Bifunctional crosslinking ligands for transthyretin

Wild-type and variant forms of transthyretin (TTR), a normal plasma protein, are amyloidogenic and can be deposited in the tissues as amyloid fibrils causing acquired and hereditary systemic TTR amyloidosis, a debilitating and usually fatal disease. Reduction in the abundance of amyloid fibril precursor proteins arrests amyloid deposition and halts disease progression in all forms of amyloidosis including TTR type. Our previous demonstration that circulating serum amyloid P component (SAP) is efficiently depleted by administration of a specific small molecule ligand compound, that non-covalently crosslinks pairs of SAP molecules, suggested that TTR may be also amenable to this approach. We first confirmed that chemically crosslinked human TTR is rapidly cleared from the circulation in mice. In order to crosslink pairs of TTR molecules, promote their accelerated clearance and thus therapeutically deplete plasma TTR, we prepared a range of bivalent specific ligands for the thyroxine binding sites of TTR. Non-covalently bound human TTR–ligand complexes were formed that were stable in vitro and in vivo, but they were not cleared from the plasma of mice in vivo more rapidly than native uncomplexed TTR. Therapeutic depletion of circulating TTR will require additional mechanisms.

The reaction was degassed with vacuum/nitrogen x 3, then heated to reflux and stirred under nitrogen for 18 h. The reaction was allowed to cool to room temperature, then concentrated in vacuo. The mixture was then partitioned between water and EtOAc. The aqueous phase was Hz), 7.33 (1H, d, J = 9.28 Hz), 7.08 (1H, dd, J = 2.87, 9.28 Hz), 7.04 (2H, s), 6.94 (1H, s), 3.93 (3H, s), 3.83 (3H, s). To an anhydrous solution of 2-(3,5-dichloro-phenylamino)-5-methoxy-benzoic acid methyl ester (6 g, 18.4 mmol) in DCM (50 ml) at -5 o C, was added BBr 3 (5.2 ml, 55.2 mmol) dropwise under nitrogen. The resulting mixture was left to stir at -5 o C for 30 mins. TLC analysis showed no starting material present. Water was carefully added and the resulting mixture was extracted with DCM. The combined organic layer was washed with brine, dried over Na 2 SO 4 and filtered. The filtrate was evaporated under reduced pressure to give a crude oil. This was left under vacuum to dry and then taken into the next step.
The solution was heated under reflux for 8 h. The solution was then evaporated to give colourless oil. This was partitioned between EtOAc and water. The aqueous layer extracted with additional EtOAc. The combined organic layer washed with brine, dried over sodium sulfate and filtered. The filtrate was evaporated under reduced pressure followed by drying under vacuum to give the title compound as yellow solid (  Under N 2 , 2-(3,5-dichloro-phenylamino)-5-hydroxy-benzoic acid methyl ester (2 g, 6.4 mmol) was dissolved in THF (25 ml). t-Butyl-N-(3-hydroxypropyl)carbamate (1.31 ml, 7.68 mmol) and triphenylphosphine (2.52 g, 9.6 mmol) were then added, followed by DIAD (1.51 ml, 7.7 mmol). The reaction was allowed to stir overnight, then quenched by addition of NaHCO 3 (sat. aq.), extracted x 3 with ethyl acetate, the combined organics washed with brine, dried over Na 2 SO 4 , filtered and concentrated in vacuo. The residue was purified by flash column chromatography with 10-20% EtOAc/i-hexanes to afford 2.7 g of yellow foam.
The mixture was taken into the next step.
An additional 0.1 eq. phosgene was then added (1.46 mL) and the reaction was shown to be complete by TLC.

5N HCl
Bis-(1,4-dioxa-8-aza-spiro [4.5]dec-8-yl)-methanone (3.81 g, 12.2 mmol) was dissolved in 5N HCl and allowed to stir for 24 h. The reaction was then cooled to 0 o C and quenched with 5N NaOH until pH 9, as judged by universal pH paper. The mixture was then extracted 3 x EtOAc, dried over Na 2 SO 4 , filtered and concentrated in vacuo to afford a colourless solid.

IIc
IIc dimethyl ester (39 mg, 0.0273 mmol) was suspended in THF (1.2 mL). The mixture was heated to aid solubility, however the compound did not dissolve. After cooling to RT, LiOH

IIa
IIa methyl ester (formate salt, 62 mg, 0.043 mmol) was dissolved in dimethoxyethane (1 mL), tetrahydrofuran (1 mL) and methanol (1 mL) and lithium hydroxide added (0.5 M in water, 191 µL, 0.095 mmol) was added. The mixture was heated to 70°C for 3 h. LCMS showed no change, so additional LiOH (0.5 M in water, 191 µL, 0.095 mmol) was added and the mixture allowed to stir at 70°C overnight. LCMS showed no change so additional LiOH (0.5 M in water, 1 mL) was added. After 3 h the reaction showed complete conversion to the di-acid.
The mixture was allowed to cool to room temperature. The mixture was quenched with 2N HCl and a yellow precipitate appeared. The mixture was diluted with MeOH and loaded onto a SCX cartridge. The cartridge was washed with methanol, then the product was eluted with

Group I ligands:
Amino acids and coupling agents were purchased from Novabiochem, and other reagents and solvents were purchased from Sigma-Aldrich or VWR. Succinimido-(Pro) 5 -Gly-OH was synthesised by Fmoc peptide chemistry using a 3 fold molar excess of amino acid and HBTU, and 9 fold molar excess of DIPEA, on a Symphony automated peptide synthesiser (Protein Technologies). The peptide was cleaved using TFA:TIS:H 2 0 95:2:2 and purified by RP-HPLC (C18 Gemini Axia, 5u 110A 250 x 21.2mm 20ml/min at