HK1229792A1 - Benzoxazinone derivatives for treatment of skin diseases - Google Patents

Description

Benzoxazinone derivatives for the treatment of skin diseases

Technical Field

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

Background

KLK7(hK7, or keratinized layer chymotrypsin (SCCE), SwissprotP49862) is a S1 serine protease of the kallikrein gene family that shows chymotrypsin-like activity. KLK7 is expressed primarily in the skin and appears to play an important role in skin physiology (Egelrud.1993.purification and purification of a ceramic skin collagen enzyme: a protease which can be used as a protein in a fermented formulation).J.Invest.Dermatol.101,200-204；Skytt et al 1995.Primary substratespecificity of recombinant human stratum corneum chymotryptic enzyme.Biochem Biophys Res Commun.211,586-589；Yousef et al.2000.The KLK7(PRSS6)gene,encodingfor the stratum corneum chymotryptic enzyme is a new member of the humankallikrein gene family-genomic characterization,mapping,tissue expression andhormonal regulation.Gene.254,119-1281)。

KLK7 is involved in the degradation of the cohesive structure (coherent structure) between keratinized squamous epithelial cells during desquamation. The desquamation process is well regulated and in delicate balance with the production of corneocytes from the head to maintain a constant thickness of the stratum corneum. In this regard, KLK7 was reported to be capable of cleaving the stratum corneum desmoplamin (corneodesmosomal proteins) keratinocyte and desmocollin 1(Simon et al 2001. referred to a modified chromatography of comedogenesin proteins degradation of human epitopic derivatives to degradation).J.Biol.Chem.276,20292-20299；Caubet etal.2004.Degradation of corneodesmosome proteins by two serine proteases ofthe kallikrein family,SCTE/KLK5/hK5and SCCE/KLK7/hK7.J.Invest.Dermatol.122,1235-1244；Brattsand et al.2005.A proteolytic cascade of kallikreins in thestratum corneum.J.Invest.Dermatol.124,198-). furthermore, it has been shown that these two lipid processing enzymes β -glucocerebrosidase and acid sphingomyelinase can be degraded by KLK7 (Hachem et al 2005. Sustanated serum proteases activity by protein amplified in pH leader degradation of lipid processing enzymes and protein analytes across barrier function and strand corn integration.J.Invest.Dermatol.125,510-520) these two lipid processing enzymes co-secrete with their substrates glucosylceramides and sphingomyelin and process these polar lipid precursors into their more apolar products such as ceramides which are then incorporated into the extracellular lamellar membrane&Egelrud.1997.Formation of active IL-1βfrom pro-IL-1βcatalyzed by stratum corneum chymotryptic enzyme in vitro.Acta Derm.Venereol.77,203-206).

Several studies have linked enhanced KLK7 activity to inflammatory skin diseases such as atopic dermatitis, psoriasis or netherton syndrome. Increased KLK7 activity may lead to uncontrolled degradation of the corneosomes (corneodesmosome) leading to disregulated desquamation, resulting in enhanced degradation of lipid processing enzymes leading to disorganized lamellar membrane structure or uncontrolled activation of the pro-inflammatory cytokine IL-1 β. It has previously been shown that this may lead to an impaired barrier function of the skin and inflammation (WO 2004/108139).

The KLK7 activity was controlled at several levels. Various factors may be responsible for increased KLK7 activity in inflammatory skin diseases. First, the amount of protease expressed may be influenced by genetic factors. Such a genetic linkage, a polymorphism in the 3'-UTR in the KLK7 gene, has been described recently (Valipoulos et al 2004.genetic association between an AACC insertion in the 3' UTR of the stratum corneum gene and atomic genetics.J.Invest,Dermatol.123,62-66.). The authors hypothesized that the described 4 base pair insertion in the 3' -UTR of the kallikrein 7 gene stabilized KLK7mRNA, resulting in overexpression of KLK 7. Second, KLK7 cannot be activated automatically because it is secreted as a zymogen into the stratum corneum extracellular space via lamellar bodies, which require activation by another protease, such as active KLK5 (Caubet et al supra). The uncontrolled activity of this activating enzyme may lead to over-activation of KLK 7. Third, activated KLK7 can be inhibited by natural inhibitors such as LEKTI, ALP, or elastase inhibitors (elafin) (Schechter et al 2005.inhibition of human kallikreins 5and 7byth serine protease inhibitors lysine-epidermal Kcizal-type inhibitor (LEKTI).Biol.Chem.386,1173-1184；Franzke et al.1996.Antilenkoprotease inhibitsstratum corneum chymotryptic enzyme-Evidence for a regulative function indesquamation.J.Biol.Chem.271, 21886-21890). Reduced expression or lack of such inhibitors may result in enhanced activity of KLK 7.

It has been found that mutations in the spin gene encoding LEKTI induce Netherton syndrome (Descagues et al 2005. Spink5-specific microorganism mication Netherton syndrome through mutation of Desmogein 1by epicormal) protease hyperactivity.Nat.Genet.37,56-65), a single point mutation in this gene is associated with atopic dermatitis (Walley et al 2001.gene polymorphism in nethertona and common atopic disease.Nat.Genet.29,175-178；Nishio et al.2003.Associationbetween polymorphisms in the SPINK5gene and atopic dermatitis in theJapanese.Genes Immun.4,515-517). Finally, another aspect controlling the activity of KLK7 is the pH. The optimum pH conditions for KLK7 are neutral to slightly basic and there is a pH gradient from neutral to acidic from the innermost layer to the outermost layer of the skin. Environmental factors such as soap may cause the pH of the outermost layer of the stratum corneum to rise, approaching the optimum pH of KLK7, thereby enhancing KLK7 activity.

The hypothesis that increased KLK7 activity is associated with inflammatory skin diseases was confirmed by subsequent studies that, first, patients with Netherton syndrome show a phenotypically dependent increase in serine protease activity, a decrease in keratinocytes, a decrease in the lipid processing enzyme β -glucocerebrosidase and acid sphingomyelinase, and an impaired barrier function (Descriptions et al 2006. Cormodessmosomal a cadherin specific targets of stratum cornum typsin-and chromophorin-lipidosis in a herenylon.J.Invest.Dermatol.126,1622-1632；Hachem et al.2006.Serine proteaseactivity and residual LEKTI expression determine phenotype in Nethertonsyndrome.J.Invest,Dermatol.126,1609-1621.). Secondly, transgenic Mice overexpressing KLK7 displayed a phenotype similar to that of the skin found in atopic Dermatitis patients (Hansson et al 2002. Epidermalcoverxpression of Stratum Corneuum Chymotryptic Enzyme in Mice: A Model for Chronic Itchytitics).J.Invest.Dermatol.118,444-449；Ny&Egelrud.2003.Transgenic mice over-expressing a serine protease in the skin:evidence of interferon gamma-independent MHC II expression by epidermalkeratinocytes.Acta Derm.Venereol.83,322-327；Ny&Egelrud.2004.Epidermalhyperproliferation and decreased skin barrier function in mice overexpressingstratum corneum chymohyptic enzyme.Acta Derm.Venereol.84,18-22). Third, there is a report on KLK7 in atopic skinIncreased levels in the skin of patients with inflammation and psoriasis (Ekholm)&Egelrud.1999.Stratumcorneam chymotryptic enzyme in psoriasis.Arch.Dermatol.Res.291,195-200). KLK7 is therefore considered a target for the treatment of inflammatory skin diseases, such as atopic dermatitis, psoriasis or netherton syndrome, and specific inhibitors are therefore required.

Since patients with netherton syndrome have a severely compromised skin barrier, topical administration of therapeutically active compounds will result in systemic exposure of the compound to the patient. Thus, there is a need to identify inhibitors that are selective for skin proteases and that can be used in the treatment of netherton syndrome without the risk of unwanted systemic effects due to systemic protease inhibition.

KLK7, KLK5, and KLK14 are believed to be part of the proteolytic cascade in the stratum corneum of human skin (Brattsand et al 2005.A proteolytic cascade of kallikreins in the stratum corneum.J Invest Dermatol.124,198-203)。

Therefore, it would be beneficial to identify inhibitors that are not only active against KLK7, but are also active against KLK 5and KLK14 at the same time.

WO2004/108139 describes certain substituted benzoxazinone and thienooxazinone (thienoxazinone) compounds as inhibitors of KLK7, but does not report any selectivity data for the compounds described.

Disclosure of Invention

The present inventors have been able to identify selective inhibitors of skin proteases and have found that the activity of KLK7, KLK 5and KLK14 can be selectively inhibited by compounds of formula I,

wherein R is-S-CH₃or-Cl.

The compounds show several advantageous properties, such as selectivity for the skin proteases KLK7, KLK 5and KLK14, are involved in the pathophysiology of inflammatory skin diseases such as netherton syndrome, and have no or only low inhibitory activity against other proteases.

Accordingly, the present invention provides a compound of formula I, or a pharmaceutically acceptable salt thereof,

wherein R is-S-CH₃or-Cl.

The invention also provides a compound shown in the formula I or pharmaceutically acceptable salt thereof, which is used for medicine.

The compound can be 6-ethoxy-7-methoxy-2- (2-methylmercaptophenyl) -3, 1-benzoxazine-4-one or 2- (2-chlorphenyl) -6-ethoxy-7-methoxy-3, l-benzoxazine-4-one.

The invention also provides a compound shown as the formula I or pharmaceutically acceptable salt thereof, which is used for preventing, preventing and/or treating skin diseases.

The present invention also provides a pharmaceutical composition comprising a compound according to formula I in admixture with pharmaceutically acceptable adjuvants, diluents and/or carriers.

The invention further provides a pharmaceutical composition based on the invention, which can be used for preventing, preventing and/or treating skin diseases.

The invention also relates to the use of a compound of formula I or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment of a skin disorder.

The present invention also provides a method for preventing, preventing and/or treating skin diseases, which comprises administering to a subject in need of such treatment a therapeutically effective amount of a compound represented by formula I or a pharmaceutically acceptable salt thereof.

The skin disease may be an inflammatory skin disease. The skin disease may be selected from netherton syndrome, atopic dermatitis, contact dermatitis, eczema, psoriasis, acne, epidermal hyperkeratosis, acanthosis, epidermal inflammation, dermal inflammation and pruritus. The subject of treatment may be a mammal, such as a human, dog, cat, or horse.

Definition of

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

In the context of this specification, the term "treatment" also includes "prevention", unless there is a specific indication to the contrary. The term "treating" in the context of the present invention also includes administering an effective amount of a compound of the present invention to alleviate an existing acute or chronic disease state or recurring disorder. The definition also includes prophylactic therapies for preventing recurring conditions and persistent therapies for chronic disorders.

The compounds of the present invention may be administered by any route, including oral, intramuscular, subcutaneous, topical, intranasal, intraperitoneal, intrathecal (intrathecal), intravenous, epidural, intrathecal, intracerebroventricular administration and injection into the joints, in the form of conventional pharmaceutical compositions.

In one embodiment of the invention, the route of administration may be topical.

In determining the individual regimen and the dose level best suited for a particular patient, the dosage will depend upon the route of administration, the severity of the disease, the age, the weight of the patient and other factors normally considered by the attending physician.

For the preparation of pharmaceutical compositions by the compounds of the present invention, inert, pharmaceutically acceptable carriers may be either solid or liquid. Solid form preparations include powders, tablets, dispersible granules, capsules, cachets, and suppositories.

A solid carrier can be one or more substances which may also act as diluents, flavoring agents, solubilizers, lubricants, suspending agents, binders, or tablet disintegrating agents; it may also be a capsule material.

In powders, the carrier is a finely divided solid which is in admixture with the finely divided compound or active ingredient of the invention. In tablets, the active ingredient is mixed with a suitable proportion of a carrier having the necessary binding properties and compacted in the shape and size desired.

To prepare suppository compositions, a low melting wax, such as a mixture of fatty acid glycerides and cocoa butter, is first melted and the active ingredient dispersed therein, for example, by stirring. The molten homogeneous mixture is then poured into a conveniently sized mold and allowed to cool and solidify.

Suitable carriers are magnesium carbonate, magnesium stearate, talc, lactose, sugar, pectin, dextrin, starch, methylcellulose, sodium carboxymethylcellulose, low melting waxes, cocoa butter, and the like.

The term composition also includes formulations of the active ingredient with encapsulating material as a carrier, which provides a capsule in which the active ingredient is encapsulated by a carrier material (with or without other carriers). Similarly, cachets are included.

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

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

Aqueous solutions for oral administration may be prepared by dissolving the active component in water and adding suitable coloring, flavoring, stabilizing and thickening agents as desired. Aqueous solutions for oral use can be prepared by dispersing the finely divided active component in water together with viscous materials such as natural synthetic gums, resins, methylcellulose, sodium carboxymethylcellulose and other suspending agents well known in the pharmaceutical formulation art.

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

A therapeutically effective amount in the practice of the present invention can be determined by using known criteria including age, weight and response of the individual patient and understood by those skilled in the art in the context of treating or preventing the disease.

The above-mentioned subject matter is a pharmaceutical composition comprising a compound according to the invention, similarly applicable to a pharmaceutical composition comprising a combination according to the invention.

It is contemplated that any method or composition described herein can be practiced with respect to any other method or composition described herein.

In the claims and/or the specification, the words "a" or "an", when used in conjunction with the term "comprising", may mean "one", but it also conforms to the meaning of "one or more", "at least one", "one or more".

These and other embodiments of the invention will be better understood when considered in conjunction with the following description and the accompanying drawings. However, the following description, given for several embodiments of the invention and numerous specific details, is to be construed as provided for purposes 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 set forth herein, and the invention includes all such substitutions, modifications, additions and/or rearrangements.

Examples

Example 1

Substituted 3, 1-benzoxazine-4-ones as inhibitors of human proteases

The IC of some substituted 3, 1-benzoxazine-4-ones on a panel of human proteases was determined₅₀The value is obtained.

KLK7 test

Materials: recombinant human KLK7, substrate S-2586 (chromogenic substrate, cat. No.820894)

KLK7 activity was determined under the following conditions: absorbance was measured at 405nM using a 96-well plate by a microplate reader (Spectramax) at 37 ℃ in 10mM sodium phosphate pH7.2, 0.5M NaCl, KLK7 final concentration 2.5. mu.g/mL (100nM), 1.0mM substrate, 5% DMSO inhibitor concentration 0. mu.M, 0.1. mu.M, 0.5. mu.M, 1.0. mu.M and 5.0. mu.M, respectively.

KLK5 test

Materials: recombinant human KLK5, substrate S-2288 (chromogenic substrate, cat. No.820852)

KLK5 activity was determined under the following conditions: absorbance was measured at 405nm using a microplate reader (Spectramax) in 96-well plates at 37 ℃ in 0.1mM Tris (Tris), pH8.0, 0.15M NaCl, KLK5 final concentration 2.5. mu.g/mL, 1mM substrate, 5% DMSO, and inhibitor concentrations of 0. mu.M, 0.1. mu.M, 1.0. mu.M and 10. mu.M, respectively.

KLK14 test

Materials: recombinant human KLK14, substrate S-2302 (chromogenic substrate, cat. No. 820340).

KLK14 activity was determined under the following conditions: absorbance was measured at 405nM using a 96 well plate by a microplate reader (Spectramax) at 37 ℃ in 0.1mM Tris (Tris), pH8.0, 0.15M NaCl, KLK14 final concentration 0.26. mu.g/mL (9.4nM), 0.75mM substrate, 5% DMSO, and 0. mu.M, 0.1. mu.M, 1.0. mu.M, and 10. mu.M inhibitor concentrations, respectively.

Cathepsin G assay

Materials: cathepsin G, 100mU (VWR, Calbiochem, cat No.219373), substrate cathepsin G substrate (VWR, Calbiochem, cat No. 219407).

Cathepsin activity was determined under the following conditions: the absorbance was measured at 405nM by a microplate reader (Spectramax) using 96-well plates at 37 ℃ in 10mM sodium phosphate pH7.2, 0.5M NaCl, 1.5M cathepsin G final concentration of 1.5mU/mL (0.75. mu.g/mL, 32nM), 0.75mM substrate, 5% DMSO inhibitor concentration of 0. mu.M, 0.1. mu.M, 1.0. mu.M and 10. mu.M, respectively.

Chymotrypsin test

Materials: chymotrypsin, bovine, 25. mu.g (Roche, sequencing grade), substrate S-2586 (luminescent substrate, cat. No.820894)

Chymotrypsin activity was determined under the following conditions: absorbance was measured at 405nM using a 96-well plate by a microplate reader (Spectramax) at 37 ℃ in 10mM sodium phosphate pH7.2, 0.5M NaCl, final chymotrypsin concentration of 0.2. mu.g/mL (6,8nM), 1mM substrate, inhibitor concentration in 5% DMSO of 0. mu.M, 0.1. mu.M, 1.0. mu.M and 10. mu.M, respectively.

Trypsin assay

Materials: trypsin, 100. mu.g (Roche, sequencing grade, molecular weight 23500), substrate S-2288 (luminescent substrate, cat. No.820852)

Trypsin activity was determined under the following conditions: absorbance was measured at 405nM using a microplate reader (Spectramax) in 96-well plates at 37 ℃ in 10mM sodium phosphate pH7.2, 0.5M NaCl, trypsin at a final concentration of 0.8. mu.g/mL (34nM), 1mM substrate, and 5% DMSO at an inhibitor concentration of 0. mu.M, 0.1. mu.M, 1.0. mu.M, and 10. mu.M, respectively.

Thrombin assay

Materials: thrombin (luminescent substrate, cat No.820712), substrate S-2288 (luminescent substrate, cat No.820852)

Thrombin activity was determined under the following conditions: absorbance was measured at 37 ℃ by a microplate reader (Spectramax) using 96-well plates at 405nm in 50mM Tris (Tris) pH8.3, 130mM sodium chloride, 1pkat/mL thrombin (0.03. mu.g/mL, 88pM), 0.5mM substrate, 0. mu.M inhibitor concentration in 5% DMSO, 0.1. mu.M, 1.0. mu.M and 10. mu.M, respectively.

TABLE 1 Selectivity of substituted 3, 1-benzoxazin-4-ones-IC₅₀(μM)

As shown in Table 1, only the compound 1 (6-ethoxy-7-methoxy-2- (2-methylmercaptophenyl) -3, 1-benzoxazin-4-one) and the compound 2(2- (2-chlorophenyl) -6-ethoxy-7-methoxy-3, l-benzoxazin-4-one) were found to have the desired selectivity, IC of KLK7₅₀IC of less than 0.1. mu.M, KLK 5and KLK14₅₀IC of less than 1. mu.M, other proteases tested₅₀Above 10. mu.M.

It should be noted that although several compounds were found to have strong inhibitory effects on KLK7, as well as KLK 5and KLK14, only compounds 1 and 2, i.e. the compounds of the present invention, proved to have sufficiently low inhibitory activity on other proteases.

Even small changes in the substitution pattern of a compound can have a significant effect on the selectivity of the compound. For example, compound 12 substituted with methoxy at the 6-position, compared to compound 1 substituted with ethoxy at the 6-position, showed more than ten times higher inhibitory activity (considered as a 10-fold lower IC) for cathepsin G and chymotrypsin than compound 1₅₀) Making compound 12 unsuitable for the treatment of skin diseases.

In summary, the data presented in table 1 show that only compounds 1 and 2, i.e. the compounds according to the invention, are sufficiently selective, have high inhibitory activity against KLK7, KLK 5and KLK14, while having sufficiently low inhibitory activity against other proteases, making them suitable for the treatment of skin diseases.

Example 2

Synthesis of 6-ethoxy-7-methoxy-2- (2-methylmercaptophenyl) -3, 1-benzoxazine-4-one

Example 3

Synthesis of 2- (2-chlorophenyl) -6-ethoxy-7-methoxy-3, l-benzoxazine-4-one

Same as example 2 but used in the third step

Claims (12)

1. A compound according to formula I, or a pharmaceutically acceptable salt thereof,

wherein R is-S-CH₃or-Cl.

2. The compound 6-ethoxy-7-methoxy-2- (2-methylmercaptophenyl) -3, 1-benzoxazine-4-one.

3. The compound 2- (2-chlorophenyl) -6-ethoxy-7-methoxy-3, l-benzoxazin-4-one.

4. A compound as claimed in any one of claims 1 to 3 for use in medicine.

5.A compound according to any one of claims 1 to 3 for use in the prevention, prevention and/or treatment of a skin disease.

6. A pharmaceutical composition comprising a compound according to any one of claims 1 to 3 in admixture with pharmaceutically acceptable adjuvants, diluents and/or carriers.

7. A pharmaceutical composition according to claim 6 for the prevention, prevention and/or treatment of skin diseases.

8. The invention also relates to the use of a compound according to any one of claims 1 to 3 for the preparation of a medicament for the treatment of skin diseases.

9. The present invention also provides a method for the prevention, prevention and/or treatment of skin diseases comprising administering to a subject in need of such treatment a therapeutically effective amount of a compound as claimed in any one of claims 1 to 3.

10. The composition of claim 7, the use of claim 8, or the method of claim 9, wherein the skin disease is an inflammatory skin disease.

11. The composition for use according to claim 7, the use according to claim 8, or the method according to claim 9, wherein the skin disease is selected from netherton syndrome, atopic dermatitis, contact dermatitis, eczema, psoriasis, acne, epidermal hyperkeratosis, acanthosis, epidermal inflammation, dermal inflammation and pruritus.

12. The composition of claim 7, the use of claim 8, or the method of claim 9, wherein the skin disease is Netherton syndrome.