Allosteric Partial Inhibition of Monomeric Proteases. Sulfated Coumarins Induce Regulation, not just Inhibition, of Thrombin

Allosteric partial inhibition of soluble, monomeric proteases can offer major regulatory advantages, but remains a concept on paper to date; although it has been routinely documented for receptors and oligomeric proteins. Thrombin, a key protease of the coagulation cascade, displays significant conformational plasticity, which presents an attractive opportunity to discover small molecule probes that induce sub-maximal allosteric inhibition. We synthesized a focused library of some 36 sulfated coumarins to discover two agents that display sub-maximal efficacy (~50%), high potency (<500 nM) and high selectivity for thrombin (>150-fold). Michaelis-Menten, competitive inhibition, and site-directed mutagenesis studies identified exosite 2 as the site of binding for the most potent sulfated coumarin. Stern-Volmer quenching of active site-labeled fluorophore suggested that the allosteric regulators induce intermediate structural changes in the active site as compared to those that display ~80–100% efficacy. Antithrombin inactivation of thrombin was impaired in the presence of the sulfated coumarins suggesting that allosteric partial inhibition arises from catalytic dysfunction of the active site. Overall, sulfated coumarins represent first-in-class, sub-maximal inhibitors of thrombin. The probes establish the concept of allosteric partial inhibition of soluble, monomeric proteins. This concept may lead to a new class of anticoagulants that are completely devoid of bleeding.

General Procedure for O-Demethylation -O-methylated analogs were suspended in dry DCM and subjected to 1.5 eq. per methoxy group of 1.0 M BBr 3 solution at -78 ˚C under N 2 atmosphere. The solutions were stirred for 5 h, allowed to reach RT, following which the solutions were re-cooled to 0 ˚C and water (or 1:1 water/methanol) were slowly added with stirring to quench the reaction. The biphasic solution was then condensed under vacuum and 20 mL ethyl acetate and 25 mL saturated ammonium chloride solutions were sequentially added to the mixture. The aqueous phase was washed twice with 10-20 mL ethyl acetate and the organic layers were pooled, washed with brine, dried over an. Na 2 SO 4 and then condensed under vacuum, followed by purification via flash chromatography (0-70% ethyl acetate/hexanes). Fractions that contained the demethylated products were pooled to yield white or yellow solids in 87 -98% yields in purity greater than 95%, confirmed by UPLC. Products of 2w-2y were then directly subjected to sulfation. (2w-2y, 3a1-3g1, see Figure  General Procedure for Dimer Synthesis -To prepare dimeric C-SAMs 3a-3g (Table 1), we employed copper (I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction, as described earlier. 34 Supplementary Figure S3 shows the preparation of the alkylazide scaffold. Scaffolds 3a1-3g1 containing two phenolic -OH groups (at the 7-hydroxy position and 3-[4'-hydroxyphenyl]) were treated with 0.9 -1.0 molar eq. of 1-bromo-3-chloropropane/1-bromo-4-chlorobutane and 0.5 eq. of Cs 2 CO 3 in DMF for 24 h at room temperature. Upon completion of the reaction, an acidic work up was followed by extraction with ethyl acetate and standard chromatographic purification to give the intermediates 3a2-3g2 in 66-83% yields.
The resulting alkylchloride intermediate was dissolved in 3:1 DMF/ACN, transferred to a 10 mL microwave vessel containing 1.5 eq. of sodium azide. The vessel was sealed, placed in the microwave reactor, and heated for 5 h at 70 ºC. Upon cooling to RT, the solution was mixed with 10-20 mL ethyl acetate and 10 mL 1.0 M HCl and the organic layer separated and worked up to yield corresponding the alkyl azide derivatives 3a3-3g3 in 90 -97% yields after flash chromatography and >94% purity confirmed by UPLC. The products were characterized using 1 H-NMR, ROESY, and IR (see Supplementary Figure S3-S4 and Spectra Data S1).
Supplementary Figure S3 also shows the preparation of the propargyl scaffold. To produce the alkyne reactant, the analogs displayed two OH groups as stated above. 3a-3g and 0.9-1.0 eq. of 80% propargyl bromide solution were added to a stirring solution of 0.5 eq. of Cs 2 CO 3 in DMF and continued to stir at room temperature for 24 h. Upon completion, 20 mL of ethyl acetate and 15 mL of 1.0 M HCl were added to the reaction and the organic layer was separated and worked up to yield the propargyl derivatives 3a4-3g4 in 58-89% yields after flash chromatography and >95% purity confirmed by UPLC.
The structure and propargyl positioning were confirmed by 1 H-NMR and ROESY (see Supplementary Figure S3 and S5 and Spectra Data S1).
The intermediates 3a3-3g3 and 3a4-3g4 were coupled together, respectively under CuAAC condition. 1.1 eq of azide intermediate and 1.0 eq of alkyne intermediate were combined and stirred in DMF with 7 mol% of sodium ascorbate solution in water and 7 mol% 1:1 CuSO 4 •5H 2 O : tris-(benzyltriazolylmethyl)amine (TBTA) in 55% DMSO for 24 h at RT. The reaction was then quenched with 15 mL of chilled water to form precipitants, then filtered, and washed 20 mL of water twice. The precipitant collected was then dissolved in 25 mL ethyl acetate, which was washed with brine, dried over Na 2 SO 4 , and purified via flash chromatography giving the coupled products 3a5-3g5 in 72-94% yields with >95% purity, confirmed by UPLC. Structural identification was performed by 1 H-NMR (see Supplementary Figure S3 and Spectra Data S1).
General Procedure for Sulfation of Coumarin Derivatives -Derivatives composed of the free phenolic-OH were then subject to microwave assisted sulfation, Supplementary Figure S1-S3 shows the preparation of sulfation of 1a-1d, 2a-2y, and 3a-3g. The compounds were added to a microwave reaction vessel with 6 eq of SO 3 •N(CH 3 ) 3 per OH, 10 eq of TEA per OH, and 2-5 mL of ACN (or ACN/DMF).
The vessel was then placed in the microwave reactor for 1 h per OH at 90-100 ºC. The solution and vessel were washed out with 10 mL of DCM and purified by flash chromatography (0-30% methanol/DCM), affording the sulfate containing the trimethylamine counter ion. Cation exchange chromatography was performed on the purified compounds and lyophilized to give the sodium sulfate final products in high yields (82-99%) with >95% purity confirmed by UPLC-ESI-MS, along with structural identification by Table S1. Inhibition of thrombin, factor Xa and factor XIa by sulfated coumarins. a