Benzoxazinone derivatives for treatment of skin diseases

Abstract

The present invention relates to methods and compositions for inhibiting the activity of skin proteases, especially human kallikrein 7 (KLK7), human kallikrein 5 (KLK5), and human kallikrein 14 (KLK14). More specifically, the invention relates to the use of substituted 3,1-benzoxazin-4-ones being selective inhibitors of human skin kallikreins for the treatment of skin diseases, more specifically for the treatment of inflammatory skins diseases, especially Netherton syndrome.

Claims

1. A method for the treatment of an inflammatory skin disease which comprises the administration of a therapeutically active amount of a compound according to Formula I ##STR00024## wherein R is SCH3 or Cl, or a pharmaceutically acceptable salt thereof, to a subject in need of such treatment.

2. The method according to claim 1, wherein the compound is 6-Ethoxy-7-methoxy-2-(2-methylsulfanylphenyl)-3,1-benzoxazin-4-one.

3. The method according to claim 1, wherein the compound is 2-(2-Chlorophenyl)-6-ethoxy-7-methoxy-3,1-benzoxazin-4-one.

4. The method according to claim 1, wherein the inflammatory skin disease is selected from Netherton syndrome, atopic dermatitis, contact dermatitis, eczema, psoriasis, acne, epidermal hyperkeratosis, acanthosis, epidermal inflammation, dermal inflammation and pruritus.

5. The method according to claim 1, wherein the inflammatory skin disease is Netherton syndrome.

6. The method according to claim 1, wherein the treatment is given to mitigate a pre-existing acute or chronic disease state.

7. The method according to claim 1, wherein the treatment is given to mitigate a recurring disease condition.

8. The method according to claim 1, wherein the treatment is a prophylactic treatment for prevention of a recurring condition.

9. The method according to claim 1, wherein the treatment is a continued treatment for a chronic disorder.

10. A composition for the treatment of an inflammatory skin disease comprising: 0.05% to 99% wt. of a compound according to Formula I ##STR00025## wherein R is SCH.sub.3 or Cl, or a pharmaceutically acceptable salt thereof.

11. The composition of claim 10, wherein the compound is 6-Ethoxy-7-methoxy-2-(2-methylsulfanylphenyl)-3,1-benzoxazin-4-one.

12. The composition of claim 10, wherein the compound is 2-(2-Chlorophenyl)-6-ethoxy-7-methoxy-3,1-benzoxazin-4-one.

13. The composition of claim 10, wherein the composition is formulated for topical administration.

14. The composition of claim 10, wherein the composition is formulated for oral administration.

15. The composition of claim 10, wherein the composition is formulated for injection.

16. The composition of claim 10, wherein the composition is in the form of a powder, tablet, dispersible granule, capsule, cachet, or suppository.

Description

DESCRIPTION OF THE INVENTION

(1) The present inventors have been able to identify inhibitors being selective for skin protease and have found that the activity of KLK7, KLK5, and KLK14 can be selectively inhibited by compounds according to Formula I,

(2) ##STR00001##
wherein R is SCH.sub.3 or Cl.

(3) Said compounds exhibit several advantageous properties, such as selectivity for the skin proteases KLK7, KLK5, and KLK14, involved in the pathophysiology of inflammatory skin diseases such as Netherton syndrome, having no or only low inhibitory activity on other proteases.

(4) Accordingly, the present invention provides compounds according to Formula 1

(5) ##STR00002##
wherein R is SCH.sub.3 or Cl, or a pharmaceutically acceptable salt thereof

(6) The present invention further provides compounds according to Formula I or a pharmaceutical acceptable salt thereof, for use in in medicine.

(7) The compound can be 6-Ethoxy-7-methoxy-2-(2-methylsulfanylphenyl)-3, 1-benzoxazin-4-one, or 2-(2-Chlorophenyl)-6-ethoxy-7-methoxy-3,1-benzoxazin-4-one

(8) The present invention further provides compounds according to Formula I or a pharmaceutical acceptable salt thereof, for use in the prophylaxis, prevention and/or treatment of a skin disease.

(9) The invention further provides pharmaceutical compositions comprising a compound according to Formula I in admixture with pharmaceutically acceptable adjuvants, diluents and/or carriers.

(10) The invention further provides a pharmaceutical composition according to the invention for use in prophylaxis, prevention and/or treatment of a skin disease.

(11) The invention further relates to the use of a compound according to the Formula I or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment of a skin disease.

(12) The present invention further provides a method for the prophylaxis, prevention and/or treatment of a skin disease which comprises the administration of a therapeutically active amount of a compound according to Formula I or a pharmaceutical acceptable salt thereof, to a subject in need of such treatment.

(13) The skin disease may be an inflammatory skin disease. The skin disease can be selected from Netherton syndrome, atopic dermatitis, contact dermatitis, eczema, psoriasis, acne, epidermal hyperkeratosis, acanthosis, epidermal inflammation, dermal inflammation and pruritus. The subject to be treated can be a mammal, such as a human, a dog, a cat, or a horse.

(14) Definitions

(15) As used herein, the term pharmaceutically acceptable salts includes acid addition salts and base addition salts. Such salts may be formed by conventional means, for example by reaction of a free acid or a free base form of the compound of the invention with one or more equivalents of an appropriate acid or base, optionally in a solvent, or in a medium in which the salt is insoluble, followed by removal of said solvent, or said medium, using standard techniques (e.g. in vacuo or by freeze-drying). Salts may also be prepared by exchanging a counter-ion of a compound of the invention in the form of a salt with another counter-ion using a suitable ion exchange resin.

(16) In the context of the present specification, the term treat also includes prophylaxis unless there are specific indications to the contrary. The term treat within the context of the present invention further encompasses to administer an effective amount of a compound of the present invention, to mitigate either a pre-existing disease state, acute or chronic, or a recurring condition. This definition also encompasses prophylactic therapies for prevention of recurring condition and continued therapy for chronic disorders.

(17) The compound of the present invention may be administered in the form of a conventional pharmaceutical composition by any route including orally, intramuscularly, subcutaneously, topically, intranasally, intraperitoneally, intrathoracially, intravenously, epidurally, intrathecally, intracerebroventricularly and by injection into the joints.

(18) In one embodiment of the present invention, the route of administration may be topical.

(19) The dosage will depend on the route of administration, the severity of the disease, age and weight of the patient and other factors normally considered by the attending physician, when determining the individual regimen and dosage level at the most appropriate for a particular patient.

(20) For preparing pharmaceutical compositions from the compound of the present invention, inert, pharmaceutically acceptable carriers can be either solid or liquid. Solid form preparations include powders, tablets, dispersable granules, capsules, cachets, and suppositories.

(21) A solid carrier can be one or more substances, which may also act as diluents, flavouring agents, solubilizers, lubricants, suspending agents, binders, or tablet disintegrating agents; it can also be an encapsulating material.

(22) In powders, the carrier is a finely divided solid, which is in mixture with the finely divided compound of the present invention, or the active component. In tablets, the active component is mixed with the carrier having the necessary binding properties in suitable proportions and compacted in the shape and size desired.

(23) For preparing suppository compositions, a low-melting wax such as a mixture of fatty acid glycerides and cocoa butter is first melted and the active ingredient is dispersed therein by, for example, stirring. The molten homogenous mixture is then poured into conveniently sized molds and allowed to cool and solidify.

(24) Suitable carriers are magnesium carbonate, magnesium stearate, talc, lactose, sugar, pectin, dextrin, starch, methyl cellulose, sodium carboxymethyl cellulose, a low-melting wax, cocoa butter, and the like.

(25) The term composition is also intended to include the formulation of the active component with encapsulating material as a carrier providing a capsule in which the active component (with or without other carriers) is surrounded by a carrier which is thus in association with it. Similarly, cachets are included.

(26) Tablets, powders, cachets, and capsules can be used as solid dosage forms suitable for oral administration.

(27) Liquid form compositions include solutions, suspensions, and emulsions. For example, sterile water or propylene glycol solutions of the active compounds may be liquid preparations suitable for parenteral administration. Liquid compositions can also be formulated in solution in aqueous polyethylene glycol solution.

(28) Aqueous solutions for oral administration can be prepared by dissolving the active component in water and adding suitable colorants, flavouring agents, stabilizers, and thickening agents as desired. Aqueous solutions for oral use can be made by dispersing the finely divided active component in water together with a viscous material such as natural synthetic gums, resins, methyl cellulose, sodium carboxymethyl cellulose, and other suspending agents known to the pharmaceutical formulation art.

(29) Depending on the mode of administration, the pharmaceutical composition will according to one embodiment of the present invention include 0.05% to 99% weight (percent by weight), according to an alternative embodiment from 0.10 to 50% weight, of the compound of the present invention, all percentages by weight being based on total composition.

(30) A therapeutically effective amount for the practice of the present invention may be determined, by the use of known criteria including the age, weight and response of the individual patient, and interpreted within the context of the disease which is being treated or which is being prevented, by one of ordinary skills in the art.

(31) The above-mentioned subject-matter for a pharmaceutical composition comprising a compound according to the present invention is applied analogously for a pharmaceutical composition comprising a combination according to the present invention.

(32) It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein.

(33) The use of the word a or an when used in conjunction with the term comprising in the claims and/or the specification may mean one, but it is also consistent with the meaning of one or more, at least one, and one or more than one.

(34) These, and other, embodiments of the invention will be better appreciated and understood when considered in conjunction with the following description and the accompanying drawings. It should be understood, however, that the following description, while indicating various embodiments of the invention and numerous specific details thereof, is given by way of illustration and not of limitation. Many substitutions, modifications, additions and/or rearrangements may be made within the scope of the invention without departing from the spirit thereof, and the invention includes all such substitutions, modifications, additions and/or rearrangements.

EXAMPLES

Example 1

Selectivity of Substituted 3,1-benzoxazin-4-ones as Inhibitors of Human Proteases

(35) IC.sub.50 values for a number of substituted 3,1-benzoxazin-4-ones on a panel of human proteases were determined.

(36) KLK7 Assay

(37) Materials: Recombinant human KLK7, Substrate S-2586 (Chromogenics, cat. no. 820894) KLK7 activity was determined at 37 C. in 10 mM Na phosphate pH 7.2, 0.5 M NaCl, with final concentrations of 2.5 g/ml (100 nM) of KLK7, 1.0 mM substrate, 5% DMSO in the presence of 0 M, 0.1 M, 0.5 M, 1.0 M and 5 M of inhibitor, in 96 well plates by measuring absorbance at 405 nm in a plate reader (Spectramax).

(38) KLK5 Assay

(39) Materials: Recombinant human KLK5, Substrate S-2288 (Chromogenics, cat. no. 820852) KLK5 activity was determined at 37 C. in 0.1 M Tris, pH 8.0, 0.15 M NaCl, with final concentrations of 2.5 g/ml KLK5, 1 mM substrate, 5% DMSO in the presence of 0 M, 0.1 M, 1.0 M and 10 M of inhibitor, in 96 well plates by measuring absorbance at 405 nm in a plate reader (Spectramax).

(40) KLK14 Assay

(41) Materials: Recombinant human KLK14, Substrate S-2302 (Chromogenics, cat. no. 820340). KLK14 activity was determined at 37 C. in 0.1 mM Tris, pH 8.0, 0.15 M NaCl, with final concentrations of 0.26 g/ml (9.4 nM) of KLK14, 0.75 mM substrate, 5% DMSO, in the presence of 0 M, 0.1 M, 1.0 M and 10 M of inhibitor, in 96 well plates by measuring absorbance at 405 nm in a plate reader (Spectramax).

(42) Cathepsin G Assay

(43) Materials: Cathepsin G, 100 mU (VWR, Calbiochem, cat. no. 219373), Substrate Cathepsin G substrate (VWR, Calbiochem, cat. no. 219407)

(44) Cathepsin activity was determined at 37 C. in 10 mM Na phosphate pH 7.2, 0.5 M NaCl, with final concentrations of 1.5 mU/ml (0.75 g/ml, 32 nM) of Cathepsin U, 0.75 mM substrate, 5% DMSO, in the presence of 0 M, 0.1 M, 1.0 M and 10 M of inhibitor, in 96 well plates by measuring absorbance at 405 nm in a plate reader (Spectramax).

(45) Chymotrypsin Assay

(46) Materials: Chymotrypsin, bovine, 25 g (Roche; sequence grade), Substrate S-2586 (Chromogenics, cat. no. 82 08 94)

(47) Chymotrypsin activity was determined at 37 C. in 10 mM Na phosphate pH 7.2, 0.5M NaCl, with final concentrations of 0.2 g/ml (6.8 nM) of Chymotrypsin, 1 mM substrate, 5% DMSO in the presence of 0 M, 0.1 M, 1.0 M and 10 M of inhibitor, in 96 well plates by measuring absorbance at 405 nm in a plate reader (Spectramax).

(48) Trypsin Assay

(49) Materials: Trypsin, 100 g (Roche, sequence grade, Mw 23500), Substrate S-2288 (Chromogenics, cat. no. 820852)

(50) Trypsin activity was determined at 37 C. in 10 mM Na phosphate pH 7.2, 0.5 M NaCl, with final concentrations of 0.8 g/ml (34 nM) of Trypsin, 1 mM substrate, 5% DMSO in the presence of 0 M, 0.1 M, 1.0 M and 10 M of inhibitor, in 96 well plates by measuring absorbance at 405 nm in a plate reader (Spectramax).

(51) Thrombin Assay

(52) Materials: Thrombin (Chromogenics, cat. no. 820712), Substrate S-2288 (Chromogenics, cat. no. 82 08 52)

(53) Thrombin activity was determined at 37 C. in 50 mM Tris, pH 8.3, 130 mM NaCl, with final concentrations of 1 pkat/ml (0.03 g/ml, 88 pM) of thrombin, 0.5 mM substrate, 5% DMSO in the presence of 0 M, 0.1 M, 1.0 M and 10 M of inhibitor, in 96 well plates by measuring absorbance at 405 nm in a plate reader (Spectramax).

(54) TABLE-US-00001 TABLE 1 Selectivity of substituted 3,1-benzoxazin-4-ones-IC.sub.50 (M) Cathep- Chymo- Compound KLK7 KLK5 KLK14 sin G trypsin Trypsin Thrombin 1 0.048 0.2 0.1 >>10 >>10 >>10 >10 2 0.073 0.5 0.1 >10 >>10 >>10 >10 3 0.116 0.6 0.5 1.1 >>10 >>10 >10 4 0.230 1.0 1.0 2.8 >>10 >>10 >10 5 0.085 0.5 0.3 1.2 >10 >>10 >10 6 0.080 1.0 1.4 0.7 <0.1 >>10 0.6 7 0.090 2.2 2.7 1.9 <0.1 >>10 0.5 8 0 0.065 1.0 2.3 0.6 <0.1 >>10 >10 9 0.228 1.3 1.5 1.3 0.4 >>10 0.9 10 0.188 1.9 5.2 0.5 0.2 >>10 0.5 11 1.370 >10 >10 >>10 1.0 >>10 >10 12 0.147 1.0 1.1 0.7 0.6 >>10 >>10 13 0.155 0.6 0.8 0.7 <0.1 1.8 3.0 14 0.183 0.6 1.5 0.2 <0.1 1.8 3.0 15 0.105 0.5 0.5 0.5 <0.1 3.7 0.6 16 0.149 1.0 1.3 0.4 <0.1 3.7 0.6 17 0.175 1.0 1.1 0.1 1.2 >>10 >>10 18 0 0.10 4.5 2.1 1.9 >10 >>10 >10

(55) As seen in Table 1 only compound 1 (6-ethoxy-7-methoxy-2-(2-methylsulfanylphenyl)-3,1-benzoxazin-4-one) and compound 2 (2-(2-chlorophenyl)-6-ethoxy-7-methoxy-3,1-benzoxazin-4-one) were found to have the desired selectivity, i.e. an IC.sub.50 below 0.1 M for KLK7, an IC.sub.50 below 1 M for KLK5 and KLK14, and an IC.sub.50 above 10 M for the other proteases tested.

(56) It should be noted that although several compounds were found to have a strong inhibitory effect on KLK7, as well as KLK5 and KLK14, only compounds 1 and 2, i.e. the compounds according to the invention, could be demonstrated to have a sufficiently low inhibitory activity for other proteases.

(57) Even small changes in the substation pattern of the compounds have a dramatic effect on the selectivity of the compounds. For example, compound 12 being methoxy substituted in position 6, as compared to compound 1 being ethoxy substituted in position 6, shows a more than 10 fold higher inhibitory activity (seen as a 10 fold lower IC.sub.50) on Cathepsin G and Chymotrypsin compared to compound 1, making compound 12 unsuitable for use in the treatment of skin diseases.

(58) In summary, the data presented in Table 1 demonstrates that only compounds 1 and 2, i.e. the compounds according to the invention, are sufficiently selective having a high inhibitory activity for the skin proteases KLK7, KLK5, and KLK14, while having a sufficiently low inhibitory activity for other proteases, making them suitable for use in the treatment of skin diseases.

Example 2

Synthesis of 6-ethoxy-7-methoxy-2-(2-methylsulfanylphenyl)-3, 1-benzoxazin-4-one

(59) ##STR00021## ##STR00022##

Example 3

Synthesis of 2-(2-chlorophenyl)-6-ethoxy-7-methoxy-3,1-benzoxazin-4-one

(60) Same as in Example 2 but with use of

(61) ##STR00023##
in Step 3.