MATRIPTASE INHIBITORS AND USES THEREOF

Abstract

The present application relates to a compound of formulae (I), (II) or (III) or a pharmaceutically acceptable salt thereof, methods and uses thereof for treating disorders associated with matriptase activity. (I), (II), (III)

##STR00001##

Claims

1. A compound of formula: ##STR00028## or a pharmaceutically acceptable salt thereof.

2.-4. (canceled)

5. The compound of claim 1, wherein said compound is of formula: ##STR00029## or a pharmaceutically acceptable salt thereof.

6. The compound of claim 1, wherein said compound is formula: ##STR00030## or a pharmaceutically acceptable salt thereof.

7. The compound of claim 1, wherein said compound is formula: ##STR00031## or a pharmaceutically acceptable salt thereof.

8. The compound of claim 1, wherein said compound is formula: ##STR00032## or a pharmaceutically acceptable salt thereof.

9. A pharmaceutical composition comprising a compound according to claim 1 and a pharmaceutically acceptable carrier, diluent and excipient.

10.-24. (canceled)

25. A method for treating hyperproliferative disorders, tissue disorders, inflammatory disorders, respiratory disorders, viral infections or disorders associated with iron overload in a subject in need thereof which comprises administering a therapeutically effective amount of a compound according to claim 1 or a composition comprising said compound.

26. The method according to claim 25, for the treatment or prevention of oral squamous cell carcinoma.

27. The method according to claim 25, for the treatment or prevention of osteoarthritis.

28. The method according to claim 25, for the treatment or prevention of idiopathic pulmonary fibrosis.

29. The method according to claim 25, for the treatment or prevention of influenza type A, B or C.

30. The method according to claim 25, for the treatment or prevention of coronaviruses infections.

31. The method according to claim 25, for the treatment or prevention of human coronavirus HCoV-NL63, HCoV-OC43, HCoV-229E, HCoV-HKUI, SARS-CoV (Severe Acute Respiratory Syndrome-Corona Virus), or CoV MERS (Middle East Respiratory Syndrome virus.

32. The method according to claim 25, for the treatment or prevention of parainfluenza viruses infections.

33. The method according to claim 25, for the treatment or prevention of HPIV type 1, HPIV type 2, HPIV type 3 or HPIV type 4.

34. The method according to claim 25, wherein the subject is a human subject.

35. The method according to claim 25, wherein said compound is of formula: ##STR00033## or a pharmaceutically acceptable salt thereof.

36. The method of claim 35, for the treatment or prevention of oral squamous cell carcinoma in a human subject.

37. The method of claim 35, for the treatment or prevention of osteoarthritis in a human subject.

38. The method of claim 35, for the treatment or prevention of idiopathic pulmonary fibrosis in a human subject.

Description

EXAMPLES

[0158] As used herein, the following abbreviations may have the following meanings:

TABLE-US-00001 Abbreviation Term ACN Acetonitrile DCM Dichloromethane DIPEA N,N-Diisopropylethylamine DMF N,N-dimethyl formamide DPM Dess-Martin periodinane EtOAc Ethyl acetate HATU (dimethylamino)-N,N-dimethyl(3H- [1,2,3]triazolo[4,5-b]pyridin-3- yloxy)methaniminium hexafluorophosphate HCl Hydrochloric acid HFIP Hexafluoroisopropanol iPrOH Isopropanol UPLC-Ms Ultra perfomance liquid chromatography mass spectrum min Minute(s) MeOH Methanol MsCl Methanesulfonyl chloride Mtr 4-Methoxy-2,3,6- trimethylbenzenesulphonyl NMR Nuclear magnetic resonance SFC Supercritical fluid chromatography THF Tetrahydrofuran TFA Trifluoroacetic acid

Example 1: Synthesis of Compound 1

[0159] ##STR00012##

Fmoc-Phe-Resin, Intermediate 4:

[0160] To 10 g of CTC Resin with a loading of 1.2 mmol/g were added Fmoc-Phe-OH (9.3 g, 24 mmol, 2 eq) dissolved in DCM (approximately 10 mL per gram of resin), and DIPEA (6.3 mL, 3 eq). The mixture was shaken vigorously for 30-60 min. To endcap any remaining reactive trityl chloride groups, HPLC grade methanol was added (0.8 mL per gram of resin), and mixed for 15 minutes. The resin was filtered and washed with 3DCM, 2DMF, 2DCM, 3iPrOH, 3DCM, then dried in vacuo.

NH.SUB.2.-Gln(Trt)Phe-Resin, Intermediate 5:

[0161] A solution of DMF/piperidine (20%) was added to the resin, which was then gently shaken for 30 minutes. The resin was filtered and washed with 3DMF, iPrOH, 3DCM then dried in vacuo. A solution of Fmoc-Gln(Trt)-OH (14.6 g, 24 mmol, 2 eq), HATU (9.3 g, 24 mmol, 2 eq) and DIPEA (1.05 mL, 6 mmol, 5 eq) were dissolved in DMF (approximately 10 mL per gram of resin), was added on resin. The resin was shaken for 2 h, filtered, then with 3DMF, iPrOH, 3DCM then dried in vacuo. A solution of DMF/piperidine (20%) was added to the resin, which was then gently shaken for 30 minutes. The resin was filtered and washed with 3DMF, iPrOH, 3DCM then dried in vacuo.

(H)Arg(Boc).SUB.2.-Gln(Trt)-Phe-Resin, Intermediate 6:

[0162] A solution of (H)Arg(Boc).sub.2-OH 8 (8.7 g, 24 mmol, 3 eq), HATU (9.3 g, 24 mmol, 3 eq) and DIPEA (1.05 mL, 6 mmol, 5 eq) were dissolved in DMF (approximately 10 mL per gram of resin), and was added on to the resin. The resin was shaken for 2 h, filtered, washed with 3DMF, iPrOH, 3DCM then dried in vacuo.

(H)Arg(Boc).SUB.2.-Gln(Trt)-Phe-OH Intermediate 7:

[0163] To 10 g of derivatized resin was added a solution 20% HFIP in DCM and shaken for 45 minutes. After removal of the solution, the resin was washed with DCM/HFIP (20%), 3DCM. After suspension and co-evaporation in diethylether, the white solid was filtrated and dried in vacuo to give tripeptide 7 as a white solid (8.8 g). The compound was used as it in the next step without purification. Purity: >95% by UPLC.

##STR00013##

[0164] To a solution of Intermediate 7 (3.72 g, 4.25 mmol, 1 eq) in anhydrous DMF were added HATU (1.61 g, 4.25 mmol, 1.1 eq), amine 3 (2.55 g, 4.68 mmol, 1.1 eq), and DIPEA (2.2 mL, 12.7 mmol, 3 eq) at 0 C. The mixture was stirred 15 minutes. The tetrapeptide was precipitated in cold water (0 C.), filtrated and washed twice with cold water. The filtrate was dissolved in ethyl acetate, washed with aqueous citric acid (10%) and brine. The organic phase was dried with sodium sulfate, filtrated and evaporated. The white solid was triturated in ether and purified by flash chromatography (MeOH/DCM 1:99 to MeOH/DCM 5:95). Intermediate 22 was obtained as a white solid (3.2 g, 52%).

[0165] DMP (1.2 g, 2.8 mmol, 1.4 eq) was added to a solution of protected tetrapeptide 22 (2.8 g, 2 mmol, 1 eq) in DCM for 15 minutes. The solution was washed with water, aqueous citric acid 10% and brine. The organic phase was dried with sodium sulfate and evaporated. The residue was triturated in cold ether and purified by flash chromatography (MeOH/DCM 1:99 to MeOH/DCM 5:95) to give the desired intermediate 23 as a white solid (2.4 g, 86%).

[0166] 2.4 g of intermediate 23 is dissolved in a mixture of 20 mL of TFA/H.sub.2O (95:5) and stirred for 1 hour, until completion of the reaction by UPLC-MS. The TFA/H.sub.2O solution was added dropwise to 235 ml of cold water (0 C.) in two centrifugation tubes and then centrifuged at 4000 rpm for 30 minutes. The supernatant was removed and the white precipitated dissolved in water, washed with ether and lyophilized. A >95:5 mixture of diastereomer in favor of the S diastereomer of the arginine alpha carbon was obtained (1.3 g).

[0167] Compound is purified by reverse phase prep-HPLC MS (C18) using a ACN/water gradient (0.1% TFA) from 10 to 30% of ACN. As an example, 27 mg of pure compound was obtained from 50 mg of crude.

[0168] UPLC-Ms Retention time: 1.19 min

[0169] Purity: >99%

[0170] HRMS: Calculated for C.sub.33H.sub.45N.sub.11O.sub.5S: 708.3404 (MH.sup.+); Found: 708.3534 (MH.sup.+).

Example 2: Synthesis of Compound 2

[0171] ##STR00014##

Fmoc-Phe-Resin, Intermediate 4:

[0172] To 10 g of CTC Resin with a loading of 1.2 mmol/g were added Fmoc-Phe-OH (9.3 g, 24 mmol, 2 eq) dissolved in DCM (approximately 10 mL per gram of resin), and DIPEA (6.3 mL, 3 eq). The mixture was shaken vigorously for 30-60 min. To endcap any remaining reactive trityl chloride groups, HPLC grade methanol was added (0.8 mL per gram of resin), and mixed for 15 minutes. The resin was filtered and washed with 3DCM, 2DMF, 2DCM, 3iPrOH, 3DCM, then dried in vacuo.

NH.SUB.2.-Gln(Trt)Phe-Resin, Intermediate 5:

[0173] A solution of DMF/piperidine (20%) was added to the resin, which was then gently shaken for 30 minutes. The resin was filtered and washed with 3DMF, iPrOH, 3DCM then dried in vacuo. A solution of Fmoc-Gln(Trt)-OH (14.6 g, 24 mmol, 2 eq), HATU (9.3 g, 24 mmol, 2 eq) and DIPEA (1.05 mL, 6 mmol, 5 eq) were dissolved in DMF (approximately 10 mL per gram of resin), and added onto the resin. The resin was shaken for 2 h, filtered, then with 3DMF, iPrOH, 3DCM then dried in vacuo. A solution of DMF/piperidine (20%) was added to the resin, which was then gently shaken for 30 minutes. The resin was filtered and washed with 3DMF, iPrOH, 3DCM then dried in vacuo.

Ac-Gln(Trt)-Phe-Resin, Intermediate 6:

[0174] A solution of acetic acid (1.8 mL, 30 mmol, 2.5 eq), HATU (12 g, 30 mmol, 2.5 eq) and DIPEA (10 mL, 60 mmol, 5 eq) were dissolved in DMF (approximately 10 mL per gram of resin), and was added onto the resin. The resin was shaken for 2 h, filtered, washed with 3DMF, iPrOH, 3DCM then dried in vacuo.

Ac-Gln(Trt)-Phe-OH Intermediate 7:

[0175] To 10 g of derivatized resin was added a solution 20% HFIP in DCM and shaken for 45 minutes. After removal of the solution, the resin was washed with DCM/HFIP (20%), 3DCM. After suspension and co-evaporation in diethylether, the white solid was filtrated and dried in vacuo to give tripeptide 7 as a white solid (6.7 g). The compound was used as it in the next step without purification. Purity: >95%.

##STR00015##

[0176] To a solution of Intermediate 7 (3.5 g, 6.1 mmol, 1 eq) in anhydrous DMF (45 mL) was added HATU (2.6 g, 6.9 mmol, 1.1 eq), amine 3 (3.7 g, 6.9 mmol, 1.1 eq), and DIPEA (3.25 mL, 10.2 mmol, 3 eq) at 0 C. The mixture was stirred 15 minutes. The solution was poured in cold water (0 C.), filtrated and washed twice with cold water. The filtrate was dissolved in ether and DCM, washed with aqueous citric acid (10%) and brine. The organic phase was dried with sodium sulfate, filtrated and evaporated. The white solid was triturated in ether/hexane. The white solid was filtrated and used as it without purification. (5.3 g)

[0177] DMP (3 g, 7 mmol, 1.5 eq) was added to a solution of tetrapeptide 22 (5.2 g, 4.7 mmol, 1 eq) in DCM for 15 minutes. The solution was washed with water, aqueous citric acid 10% and brine. The organic phase was dried with sodium sulfate and evaporated. The residue was triturated in cold ether and purified by flash chromatography (EtOAc/Hexane 10:90 0:100) to give the desired intermediate 23 as a white solid (3.5 g)

[0178] 3 g of intermediate 23 is dissolved in a mixture of 20 mL of TFA/H.sub.2O (95:5) and stirred for 1 hours, until completion of the reaction by UPLC-MS. The TFA/H.sub.2O solution is added dropwise to 235 ml of cold water (0 C.) in two centrifugation tubes and then centrifuged at 4000 rpm for 30 minutes. The supernatant is removed and the white precipitated is dissolved in water and washed with ether. A 90:10 mixture of diastereomer in favor of the S diastereomer of the arginine alpha carbon is obtained (2.0 g)

[0179] Compound is purified by reverse phase prep-HPLC MS (C18) using a ACN/water gradient (0.1% TFA) from 20 to 40% of ACN. For example, 38 mg of pure compound was obtained from 50 mg of crude.

[0180] UPLC-Ms Retention time: 1.28 min

[0181] Purity: 99%

[0182] HRMS: Calculated for C.sub.29H.sub.36N.sub.8O.sub.5S: 609.2602 (MH.sup.+); Found: 609.2621 (MH.sup.+).

Example 3: Synthesis of Compound 3

[0183] ##STR00016##

[0184] Gln(Trt)-OH (10 g, 25.7 mmol, 1 eq.) was dissolved in 1.5N NaOH (25 mL) and dioxane (75 mL) was added and cooled at 0 C. MsCl (2.7 ml, 25.7 mmol, 1 eq.), and NaOH 1.5N was added dropwise to maintain the pH to 9-10 for 2 h and at room temp. for 2 h. Dioxane is evaporated and ether is added. The precipitate is filtrated and the solid dissolved in DCM/Ether and dried with sodium sulfate. A white solid is obtained (7.2 g) and used in next step without purification.

##STR00017##

Fmoc-Phe-Resin, Intermediate 4:

[0185] To 4 g of CTC Resin with a loading of 1.2 mmol/g was added Fmoc-Phe-OH (4.6 g, 12 mmol, 2 eq) dissolved in DCM (approximately 10 mL per gram of resin), and DIPEA (3.2 mL, 3 eq). The mixture was shaken vigorously for 30-60 min. To endcap any remaining reactive trityl chloride groups, HPLC grade methanol was added (0.8 mL per gram of resin), and mixed for 15 minutes. The resin was filtered and washed with 3DCM, 2DMF, 2DCM, 3iPrOH, 3DCM, then dried in vacuo.

[0186] Ms-Gln(Trt)Phe-Resin, Intermediate 5:

[0187] A solution of DMF/piperidine (20%) was added to the resin, which was then gently shaken for 30 minutes. The resin was filtered and washed with 3DMF, iPrOH, 3DCM then dried in vacuo. A solution of Ms-Gln(Trt)-OH (5.35, 12 mmol, 3 eq), HATU (4.6 g, 12 mmol, 3 eq) and DIPEA (3.5 mL, 20 mmol, 5 eq) were dissolved in DMF (approximately 10 mL per gram of resin), and was added onto the resin. The resin was shaken for 2 h, filtered, then with 3DMF, iPrOH, 3DCM then dried in vacuo.

Ms-Gln(Trt)-Phe-OH Intermediate 7:

[0188] To 4 g of derivatized resin was added a solution 20% HFIP in DCM and shaken for 45 minutes. After removal of the solution, the resin was washed with DCM/HFIP (20%), 3DCM. After suspension and co-evaporation in diethylether, a white solid was obtained after flash chromatography purification (1.9 g). Purity: >95%.

##STR00018##

[0189] To a solution of Intermediate 7 (1.8 g, 2.9 mmol, 1 eq) in anhydrous DMF was added HATU (1.1 g, 2.9 mmol, 1 eq), amine 3 (1.74 g, 3.2 mmol, 1.1 eq), and DIPEA (1.5 m L, 8.7 mmol, 3 eq) at 0 C. The mixture was stirred 15 minutes. The solution was poured into cold water (0 C.), filtrated, and washed with cold water twice. The filtrate was dissolved in ethyl acetate, washed with aqueous citric acid (10%) and brine. The organic phase was dried with sodium sulfate, filtrated and evaporated. The white solid was triturated in ether and filtrated to give intermediate 22 as a white solid (3.1 g).

[0190] DMP (1.6 g, 5.25 mmol, 1.5 eq) was added to a solution of tetrapeptide 22 (2.9 g, 3.5 mmol, 1 eq) in DCM for 15 minutes. The solution was washed with water, aqueous citric acid 10% and brine. The organic phase was dried with sodium sulfate and evaporated. The residue was triturated in cold ether and purified by flash chromatography (EtOAc/Hexane 10:90 0:100) to give the Intermediate 23 as a white solid (1.6 g)

[0191] 1.6 g of intermediate 23 was dissolved in a mixture of 20 mL of TFA/H.sub.2O (95:5) and stirred for 1 hour, until completion of the reaction by UPLC-MS. The TFA/H.sub.2O solution is added dropwise to 235 ml of cold water (0 C.) in two centrifugation tubes and then centrifuged at 4000 rpm for 30 minutes. The supernatant is removed and the white precipitated is dissolved in water, washed with ether and lyophilized. A >95:5 mixture of diastereomer in favor of the S diastereomer of the arginine alpha carbon is obtained (0.8 g).

[0192] The compound was purified by reverse phase prep-HPLC MS (C18) using a ACN/water gradient (0.1% TFA) from 20 to 40% of ACN. For example, 32 mg of pure compound was obtained from 50 mg of crude.

[0193] UPLC-Ms Retention time: 1.27 min

[0194] Purity: 99%

[0195] HRMS: Calculated for C.sub.28H.sub.36N.sub.8O.sub.6S.sub.2: 645.2272 (MH.sup.+); Found: 645.2305 (MH.sup.+).

Building Block Synthesis

[0196] ##STR00019##

Synthesis of Intermediate: 2

[0197] ##STR00020##

Procedure

[0198] To a stirred solution of Intermediate 1 (35 g, 66.4 mmol) in anhydrous THF (700 mL) was added HATU (37.9 g, 99.6 mmol), N,O-dimethylhydroxylamine.HCl (7.77 g, 79.7 mmol) and DIPEA (35.7 mL, 199.3 mmol) at room temperature and the reaction mixture was allowed to stir overnight. The solvent was evaporated and the crude material was purified by column chromatography using silica gel, eluting with 60-65% ethyl acetate in hexanes. The pure product fractions were collected to afford 35 g of pure product as a white solid in 92% yield. Chiral HPLC purity: 98.88%, MH.sup.+ 569.72

Synthesis of Intermediate: 3

[0199] ##STR00021##

Procedure

[0200] To a stirred solution of benzothiazole (21 mL, 189.56 mmol) in anhydrous THF (125 mL) was added n-BuLi (1M in hexane) (110 mL, 112.33 mmol) at 78 C. by cannula over a period of 20 minutes and stirred for 30 minutes, followed by the addition of solution of Intermediate 2 (12 g, 21.06 mmol) in anhydrous THF (75 mL) within a minute. After 5 minutes, a saturated solution of ammonium chloride (100 mL) was added and the reaction extracted with ethyl acetate (500 mL3). The combined the organic layers were washed with brine (100 mL), dried over anhydrous sodium sulphate and evaporated under vacuum to afford the crude product. The crude material was purified by silica gel column chromatography, eluting with 2% methanol in dichloromethane. The pure product fractions were collected and evaporated to afford 6.5 g of pure compound in 48% yield. Chiral HPLC SFC purity: S-isomer (82.09%), R-isomer (16.59%). MH.sup.+ 643.95

Synthesis of Intermediate A

[0201] ##STR00022##

Procedure

[0202] To a stirred solution of Intermediate 3 (13 g, 20.18 mmol) in MeOH (150 mL) at 20 C. was added sodium borohydride (4.58 g, 121.12 mmol) portion wise and stirred for 30 min. After 30 min, acetone (150 mL) was added and the reaction mixture was stirred for 30 minutes. The solvent was evaporated under reduced pressure and water (300 mL) was added to the residue and then extracted with ethyl acetate (500 mL3). The combined organic layer was washed with brine (500 mL), dried over anhydrous sodium sulphate and evaporated under vacuum to afford the crude product. The crude material was purified by silica gel column chromatography, eluting with 3% methanol in dichloromethane. The pure product fractions were collected and evaporated to afford 9.6 g of pure intermediate A in 74% yield. .sup.1H-NMR (DMSO-d.sub.6) 8.07-8.05 (d, 1H, ArH), 7.94-7.92 (d, 1H, ArH), 7.50-7.46 (dd, 1H, ArH), 7.46-7.38 (dd, 1H, ArH), 6.79-6.35 (bs, 3H, NH), 4.86-4.84 (m, 1H, CH), 3.39 (m, 1H, CH), 2.95 (bs, 4H, CH.sub.2), 2.49-2.41 (m, 6H, CH.sub.3), 1.99 (s, 3H, CH.sub.3) and 1.44-1.11 (m, 17H, CH.sub.2, CH.sub.3). Chiral HPLC purity (in 4 peaks): 100%, MH.sup.+645.83.

Example 4: Matriptase Inhibition

Materials

[0203] Purified recombinant human matriptase, was prepared as described in Dsilets A et. al 2006, Inhibition of human matriptase by eglin c variants. FEBS Lett. April 17; 580(9):2227-32. Matriptase was active-site titrated with the burst titrant 4-methylumbelliferyl-p-guanidino benzoate (MUGB).

General Kinetic Methods

K.SUB.i .Determination Using Steady-State Velocities

[0204] Enzymatic assays and K.sub.i determination were performed at room temperature in an assay buffer containing 50 mM Tris-HCl, 150 mM NaCl and 500 g/mL BSA at pH 7.4. To determine which method to use for the evaluation of inhibition, 0.25 nM protease was added to a reaction buffer containing 0 nM, 2.5 nM or 1 mM of inhibitors and 200 M of a fluorogenic substrate (Boc-Gln-Ala-Arg-AMC). Proteolytic activity was monitored by measuring the release of fluorescence (excitation; 360 nm, emission; 441 nm) in a FLX800 TBE microplate reader (Bio-Tek Instruments, Winooski, Vt., USA).

[0205] If inhibition occurs only at I/E >10, data generated from plots of enzyme velocity as a function of substrate concentration at several inhibitor concentrations were fitted by nonlinear regression to equations describing different models of reversible inhibition (competitive, uncompetitive, non-competitive and mixed model). The preferred model was used for K.sub.i determination.

[0206] If substantial inhibition occurred using a ratio I/E10, compounds were treated as tight-binding inhibitors. Plots of enzyme velocity as a function of inhibitor concentrations were fitted by nonlinear regression analysis to the Morrison equation for K.sub.i determination of tight-binding inhibitors.

[0207] All assays were performed at least three times in duplicates, and data were presented as meanstandard error of the mean (SEM). Nonlinear regression and statistical analysis were performed using GraphPad Prism version 6.02 for Windows (GraphPad Software, San Diego, Calif., USA).

Kinetic Parameters Determination Using Progress Curve Analysis

[0208] Matriptase cleavage of Boc-Gln-Ala-Arg-AMC was monitored (excitation; 360 nm, emission; 460 nm) 1200 min using a FLX-800 TBE microplate reader (Bio-Tek Instruments, Winooski, Vt., USA).

[0209] Equations representing one- and two-step mechanisms of reaction were used to fit the data from the progress curves obtained in the presence of different inhibitor concentrations. Data fitting was performed using Dynafit version 4.07.066.

[0210] When rapid equilibrium was assumed, the ON rates for ES and EI formation (k.sub.1 and k.sub.3) were fixed at 100 M.sup.1 s.sup.1, and k.sub.2 at 8,400 s.sup.1 for matriptase to satisfy experimental K.sub.m value. Calculated K.sub.cat was fixed to 9.52 s.sup.1.

[0211] Enzyme inactivation rate (k.sub.iE) and enzyme concentration ([E]) were determined by curve fitting in the absence of inhibitor when fixing substrate concentration ([S]). Determined values were used as fixed values to determine k.sub.3, k.sub.4, k.sub.5 and k.sub.6 values with kinetics in presence of inhibitors. Inhibitor concentrations ([I]) were fitted except for the lowest concentration that was fixed.

[0212] For the two-step model, the inhibition constants were calculated as: K.sub.i=k.sub.4/k.sub.3, K.sub.i*=K.sub.i k.sub.6/(k.sub.5+k.sub.6), k.sub.on=k.sub.5 and k.sub.off=k.sub.4 k.sub.6/(k.sub.4+k.sub.5+k.sub.6) The dissociation half-life of the enzyme-inhibitor complex was calculated as t.sub.1/2=0.693/k.sub.off. For the one-step model, k.sub.on and k.sub.off were equal to k.sub.3 and k.sub.4, respectively.

[0213] The results are shown in Table 1.

Example 5: Cellular Assay-Influenza Virus Replication PR8 and X31 in Calu-3 Human Bronchial Epithelial Cells

[0214] The ability of the tested compound to block influenza virus replication (PR8 and X31) in Calu-3 human bronchial epithelial cells was evaluated as described by Beaulieu A. et al. J Virol. 2013 April; 87(8):4237-51.

[0215] Calu-3 cells were washed with Dulbecco's phosphate-buffered saline (D-PBS) and exposed to influenza virus (diluted in incomplete medium; 0.2% bovine serum albumin [BSA] instead of FBS). After virus adsorption (1 h at 37 C.), cells were washed once with D-PBS, and cells were incubated in incomplete culture medium containing increasing concentrations of the tested compound for 48 h.

[0216] Viral titers were determined in the supernatants of infected cells by viral plaque assays as described by Cloutier et al. J Infect Dis. 2012 Feb. 15; 205(4):621-30. Serial 10-fold dilutions of clarified supernatants were prepared in incomplete Eagle's minimal essential medium (EMEM) (containing 0.1% bovine serum albumin instead of fetal bovine serum) and were titered on Madin-Darby canine kidney (MDCK) cells according to standard viral plaque assays. Confluent MDCK cells were exposed to lung supernatant dilutions for 1 hour to allow virus adsorption. Cells were then washed, and a semifluid medium containing Avicel RC-581 (FMC BioPolymer), incomplete EMEM, and 1 g/mL Tosyl phenylalanyl chloromethyl ketone (TPCK)-treated trypsin (Sigma-Aldrich) was added to the cells. Cells were incubated for 48 hours, and viral plaques were revealed with 2% crystal violet after Carnoy fixation.

[0217] The data shows that the tested compound inhibited PR8 H1N1 and X31 H3N2 influenza virus replication in a dose dependent manner. The results are shown in Table 1.

TABLE-US-00002 TABLE 1 Matriptase calu3_pr8_H1N1 calu3_x31_H3N2 Tested Ki EC.sub.50 EC.sub.50 compound Structure (avg) nM (avg) nM (avg) nM Comparative #1 [00023] 0.0114 5069.3 7698.3 Comparative #2 [00024] 0.0877 N/A 5689 Example 1 [00025] 0.1341 27.333 20.867 Example 2 [00026] 2.6303 1.5583 9.3333 Example 3 [00027] 0.5063 2.2813 34

Example 6: Osteoarthritis (OA) Model

[0218] The ability of the tested compound to protects against in vivo aggrecan loss in an osteoarthritis (OA) model was evaluated as described by Litherland G J. et al. Arthritis Rheumatol 2014; 66:2175-87.

[0219] OA was induced in C57BL/6J mice following destabilization of the medial meniscus (DMM) surgery. Mice received 5 microlitre intraarticular injections of either saline or Compound 1 (10 mg/ml in saline) on days 7 and 28 post surgery. At day 56 post-surgery, mice were killed, knee joints isolated and fixed overnight in 7% formaldehyde in PBS. Joints were decalcified in EDTA in PBS for 10 days and then wax embedded. Consecutive sections (6 m) were stained with safranin O with hematoxylin counterstaining and then cartilage damage graded (by two blinded, independent observers) according to a published grading system [Litherland, 2014]: 0 (normal) to 6 (vertical clefts/erosion to the calcified cartilage extending to 0.75% of the articular surface). Bars show the mean+/SD of the highest scores in the medial tibial and femoral condyles for each joint. Compound 1 decreased the cartilage damage score. The results are depicted in FIG. 1.

[0220] **P<0.01 for the control and ***P<0.001 for Compound 1, by Kruskal-Wallis test with Dunn's multiple comparison test.

Example 7: Bleomycin-Induced Pulmonary Fibrosis (IPF) Model

[0221] Idiopathic Pulmonary Fibrosis (IPF) is a chronic progressive and usually fatal lung disease with no identifiable etiology. Evolving fibrotic process is responsible for an Interstitial Lung Diseases, with abnormal pulmonary functions; including evidence of restriction and impaired gas exchange.

[0222] The activity of the compounds of formula I, II or III in IPF is evaluated as follows.

[0223] Bleomycin (0.08 U/kg) or PBS is instilled intratracheally into male C57/BL6 mice. Mice are treated intranasally with a tested compound (5 mg/kg/day) starting either the next day after bleomycin instillation (tested compound d1-d10, pre-treatment) or 10 days after bleomycin instillation (tested compound d10-d20, treatment). In both experimental settings (pre-treatment or treatment), saline control groups are included. At day 21 after bleomycin instillation, mice are euthanized and their lungs are harvested. The right lobe is fixed with 10% neutral buffered formalin for 24 hours, transferred to 70% ethanol, and embedded in parafin. Five-micrometer sections are processed for histopathology with Masson's trichrome stain. Fibrosis is quantified using the modified Ashcroft scoring system. n=1, 4-6 mice per group