Synthesis and Assessment of Novel Probes for Imaging Tau Pathology in Transgenic Mouse and Rat Models

Aggregated tau protein is a core pathology present in several neurodegenerative diseases. Therefore, the development and application of positron emission tomography (PET) imaging radiotracers that selectively bind to aggregated tau in fibril form is of importance in furthering the understanding of these disorders. While radiotracers used in human PET studies offer invaluable insight, radiotracers that are also capable of visualizing tau fibrils in animal models are important tools for translational research into these diseases. Herein, we report the synthesis and characterization of a novel library of compounds based on the phenyl/pyridinylbutadienylbenzothiazoles/benzothiazolium (PBB3) backbone developed for this application. From this library, we selected the compound LM229, which binds to recombinant tau fibrils with high affinity (Kd = 3.6 nM) and detects with high specificity (a) pathological 4R tau aggregates in living cultured neurons and mouse brain sections from transgenic human P301S tau mice, (b) truncated human 151-351 3R (SHR24) and 4R (SHR72) tau aggregates in transgenic rat brain sections, and (c) tau neurofibrillary tangles in brain sections from Alzheimer’s disease (3R/4R tau) and progressive supranuclear palsy (4R tau). With LM229 also shown to cross the blood–brain barrier in vivo and its effective radiolabeling with the radioisotope carbon-11, we have established a novel platform for PET translational studies using rodent transgenic tau models.

The solution was cooled to -78 ˚C and methanol (1.00 mL) was added. The solution was warmed to room temperature and aqueous saturated Rochelle salts solution (100 mL) was added and the resulting solution stirred at room temperature for 1 h after which the aqueous layer was extracted with dichloromethane. The combined organic layers were dried (MgSO4) and concentrated in vacuo.
Step 2: The crude alcohol from step 1 was dissolved in tetrahydrofuran (5 mL) and activated manganese dioxide (590 mg, 6.78 mmol) was added. The reaction mixture was stirred at room temperature for 16 h after which the mixture was filtered. The filtrate was concentrated in vacuo. The crude product was purified by flash column chromatography (70% ethyl acetate/petroleum ether) to afford the title compound (95 mg, 0.586 mmol, 87%).
Once 70 ˚C was reached concentrated sulfuric acid (632 µL) in acetonitrile (1.3 mL) was added dropwise over 15 min. The solution was then allowed to stir at 70 ˚C for 4 h before being cooled to room temperature and quenched with 25% aqueous Na2SO3 solution. The aqueous layer extracted with ethyl acetate and the combined organics were dried (Na2SO4) and concentrated in vacuo. The crude product was purified by flash column chromatography (30-50% ethyl acetate/petroleum ether) to afford the title compound

5-fluoro-6-methoxy-2-methylbenzo[d]thiazole
To a mixture of N-(5-fluoro-2-iodo-4-methoxyphenyl)acetamide (2.54 g, 8.22 mmol), copper(I) iodide (163 mg, 0.858 mmol), and sodium sulfide nonahydrate (6.19 g, 25.8 mmol) was added dimethylformamide (20 mL) and the resulting reaction mixture was heated to 80 ˚C for 18 h. The solution was then cooled to room temperature and concentrated hydrochloric acid (6.70 mL) was added. After stirring at room temperature for 7 h, saturated aqueous sodium bicarbonate solution was added and the aqueous layer was extracted with ethyl acetate. The combined organics were washed with water and brine, dried (Na2SO4) and concentrated in vacuo. The crude product was purified by flash column chromatography (10% ethyl acetate/petroleum ether) to afford the title compound (1.20 g, 6.08 mmol, 74%).
The aqueous layer extracted with ethyl acetate and the combined organic layers were washed with saturated aqueous sodium bicarbonate, dried (Na2SO4) and concentrated in vacuo. The crude product was purified by flash column chromatography (10-20% ethyl acetate/petroleum ether) to afford the title compound (90 mg, 0.172 mmol, 64%). Nb. TBS group can cleave in reaction therefore may be purified off and re-protected under standard conditions.

Binding affinity of all compounds with tau fibrils
Microscale Thermophoresis was first used to screen whether there were interaction between the compounds and tau fibrils. For this procedure 200 µL, 5 µM tau fibrils were mixed with 200 µL, 10 µM NT647-NHS dye and then incubated in dark for 30 minutes. Further dialysis (18 hours, changing the buffer after the first 4 hours) was used to remove the free dye giving a 2.5 µM solution of labelled protein. The labelling ratio of dye unit per tau monomer was then tested by a method recommended by the instrument manufacturer, NanoTemper Technologies, which is based on a calibration curve. The 2.5 µM tau fibril solution was then aliquoted prior to use. The unused samples were frozen at -20 °C. MST measurements were conducted using Monolith NT.115 instrument (NanoTemper Technologies, Germany) at 25 °C. Assays were conducted at 40 % MST Power and 90 % IR-laser power. With each compound, two concentrations (1µM and 1nM) were used to mix with 100nM tau fibrils and then loaded to the MST instrument to read out the MST data curve. Only three compounds 166, 164 and 169 showed no changes with the MST data curve. All other compounds binding affinities towards tau fibrils were then tested by fluorescent binding assay.

Binding to cultured neurons from P301S tau transgenic mice
DRG were incubated with the indicated concentrations of LM229 or PBB3 for 20 minutes at room temperature, washed 3 times in PBS, and imaged on a Leica DMI 4000B microscope using a Leica DFC3000 G camera and the Leica application suite 4.0.0.11706. Fluorescence intensity was measured in 10-16 neurons each from 3 independent cultures using Image J (imagej.nih.gov/ij/). Results shown are mean ± SD. Non linear least squares curve fitting was applied using http://faculty.gvsu.edu/carlsont/232lab/nonlin2.html. No Km value was determined for PBB3.