Synthesis of compound 6
To a solution of Boc-Cys-OMe (0.88g, 3.74 mmol) in DMF (30 mL) at 0 °C was added K2CO3 (0.51 g, 3.74 mmol) and MeI (0.23 mL, 3.74 mmol) warmed to room temperature and stirred for 20 h. The reaction was diluted with a 9:1 water/NH4Cl (2 M aq. Solu.) and extracted with EtOAc (×2). The organic extracts were washed with 5 % brine, dried over Na2SO4 and concentrated. Dissolved in MeOH (10 mL) and added to a 2 N methanolic-HCl solution and stirred at room temperature for 3 h. The mixture was concentrated and the solid was washed with MTBE (50 mL ×3) to afford (S)-methyl 2-amino-3-(methylthio)propanoate hydrochloride) as a clear light yellow oil (0.62 g, 90 %). Dissolved in MeOH (8 mL) and cooled to -78 °C, added 2,2,2,4′-trifluoroacetophenone (0.60 g, 3.35 mmol) and MeOK (0.47 g, 6.70 mmol). The reaction was slowly warm to room temperature whilst stirring for 20 h – solution A. To a solution of NaBH4 (0.51 g, 13.4 mmol) in DME (10 mL) at 0 °C was added a 2 M solution of ZnCl2 in Et2O (8.03 mL). The mixture was allowed to age for 20 h –solution B. Solution A was cooled to -40 °C, diluted with MeCN (28 mL) and slowly added solution B over 20 min, then stirred at -40 °C for 2.5 h. The reaction was quenched with acetone (40 mL) over 20 min and then warmed to room temperature. The mixture was poured into an ice/water mix (250 mL), the pH was adjusted to 5 with 1 N HCl and extracted with EtOAc (200 mL ×3). The organic extracts were washed with brine (400 mL), dried over Na2SO4 and concentrated. Dissolved in DMF (15 mL), added 1-amino-1-cyclopropanecarbonitrile hydrochloride (0.39 g, 3.35 mmol), HBTU (1.59 g, 4.20 mmol) and NMM (0.54 mL, 4.00 mmol) and stirred at room temperature for 20 h. Diluted the reaction with water (120 mL) and extracted with EtOAc (100 mL ×2). The combined organic extracts were washed with 5 % brine (120 mL ×2) and brine (140 mL), dried over Na2SO4 and concentrated. The residue was purified by column chromatography (40 g column) using 0 to 75 % EtOAc in hexanes to afford (S)-N-(1-cyanocyclopropyl)-3-(methylthio)-2-(((S)-2,2,2-trifluoro-1-(4-fluorophenyl)ethyl)amino)propanoate as a clear colourless oil (0.36 g, 29 %). To a solution of (S)-N-(1-cyanocyclopropyl)-3-(methylthio)-2-(((S)-2,2,2-trifluoro-1-(4-fluorophenyl)ethyl)amino)propanoate (0.34 g, 0.91 mmol) in EtOAc (8.78 mL) was added NaTg•2H2O (3.56 mg, 0.01 mmol) and TBAH (15.6 mg, 0.05 mmol) followed by addition of H2O2 (30 % w/w in water, 0.23 mL). The reaction was stirred at room temperature for 3 h. Diluted the reaction mixture with EtOAc (70 mL) and washed with a solution of 2 M Na2S2O3 (100 mL ×2) and brine (100 mL), dried over Na2SO4 and concentrated to afford (S)-N-(1-cyanocyclopropyl)-3-(methylsulfonyl)-2-(((S)-2,2,2-trifluoro-1-(4-fluorophenyl)ethyl)amino) propanoate (compound 6) as a white powder (0.29 g, 79 %); 1H-NMR (400 MHz, d
6
-DMSO) δ 9.05 (s, 1H), 7.45 (dd, J = 8.0, 6.0 Hz, 2H), 7.25 (dd, J = 8.0, 6.0 Hz, 2H), 4.37 (quintet, J = 8.0 Hz, 1H), 3.74-3.66 (m, 1H), 3.54-3.46 (m, 2H), 3.17 (dd, J = 16.0, 4.0 Hz, 1H), 3.12 (s, 3H), 1.42-1.33 (m, 2H), 1.05-0.98 (m, 1H), 0.79-0.74 (m, 1H); LC-MS > 98 %, m/z = 408.12 [M + H].
Inhibition of cathepsin activity using compound 6
Inhibition of cathepsin activity using compound 6 Recombinant cathepsin activity: Analysis of recombinant cathepsin activity was performed in a 96-well plate. All assays were performed in triplicate in the presence of sodium acetate assay buffer (sodium acetate 100 mM, EDTA 1 mM, Brij 0.1 %, and Dithiothreitol 2 mM, pH 5.5). Recombinant CTSS (4 nM), K (4.25 nM), V (4 nM), L (4 nM) and B (3.5 nM) (Calbiochem, UK) was incubated with compound 6 at a range of concentrations. The concentration of recombinant protein used in these assays was assumed to be equivalent to the active enzyme concentrations. Cathepsin activity was monitored using peptidyl fluorescent substrates; Cbz-VVR-AMC (20 μM, CTSS), Cbz-FR-AMC (20 μM, Cathepsins K, V and at 5 μM for cathepsin L) and Cbz-RR-AMC (20 μM, cathepsin B). Protease activity was monitored over the period of 1 h using a fluorometer (Flurostar Optima) with excitation at 390 nm and emission at 460 nm. Progress curve data points generated by compound 6 were fitted to equation
1 using GraphFit software.
$$ v={v}_o\ \frac{\left[E\right]-\left[I\right]-{K}_{i\left(\mathrm{app}\right)}+\sqrt{{\left(\left[E\right]-\left[I\right]-{K}_{i\ \left(\mathrm{app}\right)}\right)}^2+4\left[E\right].{K}_{i\ \left(\mathrm{app}\right)}}}{2\left[E\right]} $$
(1)
Where,
\( v \)
o is the steady state rate,
\( E \) is the enzyme concentration and
\( I \) is the inhibitor concentration. Using this equation values of
\( v \) were generated for each concentration of inhibitor. Using these values, Morrison Plots were subsequently produced (
\( v \) versus [I]), allowing determination of
K
i (app) values. To account for any completing substrate these values were corrected using equation
2, allowing the generation of true
K
i values for compound 6 versus cathepsins S, K, V, L and B.
$$ {K}_i = {K}_{i\ \left(\mathrm{app}\right)}/\left(1+\frac{\left[S\right]}{K_m}\right) $$
(2)
Cathepsin activity in lysates: For analysis of CTSS-like activity in MC38 cell lysates, MC38 cells were grown to confluency, harvested and lysed on ice using sodium acetate lysis buffer (Sodium acetate 100 mM, sodium chloride 100mM, Triton X-100 0.1 %, pH 5.5). Lysates were quantified by BCA, before addition to black-bottom 96-well plate at 100 μg per well. The lysates were incubated in 100 mM phosphate buffer (pH 7.5) at 37 °C for 1 h to inactivate cathepsins B and L. Following this step, the lysates were incubated in MES buffer (MES 500 mM, EDTA 1mM, Dithiothreitol 2 mM, pH 5.5) to return the pH to 6, compound 6 added, and the lysates incubated for 30 min at 37 °C. The lysates were then evaluated for CTSS-like activity using 20 μM Cbz-VVR-AMC and fluorogenic substrate turnover monitored over the period of 1 h using a fluorometer (Flurostar Optima) as described above. The result was presented in triplicate, expressing relative fluorescent units versus time in minute ± SEM.
Western blotting
Western blotting was carried out as previously described [
29] using the following primary antibodies: mouse anti-human CD74 (1:400) (sc-47741, Santa Cruz, USA [
30]), rat anti-mouse CD74 (1:1000) (555317, BD Biosciences, USA [
31]) and rat anti-α-tubulin (1:10000) (ab6160, Abcam, UK [
32]). The membrane was subsequently incubated with appropriate secondary antibody; goat anti-mouse HRP conjugate (1:10000) (172-1011, BioRad, UK [
29]) or rabbit anti-rat HRP conjugate (1:40000) (ab102199, Abcam, UK [
29]). Proteins were detected by chemiluminescence protocol and exposed using the BioRad Molecular Imager ChemiDoc XRS+ Imaging System (BioRad, USA).
MC38 syngeneic model
Six to eight week old C57BL/6 mice were subcutaneously injected with 5.0×10
6 MC38 cells resuspended in growth factor reduced matrigel (4 mg/ml, diluted in sterile PBS) into the right flank on day 0. Upon tumors reaching 100 mm
3, mice were then treated with compound 6 (2 mg compound 6 in 4 % DMSO: 96 % peanut oil; 100 mg/kg) or vehicle control (4 % DMSO: 96 % peanut oil) via intraperitoneal injection every 2 to 3 days. Tumor volumes were calculated as Volume = length × breadth ×
π/6 (
n = 5/group). Data was presented as mean tumor volume per group ± SEM. Statistical analysis carried out by student’s
t test. Blood serums were sampled at day 13 from mice and subjected to CCL2 ELISA (R&D Systems, UK) as previously described [
6].