that the incubation with the compounds modulates the aggregation states of preformed tau oligomers as compared to the untreated TauO. (B) Filter Trap and Dot blot analyses of tau oligomers alone and pretreated with curcumin and CH analogs probed with T22 and Tau 5. Some of the compounds are able to alter the aggregation states of preformed tau oligomers, resulting in decreased tau oligomer levels as compared to tau oligomers alone. CH analogs reduce TOMA1 immunoreactivity. (C) ELISA analysis of oligomeric tau with and without CH derivatives show decreased T22 immunoreactivity after treatment with the compounds and no changes in total tau protein as assessed using Tau 5 antibody. Data were compared by one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparison test: *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. Bars and error bars represent the mean and standard deviation. Figure S4. Biochemical analysis of oligomeric tau with and without Calebin-A (Cal) derivatives treatment. (A) Western blot analysis of tau oligomers alone and those pretreated with curcumin and Calebin-A analogs probed with T22, shows that the incubation with the compounds modulates the aggregation states of tau oligomers as compared to the untreated TauO. (B) Filter Trap assay, probed with T22 and Tau 5, show that some of the compounds decrease T22 immunoreactivity as compared to the untreated TauO. (C) ELISA analysis of oligomeric tau after treatment with Cal derivatives shows that some of the compounds decrease tau oligomer levels as seen by the reduced T22 immunoreactivity and no changes in total tau protein using Tau 5. Data were compared by one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparison test: **p<0.01; ***p<0.001; ****p<0.0001. Bars and error bars represent the mean and standard deviation.

Experimental Section
All solvent and reagents were used as received, unless otherwise stated. Melting points were determined on a hot-stage apparatus. 1 H-NMR and 13 C-NMR spectra were recorded at indicated frequencies, residual solvent peak was used as reference. Chromatography was performed by using silica gel (0.040-0.063 mm) and mixtures of ethyl acetate and petroleum ether (fraction boiling in the range of 40-60 °C) in various ratios (v/v). All solvent and reagents were used as received. Compounds 2a,b,e,g 1 , 2c 2 , 2d 3 , 3a,b,e,g 1 , 4b-e,j 4 , 4k 5 , 5a 6 , 5h 7 , CL1-3,5 8 , 7 5,9 , 8 5,10 , CH4 5 were prepared as previously reported. Other already known and new compounds were prepared adapting previously reported methods as indicated below.

Hemi-curcuminoid derivatives 2-5
These compounds were obtained by adapting previously reported condensation reactions (Scheme S1).,-Unsaturated ketones 2 were obtained through Claisen-Schmidt Aldol condensation 11 by treating commercial aldehydes 1 with acetone under basic conditions. In turn, reaction of compounds 2 with iodine, in the presence of CuO as catalyst, yields to iodo-derivatives 3 1 E-Cinnamic acids 4 were obtained performing Doebner modification of Knoevenagel condensation 4 , ethyl cinnamate 4k was similarly obtained 5 . Cinnamonitriles 5a,h were obtained from benzaldehyde 1 condensation with acetonitrile, as previously reported 7 .

S7
Among obtained compounds 2-5 were selected Hemi-curcuminoid compounds HemiC1-10 (Table   S1) which were tested as representative example of variously substituted derivatives. On the other hand, compounds 2-5 were used as building-block for the obtainment of other target compounds (see below).

Calebin-A analogs Cal1-9
The synthesis of Calebin-A and its analogs Cal1-9 was accomplished by coupling, through a nucleophilic substitution reaction, iodo-derivatives 3 and cinnamic acids 4 14 , avoiding the use of protective groups.

General Procedure for Claisen-Schmidt Aldol condensation.
A mixture of aldehyde 1f (0.1 mol) and acetone (0.11 mol, 8 mL) in 150 mL EtOH/H2O (1:1) was stirred at room temperature for about 5 min followed by the addition of NaOH (5.2 g, 0.13 mol). The reaction was monitored by TLC. 1 M HCl was used to neutralize the system before extraction three times with EtOAc. The combined organic layer was dried with Na2SO4 and then concentrated in vacuo gave the crude product, which was employed without further purification.

General procedure for the Doebner condensation
To a stirred solution of aldehyde 1 (1.00 mmol) in toluene (5 mL) were added malonic acid (156 mg, 1.50 mmol), pyridine (0.12 mL, 1.54 mmol), and aniline (0.01 mL, 0.12 mmol), and the resulting S11 mixture was refluxed for 18 h. After cooling, the reaction mixture was diluted with AcOEt and 10% HCl aq. and organic phase was separated, dried over Na2SO4, and concentrated in vacuo. The residue was chromatographed to give corresponding carboxylic acid 4. According to this procedure, the following known carboxylic acids were prepared. General procedure for the preparation of Cinnamils CL1-12.
Cinnamils CL were prepared according to the known literature procedure. 8 In a 50 mL round bottom flask to a solution of diacetyl 6 (2.15 g, 25 mmol) in 10 mL methanol were added aldehyde 1 (100 mmol, 4 equiv), acetic acid (0.03 equiv) and piperidine (0.03 equiv). The reaction mixture was refluxed at 83°C for 3h with stirring. The reaction mixture was cooled to room temperature, solvent was removed and cooled in an ice bath and the precipitate formed was filtered, washed with cold methanol and dried.   19 .

General method of preparation of 1,3,4-oxadiazole
To a solution of cinnamic acid 4 (1.2 mmol) in acetonitrile (7 mL At the end of the reaction the crude mixture was concentrated in vacuo end treated with water and NaOH. The aqueous phase was extracted with dichloromethane, dried over sodium sulfate and concentrated in vacuo The resulting crude was purified by chromatography.