Synthesis of Novel 5-( N-Boc-N-Benzyl-2-aminoethyl )-7-oxo-4 , 7-dihydropyrazolo [ 1 , 5-a ] pyrimidin-3-carboxamides and Their Inhibition of Cathepsins B and K

Eight novel 5-(N-Boc-N-benzyl-2-aminoethyl)-7-oxo-4,7-dihydropyrazolo[1,5-a]pyrimidin-3-carboxamides were prepa red in three steps from methyl 3-amino-1H-pyrazole-4-carboxylate and methyl 5-(benzyl(tert-butoxycarbonyl)amino)-3-oxopentanoate. The synthetic procedure comprises cyclocondensation of the above starting compounds, hydrolysis of the ester, and bis(pentafluorophenyl) carbonate (BPC)-mediated amidation. Title carboxamides were tested for inhibition of cathepsins K and B. The N-butylcarboxamide 5a exhibited appreciable inhibition of cathepsin K (IC50 ~ 25 μM), while the strongest inhibition of cathepsin B was achieved with N-(2-picolyl)carboxamide 5c (IC50 ~ 45 μM).

The biological activity of compounds 3, 4, and 5a-h was tested against the cysteine peptidases cathepsins B and K, which are both important drug targets. 31All compounds were initially tested for their activity at a concentration of 100 µM.As shown in Table 2, compound 5a had the strongest inhibitory effect on cathepsin K, with an IC 50 value of 25 ± 5 µM under the experimental conditions used in the assay and complete (100%) inhibition was observed at concentrations of 600 µM or higher.The effect of other compounds was significantly weaker and resulted in less than 50% inhibition.Cathepsin B was most strongly inhibited by compound 5c (IC 50 value of 45 ± 15 µM) and to a lesser extent by compounds 5a and 5d.Altogether these results identify three compounds, 5a, 5c and 5d, as potential lead compounds for further development (Table 2).

1. General Methods
Melting points were determined on a Stanford Research Systems MPA100 OptiMelt automated melting point system.The NMR spectra were obtained on a Bruker Avance III UltraShield 500 plus at 500 MHz for 1 H and 126 MHz for 13 C, using CDCl 3 and DMSO-d 6 (with TMS as the internal standard) as solvents.Mass spectra were recorded on an Agilent 6224 Accurate Mass TOF LC/MS spectrometer, IR spectra on a Bruker FTIR Alpha Platinum ATR spectrophotometer.Flash column chromatography (FC) was performed on silica gel (Fluka, Silica gel 60, particle size 35-70 μm).

-carboxamides 5a-h
A mixture of carboxylic acid 4 (207 mg, 0.5 mmol), MeCN (5 mL), and Et 3 N (70 μL, 0.5 mmol) was stirred at room temperature for 5 minutes.Then, BPC (197 mg, 0.5 mmol) was added and the reaction mixture was stirred at r.t. for 2 h (activation of carboxylic acid 4 via formation of the pentafluorophenyl ester 4').Next, amine 6 (0.5 mmol) and Et 3 N (70 μL, 0.5 mmol) were added and stirring at room temperature was continued for 24 h.The reaction mixture was evaporated in vacuo (60 °C/2 mbar) and the crude semi-solid carboxamide 5 was purified by FC on silica gel (first EtOAc to elute the non-polar impurities, then CH 2 Cl 2 -MeOH, 10:1, to elute the product).Fractions containing the product were combined and evaporated in vacuo to give carboxamides 5a-h.

5. Activity assays against cathepsins K and B
The activity of all compounds was tested against recombinant human cathepsins K and B produced in-house according to the known protocol. 32All assays were performed in 50 mM sodium acetate buffer pH 5.5 containing 1 µM EDTA and 2.5 mM DTT.The hydrolysis of the synthetic substrate Z-Phe-Arg-AMC (5 µM final concentration) was followed fluorimetrically at an excitation wavelength of 370 nm and an emission wavelength of 455 nm.Final concentrations of the enzymes in the reaction mixtures were 1 nM.Experiments were first performed at a fixed compound concentration of 100 µM.Compounds with significant inhibitory activity were re-tested by measuring residual enzyme activity in the presence of increasing concentrations of the compounds and IC 50 values were calculated from these titration curves.

Conclusions
Eight novel 5-(N-Boc-N-benzyl-2-aminoethyl)-7oxo-4,7-dihydropyrazolo[1,5-a]pyrimidin-3-carboxamides 5a-h were prepared in three synthetic steps from methyl 3-amino-1H-pyrazole-4-carboxylate (1) and methyl 5-(benzyl(tert-butoxycarbonyl)amino)-3-oxopentanoate (2).The synthetic procedure comprises cyclocondensation of the above starting compounds, hydrolysis of the ester function, and BPC-mediated amidation.This method offers a quick access to various 5-(2-aminoethyl) substituted pyrazolo[1,5-a]pyrimidin-3-carboxamides 5 from easily available starting materials.Testing of the intermediates 3 and 4 and title compounds 5a-h for inhibition of cathepsins B and K revealed that most of them were weak inhibitors at 100 mM concentration.Carboxamide 5a had the strongest inhibitory effect on cathepsin K, with an IC 50 value of 25 ± 5 µM.Cathepsin B was most strongly inhibited by compounds 5c and 5d with the respective IC 50 values of 45 ± 15 µM and 150 ± 50 µM and to a lesser extent by compound 5a as well.Inhibitory activities of compounds 5a, 5c, and 5d against cysteine peptidases cathepsins B and K identify them as potential leads for drug development.In summary, the synthetic method allows for a simple preparation of libraries of title compounds that could be useful for medicinal and pharmaceutical applications.

a)
All experiments were performed in 50 mM sodium acetate buffer pH 5.5 containing 1 mM EDTA, 2.5 mM DTT and the fluorigenic substrate Z-Phe-Arg-AMC (5 µM final concentration).Final enzyme concentrations were 1 nM.IC 50 values were determined from titration curves.b ) Residual activity at saturation.c ) Activity of 5b could not be determined fluorometrically due to strong absorption of the compound at the excitation wavelength.Lukić et al.: Synthesis of novel 5-(N-Boc-N-benzyl-2-aminoethyl)-7-oxo-...

Table 2 :
Effect of compounds 3, 4 and 5a-h on the activity of cathepsins K and B. a