USE OF COUMARIN DERIVATIVES FOR THE PREPARATION OF DRUGS FOR TREATING SKIN DISEASES

Abstract

A compound of formula (I-1)

##STR00001##

wherein n equals 0 or 1, Z represents O or S, R1 represents one group chosen among the group consisting of hydrogen, C1-C7 alkyl, substituted, or not, by a halogen, a hydroxyl or a OR12 group, wherein R12 is a C1-C7 alkyl, a group CH.sub.2OCOR5 wherein R5 is chosen among a hydrogen atom and a C1-C7 alkyl, substituted or not by at least one halogen, a group OR13, wherein R13 is chosen among hydrogen and a C1-C7 alkyl, an amine or a CH.sub.2-amine, R1 represents a group chosen among hydrogen and OR14, wherein R14 is chosen among hydrogen and a C1-C7 alkyl, and R2 is chosen among the group consisting of a C1-C7 alkyl, a C3-C6 cycloalkyl, an aryl group, and an heteroaryl group for the treatment of pathologies involving excess activity of at least one member of the kallikrein family.

Claims

1. A method for the treatment of pathologies involving an excess of activity of at least one member of the kallikrein family, comprising administering to a patient in need thereof a composition comprising an effective amount of a compound selected from the group consisting of: ##STR00185##

2. The method according to claim 1, wherein said at least one member of the kallikrein family is selected from the group consisting of kallikrein-5 and kallikrein-7, and said pathology is selected from the group consisting of Netherton syndrome, psoriasis, atopic eczema and allergic contact dermatitis.

3. The method according to claim 1, wherein said compound is ##STR00186##

4. The method according to claim 2, wherein said compound is ##STR00187##

5. The method according to claim 1, wherein said compound is ##STR00188##

6. The method according to claim 2, wherein said compound is ##STR00189##

7. The method according to claim 1, wherein said compound is ##STR00190##

8. The method according to claim 2, wherein said compound is ##STR00191##

9. The method according to claim 1, wherein said pathology is Netherton syndrome.

10. The method according to claim 2, wherein said pathology is Netherton syndrome.

Description

[1171] FIG. 1: This figure presents the activation cascade involving kallikreins. In Netherton syndrome LEKTI loses its faculty to efficiently control the KLK5, leading to an abnormal skin desquamation due to an increase of the degradation of the corneodesmosomes proteins. Synthetic inhibitors are susceptible to control the double KLK5 function, that is direct degradation and other proteases activation, KLK7 and KLK14, this one activating pro-elastase.

[1172] FIG. 2 and FIG. 3 present cell viability of normal human keratinocytes after treatment with some compounds of the present invention, respectively at 1 M (FIG. 2) and 10 M (FIG. 3).

[1173] FIG. 4 shows the mean+/SD of the expression of the target genes relative to the expression of the housekeeping gene: (A) TSLP, (B) IL-8, (C) TNF, (D) MDC and (E) TARC.

[1174] FIG. 5 presents the evaluation of the cytotoxic effect of coumarin compounds JFR5, JFR8, JFR9 and JFR11 on healthy human keratinocytes at 1 (hatched) and 10 (cross hatched) M after a 48 h treatment and neutral red staining. Three independent experiments were performed for each compound.

[1175] FIG. 6 illustrates the effect of JFR9 compound (5 M) efficacy on the total protease activity of transgenic KLK5 mice skin section, by in situ zymography.

[1176] The protease activity is shown with a gradient of fluorescence. The fluorescence intensity represents the cleavage efficiency of a casein substrate coupled to a FITC group.

[1177] Control (+): no inhibitor added; control (): addition of a coumarin compound that does not inhibit proteases.

EXAMPLES

Example 1

synthesis of CFL33, isoquinolin-1-yl 2-oxo-2H-1-benzopyran-3-carboxylate

[1178] 1.0 g of the commercially available 2-oxo-2H-1-benzopyran-3-carboxylic acid and 10 ml of thionyl chloride were refluxed for 3 h. The resulting solution was evaporated under reduced pressure. The residue was suspended in 10 ml anhydrous toluene. The solvent was eliminated by distillation under reduced pressure. The two last steps were repeated twice. The residue was dispersed in 10 ml dioxane. To this suspension were added 1-hydroxyquinoleine (1.1 q.) and anhydrous pyridine (1.1 eq.). After 90 min stirring at room temperature, the solvent was removed by distillation under reduced pressure. The residue was solubilized in chloroform and the organic phase was washed three times with HCl 0.1N, then dried over MgSO.sub.4. The solvent was evaporated under reduced pressure and the residue obtained was recrystallized in ethyl acetate.

[1179] white solid; m.p. 197-200 C.

[1180] .sup.1H NMR (500 MHz) DMSO-d.sub.6: 6.87 (d, 1H, 4-H isoquin.), 7.47 (t, 1H, 6-H coumar.), 7.52 (d, 1H, 8-H coumar.), 7.58 (t, 1H, 7-H isoquin.), 7.75 (m, 2H, 7-H coumar.+5-H isoquin.), 7.84 (m, 2H, 6-H+3-H isoquin.), 7.93 (d, 1H, 5-H coumar.), 8.16 (d, 1H, 8-H isoquin.), 8.56 (s, 1H, 4-H coumar.).

Example 1.2

synthesis of SMB28, 2-iodophenyl 6-acetoxymethyl-2-oxo-2H-1-benzopyran-3-carboxylate

[1181] To the suspension of the acid chloride of 6-acetoxymethyl-2-oxo-2H-1-benzopyran-3-carboxylic acid (4 mmol) obtained according to Pochet et al. (Bioorg. Med. Chem. 2000, 8, 1489) in anhydrous dioxane (10 mL) was added 2-iodophenol (5 mmol) and pyridine (0.4 mL). After 12 h stirring at room temperature, the solvent was evaporated under vacuum and the residue was suspended in methanol. The resulting precipitate was collected by filtration, washed with methanol and dried. The solid was crystallized in ethyl acetate (45%); mp: 156-158 C.

Example 1.3

synthesis of SMB33, 2-cyanophenyl 6-acetoxymethyl-2-oxo-2H-1-benzopyran-3-carboxylate

[1182] To the suspension of the acid chloride of 6-acetoxymethyl-2-oxo-2H-1-benzopyran-3-carboxylic acid (4 mmol) obtained according to Pochet et al. (Bioorg. Med. Chem. 2000, 8, 1489) in anhydrous dioxane (10 mL) was added 2-cyanophenol (5 mmol) and pyridine (0.4 mL). After 12 h stirring at room temperature, the solvent was evaporated under vacuum and the residue was suspended in methanol. The resulting precipitate was collected by filtration, washed with methanol and dried. The solid was crystallized in ethyl acetate (40%); mp: 181-183 C.

General synthetic pathway to naphthyl 2-oxo-2H-1-benzopyran-3-carboxylates

[1183] ##STR00114##

Synthesis of Dinaphthyl Esters of Malonic Acid

[1184] The mixture of malonic acid (2 g, 19.2 mmol), the appropriate naphthol (2 eq., 38.4 mmol) and phosphoryl chloride (2 eq., 38.4 mmol) was heated at 100 C. for 90 minutes. The reaction mixture was then carefully poured on a cooled 1% w/v aqueous solution of sodium hydroxide (50 mL). The resulting precipitate of the title compound was collected by filtration, washed with water, dried and recrystallized in methanol (yields: 25-30%). The white solid of the title compound was used in the next step without further purification.

Synthesis of naphthyl 2-oxo-2H-1-benzopyran-3-carboxylates

[1185] The solution of salicylaldehyde (0.4 g, 3.28 mmol) and the appropriate dinaphthyl malonate (1.5 eq., 4.92 mmol) in dioxane (12 mL) was supplemented with 8 drops of piperidine and 4 drops of glacial acetic acid and stirred for 30 minutes at room temperature. At the end of the reaction, the solvent was removed by distillation under reduced pressure and the residue was triturated with cold methanol (20 mL). The resulting precipitate was collected by filtration, washed with cold methanol and dried. The white solid of the title compound was recrystallized in a mixture of methylene chloride and hexane (yields: 75-80%).

Example 1.4

1-Naphthyl 2-oxo-2H-1-benzopyran-3-carboxylate (JFR1)

[1186] 1-Naphthyl 2-oxo-2H-1-benzopyran-3-carboxylate (JFR1) was obtained according to the above-mentioned general procedure, starting from 1-naphthol.

[1187] m.p.: 159-160 C.

Example 1.5

2-Naphthyl 2-oxo-2H-1-benzopyran-3-carboxylate (JFR2)

[1188] 2-Naphthyl 2-oxo-2H-1-benzopyran-3-carboxylate (JFR2) was obtained according to the above-mentioned general procedure, starting from 2-naphthol.

[1189] m.p.: 166-167 C.

General synthetic pathway to halophenyl 6-halomethyl-2-oxo-2H-1-benzopyran-3-carboxylates

[1190] ##STR00115##

Synthesis of Di(Halo)Phenyl Esters of Malonic Acid

[1191] The mixture of malonic acid (2 g, 19.2 mmol), the appropriate halophenol (2 eq., 38.4 mmol) and phosphoryl chloride (2 eq., 38.4 mmol) was heated at 100 C. for 90 minutes. The reaction mixture was then carefully poured on a cooled 1% w/v aqueous solution of sodium hydroxide (50 mL). The resulting precipitate of the title compound was collected by filtration, washed with water, dried and recrystallized in methanol (yields: 65-80%). The white solid of the title compound was used in the next step without further purification.

Synthesis of (halo)phenyl 6-hydroxymethyl-2-oxo-2H-1-benzopyran-3-carboxylates

[1192] The solution of 5-hydroxymethylsalicylaldehyde (Stoermer et al. Ber. 1901, 34, 2455-2460) (0.4 g, 2.62 mmol) and the appropriate di(halo)phenyl malonate (1.5 eq., 3.94 mmol) in dioxane (12 mL) was supplemented with 8 drops of piperidine and 4 drops of glacial acetic acid and stirred for 30 minutes at room temperature. At the end of the reaction, the solvent was removed by distillation under reduced pressure and the residue was triturated with cold methanol (10-20 mL). The resulting precipitate was collected by filtration, washed with cold methanol and dried (yields: 70-85%). The white solid of the title compound was used in the next step without further purification.

Synthesis of (halo)phenyl 6-bromomethyl-2-oxo-2H-1-benzopyran-3-carboxylates

[1193] The appropriate (halo)phenyl 6-hydroxymethyl-2-oxo-2H-1-benzopyran-3-carboxylate (0.4 g, 0.7 mmol) was dissolved in methylene chloride (10 mL) and then supplemented with pyridine (1.0 eq.) and thionyl bromide (1.15 eq.). The reaction mixture was heated under reflux for 90 minutes. After cooling, the reaction medium was poured onto water (20 mL). The organic layer was collected, dried over magnesium sulfate, filtered and concentrated under reduced pressure. The residue of the title compound was recrystallized in a mixture of methylene chloride and hexane (yields: 15-50%).

Example 1.6

Phenyl 6-bromomethyl-2-oxo-2H-1-benzopyran-3-carboxylate (JFR5)

[1194] Phenyl 6-bromomethyl-2-oxo-2H-1-benzopyran-3-carboxylate (JFR5) was obtained according to the above-mentioned general procedure, starting from phenol.

[1195] m.p.: 186-187 C.

Example 1.7

3-Fluorophenyl 6-bromomethyl-2-oxo-2H-1-benzopyran-3-carboxylate (JFR11)

[1196] 3-Fluorophenyl 6-bromomethyl-2-oxo-2H-1-benzopyran-3-carboxylate (JFR11) was obtained according to the above-mentioned general procedure, starting from 3-fluorophenol.

[1197] m.p.: 189-190 C.

Example 1.8

3-Chlorophenyl 6-bromomethyl-2-oxo-2H-1-benzopyran-3-carboxylate (JFR9)

[1198] 3-Chlorophenyl 6-bromomethyl-2-oxo-2H-1-benzopyran-3-carboxylate (JFR9) was obtained according to the above-mentioned general procedure, starting from 3-chlorophenol.

[1199] m.p.: 186-187 C.

Example 1.9

3-Bromophenyl 6-bromomethyl-2-oxo-2H-1-benzopyran-3-carboxylate (JFR8)

[1200] 3-Bromophenyl 6-bromomethyl-2-oxo-2H-1-benzopyran-3-carboxylate (JFR8) was obtained according to the above-mentioned general procedure, starting from 3-bromophenol.

[1201] m.p.: 185-186 C.

Synthesis of (halo)phenyl 6-fluoromethyl-2-oxo-2H-1-benzopyran-3-carboxylates

[1202] The appropriate (halo)phenyl 6-hydroxymethyl-2-oxo-2H-1-benzopyran-3-carboxylate (0.4 g, 0.7 mmol) was dissolved in methylene chloride (10 mL) and the resulting solution cooled on an ice bath was supplemented with Ishikawa's reagent (2.5 eq.). The reaction mixture was stirred for 30 minutes at 0 C. and then heated under reflux for 90 minutes. After cooling, the reaction medium was poured onto water (20 mL). The organic layer was collected, dried over magnesium sulfate, filtered and concentrated under reduced pressure. The residue of the title compound was purified by column chromatography on silicagel (eluent: methylene chloride) and then recrystallized in a mixture of methylene chloride and hexane (yields: 15-55%).

Example 1.10

4-Chlorophenyl 6-fluoromethyl-2-oxo-2H-1-benzopyran-3-carboxylate (JFR7)

[1203] 4-Chlorophenyl 6-fluoromethyl-2-oxo-2H-1-benzopyran-3-carboxylate (JFR7) was obtained according to the above-mentioned general procedure, starting from 4-chlorophenol.

[1204] m.p.: 216-218 C.

Example 2

Biological Results, Enzyme and Inhibition Assays

[1205] In the examples described below: [1206] DMSO means dimethylsulfoxide [1207] Tris-HCl means tris(hydroxymethyl)aminomethane, hydrogen chloride>> [1208] Tween 20 refers to polysorbate 20 or polyoxyethylene (20) sorbitan monolaurate [1209] The Hill number provides a way to characterize the binding of a ligand to a macromolecule.

[1210] Kallikreins KLK5, KLK7 and KLK14 activities were determined by monitoring the hydrolysis of the appropriate fluorogenic substrate (.sub.exc=355, .sub.em=460 nm) for 15 min at 37 C. in the presence of the untreated kallikrein (control) or kallikrein that had been incubated with a test compound. Substrate and compounds were previously dissolved in DMSO, with the final solvent concentration kept constant at 2% (v/v) (KLK 5 and 14), and 4% (v/v) (KLK7). The composition of the activity buffers (pH 8.0) was 100 mM Tris-HCl, 150 mM NaCl, 0.01% (v/v) Tween 20 for the KLK5 assay, and 100 mM Tris-HCl, 1M NaCl, 0.01% (v/v) Tween 20 in the KLK 7 and 14 assays. The final concentrations were 0.6 nM (KLK5) and 100 M (Boc-Val-Pro-Arg-AMC) (KLK5 assay), 8 nM (KLK7) and 40 M (Suc-Leu-Leu-Val-Tyr-AMC, AMC: 7-amino-4-methyl-coumarin) (KLK7 assay), and 0.17 nM (KLK14) and 10 M (Boc-Val-Pro-Arg-AMC) (KLK14 assay). Using the appropriate substrate, the coumarinic compounds (0.01-100 M) were tested in duplicate for each inhibitor concentration to detect their inhibitory potential. The enzyme and the inhibitors were incubated for 15 min before the determination of the enzyme activity. Initial rates determined in control experiments (V.sub.0) were considered to be 100% of the peptidase activity; initial rates below 100% were considered to be inhibitions. The inhibitory activity of compounds was expressed as IC.sub.50 (inhibitor concentrations giving 50% inhibition). The values of IC.sub.50 were calculated by fitting the experimental data to the equation:

% Inhibition=100(1V.sub.i/V.sub.0)=100[I].sub.0/(IC.sub.50+[I].sub.0),

or equation:

% Inhibition=100[I].sub.0.sup.nH/(IC.sub.50.sup.nH+[I].sub.0.sup.nH), n.sub.H is the Hill number.

Example 3

Characterization of the Inhibition Reversibility

[1211] To examine the putative covalent nature of the inhibition by coumarins, percentage of inhibition was monitored as function of time; a putative enzyme reactivation by hydroxylamine was detected. The reaction mixtures containing the inhibited enzyme were treated with 0.5 M hydroxylamine at pH 7.5 and 37 C. during 30 min. Enzyme activity of aliquots was monitored and compared to a control. The fast reactivation in the presence of hydroxylamine indicated the formation of a stable acyl-enzyme; the absence of reactivation was in agreement with a suicide inactivation.

[1212] The results obtained for the compounds of the invention are shown in tables A and A2.

[1213] The tests were realized at 37 C. after 15 min of incubation at pH 8.

[1214] In these tables, <<NI>> means non-inhibitor and ND non-determined.

TABLE-US-00001 TABLE A IC.sub.50 (M) or % Inhibition molecule name KLK5 KLK7 KLK14 [00116] MFS7 26% (50 M) 0.235 0.009 45% (100 M) [00117] MFS36 50% (100 M) 0.209 0.006 100% (100 M) [00118] LP15 NI 1.9 0.1 35% (100 M) [00119] IK11 37.4% (50 M) 63% (100 M) 43 4 [00120] IK1 26.3 1.4 2.7 0.1 38 5 [00121] IK3 NI NI 77% (100 M) [00122] LP60 112 6 1.44 0.02 46% (100 M) [00123] IK5 36.9 4.2 5.8 0.2 89 10 [00124] LP73 NI 0.063 0.004 NI [00125] LP74 1.3 0.2 0.195 0.024 54 6 M [00126] LP72 59 9 0.495 0.037 50% (100 M) [00127] LP51 26% (50 M) 0.25 0.01 30% (100 M) [00128] LP8 49% (100 M) 0.103 0.005 40% (100 M) [00129] LP53 34% (50 M) 0.065 0.003 41% (100 M) [00130] LP14 48% (100 M) 0.077 0.003 70% (100 M) [00131] IK4 21.6 5 7.8 0.6 30 8 [00132] IK2 NI 0.68 0.01 67% (100 M) [00133] LP46 31% (50 M) ND 31 6 [00134] LP55 NI 8.4 0.2 NI [00135] LP75 80 8 0.136 0.012 NI [00136] LP61 NI 0.130 0.005 NI [00137] CFL16 40% (100 M) 0.9 0.4 73% (100 M) [00138] CFL5 35% (100 M) 0.50 0.04 39 4 [00139] CFL21 [121 5] 22% (100 M) 43% (100 M) [00140] CFL25 44 10 ND 68 9 [00141] CFL15 47% (100 M) 0.352 0.055 45% (100 M) [00142] CFL7 NI 33% (100 M) 33% (100 M) [00143] MFS35 NI 55% (100 M) 71% (100 M) [00144] IK8 40 64.3 2.7 64% (100 M) [00145] IK10 23% (50 M) NI NI [00146] IK9 22% (50 M) NI NI [00147] CFL4 NI 5.01 0.4 60% (50 M) [00148] CFL17 NI 33% (100 M) 50% (50 M) [00149] LP7 NI 0.463 0.007 NI [00150] LP16 NI 47% (50 M) NI [00151] IK13 NI 34% (100 M) 72% (100 M) [00152] LP71 182 14 NI NI [00153] LP18 78 12 4.9 0.3 30 2 [00154] LP42 22% (50 M) 52% (100 M) NI [00155] LP76 30% (50 M) 25% (50 M) 35% (50 M) [00156] LP2 NI 0.128 0.030 NI [00157] D5 90% (100 M) 61% (100 M) 38% (100 M) [00158] MFS2 54 2 26.8 1.5 73 14 [00159] MFS3 39% (50 M) 20.9 0.9 48% (100 M) [00160] LP43 49% (100 M) 30% (100 M) 49% (100 M) [00161] D9 80% (100 M) NI 40% (100 M) [00162] CFL33 7.0 0.4 NI 61% (100 M) [00163] LP41 32% (100 M) 0.39 0.03 67% (100 M) [00164] IK48 27% (50 M) 9.7 1.3 32% (50 M) [00165] MH30 25% (50 M) 33.2 1.4 32% (50 M) [00166] SMB27 NI 42% (50 M) 27% (50 M) [00167] SMB26 NI 52% (10 M) NI [00168] SMB28 NI 82.2 13.2 NI [00169] IK53 NI 54% (10 M) NI [00170] MH52 NI 58% (10 M) NI [00171] IK49 NI 54% (10 M) NI [00172] MH24 NI 33.2 1.4 42% (50 M) [00173] MH8 NI 25% (50 M) NI [00174] MH14 NI 35% (50 M) NI [00175] SMB33 NI NI 27% (50 M) [00176] MH22 32% (50 M) 100% (50 M) 85% (50 M)

TABLE-US-00002 TABLE A2 IC.sub.50 (nM) or % d'inhibition Compound KLK5 KLK7 KLK14 [00177] JFR1 ni 53% (50 M) ni [00178] JFR2 ni 30% (50 M) ni [00179] JFR5 34% (50 M) 249 3.5 53% (50 M) [00180] JFR7 ni 30% (50 M) ni [00181] JFR8 1470 60 57 2 3000 100 [00182] JFR9 920 70 63.7 1.7 2900 100 [00183] JFR11 42% (50 M) 198 8 48% (50 M) [00184] MFS33 62% (100 M) 5.7 0.3 60% (100 M)

[1215] Examples of coumarin derivatives presenting a selective activity against KLK5, KLK7 and KLK14 are listed in Table B.

[1216] An acceptable threshold of inhibition corresponds for example to 30% at 100 M.

TABLE-US-00003 TABLE B Enzyme Molecule KLK5 LP71, CFL33, IK9, IK10, D9 KLK7 LP2, LP42, LP51, LP55, LP72, LP74, MFS7, CFL15, SMB26, SMB28, IK53, MH52, IK49, MH8, MH14, JFR1, JFR2, JFR7, LP14, LP8, IK2, MFS36, LP53, LP73, JFR9, JFR8 KLK14 MFS35, CFL21, CFL17, IK3, IK13, SMB33

Example 4

Cytotoxicity in Human Keratinocytes Using the Neutral Red Uptake (NRU) Cytotoxicity Test

[1217] The NRU cytotoxicity assay procedure is a cell survival/viability chemosensitivity assay based on the ability of viable cells to incorporate and bind neutral red (NR), a supravital dye. NR is a weak cationic dye that readily penetrates cell membranes by non-ionic diffusion and accumulates intracellularly in lysosomes. Alterations of the cell surface or the sensitive lysosomal membrane lead to lysosomal fragility and other changes that gradually become irreversible. Such changes caused by the action of xenobiotics result in a decreased uptake and binding of NR. It is thus possible to distinguish between viable, damaged, or dead cells, which is the basis of this assay. After treatment with the small-molecules inhibitors, NR uptake in cells will be measured by spectrophotometry at 54010 nm.

Day 0: Cell Preparation

[1218] Keratinocytes from healthy persons or patients with Netherton syndrome are seeded at the density of 4000 cells in 125 L of medium per well in 96-well plates.

[1219] The culture medium is made of: [1220] half of complete Green medium: 60% (Dulbecco's Modified Eagle Media), 30% Ham's F12, 10% FCS, 180 mM adenin, 5 g/mL insulin, 0.4 g/mL hydrocortisone, 10 nM cholera toxin, 2 nM triiodothyronin, 10 ng/mL human recombinant EGF, 100 U/mL penicillin G/streptomycin. [1221] half of a basal medium (EpilifeCascade Biologics) allowing keratinocytes culture without feeders.

2Day 2: Cell Treatment

[1222] After seeding, cells are incubated for 48 hours in the following experimental conditions: 37 C.1 C., 90%5.0% humidity, and 5%1% CO.sub.2/air. This incubation period allows for cell recovery and adherence and progression to exponential growth phase.

[1223] Prepare inhibitors solutions 2-fold more concentrated than desired. Add 125 L of these solutions to the wells without changing or removing the medium (125 L).

[1224] A control with the vehicle alone (DMSO) is made.

[1225] A minimum of 3 wells per each experimental condition is done.

3Day 4: NRU Measurement

[1226] 48 h hours after the beginning of the treatment, carefully remove the medium (containing the tested inhibitor) and rinse the cells very carefully with 250 L pre-warmed PBS.

[1227] Add 250 L NR medium and incubate (37 C.1 C., 90%5% humidity, and 5.0%1% CO.sub.2/air) for 30.1 h. NR medium is the culture medium containing 33 g/mL of NR. After incubation, remove the NR medium, and carefully rinse cells with 250 L pre-warmed PBS.

[1228] Decant and blot PBS from the plate.

[1229] Add exactly 100 L NR Desorb (50% ETOH/1% acetic acid) solution to all wells, including blanks.

[1230] Shake microtiter plate rapidly on a microtiter plate shaker for 20-45 min to extract NR from the cells and form a homogeneous solution. Plates should be protected from light by using a cover during shaking.

[1231] Plates should be still for at least five minutes after removal from the plate shaker.

[1232] Measure the absorption (within 60 minutes after adding NR Desorb solution) of the resulting colored solution at 540 nm10 nm in a microtiter plate reader (spectrophotometer), using the blank as a reference.

4Data Analysis

[1233] The experiments are performed three times on three different days for each inhibitor.

[1234] A calculation of cell viability expressed as NRU is made for each concentration of the tested inhibitor by using the mean NRU of the three replicate values per each concentration. This value is compared with the mean NRU of all vehicle values. Relative cell viability is then expressed as percent of untreated vehicle control (DMSO).

Example 5

Cell Viability

[1235] Normal human keratinocytes were seeded in a 96-well tissue culture plate. At 20% of confluence, molecules were added in the culture medium and incubated for 48 h. Neutral red medium was added to every well for an incubation period of 3 h, then the dye was extracted with acidified ethanol solution. Data (FIGS. 2 and 3) represent the mean cell viability after treatment +/SD compared to mean viability of cells treated with the vehicle only (DMSO). Data represent the mean of 5 individual experiments.

[1236] No noticeable cytotoxicity on human normal keratinocytes was observed at 1 and 10 M when assayed using the neutral red uptake test for LP7, LP14, LP41, LP53, LP55, LP61, LP73, LP76, MFS7, MFS33, CFL5, CFL33, IK2 and IK4.

[1237] No noticeable cytotoxicity on human normal keratmocytes was observed at 1 and 10 mM when assayed using the neutral red uptake test for JFR5, 8, 9 and 11 (FIG. 5).

[1238] Toxicity was observed for IK3 at 1 and 10 M reaching 16.098.32% at 1 M and 47.14.43% at 10 M.

Example 6

Inhibitory Effect of Compounds of the Invention on the Expression of Pro-Allergic and Inflammatory Molecules in Netherton Syndrome Patient Keratinocytes

1Cell Preparation:

[1239] 250,000 keratinocytes from a Netherton syndrome patient (NSK) were seeded in 6 well plates in 2 mL of medium made of: (a) half of complete Green medium: (Dulbecco's Modified Eagle Media) 60%; Ham's F12 30%; FCS 10%; adenin 180 mM; insulin 5 g/mL; hydrocortisone 0.4 g/mL; cholera toxin 10 nM; triiodothyronin 2 nM; human recombinant EGF 10 ng/mL; penicillin G/streptomycin 100 U/mL; (b) half of a basal medium (EpilifeCascade Biologics).

[1240] When confluent, cells were washed 5 times with PBS and 2 mL of complete Green medium without FCS (fetal calf serum) were added per well.

2Treatment of Cells with Inhibitors:

[1241] Inhibitors were added in the culture medium 24 h after medium renewal.

3Arrest of the Cell Culture and RNA Extraction 72 h after Beginning of the Treatment

[1242] The supernatant was removed and cells were washed once with PBS.

[1243] 350 L of RLT lysis buffer (Mini Kit QIAGEN)+-mercapto-ethanol (0.1%) were added per well to extract total RNA. The cell lysate was kept at 80 C. before RNA extraction (Mini Kit QIAGEN).

4Measurement of KLK5 Activity Inhibition

[1244] In this in cellulo test, KLK5 activity was evaluated by an indirect method. Indeed KLK5 induces TSLP (thymic stromal lymphopoietin), TNF (tumor necrosis factor alpha) and IL-8 (interleukin 8) expression. If KLK5 activity is inhibited, the expression of these cytokines will be reduced.

[1245] The relative amount of TSLP, TNF and IL-8 transcripts [relative to HPRT (hypoxanthine phosphoribosyltransferase 1) housekeeping gene] was compared between cells treated or not treated with the inhibitors. The ratio <<treated/not treated>> relative to housekeeping gene reflects KLK5 inhibition.

[1246] Two other genes showed increased expression in NSK i.e. MDC (macrophage derived cytokine or CCL22) and TARC (thymus and activation regulated chemokine or CCL17). Their expression had also been studied even if their mechanism of induction is not known yet.

5Results

[1247] The effect of LP73 and CFL33 on the expression of the described pro-allergic and inflammatory cytokines was analyzed on keratinocytes from two different NS patients. Each experiment was performed twice (FIG. 4).

[1248] LP73 at the dose of 1 M significantly inhibited the expression of TSLP (p=0.0088), IL-8 (p=0.0269), TNF (p=0.0004), MDC (p<0.0001) and TARC (p=0.0004) whereas at the dose of 10 M, it only inhibited the expression of TSLP (p=0.002) and TARC (p=0.0461).

[1249] CFL33 at the dose of 1 M significantly inhibited the expression of IL-8 (p=0.0309), MDC (p<0.0001) and TARC (p=0.0078) only.

Example 6bis

Study on a Mouse Model of Netherton Syndrome

[1250] JFR9 efficacy has been assayed on transgenic KLK5 mice skin section by in situ zymography (FIG. 6). These mice overexpress KLK5 and reproduce the phenotypic characteristics of Netherton syndrome. It has been shown that JFR9 molecule (5 M) decreased significantly the total protease activity of the transgenic KLK5 mice skin section.

Example 7

Evaluation of the Efficacy of Coumarin Derivatives of the Invention on Psoriasis, Atopic Eczema and Allergic Contact Dermatitis Skin Pathologies

[1251] To analyze whether coumarin derivatives have an efficacy on psoriasis, atopic eczema and allergic contact dermatitis skin pathologies, in situ zymographies on skin sections can be performed. The total protease activity can be visualized, as well as specific KLK5 and KLK7 activities directly in the tissue.

[1252] In situ zymography allows the study of protease activity in tissue. Skin sections of lesional skin are incubated in the presence, or not, of the tested compound and incubated O/N with a specific substrate coupled to a fluorochrome (casein can be used as substrate to assess total protease activity or KLK5 and KLK7 specific substrates can be used to evaluate KLK5 and KLK7 activities. When the substrate is cleaved, the fluorochrome is released. The inhibition of the fluorochrome release is then analysed and quantified by confocal microscopy analysis.

[1253] The protocol is the following: frozen sections of lesional skin (5-m thickness) are rinsed with a washing solution (2% Tween 20 in PBS, 2 min, followed by 5 min in PBS) and incubated or not with the tested compound and incubated at 37 C. overnight with 100 l of BODIPY FL casein (10 g/ml) using the EnzChek Ultra Protease Assay kit (Invitrogen) in 100 mM Tris-C1, pH 8, in order to visualize global protease activity.

[1254] To assess KLK5 and KLK7 activities, cryostat sections after incubation or not with the tested compound, are incubated in the same conditions with 100 l of Boc-Val-Pro-Arg-AMC or Suc-Leu-Leu-Val-Tyr-AMC (Sigma-Aldrich) at 100 M and 40 M respectively in Tris 50 mM, CaCl2 10 mM for the detection of trypsin-(KLK5) and chymotrypsin-like (KLK7) activity, respectively.

[1255] After incubation overnight at 37 C. with the substrate, sections are rinsed with PBS solution and visualized with the Axiovert 200 inverted high-end microscope (Zeiss) for KLK5 and KLK7 activities and Leica TCS SP5 AOBS for global protease activity. Images are analyzed with Image J software.

[1256] Should the coumarin derivatives inhibit KLK5 and KLK7 activities, a decrease of the release of the fluorochrome will be observed.