Benzoxazinone compounds as dual KLK5 / 7 inhibitors

Abstract

The present invention provides a compound and a pharmaceutical composition comprising the compound, wherein the compound and the pharmaceutical composition are used to treat skin diseases associated with the proteolytic activity of one or more KLK proteases and the pharmaceutical composition comprising the compound, wherein the compound has a structure shown in formula (I), wherein R has the definition described in the specification.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 62 / 909,006, filed on October 1, 2019, the contents of which are hereby incorporated by reference in their entirety for all purposes.

[0003] STATEMENT REGARDING RIGHTS TO INVENTS UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

[0004] not applicable

[0005] Reference to a Sequence Listing, Table or Computer Program Listing Appendix Submitted on CD-ROM

[0006] not applicable Background Art

[0007] Genetic skin disorders are inherited diseases that manifest as skin symptoms. These disorders are usually detectable at birth or shortly after birth. The type of disorder depends on how the genes are affected, but the resulting symptoms can be severe, rare, and can seriously affect the patient's life by causing disability, shortening lifespan, and the development of other chronic diseases and cancers. There are approximately 400 different kinds of inherited skin disorders, with incidence ranging from 1 in 6000 to 1 in 500,000 people. The burden of the disease can be heavy for patients and families who may only have access to a very limited range of expensive medications to help manage the disease. Genetic skin disorders can be divided into monogenic disorders, where the skin disorder is caused by an abnormality in a single gene, and polygenic disorders, where mutations (defects) in several genes influence the type of disorder a patient has. Monogenic disorders include acute intermittent porphyria, epidermolysis bullosa, ichthyosis, Fabry disease, anhidrotic ectodermal dysplasia, and incontinence pigmenti. Genetic disorders with ichthyosis symptoms include Netherton Syndrome.

[0008] Netherton syndrome (NS) is a rare autosomal recessive skin disorder characterized by congenital ichthyosis, atopic diathesis, and a characteristic hair shaft abnormality called invaginated alopecia. Netherton syndrome is one of the most severe keratinization disorders. Infants usually present with generalized scaly erythroderma at birth and are at higher risk for life-threatening complications such as hypernatremic dehydration, developmental delay, and sepsis. In older children, a variety of allergic manifestations may develop, including severe atopic dermatitis, asthma, hay fever, and markedly elevated serum immunoglobulin E levels. In older children and adults, the scaling may have a distinctive circular pattern (convoluted ichthyosis). But in infants and young children, the skin is usually red with generalized scaling without a distinctive circular pattern. The hair shaft is fragile and breaks easily due to invaginated follicles or "bamboo hairs," resulting in short, sparse hair. Another characteristic of Netherton syndrome is a predisposition to allergies, asthma, and eczema. In addition to bacterial skin and systemic infections, adults and children with Netherton syndrome are also susceptible to viral skin infections from herpes and human papillomavirus. The prevalence of Netherton syndrome is estimated to be 1 / 200,000 births, with an incidence estimated to be 1-9 / 1,000,000. However, regional studies suggest that the incidence may be higher due to diagnostic difficulties in infancy and young children, including overlapping features with atopic dermatitis and other occult ichthyoses. Currently, there is no specific treatment for NS, only palliative care to treat skin infections and reduce itching and pain.

[0009] NS is caused by loss-of-function mutations in the serine protease inhibitor Kazal type 5 (SPINK5) gene, which encodes LEKTI (lymphoepithelial Kazal-type inhibitor). LEKTI loss of function results in uninhibited protease activity and excessive digestion of epidermal structural and barrier proteins.

[0010] LETKI is a specific inhibitor of members of the kallikrein (KLK) serine protease family (KLK5, KLK7 and KLK14) (Chavanas et al., Nat Genet 2000, 25: 141-142). KLK5 is characterized as a promoter upstream of KLK7. According to the current hypothesis, pro-KLK is synthesized and activated in the stratum granulosum, and the active KLK enzyme rapidly complexes with LEKTI, thereby preventing premature degradation of desmosomes at the stratum corneum / stratum granulosum interface (Borgono et al., J Biol Chem 2007, 282: 3640-3652; Deraison et al., Mol Biol Cell 2007, 18: 3607-3619; Ovaere et al., Trends Biochem Sci 2009, 34: 453-463). The KLK-LEKTI complex diffuses into the outer stratum corneum, where the acidic microenvironment causes LEKTI to release active KLK. Subsequently, active KLK cleaves corneal desmosomes in the most superficial layer of the stratum corneum. This ensures a finely balanced regulation of the desquamation process (Ovaere et al., Trends Biochem Sci 2009, 34: 4S3-463).

[0011] Spink5 - / - Mice recapitulate NS disease (Descargues et al., Nat Genet 200S, 37: S6-6S; Hewett et al., Hum Mol Genet 200S, 14: 33S-346; Yang et al., Genes Dev 2004, 18: 23S4-23S8). A key finding is that, similar to what is observed in subjects with NS, Spink5 - / - Mouse epidermis displays unopposed KLK5 and KLK7 protease activity (Descargues et al., Nat Genet 200S, 37: S6-6S).

[0012] Conversely, loss of KLK5 rescues Spink5 - / - Neonatal mortality in newborn mice was reduced and the skin characteristics of NS, including skin barrier defects, epidermal structural disorder and skin inflammation, were reversed (Furio et al., PLoS Genet 201S, Sep; 11(9): el00S389). Notably, loss of KLK5 resulted in a decrease in epidermal proteolytic activity, especially KLK7 and KLK14. Subsequently, the structural integrity of desmosomes and angular desmosomes was restored, normal epidermal differentiation and normalized expression of IL-1β, IL-17A and TSLP (thymic stromal hematopoietic factor) were restored.

[0013] In addition to animal models, organotypic 3D cultures were generated using collagen gels filled with normal human keratinocytes and fibroblasts transfected with SPINK5-targeting small interfering RNA (siRNA), and epidermal defects were observed in these culture models (Wang et al., Exp Dermatol 2014, Jul; 23(7): S24-6). Gene silencing of KLK5 or KLK7 significantly improved the epidermal structure impaired by reduced SPINK5 expression. In summary, these studies confirm the major role of KLK5 and its upstream and downstream regulators in NS.

[0014] KLK7 is expressed by keratinocytes in the stratum granulosum and secreted into the extracellular space of the stratum corneum (i.e., the outermost layer of the skin) as a zymogen that is subsequently activated by KLK5. Once activated, KLK7 plays a central role in the desquamation process of the skin. Proteolytic events of KLK7 are essential for the desquamation process and the controlled regeneration of the skin. A tight balance between the production of new keratinocytes and desquamation is essential to maintain skin homeostasis, and conversely, an imbalance between these two processes can lead to impaired skin function and ultimately to skin diseases such as Netherton syndrome. Under normal circumstances, this balance is maintained by the expression of an endogenous protein inhibitor of KLK7 (LEKTI) to counteract its proteolytic activity. In skin diseases, overexpression and / or increased activity of KLK7 can lead to excessive desquamation. Genetic associations in animal models and humans further support the theory that KLK7 is involved in the development of skin diseases. It has been shown that transgenic mice that overexpress human KLK7 have skin features similar to those seen in patients with chronic atopic dermatitis, who share certain features with patients with Netherton syndrome.

[0015] Given the roles of KLK5 and KLK7 in regulating desquamation, and combined with the reported association between KLK5 and KLK7 in regulating desquamation, - / - / Klk5 - / - / Klk7 - / - The results of complete phenotypic rescue in knockout mouse models (Kasparek et al., PLoS Genet 2017, Jan;13(1):e1006566) suggest that potent and selective dual KLK5 / KLK7 inhibitors are needed to treat genetic skin diseases such as Netherton syndrome by modulating the proteolytic activity of these proteases. Summary of the invention

[0016] In the first aspect of the present invention, there is provided a compound represented by formula (I), or a pharmaceutically acceptable complex thereof:

[0017]

[0018] Wherein, R is H or is selected from the following group: -R1 and -C(O)R 1a , where R 1 C 1-12 Alkyl, R 1a H or C 1-12 alkyl.

[0019] In a second aspect of the present invention, a pharmaceutical composition is provided, comprising a compound represented by formula (I) or a pharmaceutically acceptable complex thereof, and a pharmaceutically acceptable carrier.

[0020] In a third aspect of the present invention, a method for treating a skin disease associated with the proteolytic activity of one or more KLK proteases in a subject in need thereof is provided. The method comprises administering to the subject an effective amount of a compound of formula (I) or a pharmaceutically acceptable complex thereof, or a pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable complex thereof.

[0021] In a fourth aspect of the present invention, an in vitro assay method for determining the proteolytic activity of one or more KLK proteases in the skin is provided. The method comprises 1) preparing a skin extract; 2) exposing a substrate and a compound of formula (I) or a pharmaceutically acceptable complex thereof to the skin extract; 3) determining the proteolytic cleavage rate of the substrate. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 A scheme for the preparation of compound 1.001 is shown.

[0023] Figure 2 A scheme for the preparation of compound 1.002 is shown.

[0024] Figure 3 Shown is a scheme for the preparation of intermediate 100, which was used to prepare compounds 1.001 and 1.002.

[0025] Figures 4A-4D The proteolytic cleavage rates of KLK5 selective substrates in the skin extract assay by compounds 1.001, 1.002 or known compound 14 are shown. Figure 4A : control group; Figure 4B : Compound 14; Figure 4C : Compound 1.001; Figure 4D :Compound 1.002. Wherein, NSK refers to freshly prepared skin extract; OSK refers to freeze-thawed skin extract. DETAILED DESCRIPTION

[0026] I. Overview

[0027] The present invention provides compounds of formula (I) and pharmaceutical compositions comprising compounds of formula (I) for use in treating skin diseases associated with the proteolytic activity of one or more KLK proteases. In particular, the pharmaceutical composition can be a topical preparation comprising a compound of formula (I) for topical use in treating hereditary skin diseases. The present invention also provides methods for treating skin diseases associated with the proteolytic activity of one or more KLK proteases by administering a compound of formula (I) or a pharmaceutical composition thereof. In particular, a compound of formula (I) or a pharmaceutical composition thereof can be administered topically. Specifically, the skin disease is a hereditary skin disease, such as Netherton syndrome.

[0028] II. Definitions

[0029] Abbreviations used herein have their conventional meanings in the fields of chemistry and biology.

[0030] "Alkyl" refers to a straight or branched chain saturated aliphatic group (i.e., C 1-12 represents 1 to 12 carbons). The alkyl group may include any number of carbons, for example, C 1-2 , C 1-3 , C 1-4 , C 1-5 , C 1-6 , C 1-7 , C 1-8 , C 1-9 , C 1-10 , C 1-11 , C 1-12 , C 2-3 , C 2-4 , C 2-5 , C 2-6 , C 2-7 , C 2-8 , C 2-9 , C 2-10 , C 2-11 , C 2-12 , C 3-4 , C 3-5 , C 3-6 , C 3-7 , C 3-8 , C 3-9 , C 3-10 , C 3-11 , C 3-12 , C 4-5 , C 4-6 , C 4-7 , C 4-8 , C 4-9 , C 4-10 , C 4-11 , C 4-12 , C 5-6 , C 5-7 , C 5-8 , C5-9 , C 5-10 , C 5-11 , C 5-12 , C 6-7 , C 6-8 , C 6-9 , C 6-10 , C 6-11 , C 6-12 , C 7-8 , C 7-9 , C 7-10 , C 7-11 , C 7-12 , C 8-9 , C 8-10 , C 8-11 , C 8-12 , C 9-10 , C 9-11 , C 9-12 , C 10-11 , C 10-12 and C 11-12 For example, C 1-12 Alkyl includes, but is not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, n-hexyl, n-heptyl, n-octyl, n-nonyl, n-decyl, n-undecyl, n-dodecyl, etc. Alkyl may also refer to an alkyl group having up to 20 carbon atoms, such as, but not limited to, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, etc.

[0031] As used herein, "pharmaceutically acceptable complex" refers to a complex comprising a compound of formula (I) and at least one complexing agent. The term "pharmaceutically acceptable" refers to a complex approved by a federal or state regulatory agency or listed in the U.S. Pharmacopoeia or other recognized pharmacopoeia for use in animals, especially in humans. The complexing agent has the function of forming a complex structure with the compound of formula (I) through non-covalent secondary interactions. Secondary interactions can be formed by electrostatic interactions, such as ionic interactions, hydrogen bonds, dipole-dipole interactions, dipole-induced dipole interactions, London dispersion forces, nn interactions, and hydrophobic interactions. Pharmaceutically acceptable complexes of compounds of formula (I) include pharmaceutically acceptable salts and / or solvates thereof.

[0032] "Solvate" refers to a compound provided herein or a salt thereof, which further includes a stoichiometric or non-stoichiometric amount of a solvent bound by non-covalent intermolecular forces. In the case where the solvent is water, the solvate is a hydrate.

[0033] "Hydrate" refers to a compound complexed with water molecules. The compounds of the present invention may be complexed with 1 / 2 water molecules or 1 to 10 water molecules.

[0034] As used herein, "peptide-p-nitroaniline" or "peptide-pNA" refers to a peptide substrate having 4 to 6 amino acids linked by peptide bonds and having a p-nitroaniline group at the C-terminus. Examples of peptide-pNA substrates in the present invention include Tyr-Arg-Ser-Arg-pNA and Lys-His-Leu-Tyr-pNA.

[0035] As used herein, "composition" is intended to encompass a product comprising the specified ingredients in the specified amounts, as well as any product which results, directly or indirectly, from combination of the specified ingredients in the specified amounts. "Pharmaceutically acceptable" means the carrier, diluent or excipient must be compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.

[0036] "Pharmaceutically acceptable carrier or excipient" refers to a substance that helps to administer the active agent to a subject and helps the subject absorb it. Pharmaceutical excipients that can be used in the present invention include, but are not limited to, binders, fillers, disintegrants, lubricants, coating agents, sweeteners, flavoring agents, and pigments. Pharmaceutical excipients used for transdermal / topical delivery in the present invention include, but are not limited to, enhancers, solubilizers, antioxidants, plasticizers, thickeners, polymers, and pressure-sensitive adhesives. Those skilled in the art will recognize that other pharmaceutical excipients can be used in the present invention.

[0037] “IC 50 ” refers to the amount, concentration or dose of a specific test compound that achieves 50% inhibition of the maximal response in an assay measuring a specific response.

[0038] "Inhibit," "inhibitor," and "inhibitor" refer to a compound or method of inhibiting a specific effect or function.

[0039] "Administering" refers to topical administration, for example, as a lotion, spray, ointment, cream, gel, paste or patch.

[0040] "Topical" refers to applying a suitable compound (e.g., active ingredient) or a composition comprising a compound (e.g., active ingredient) to the skin to treat a disease or condition, such as a genetic skin disease. In some embodiments, "topical" refers to applying a suitable compound (e.g., active ingredient) or a composition comprising a compound (e.g., active ingredient) to the skin, and fully penetrating the epidermis or dermis to treat a genetic skin disease. In some topically applied embodiments, the compound or composition penetrates the epidermis or dermis without significant systemic exposure, and is not intended to treat or prevent a disease in another organ system. In some topically applied embodiments, the compound or composition is delivered transdermally through the skin for systemic distribution. Examples include transdermal patches for drug delivery.

[0041] "Treatment" refers to any marker of success in treating or ameliorating an injury, pathology, or condition, including any objective or subjective parameter, such as relief; remission; lessening of symptoms or making the injury, pathology, or condition more tolerable to the patient; slowing the rate of degeneration or decline; making the endpoint of degeneration less debilitating; improving the patient's physical or mental well-being. Treatment or amelioration of symptoms may be based on objective or subjective parameters; including the results of a physical examination, neuropsychiatric examination, and / or psychiatric evaluation.

[0042] "Patient" or "subject" refers to a living organism suffering from or prone to suffering from a disease or condition that can be treated by administering a pharmaceutical composition provided herein. Non-limiting examples include humans, other mammals, cattle, rats, mice, dogs, monkeys, goats, sheep, cattle, deer, and other non-mammals. In some embodiments, the patient is a human.

[0043] "Therapeutically effective amount" refers to the amount of a compound or pharmaceutical composition that can be used to treat or improve an established disease or condition, or to exhibit a detectable therapeutic or inhibitory effect. The exact amount will depend on the purpose of the treatment and can be determined by those skilled in the art using known techniques (see, for example, Lieberman, Pharmaceutical Dosage Forms (Volumes 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999); and Remington: The Science and Practice of Pharmacy, 20th Edition, 2003, Gennaro, Ed., Lippincott, Williams & Wilkins)

[0044] "One", "an" or "an", when used herein to refer to a group of substituents or "substituents", means at least one. For example, when a compound is substituted with "an" alkyl or aryl group, the compound is optionally substituted with at least one alkyl group and / or at least one aryl group, wherein each alkyl group and / or aryl group is optionally different. In another example, when a compound is substituted with "a" substituent, the compound is substituted with at least one substituent, wherein each substituent is optionally different.

[0045] III. Compounds

[0046] In the first aspect of the present invention, there is provided a compound represented by formula (I), or a pharmaceutically acceptable complex thereof:

[0047]

[0048] Wherein, R is H or is selected from the following group: -R 1 and -C(O)R 1a , where R 1 C 1-12 Alkyl, R 1a H or C 1-12 alkyl.

[0049] The methanesulfonyl group (-SO2CH3) in formula (I) can be in the ortho, meta or para position of 7,8-dihydro-4H-[1,4]dioxin[2',3':4,5]benzo[1,2-d][1,3]oxazin-4-one. In some embodiments, the -SO2CH3 group is in the ortho position of 7,8-dihydro-4H-[1,4]dioxin[2',3':4,5]benzo[1,2-d][1,3]oxazin-4-one.

[0050] The -CH2OR group in formula (I) can be in the ortho, meta or para position of 7,8-dihydro-4H-[1,4]dioxin[2',3':4,5]benzo[1,2-d][1,3]oxazin-4-one. In some embodiments, the -CH2OR group is in the meta position of 7,8-dihydro-4H-[1,4]dioxin[2',3':4,5]benzo[1,2-d][1,3]oxazin-4-one. In some embodiments, the -CH2OR group is in the para position of 7,8-dihydro-4H-[1,4]dioxin[2',3':4,5]benzo[1,2-d][1,3]oxazin-4-one.

[0051] In some embodiments, the compound is as shown in formula (Ia):

[0052]

[0053] wherein R is as defined in the description of formula (I).

[0054] In some embodiments of formula (Ia), the -CH2OR group can be in the ortho position, meta position or para position of the -SO2CH3 group. In some embodiments, the -CH2OR group is in the meta position of the -SO2CH3 group. In some embodiments, the -CH2OR group is in the para position of the -SO2CH3 group.

[0055] In some embodiments, the compound is as shown in formula (Ib):

[0056]

[0057] wherein R is as defined in the description of formula (I).

[0058] In some embodiments, the compound is as shown in formula (Ic):

[0059]

[0060] wherein R is as defined in the description of formula (I).

[0061] With reference to any of Formulas (I), (Ia), (Ib), and (Ic), in some embodiments, R is H.

[0062] In some embodiments, the compound is represented by the following formula:

[0063]

[0064] In some embodiments, the compound is represented by the following formula:

[0065]

[0066] With reference to any one of formula (I), (Ia), (Ib) and (Ic), in some embodiments, R is selected from the group consisting of: -R 1 and -C(O)R 1a , where R 1 C 1-12 Alkyl, R 1a H or C 1-12 alkyl.

[0067] In any one of the embodiments of formula (I), (Ia), (Ib) and (Ic), R is C 1-12 In some embodiments, R is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, n-hexyl, n-heptyl, n-octyl, n-nonyl, n-decyl, n-undecyl or n-dodecyl. In some embodiments, R is C 1-6 In some embodiments, R is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl or n-hexyl. In some embodiments, R is methyl or ethyl. In some selected embodiments, R is methyl.

[0068] In any one of the embodiments of formula (I), (Ia), (Ib) and (Ic), R is -C(O)R 1a , where R 1a H or C 1-12 In some embodiments, R 1a is H. In some embodiments, R 1a C 1-12In some embodiments, R 1a is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, n-hexyl, n-heptyl, n-octyl, n-nonyl, n-decyl, n-undecyl or n-dodecyl. 1a C 1-6 In some embodiments, R 1a is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl or n-hexyl. 1a is methyl or ethyl. In some embodiments, R 1a It is acetyl.

[0069] In some embodiments, the compound is represented by the following formula:

[0070]

[0071] In some embodiments, the compound is represented by the following formula:

[0072]

[0073] Exemplary compounds of formula (I) are described in Table 1.

[0074] Table 1: Compounds of formula (I)

[0075]

[0076]

[0077] Other forms of compounds

[0078] The compound of the present invention can exist as a complex together with at least one complexing agent. The complexing agent has the function of forming a complex structure with the compound of formula (I) through non-covalent secondary interactions. Secondary interactions can be formed by electrostatic interactions, such as ionic interactions, hydrogen bonds, dipole-dipole interactions, dipole-induced dipole interactions, London dispersion forces, nn interactions and hydrophobic interactions. The pharmaceutically acceptable complex of the compound of formula (I) includes its pharmaceutically acceptable salt and / or solvate. In some embodiments, the pharmaceutically acceptable complex of the compound of formula (I) is its pharmaceutically acceptable salt and / or solvate. In some embodiments, the pharmaceutically acceptable complex of the compound of formula (I) is its pharmaceutically acceptable salt. In some embodiments, the pharmaceutically acceptable complex of the compound of formula (I) is its pharmaceutically acceptable salt. In some embodiments, the pharmaceutically acceptable complex of the compound of formula (I) is its pharmaceutically acceptable solvate.

[0079] Some compounds of the present invention may exist in non-solvated forms as well as solvated forms, including hydrated forms. Generally speaking, solvated forms are equivalent to non-solvated forms and are included within the scope of the present invention. Some compounds of the present invention may exist in multiple crystalline or amorphous forms. Generally speaking, all physical forms are equivalent for the purposes contemplated by the present invention and are intended to fall within the scope of the present invention.

[0080] Unless otherwise indicated, the compounds of the invention may also contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute such compounds. For example, the compounds of the invention may be labeled with radioactive or stable isotopes, such as deuterium ( 2 H), tritium (+H), iodine-125 ( 125 I), fluorine-18( 18 F), nitrogen-15 ( 15 N), oxygen-17 ( 17 O), oxygen-18 ( 18 O), carbon-13 ( 13 C) or carbon-14 ( 14 C) All isotopic variations of the compounds of the present invention, whether radioactive or not, are encompassed within the scope of the present invention.

[0081] The present invention provides compounds in the form of prodrugs. Prodrugs of the compounds described herein are those compounds that are susceptible to chemical changes under physiological conditions to provide compounds of the present invention. In addition, prodrugs can be converted into compounds of the present invention by chemical or biochemical methods in an ex vivo environment. For example, when the prodrug is placed in a transdermal patch reservoir with a suitable enzyme or chemical reagent, the prodrug can be slowly converted into a compound of the present invention.

[0082] IV. Composition

[0083] In a second aspect of the present invention, a pharmaceutical composition is provided, comprising a compound shown in formula (I) or a pharmaceutically acceptable complex thereof, and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition comprises a compound of formula (Ia) or a pharmaceutically acceptable complex thereof and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition comprises a compound of formula (Ib) or a pharmaceutically acceptable complex thereof and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition comprises a compound of formula (Ic) or a pharmaceutically acceptable complex thereof and a pharmaceutically acceptable carrier.

[0084] The pharmaceutically acceptable complexes of any of the compounds of formula (I), (Ia), (Ib) and (Ic) are defined and described as described herein. The pharmaceutically acceptable complexes of any of the compounds of formula (I), (Ia), (Ib) and (Ic) may include pharmaceutically acceptable salts and / or solvates thereof.

[0085] In some embodiments, the compound of any one of Formula (I), (Ia), (Ib) and (Ic) has the structure shown below, or a pharmaceutically acceptable salt thereof:

[0086]

[0087] In some embodiments, the compound of any one of Formula (I), (Ia), (Ib) and (Ic) has the structure shown below, or a pharmaceutically acceptable salt thereof:

[0088]

[0089] The compounds provided herein can be formulated into pharmaceutical compositions using methods available in the art and methods disclosed herein.Any compound disclosed herein can be provided in an appropriate pharmaceutical composition and administered via an appropriate route of administration.

[0090] The method of administration of the compounds described herein to a subject can be local or non-systemic, for example, topical, intradermal or intralesional. In some embodiments, the compound can be administered by topical administration. In some embodiments, the compound can be administered by intradermal administration. In some embodiments, the compound can be administered by intralesional administration, for example, by intralesional injection.

[0091] The methods provided herein include administering a pharmaceutical composition containing at least one compound as described herein, including a compound of formula (I) in salt form or complex form (if appropriate), which can be used alone or in combination with one or more compatible and pharmaceutically acceptable carriers, such as diluents or adjuvants, or if the subject needs it, together with another agent for treating a genetic skin disease.

[0092] In some embodiments, the second agent can be formulated or packaged with the compound provided herein. Of course, only when, according to the judgment of those skilled in the art, this compound formulation does not interfere with the activity or administration method of any agent, the second agent can be formulated with the compound provided herein. In some embodiments, the compound provided herein and the second agent are formulated separately. For the convenience of those skilled in the art, they can be packaged together, or packaged separately.

[0093] In clinical practice, the active ingredients provided herein can be administered by any conventional route, particularly topically, intradermally, intralesionally, orally, parenterally, rectally or by inhalation (e.g., in the form of an aerosol). In certain embodiments, the compounds provided herein are administered topically, intradermally or intralesionally. In certain embodiments, the compounds provided herein are administered topically. In certain embodiments, the compounds provided herein are administered intradermally. In certain embodiments, the compounds provided herein are administered intralesionally.

[0094] As solid compositions for oral administration, they can be used in the form of tablets, pills, hard gelatin capsules, powders or granules. In these compositions, the active product is mixed with one or more inert diluents or adjuvants, such as sucrose, lactose or starch.

[0095] These compositions may contain substances other than diluents, for example, lubricants, such as magnesium stearate, or coatings for controlled release.

[0096] As liquid compositions for oral administration, it can be used in the form of pharmaceutically acceptable solutions, suspensions, emulsions, syrups and elixirs containing inert diluents such as water or liquid paraffin. These compositions may also contain substances other than diluents, and in certain embodiments, contain moistening, sweetening or flavoring products.

[0097] Compositions for topical administration may be used in the form of lotions, tinctures, creams, emulsions, gels or ointments. In these compositions, the active product is mixed with one or more inert excipients, including water, acetone, ethanol, ethylene glycol, propylene glycol, polyethylene glycol, 1,3-butylene glycol, isopropyl myristate, isopropyl palmitate, mineral oil and mixtures thereof.

[0098] Compositions for parenteral, intralesional or intradermal administration can be emulsions or sterile solutions. As solvents or carriers, propylene glycol, polyethylene glycol, vegetable oils, particularly olive oil, or injectable organic esters can be used, and in some embodiments, ethyl oleate is used. These compositions can also include adjuvants, particularly wetting agents, isotonic agents, emulsifiers, dispersants and stabilizers. Sterilization can be carried out in a variety of ways, and in some embodiments, sterilization is carried out by using bacterial filters, radiation or heating. They can also be made into the form of sterile solid compositions, which can be dissolved in sterile water or any other injectable sterile medium when used.

[0099] Compositions for rectal administration are suppositories or rectal capsules containing, in addition to the active ingredient, excipients such as cocoa butter, semi-synthetic glycerides or polyethylene glycols.

[0100] The composition may also be an aerosol. For use in the form of a liquid aerosol, the composition may be a stable sterile solution, or a solid composition dissolved in pyrogen-free sterile water, saline or any other pharmaceutically acceptable carrier at the time of use. For use in the form of a dry aerosol intended for direct inhalation, the active ingredient is finely divided and combined with a water-soluble solid diluent or carrier, in certain embodiments, dextran, mannitol or lactose.

[0101] In certain embodiments, the compositions provided herein are pharmaceutical compositions or single unit dosage forms. The pharmaceutical compositions and single unit dosage forms provided herein include one or more preventive or therapeutic agents (e.g., compounds or other preventive or therapeutic agents provided herein) of a preventive or therapeutically effective amount, and generally one or more pharmaceutically acceptable carriers or excipients. In a specific embodiment and in this article, the term "pharmaceutically acceptable" refers to a diluent, adjuvant (e.g., Freund's adjuvant (complete and incomplete)), excipient, or carrier for administering a therapeutic agent. Such a pharmaceutical carrier can be a sterile liquid, such as water and oil, including from petroleum, animals, plants, or synthesis, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. When the pharmaceutical composition is administered intravenously, water can be used as a carrier. Saline solutions and dextrose and glycerol aqueous solutions can also be used as liquid carriers, particularly for injectable solutions. Examples of suitable pharmaceutical carriers are described in Remington: The Science and Practice of Pharmacy, Pharmaceutical Press, 22nd Edition (September 15, 2012).

[0102] Typical pharmaceutical compositions and dosage forms include one or more excipients. Suitable excipients are well known to those skilled in the art of pharmacy, and in certain embodiments, suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talcum powder, sodium chloride, skim milk powder, glycerine, propylene, ethylene glycol, water, ethanol, etc. Whether a particular excipient is suitable for incorporating into a pharmaceutical composition or dosage form depends on multiple factors well known in the art, including but not limited to the mode in which the dosage form is applied to the subject and the specific active ingredient in the dosage form. If desired, compositions or single unit dosage forms may also include a small amount of wetting agent or emulsifier, or pH buffer.

[0103] The lactose-free compositions provided by the present invention may include excipients well known in the art, and in certain embodiments, are listed in the U.S. Pharmacopoeia (USP 36-NF 31S2). Typically, the lactose-free compositions include pharmaceutically compatible and pharmaceutically acceptable amounts of active ingredients, binders / fillers, and lubricants. Exemplary lactose-free dosage forms include active ingredients, microcrystalline cellulose, pregelatinized starch, and magnesium stearate.

[0104] Further encompassed by the present invention are anhydrous pharmaceutical compositions and dosage forms comprising active ingredients, since water can promote the degradation of some compounds. For example, the addition of water (e.g., 5%) is widely accepted in the pharmaceutical art as a means of simulating long-term storage to determine properties such as shelf life or the stability of a formulation over time. See, for example, Jens T. Carstensen, Drug Stability: Principles & Practice, 2d, Marcel Dekker, ed., New York, 1995, pp. 379 80. Water and heat can effectively accelerate the decomposition of certain compounds. Therefore, the effect of water on a formulation may be of great significance, since moisture and / or humidity are typically encountered during the manufacture, handling, packaging, storage, transportation, and use of a formulation.

[0105] Anhydrous pharmaceutical compositions and dosage forms provided herein can be prepared using anhydrous or low moisture containing ingredients and low moisture or low humidity conditions. Pharmaceutical compositions and dosage forms comprising lactose and at least one active ingredient comprising a primary or secondary amine can be anhydrous if substantial contact with moisture and / or humidity is expected during manufacturing, packaging, and / or storage.

[0106] Anhydrous pharmaceutical compositions should be prepared and stored to maintain their anhydrous nature. Therefore, anhydrous compositions can be packaged using known materials that prevent exposure to water so that they can be included in suitable prescription kits. In certain embodiments, suitable packaging includes, but is not limited to, sealed foils, plastics, unit dose containers (e.g., vials), blister packs, and strip packs.

[0107] The present invention also provides pharmaceutical compositions and dosage forms comprising one or more compounds that reduce the rate of decomposition of the active ingredient. Such compounds are referred to herein as "stabilizers" and include, but are not limited to, antioxidants such as ascorbic acid, pH buffers, or salt buffers.

[0108] Pharmaceutical compositions and single unit dosage forms can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release agents, etc. Oral formulations can include standard carriers, such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. These compositions and dosage forms will contain a preventive or therapeutically effective amount of a preventive or therapeutic agent, and in certain embodiments, the preventive or therapeutic agent is in a purified form, together with an appropriate amount of a carrier, to provide a form suitable for administration to a subject. The preparation should be suitable for the mode of administration. In certain embodiments, the pharmaceutical composition or single unit dosage form is sterile and in a form suitable for administration to a subject, and in certain embodiments, the subject is an animal subject, such as a mammalian subject, and in certain embodiments, the subject is a human subject.

[0109] The pharmaceutical composition is formulated to be compatible with its expected route of administration. In certain embodiments, the route of administration includes but is not limited to parenteral, such as intravenous, intradermal, subcutaneous, intramuscular, subcutaneous, oral, oral, sublingual, inhalation, intranasal, transdermal, topical, transmucosal, intratumoral, intrasynovial and rectal administration. In certain embodiments, the route of administration is intradermal, topical or intralesional administration. In certain embodiments, the route of administration is non-systemic administration. In a specific embodiment, according to conventional procedures, the composition is formulated into a pharmaceutical composition suitable for intravenous, subcutaneous, intramuscular, oral, intranasal or topical administration to the human race. In one embodiment, the pharmaceutical composition is prepared according to the conventional procedures for subcutaneous administration to people. Usually, the composition for intravenous administration is a solution in a sterile isotonic aqueous buffer system. If necessary, the composition can also include a solubilizing agent and a local anesthetic, such as lidocaine, to alleviate the pain at the injection site.

[0110] In certain embodiments, dosage forms include, but are not limited to, tablets; caplets; capsules, such as soft elastic gelatin capsules; cachets; lozenges; lozenges; dispersions; suppositories; ointments; cataplasms (gel ointments); pastes; powders; dressings; emulsions; plasters; solutions; patches; aerosols (e.g., nasal sprays or inhalers); gels; liquid dosage forms suitable for oral or mucosal administration to a subject, including suspensions (e.g., aqueous or non-aqueous liquid suspensions, oil-in-water emulsions, or water-in-oil liquid emulsions), solutions, and elixirs; liquid dosage forms suitable for parenteral administration to a subject; and sterile solids (e.g., crystalline or amorphous solids) that can be reconstituted to provide a liquid dosage form suitable for parenteral administration to a subject.

[0111] The composition, shape and type of dosage forms provided herein will generally vary according to their use. In certain embodiments, the dosage form for the initial treatment of genetic skin diseases may include a larger amount of one or more active ingredients contained therein than the dosage form for maintaining the treatment of the same condition or disease. Similarly, compared with the oral dosage form for the treatment of the same disease or condition, the parenteral dosage form may include a smaller amount of one or more active ingredients contained therein. For those skilled in the art, these and other ways in which the specific dosage forms covered herein are different from each other will be apparent. See, for example, Remington: The Science and Practice of Pharmacy, Pharmaceutical Press, 22nd edition (September 15, 2012).

[0112] Typically, the components of the composition are provided separately or mixed together in unit dosage form, and in certain embodiments, are contained in a sealed container, such as an ampoule or a sachet, in the form of a dry lyophilized powder or anhydrous concentrate to indicate the amount of active ingredient. When the composition is administered by infusion, it can be dispensed with an infusion bottle filled with sterile pharmaceutical grade water or saline. In the case of administering the composition by injection, an ampoule of sterile water for injection or saline can be provided so that the components can be mixed before administration.

[0113] Typical dosage forms include a compound provided herein, or a pharmaceutically acceptable complex, solvate or hydrate thereof, in the range of about 0.1 mg to about 1000 mg per day, taken as a single dose once a day in the morning or divided throughout the day with meals. Specific dosage forms can have about 0.1, 0.2, 0.3, 0.4, 0.5, 1.0, 2.0, 2.5, 5.0, 10.0, 15.0, 20.0, 25.0, 50.0, 100, 200, 250, 500 or 1000 mg of active compound.

[0114] Oral dosage form

[0115] Pharmaceutical compositions suitable for oral administration can be in discrete dosage forms, such as, but not limited to, tablets (e.g., chewable tablets), caplets, capsules, and liquids (e.g., flavored syrups). Such dosage forms contain a predetermined amount of active ingredient and can be prepared by pharmaceutical methods well known to those skilled in the art. See generally, Remington: The Science and Practice of Pharmacy, Pharmaceutical Press, 22nd edition (September 15, 2012).

[0116] In certain embodiments, the oral dosage form is solid and is prepared with anhydrous ingredients under anhydrous conditions, as described herein. However, the scope of the compositions provided herein extends beyond anhydrous solid oral dosage forms. Therefore, other forms are further described herein.

[0117] By according to conventional drug mixing technology, typical oral dosage form is to prepare by closely mixing active ingredient with at least one excipient.Depending on the formulation form required for administration, excipient can adopt various forms.In some embodiments, the excipient suitable for oral liquid or aerosol dosage form includes but is not limited to water, ethylene glycol, oil, alcohol, flavoring agent, preservative and coloring agent.In some embodiments, the excipient suitable for solid oral dosage form (for example, powder, tablet, capsule and caplet) includes but is not limited to starch, sugar, microcrystalline cellulose, diluent, granulating agent, lubricant, binder and disintegrant.

[0118] Tablets and capsules represent the most advantageous oral dosage unit forms due to ease of administration, in which case solid excipients are used. Tablets may be coated, if desired, by standard aqueous or nonaqueous techniques. Such dosage forms may be prepared by any pharmaceutical method. In general, pharmaceutical compositions and dosage forms are prepared by uniformly and intimately mixing the active ingredient with a liquid carrier, a finely divided solid carrier, or both, and then, if desired, shaping the product into the desired presentation.

[0119] In certain embodiments, tablets can be prepared by compression or molding. Compressed tablets can be prepared by compressing the active ingredient in a free-flowing form such as a powder or granules in a suitable machine, optionally mixed with an excipient. Molded tablets can be prepared by molding a mixture of a powdered compound moistened with an inert liquid diluent in a suitable machine.

[0120] In certain embodiments, excipients that can be used for oral dosage forms include, but are not limited to, binders, fillers, disintegrants, and lubricants. Binders suitable for pharmaceutical compositions and dosage forms include, but are not limited to, corn starch, potato starch or other starches, gelatin, natural and synthetic gums such as gum arabic, sodium alginate, alginic acid, other alginates, guar gum, cellulose and its derivatives (e.g., ethyl cellulose, cellulose acetate, carboxymethyl cellulose calcium, sodium carboxymethyl cellulose), polyvinyl pyrrolidone, methyl cellulose, pregelatinized starch, hydroxypropyl methyl cellulose (e.g., Nos. 2208, 2906, 2910), microcrystalline cellulose and mixtures thereof.

[0121] In certain embodiments, fillers suitable for use in pharmaceutical compositions and dosage forms disclosed herein include, but are not limited to, talc, calcium carbonate (e.g., granules or powder), microcrystalline cellulose, powdered cellulose, dextran, kaolin, mannitol, silicate acid, sorbitol, starch, pregelatinized starch, and mixtures thereof. The binder or filler in the pharmaceutical composition is typically present in about 50 to about 99% by weight of the pharmaceutical composition or dosage form.

[0122] In certain embodiments, suitable forms of microcrystalline cellulose include, but are not limited to, materials sold under the trade names AVICEL PH 101, AVICEL PH 103, AVICEL RC 581, AVICEL PH 105 (available from FMC Corporation, American Viscose Division, Avicel Sales, Marcus Hook, Pennsylvania), and mixtures thereof. A particular binder is a mixture of microcrystalline cellulose and sodium carboxymethylcellulose, sold under the trade name AVICEL RC581. Suitable anhydrous or low moisture excipients or additives include AVICEL PH 103, AVICEL RC 581, AVICEL PH 105, and mixtures thereof. TM and Starch1500LM.

[0123] Disintegrants are used in compositions to provide tablets that disintegrate when exposed to an aqueous environment. Tablets with too much disintegrant may disintegrate during storage, while tablets with too little disintegrant may not disintegrate at the desired rate or under the desired conditions. Therefore, a sufficient amount of disintegrant should be used to form a solid oral dosage form, which is neither too much nor too little to deleteriously alter the release of the active ingredient. The amount of disintegrant used varies depending on the type of formulation and is easily determined by one of ordinary skill in the art. Typical pharmaceutical compositions contain about 0.5 to about 15% by weight of disintegrant, particularly about 1 to about 5% by weight of disintegrant.

[0124] Disintegrants that can be used in pharmaceutical compositions and dosage forms include, but are not limited to, agar, alginic acid, calcium carbonate, microcrystalline cellulose, croscarmellose sodium, crospovidone, polacrilin potassium, sodium starch glycolate, potato or tapioca starch, pregelatinized starch, other starches, clays, other algae, other celluloses, gums, and mixtures thereof.

[0125] Lubricants that can be used for pharmaceutical compositions and dosage forms include, but are not limited to, calcium stearate, magnesium stearate, mineral oil, light mineral oil, glycerol, sorbitol, mannitol, polyethylene glycol, other glycols, stearic acid, sodium lauryl sulfate, talc, hydrogenated vegetable oils (e.g., peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil, and soybean oil), zinc stearate, ethyl oleate, ethyl laurate, agar, and mixtures thereof. In certain embodiments, additional lubricants include syloid silica gel (AEROSIL 200, manufactured by WR Grace, Baltimore, Maryland), condensed aerosols of synthetic silica (sold by Degussa, Plano, Texas), CAB O SIL (pyrogenic silica products sold by Cabot, Boston, Massachusetts) and mixtures thereof. If a lubricant is used, the amount of the lubricant is typically less than about 1% by weight of the pharmaceutical composition or dosage form into which it is incorporated.

[0126] Delayed release dosage form

[0127] Active ingredients such as compounds provided herein can be administered by controlled release or by delivery devices well known to those of ordinary skill in the art. In certain embodiments, including but not limited to, U.S. Patent Nos.: 3,845,770; 3,916,899; 3,536,809; 3,598,123; 4,008,719; 5,674,533; 5,059,595; 5,591,767; 5,120,548; 5,073,543; 5,639,476; 5,354,556; 5,639,480; 5,733,566; 5,739,108; 5,891,474; 5, 922,356; 5,972,891; 5,980,945; 5,993,855; 6,045,830; 6,087,324; 6,113,943; 6,197,350; 6,248,363; 6,264,970; 6,267,981; 6,376,461; 6,419,961; 6,589,548; 6,613,358; and 6,699,500, each of which is incorporated herein by reference in its entirety. Such dosage forms can be used to provide slow or controlled release of one or more active ingredients, and in certain embodiments, hydroxypropyl methylcellulose, other polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, liposomes, microspheres, or combinations thereof are used to provide desired release profiles in varying proportions. Suitable controlled-release formulations known to those of ordinary skill in the art, including those described herein, can be readily selected for use with the active ingredients provided herein. Thus, single unit dosage forms suitable for oral administration, such as, but not limited to, tablets, capsules, gelatin capsules, and caplets suitable for controlled release are contemplated herein.

[0128] All controlled-release pharmaceutical products have a common goal, namely to improve the therapeutic effect of a drug compared to a non-controlled-release drug. Ideally, the use of an optimally designed controlled-release formulation in a medical treatment is characterized by using the least amount of drug in the shortest amount of time to cure or control a disease or condition. The advantages of controlled-release formulations include prolonged drug activity, reduced dosing frequency, and increased subject compliance. In addition, controlled-release formulations can be used to affect the onset time or other characteristics, such as the blood level of the drug, thereby affecting the occurrence of side effects (e.g., adverse reactions).

[0129] Most controlled-release formulations are designed to release a certain amount of drug (active ingredient) to quickly produce the desired therapeutic effect, and then gradually and continuously release the remaining amount of drug to maintain this level of therapeutic or preventive effect for a longer period of time. In order to maintain this constant level of drug in the body, the drug must be released from the dosage form at a certain rate that will replace the amount of drug metabolized and excreted from the body. The controlled release of the active ingredient can be stimulated by various diseases or conditions, including but not limited to pH, temperature, enzymes, water or other physiological diseases or conditions or compounds.

[0130] In some embodiments, intravenous infusion, implantable osmotic pumps, transdermal patches, liposomes or other modes of administration can be used to administer drugs. In some embodiments, pumps can be used (see, Sefton, CRC Crit. Ref Biomed Eng. 14: 201 (1987); Buchwald et al., Surgery 88: 507 (1980); Saudek et al., New England Journal of Medicine 321: 574 (1989)). In another embodiment, polymeric materials can be used. In another embodiment, a controlled release system can be placed in a suitable location determined by a person skilled in the art in the subject, i.e., only a portion of the systemic dose is required (see, e.g., Goodson, Medical Applications of Controlled Release, Vol. 2, pp. 115-138 (1984)). Other controlled release systems are discussed in Langer's review (Science 249: 1527-1533 (1990)). The active ingredient can be dispersed in a solid inner matrix that is insoluble in body fluids, such as polymethyl methacrylate, polybutyl methacrylate, plasticized or unplasticized polyvinyl chloride, plasticized nylon, plasticized polyethylene terephthalate, natural rubber, polyisoprene, polyisobutylene, polybutadiene, polyethylene, ethylene-vinyl acetate copolymers, silicone rubber, polydimethylsiloxane, organic silicon carbonate copolymers, hydrophilic polymers such as hydrogels of acrylic acid and methacrylate, collagen, cross-linked polyvinyl alcohol and cross-linked partially hydrolyzed polyvinyl acetate. The solid matrix is The outer polymer film that is insoluble in body fluids surrounds, for example, polyethylene, polypropylene, ethylene / propylene copolymer, ethylene / ethyl acrylate copolymer, ethylene / vinyl acetate copolymer, silicone rubber, polydimethylsiloxane, chloroprene rubber, chlorinated polyethylene, polyvinyl chloride, vinyl chloride and vinyl acetate copolymer, vinylidene chloride, the copolymer of ethylene and propylene, ionomer polyethylene terephthalate, butyl rubber epichlorohydrin rubber, ethylene / vinyl alcohol copolymer, ethylene / vinyl acetate / vinyl alcohol terpolymer and ethylene / ethyleneoxyethanol copolymer. The active ingredient then diffuses through the outer polymer film in the release rate controlling step. The percentage height of the active ingredient in this parenteral composition depends on its specific properties and the needs of the experimenter.

[0131] Parenteral Dosage Forms

[0132] In certain embodiments, provided herein is a parenteral dosage form. In certain embodiments, parenteral dosage forms can be applied to a subject by various approaches, including but not limited to subcutaneous, intravenous (including push injection), intramuscular and intra-arterial. In certain embodiments, parenteral dosage forms can be applied to a subject by various approaches, including but not limited to topical, intradermal or intralesional. Because it is used to bypass the natural defense of the subject to pollutants, parenteral dosage forms are usually sterile or can be sterilized before being applied to the subject. In certain embodiments, parenteral dosage forms include but are not limited to solutions ready for injection, dry products ready to be dissolved or suspended in a pharmaceutically acceptable carrier for injection, suspensions ready for injection and emulsions.

[0133] Suitable carriers that can be used to provide parenteral dosage forms are well known to those skilled in the art. In certain embodiments, suitable carriers include, but are not limited to, Water for Injection USP; aqueous carriers such as, but not limited to, Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, and Lactated Ringer's Injection; water-miscible carriers such as, but not limited to, ethanol, polyethylene glycol, and polypropylene glycol; and non-aqueous carriers such as, but not limited to, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate.

[0134] Compounds that increase the solubility of one or more of the active ingredients disclosed herein can also be incorporated into parenteral dosage forms.

[0135] Transdermal, topical and mucosal dosage forms

[0136] The present invention also provides transdermal, topical and mucosal dosage forms. Transdermal, topical and mucosal dosage forms include but are not limited to ophthalmic solutions, sprays, aerosols, creams, lotions, ointments, gels, solutions, emulsions, suspensions or other forms known to those skilled in the art. See, for example, Remington: The Science and Practice of Pharmacy, Pharmaceutical Press, 22nd edition (September 15, 2012); and Introduction to Pharmaceutical Dosage Forms, 4th edition, Lea and Febiger, Philadelphia (1985). Dosage forms suitable for treating oral mucosal tissues can be formulated into mouthwashes or oral gels. In addition, transdermal dosage forms include "reservoir-type" or "matrix-type" patches, which can be applied to the skin and worn for a specific length of time to allow the desired amount of active ingredients to penetrate.

[0137] The term "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition or carrier, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, for carrying or transporting any subject composition or its components. Each carrier must be "acceptable" in the sense that it is compatible with the subject composition and its components and harmless to the patient. Suitable carriers (such as excipients and diluents) and other materials that can be used to provide transdermal, topical and mucosal dosage forms covered herein are well known to those skilled in the pharmaceutical field and depend on the specific tissue to which a given pharmaceutical composition or dosage form will be applied. In view of this fact, typical carriers include, but are not limited to, water, acetone, ethanol, ethylene glycol, propylene glycol, polyethylene glycol, 1,3-butylene glycol, isopropyl myristate, isopropyl palmitate, mineral oil, and mixtures thereof to form lotions, tinctures, creams, emulsions, gels or ointments, which are non-toxic and pharmaceutically acceptable. In some embodiments, materials that can be used as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; (4) tragacanth powder; (5) malt; (6) gelatin; (7) talc; (8) excipients such as cocoa butter and suppository wax; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn starch, etc. Oil, soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerol, sorbitol, mannitol, polyethylene glycol, etc.; (12) esters, such as ethyl oleate, ethyl laurate; (13) agar; (14) buffers, such as magnesium hydroxide, aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethanol; (20) phosphate buffer solution; (21) other non-toxic compatible substances used in pharmaceutical preparations. If desired, humectants or moisturizers can also be added to the pharmaceutical compositions and dosage forms. Examples of such additional ingredients are well known in the art. See, for example, Remington: The Science and Practice of Pharmacy, Pharmaceutical Press, 22nd edition (September 15, 2012)

[0138] Depending on the specific tissue to be treated, additional components may be used before, simultaneously with, or after treatment with the active ingredient provided. In certain embodiments, a penetration enhancer may be used to help deliver the active ingredient to the tissue. Suitable penetration enhancers include, but are not limited to, acetone; various alcohols such as ethanol, oleyl alcohol, tetrahydrofuran; alkyl sulfoxides such as dimethyl sulfoxide; dimethylacetamide; dimethylformamide; polyethylene glycol; pyrrolidones such as polyvinylpyrrolidone; polyvinylpyrrolidone (povidone); urea; and various water-soluble or insoluble sugar esters such as Tween 80 (polysorbate 80) and Span 60 (sorbitol monostearate).

[0139] The pH value of pharmaceutical composition or dosage form can also be regulated, or the pH value of the tissue of pharmaceutical composition or dosage form is used, to improve the sending of one or more active components. Similarly, the polarity, ionic strength or tension of the solvent carrier can be regulated to improve sending. Compounds such as stearate can also be added in pharmaceutical composition or dosage form, to advantageously change the hydrophilicity or lipophilicity of one or more active components, thereby improve sending. In this respect, stearate can be used as lipid carrier, emulsifier or surfactant and delivery enhancer or penetration enhancer of preparation. Different complexes, salts, hydrates or solvates of active component can be used for further regulating the property of the resulting composition.

[0140] Dosage and unit dosage form

[0141] In human treatment, the physician will determine the dosage he deems most appropriate, depending on the prophylactic or curative treatment and on the age, weight, stage of the disease or disease, and other factors specific to the subject to be treated. In certain embodiments, the dosage is from about 1 to about 1000 mg per day for an adult, or from about 5 to about 250 mg per day for an adult, or from about 10 to 50 mg per day. In certain embodiments, the dosage is from about 5 to about 400 mg per day for each adult, or from 25 to 200 mg per day. In certain embodiments, dosage rates of about 50 to about 500 mg per day are also contemplated.

[0142] On the other hand, provide by applying the compound provided by the invention of treatment or prevention effective amount to the subject in need, or its pharmaceutically acceptable complex, treat the disease or illness that the subject needs to treat and / or the method for the hereditary skin disease of the subject.In treating obstacle or its one or more symptoms, the amount of effective compound or composition in treatment or prevention will change with the nature and severity of the disease or illness and the approach of applying active ingredient.Frequency and dosage will also change according to the specific factors of each subject, specifically depending on the specific therapy (for example, therapeutic agent or preventive agent) applied, illness, the severity of disease or illness, route of administration and age, health, body weight, reaction and previous medical history of the subject.Effective dose can be extrapolated from the dose-response curve from in vitro or animal model test system.

[0143] In certain embodiments, exemplary dosages of compositions include milligrams or micrograms of active compound per kilogram of subject body weight or sample weight (e.g., about 10 micrograms / kg to about 50 milligrams / kg, about 100 micrograms / kg to about 25 milligrams / kg, or about 100 micrograms / kg to about 10 milligrams / kg). For compositions provided herein, in certain embodiments, the dosage administered to a subject is, based on the weight of the active compound, 0.140 mg / kg to 3 mg / kg subject body weight. In certain embodiments, the dose administered to a subject is 0.20 mg / kg to 2.00 mg / kg, 0.30 mg / kg to 1.50 mg / kg, 1 mg / kg to 100 mg / kg, 5 mg / kg to 50 mg / kg, 10 mg / kg to 50 mg / kg, 20 mg / kg to 50 mg / kg, 15 mg / kg to 40 mg / kg, 15 mg / kg to 35 mg / kg, 15 mg / kg to 30 mg / kg, 25 mg / kg to 35 mg / kg, 10 mg / kg to 30 mg / kg, 10 mg / kg to 20 mg / kg, about 5 mg / kg, about 10 mg / kg, about 15 mg / kg, about 20 mg / kg, about 25 mg / kg, about 30 mg / kg, about 35 mg / kg, about 40 mg / kg, about 45 mg / kg, or about 50 mg / kg of the subject's body weight.

[0144] In certain embodiments, the recommended daily dose range of the compositions provided herein for the diseases or conditions described herein is in the range of about 0.1 mg to about 1000 mg / day, as a single dose once a day or divided into doses within a day. In certain embodiments, the daily dose is administered twice a day in equally divided doses. In certain embodiments, the daily dose range should be about 10 mg to about 200 mg per day, in other embodiments, about 10 mg to about 150 mg per day, and in further embodiments, about 25 to about 100 mg per day. In some cases, it may be necessary to use a dose of the active ingredient outside the disclosed range herein, which is obvious to those of ordinary skill in the art. In addition, it is noted that the clinician or treating physician will know how and when to interrupt, adjust or terminate treatment according to the subject's response.

[0145] As is readily known to those of ordinary skill in the art, different therapeutically effective amounts may be applicable to different diseases and conditions. Similarly, amounts sufficient to prevent, control, treat or improve such conditions but insufficient to cause or sufficient to reduce the side effects associated with the compositions provided herein are also included in the dosage and dosing frequency regimens described herein. In addition, when multiple doses of the compositions provided herein are administered to a subject, not all doses need to be the same. In certain embodiments, the dose administered to the subject may be increased to increase the preventive or therapeutic effect of the composition, or the dose may be reduced to reduce one or more side effects that a particular subject is experiencing.

[0146] In certain embodiments, the dose of the compositions provided herein administered to prevent, treat, manage, or improve a subject's disorder or one or more symptoms thereof is 0.1 mg / kg, 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 10 mg / kg, or 15 mg / kg of subject body weight or more, based on the weight of the active compound. In another embodiment, the dosage of the compositions provided herein administered to prevent, treat, control or improve a disorder or one or more symptoms thereof in a subject is, based on the weight of the active compound, a unit dose of 0.1 mg to 200 mg, 0.1 mg to 100 mg, 0.1 mg to 50 mg, 0.1 mg to 25 mg, 0.1 mg to 20 mg, 0.1 mg to 15 mg, 0.1 mg to 10 mg, 0.1 mg to 7.5 mg, 0.1 mg to 5 mg, 0.1 to 2.5 mg, 0.25 mg to 20 mg, 0.25 to 15 mg, 0.25 to 12 mg, 0.25 to 10 mg, 0.25 mg to 7.5 mg, 0.25 mg to 5 mg, 0.5 mg to 2.5 mg, 1 mg to 20 mg, 1 mg to 15 mg, 1 mg to 12 mg, 1 mg to 10 mg, 1 mg to 7.5 mg, 1 mg to 5 mg, or 1 mg to 2.5 mg.

[0147] In certain embodiments, treatment or prevention can be started with a loading dose of one or more compounds or compositions provided herein, followed by one or more maintenance doses. In such embodiments, the loading dose can be, for example, about 60 to about 400 mg per day, or about 100 to about 200 mg per day, for 1 day to 5 weeks. The loading dose can be followed by one or more maintenance doses. In certain embodiments, each maintenance dose is independently about 10 mg to about 200 mg per day, about 25 mg to about 150 mg per day, or about 25 mg to about 80 mg per day. The maintenance dose can be administered daily, or a single dose, or divided doses.

[0148] In some embodiments, a certain dose of compound or composition provided herein can be applied to reach the steady-state concentration of active ingredient in the blood or serum of the subject. Steady-state concentration can be determined by measurement according to the available technology of those skilled in the art, or can be based on the physical characteristics of the subject, such as height, weight and age. In some embodiments, a sufficient amount of compound or composition provided herein is applied to reach the steady-state concentration of about 300 to about 4000ng / mL, about 400 to about 1600ng / mL, about 600 to about 1200ng / mL in the blood or serum of the subject. In some embodiments, a sufficient amount of compound or composition provided herein is applied to reach the steady-state concentration of about 0.01 to about 300ng / mL, about 0.01 to about 100ng / mL, about 0.01 to about 10ng / mL, about 0.01 to about 1ng / mL, about 0.01 to about 0.1ng / mL or about 0.01 to about 0.05ng / mL in the blood or serum of the subject. In some embodiments, a loading dose may be administered to achieve a steady-state blood or serum concentration of about 1200 to about 8000 ng / mL or about 2000 to about 4000 ng / mL for 1 to 5 days. In some embodiments, a loading dose may be administered topically to achieve a steady-state blood or serum concentration of about 0.05 to about 1200 ng / mL, about 0.05 to about 100 ng / mL, about 0.05 to about 10 ng / mL, about 0.05 to about 1 ng / mL, about 0.05 to about 0.5 ng / mL, or about 0.05 to about 0.1 ng / mL for 1 to 5 days. In certain embodiments, a maintenance dose may be administered to achieve a concentration of about 300 to about 4000 ng / mL, about 400 to about 1600 ng / mL, or about 600 to about 1200 ng / mL in the blood or serum of a subject. In certain embodiments, a maintenance dose may be administered topically to achieve a steady-state concentration in the subject's blood or serum of about 0.01 to about 300 ng / mL, about 0.01 to about 100 ng / mL, about 0.01 to about 10 ng / mL, about 0.01 to about 1 ng / mL, about 0.01 to about 0.1 ng / mL, or about 0.01 to about 0.05 ng / mL.

[0149] In certain embodiments, the same composition can be administered repeatedly and the administrations can be separated by at least 1 day, 2 days, 3 days, 5 days, 10 days, 15 days, 30 days, 45 days, 2 months, 75 days, 3 months, or 6 months. In other embodiments, the same prophylactic or therapeutic agent can be administered repeatedly and the administrations can be separated by at least 1 day, 2 days, 3 days, 5 days, 10 days, 15 days, 30 days, 45 days, 2 months, 75 days, 3 months, or 6 months.

[0150] In certain aspects of the invention, provided herein is a unit dose comprising a compound or a pharmaceutically acceptable complex thereof in a form suitable for administration. For any compound, the therapeutically effective dose can be estimated initially from cell culture assays. A dose can be formulated in an animal model to achieve an IC in skin with lesions (e.g., Netherton syndrome or a skin disease mediated by a KLK protease) including those determined in cell culture. 50 The level of the test compound (i.e., the concentration that achieves half-maximal inhibition of symptoms) can be determined. Such information can be used to more accurately determine useful doses in humans. In addition, compound levels in plasma can be measured, for example, by high performance liquid chromatography, to determine systemic exposure.

[0151] It should also be understood that the specific dosage and treatment regimen for any particular patient will depend on a variety of factors, including the activity of the specific compound used, age, lesion size, number of lesions, general health, sex, diet, time of administration, drug combination, as well as the judgment of the treating physician and the severity of the specific disease being treated. The amount of the compound of formula (I) described herein in the composition will also depend on the specific compound of formula (I) in the composition.

[0152] In some embodiments, the local dose is about 0.01 μg / cm 2 , about 0.05μg / cm 2 , about 0.1μg / cm 2 , about 0.15μg / cm 2 , about 0.2μg / cm 2 , about 0.3f.lg / cm2, about 0.4f.lg / cm2, about 0.5μg / cm 2 , about 0.6μg / cm 2 , about 0.7μg / cm 2 , about 0.8μg / cm 2 , or about 0.9μg / cm 2 ; or about 0.01-0.03 μg / cm 2 , about 0.03-0.05μg / cm 2 , about 0.05-0.1μg / cm 2 , about 0.1-0.3μg / cm 2 , about 0.3-0.5μg / cm 2 , about 0.5-0.8μg / cm 2 , about 0.8-1.0μg / cm 2 , about 1-10μg / cm 2 , about 10-20μg / cm 2 , about 20-30μg / cm 2 , about 30-40μg / cm 2, about 40-50μg / cm 2 , about 50-60μg / cm 2 , about 60-70μg / cm 2 , about 70-80μg / cm 2 , about 80-90μg / cm 2 , about 90-100 μg / cm 2 , about 100-125μg / cm 2 , about 125-150μg / cm 2 , about 150-175μg / cm 2 , about 175-200 μg / cm 2 , about 200-20250μg / cm 2 , about 250-300μg / cm 2 , about 300-350μg / cm 2 , about 350-400μg / cm 2 , about 400-450μg / cm 2 , about 450-500μg / cm 2 , about 500-550μg / cm 2 , about 550-600μg / cm 2 , about 600-650μg / cm 2 , about 650-700μg / cm 2 , about 700-750μg / cm 2 , about 750-800μg / cm 2 , about 800-850μg / cm 2 , about 850-900μg / cm 2 , about 900-950μg / cm 2 or about 950-1000 μg / cm 2 within the range.

[0153] In some embodiments, the topical dose is about 0.5-1.0 mg / cm 2 1.0-1.5mg / cm 2 1.5-2.0mg / cm 2 2.5-2.5mg / cm 2 3.0-3.5mg / cm 2 , 3.5-5.0mg / cm 2 , 5.0-7.5mg / cm 2 , 7.5-10mg / cm 2 , 1-10mg / cm 2 , about 10-20mg / cm 2, about 20-30mg / cm 2 , about 30-40mg / cm 2 , about 40-50mg / cm 2 , about 50-60mg / cm 2 , about 60-70mg / cm 2 , about 70-80mg / cm 2 , about 80-90mg / cm 2 , about 90-100mg / cm 2 , about 100-125mg / cm 2 , about 125-150mg / cm 2 , about 150-175mg / cm 2 , about 175-200mg / cm 2 , about 200-250mg / cm 2 , about 250-300mg / cm 2 , about 300-350mg / cm 2 , about 350-400mg / cm 2 , about 400-450mg / cm 2 , about 450-500mg / cm 2 , about 500-550mg / cm 2 , about 550-600mg / cm 2 , about 600-650mg / cm 2 , about 650-700mg / cm 2 , about 700-750mg / cm 2 , about 750-800mg / cm 2 , about 800-850mg / cm 2 , about 850-900mg / cm 2 , about 900-950mg / cm 2 or about 950-1000mg / cm 2 .

[0154] V. Methods

[0155] In a third aspect of the present invention, a method for treating a skin disease in a subject in need thereof associated with the proteolytic activity of one or more KLK proteases is provided. The method comprises administering to the subject a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable complex thereof, or a pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable complex thereof.

[0156] In some embodiments, the compound of formula (I) is a compound represented by the following formula or a pharmaceutically acceptable complex thereof:

[0157]

[0158] In some embodiments, the compound of formula (I) is a compound represented by the following formula or a pharmaceutically acceptable complex thereof:

[0159]

[0160] Pharmaceutically acceptable complexes of the compounds of formula (I) are as defined and described herein. Pharmaceutically acceptable complexes of the compounds of formula (I) may include pharmaceutically acceptable salts and / or solvates thereof.

[0161] In some embodiments, the skin disease is a genetic skin disease. In some embodiments, the genetic skin disease is Netherton syndrome.

[0162] In some embodiments, the subject has a loss-of-function mutation of a truncated serine protease inhibitor Kazal type 5 (SPINK5). In some embodiments, the subject has a loss of function of LEKTI (lymphoepithelial Kazal-type inhibitor). In some embodiments, the subject has an overexpression of one or more kallikrein (KLK) serine proteases. In some embodiments, the subject has an overexpression of one or more KLK5, KLK7, and KLK14 proteases. In some embodiments, the subject has an overexpression of a KLK5 protease. In some embodiments, the subject has an overexpression of a KLK7 protease. In some embodiments, the subject has an overexpression of a KLK14 protease. In some embodiments, the subject has an overexpression of a KLK5 and KLK7 proteases. In some embodiments, the subject has an overexpression of a KLK5, KLK7, and KLK14 protease. In some embodiments, the subject has an elevated proteolytic activity of one or more kallikrein (KLK) serine proteases. In some embodiments, the subject has elevated proteolytic activity of one or more KLK5, KLK7, and KLK14 proteases. In some embodiments, the subject has elevated proteolytic activity of a KLK5 protease. In some embodiments, the subject has elevated proteolytic activity of a KLK7 protease. In some embodiments, the subject has elevated proteolytic activity of a KLK14 protease. In some embodiments, the subject has elevated proteolytic activity of a KLK5 and KLK7 proteases. In some embodiments, the subject has elevated proteolytic activity of a KLK5, KLK7, and KLK14 protease.

[0163] In some embodiments, the present invention provides a method for treating Netherton syndrome in a subject in need thereof. The method comprises administering to the subject a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable complex thereof, or a pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable complex thereof.

[0164] In some embodiments, provided herein is a method for treating a hereditary skin disease, wherein the subject is in need and the hereditary skin disease is mediated by one or more KLK proteases. The method comprises administering to the subject a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable complex thereof, or a pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable complex thereof. In some embodiments, the hereditary skin disease is mediated by a KLK5 protease, a KLK7 protease, a KLK14 protease, or a combination thereof. In some embodiments, the hereditary skin disease is mediated by a KLK5 protease. In some embodiments, the hereditary skin disease is mediated by a KLK7 protease. In some embodiments, the hereditary skin disease is mediated by a KLK14 protease. In some embodiments, the hereditary skin disease is mediated by both KLK5 and KLK7 proteases. In some embodiments, the hereditary skin disease is mediated by KLK5, KLK7, and KLK14 proteases. In some embodiments, the hereditary skin disease is Netherton syndrome.

[0165] In some embodiments, a method for treating a subject's genetic skin disease by inhibiting the proteolytic activity of one or more KLK proteases is provided. The method comprises administering to the subject a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable complex thereof, or a pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable complex thereof. In some embodiments, the compound of formula (I) inhibits the proteolytic activity of one or more KLK proteases. In some embodiments, the compound of formula (I) inhibits the proteolytic activity of KLK5 protease, KLK7 protease, KLK14 protease, or a combination thereof. In some embodiments, the compound of formula (I) inhibits the proteolytic activity of KLK5 protease. In some embodiments, the compound of formula (I) inhibits the proteolytic activity of KLK7 protease. In some embodiments, the compound of formula (I) inhibits the proteolytic activity of KLK14 protease. In some embodiments, the compound of formula (I) inhibits the proteolytic activity of KLK5 and KLK7 proteases. In some embodiments, the compound of formula (I) inhibits the proteolytic activity of KLK5, KLK7, and KLK14 proteases. In some embodiments, the compound of formula (I) is a KLK5 inhibitor. In some embodiments, the compound of formula (I) is a KLK7 inhibitor. In some embodiments, the compound of formula (I) is a KLK14 inhibitor. In some embodiments, the compound of formula (I) is a dual KLK5 / KLK7 inhibitor. In some embodiments, the hereditary skin disease is Netherton syndrome.

[0166] In some embodiments, provided herein is a method for treating Netherton syndrome in a subject by inhibiting the proteolytic activity of one or more KLK proteases. The method comprises administering to the subject a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable complex thereof, or a pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable complex thereof. In some embodiments, the compound of formula (I) inhibits the proteolytic activity of one or more KLK proteases. In some embodiments, the compound of formula (I) inhibits the proteolytic activity of KLK5 protease, KLK7 protease, KLK14 protease, or a combination thereof. In some embodiments, the compound of formula (I) inhibits the proteolytic activity of KLK5 protease. In some embodiments, the compound of formula (I) inhibits the proteolytic activity of KLK7 protease. In some embodiments, the compound of formula (I) inhibits the proteolytic activity of KLK14 protease. In some embodiments, the compound of formula (I) inhibits the proteolytic activity of KLK5 and KLK7 proteases. In some embodiments, the compound of formula (I) inhibits the proteolytic activity of KLK5, KLK7, and KLK14 proteases. In some embodiments, the compound of formula (I) is a KLK5 inhibitor. In some embodiments, the compound of formula (I) is a KLK7 inhibitor. In some embodiments, the compound of formula (I) is a KLK14 inhibitor. In some embodiments, the compound of formula (I) is a dual KLK5 / KLK7 inhibitor.

[0167] The compounds of the present invention can be used to protect the skin by inhibiting the proteolytic activity of one or more KLK proteases and promoting healthy skin development. Therefore, on the other hand, there is provided herein a method for treating a skin disease, wherein the subject needs treatment and the skin disease is mediated by one or more KLK proteases. The method includes administering to the subject a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable complex thereof, or a pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable complex thereof. In some embodiments, the skin disease is mediated by a KLK5 protease, a KLK7 protease, a KLK14 protease, or a combination thereof. In some embodiments, the skin disease is mediated by a KLK5 protease. In some embodiments, the skin disease is mediated by a KLK7 protease. In some embodiments, the skin disease is mediated by a KLK14 protease. In some embodiments, the skin disease is mediated by both a KLK5 and a KLK7 protease. In some embodiments, the skin disease is mediated by a KLK5, a KLK7, and a KLK14 protease. In some embodiments, the skin disease is a KLK-mediated disease selected from Netherton syndrome, peeling skin syndrome, rosacea, psoriasis, eczema, and atopic dermatitis. In some embodiments, the KLK-mediated disease is Netherton syndrome.

[0168] In some embodiments, provided herein is a method for treating a subject's skin disease by inhibiting the proteolytic activity of one or more KLK proteases. The method comprises administering to the subject a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable complex thereof, or a pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable complex thereof. In some embodiments, the compound of formula (I) inhibits the proteolytic activity of one or more KLK proteases. In some embodiments, the compound of formula (I) inhibits the proteolytic activity of KLK5 protease, KLK7 protease, KLK14 protease, or a combination thereof. In some embodiments, the compound of formula (I) inhibits the proteolytic activity of KLK5 protease. In some embodiments, the compound of formula (I) inhibits the proteolytic activity of KLK7 protease. In some embodiments, the compound of formula (I) inhibits the proteolytic activity of KLK14 protease. In some embodiments, the compound of formula (I) inhibits the proteolytic activity of KLK5 and KLK7 proteases. In some embodiments, the compound of formula (I) inhibits the proteolytic activity of KLK5, KLK7, and KLK14 proteases. In some embodiments, the compound of formula (I) is a KLK5 inhibitor. In some embodiments, the compound of formula (I) is a KLK7 inhibitor. In some embodiments, the compound of formula (I) is a KLK14 inhibitor. In some embodiments, the compound of formula (I) is a dual KLK5 / KLK7 inhibitor. In some embodiments, the skin disease is selected from Netherton syndrome, peeling syndrome, rosacea, psoriasis, eczema, and atopic dermatitis. In some embodiments, the skin disease is Netherton syndrome.

[0169] The compound of formula (I) of the present invention or the pharmaceutical composition comprising the compound of formula (I) can be used for the preventive and therapeutic treatment of skin diseases or pathologies or other adverse skin conditions in subjects. For example, the composition of the present invention can be used to prevent skin irritation, prevent rash, promote the healing of skin tissue after rash or irritation occurs (i.e., build the epidermis and / or dermis of the skin), prevent and / or delay skin atrophy, prevent and / or delay the appearance of spider blood vessels and / or red spots on the skin, prevent and / or delay skin peeling, prevent and / or relieve skin itching, regulate skin texture (e.g., improve peeling, roughness, swelling and soreness) and improve skin color (e.g., reduce redness).

[0170] The preventive or curative treatment of a subject's genetic skin disease or skin disease can be carried out by applying a compound of formula (I) or a pharmaceutical composition thereof in the form of a lotion, cream, gel, foam, ointment, paste, emulsion, spray, conditioner, tonic, patch, etc., which is preferably left on the skin. After the composition is applied to the skin, it can be left on the skin for at least about 15 minutes, or at least about 30 minutes, or at least about 1 hour, or at least several hours, for example, at least about 2 hours, 3 hours, 6 hours, 12 hours or 24 hours.

[0171] The compounds of the present invention can also be used to regulate desquamation in vitro by inhibiting the protease activity of one or more KLK proteases during cell / tissue culture. Therefore, in another aspect, the present invention provides a method of decomposing cells or tissues in vitro, the method comprising exposing the cells or tissues to a KLK protease (e.g., KLK5, KLK7 and / or KLK14) and then to a compound of formula (I) of the present invention or a pharmaceutically acceptable complex thereof.

[0172] It should be understood that the method is generally applicable to cell culture procedures, where the method can be used to dissociate and separate cells for passage. In addition, the method can be used to culture artificial skin, where cell-cell adhesion proteins are selectively cleaved by KLK proteases (e.g., KLK5, KLK7 and / or KLK14), while such cleavage is regulated by the compound of formula (I) or a pharmaceutically acceptable complex thereof, making the cells / tissues more suitable for subsequent therapeutic use.

[0173] In another aspect, the present invention provides an in vitro assay for determining the proteolytic activity of one or more KLK proteases in the skin. The method comprises 1) preparing a skin extract; 2) exposing a substrate and a compound of formula (I) or a pharmaceutically acceptable complex thereof to the skin extract; 3) determining the proteolytic cleavage rate of the substrate. In some embodiments, the substrate is peptide-p-nitroaniline (also known as peptide-pNA). In some embodiments, the substrate is dipeptide-pNA, tripeptide-pNA, tetrapeptide-pNA, pentapeptide-pNA, or hexapeptide-pNA. In some embodiments, the substrate is tetrapeptide-pNA. In some embodiments, the substrate is Tyr-Arg-Ser-Arg-pNA or Lys-HisLeu-Tyr-pNA. In some embodiments, the substrate is Tyr-Arg-Ser-Arg-pNA. In some embodiments, the substrate is Lys-His-Leu-Tyr-pNA. The skin extract can be a human skin extract or an animal skin extract (e.g., a mouse). In some embodiments, the skin extract is a human skin extract.

[0174] VI. Examples

[0175] General Synthesis Methods

[0176] As used herein, the symbols and conventions used in the processes, schemes, and examples, whether or not particular abbreviations are specifically defined, are consistent with those used in the contemporary scientific literature, for example, the Journal of the American Chemical Society or the Journal of Biological Chemistry. Specifically, the following abbreviations, but not limited to them, may be used in the examples and throughout the specification: eq. (equivalent); g (gram); mg (milligram); mL (milliliter); μL (microliter); mM (millimoles per liter); μM (micromoles per liter); Hz (hertz); MHz (megahertz); mmol (millimoles); hr or hrs (hours); min (minutes); RT (room temperature); MS (mass spectrometry); ESI (electrospray ionization); TLC (thin layer chromatography); HPLC (high pressure liquid chromatography); THF (tetrahydrofuran); CDCl3 (deuterated chloroform); AcOH (acetic acid); DCM (dichloromethane); DMW (demineralized water); DME (dimethoxyethane); DMF (N,N dimethylformamide); DMSO (dimethyl sulfoxide); DMSO-d6 (deuterated dimethyl sulfoxide); EtOAc (ethyl acetate); and MeOH (methanol).

[0177] For all of the following examples, standard post-processing and purification methods known to those skilled in the art can be used. Unless otherwise stated, all temperatures are expressed in ° C (degrees Celsius). Unless otherwise stated, all reactions are carried out at room temperature. The synthetic methods described herein are intended to illustrate applicable chemistry by using specific examples and do not represent the scope of the present disclosure.

[0178] Example 1: Synthesis of 2-(4-(hydroxymethyl)-2-(methylsulfonyl)phenyl)-7,8-dihydro-4H[1,4]dioxin[2',3':4,5]benzo[1,2-d)[1,3]oxazin-4-one (Compound 1.001)

[0179]

[0180] The synthesis of compound 1.001 is as follows Figure 1 The synthesis of intermediate 100 is as shown. Figure 3 shown.

[0181] Step 1: 4-Bromo-2-mercaptobenzoic acid (Intermediate 102)

[0182]

[0183] To a solution of 2-amino-4-bromobenzoic acid (101) (200.0 g, 926.0 mmol, 1.0 eq), NaOH (37.0 g, 926.0 mmol, 1.0 eq) and sodium nitrite (63.90 g, 926.0 mmol, 1.0 eq) in DMW (4.0 L) was slowly added a mixture of concentrated HCl (280.0 mL) and ice water (280.0 mL) at a rate such that the reaction temperature was maintained at 3-6 °C. After addition, the reaction mixture was stirred at 0 °C for 30 min and then neutralized with potassium acetate (454.0 g, 4.63 mol, 5.0 eq). This solution was added to a solution of potassium O-ethylxanthate (445.0 g, 2.78 mol, 3.0 eq) in DMW (4.0 L) preheated to 90 °C. The reaction mixture was stirred at 90°C for 30 minutes, cooled to 0°C, and acidified with concentrated HCl (600.0mL). The reaction mixture was alkalized with 20% NaOH (1750ml) and heated to 85°C for 2 hours. NaHSO (96.30g, 926.0mmol, 1.0 equivalent) was added to the mixture in small portions, and the reaction mixture was heated to 85°C for 10 minutes. The reaction mixture was filtered, cooled to 0°C, and acidified with concentrated HCl (650.0mL). The precipitated solid was collected by filtration, washed with DMW (500mL), then washed with n-hexane, and air-dried to obtain intermediate 102 (216.0g, 100.0%) as a light grey solid. 1 HNMR (300MHz, DMSO-d6): δ7.88ppm (d, J=1.9Hz, 1H), 7.81ppm (t, J=7.4Hz, 1H), 7.38ppm (dd, J=8.4, 2.0Hz, 1H). MS (ESI): m / z 232.8 (M-1).

[0184] Step 2: Methyl 4-bromo-2-(methylthio)benzoate (Intermediate 103)

[0185]

[0186] To a solution of 4-bromo-2-mercaptobenzoic acid (102) (216.0 g, 927.0 mmol, 1.0 equiv) in DMF (3.24 L) was added potassium carbonate (896.0 g, 6.49 mol, 7.0 equiv) and iodomethane (231.0 mL, 3.71 mol, 4.0 equiv) at room temperature. The reaction mixture was heated to 80°C and stirred at 80°C for 20 h. The progress of the reaction was monitored by TLC (10% EtOAc in hexane) to determine the completion of the reaction. DMW (2.5 L) was added and the product was extracted with ethyl acetate (2 x 2.5 L). The combined EtOAc extracts were washed with DMW (2 x 2.5 L), brine (2.5 L), dried over sodium sulfate, filtered, and the filtrate was concentrated in vacuo to give intermediate 103 (180 g, 74.40%) as a brown oil. 1 HNMR (300MHz, DMSO-d6): δ7.83ppm (d, J = 8.3Hz, 1H), 7.51-7.39ppm (m, 2H), 3.83ppm (s, 3H), 2.46ppm (s, 3H).

[0187] Step 3: Methyl 4-bromo-2-(methylsulfonyl)benzoate (Intermediate 104)

[0188]

[0189] To a solution of methyl 4-bromo-2-(methylthio)benzoate (103) (180.0 g, 589 mmol, 1.0 equiv) in methanol (3.60 L) was added a solution of hydrogen peroxide (1.17 kg, 3.79 mol, 5.50 equiv) in DMW (4.50 L) at 0°C. The reaction mixture was stirred at 60°C for 16 h. The progress of the reaction was monitored by TLC (30% EtOAc in n-hexane). About 40% of the solvent was removed by distillation, and then cooled DMW (4.50 L) was added to the reaction mixture and stirred for 15 minutes. The precipitated solid was collected by filtration to provide the wet product 104. The wet product was dried in a vacuum oven below 50°C to give dry intermediate 104 (200 g, 99.0%) as an off-white solid. 1 HNMR (300MHz, DMSO-d6): δ8.19-8.02ppm (m, 2H), 7.73ppm (d, J=8.1Hz, 1H), 3.87ppm (s, 3H), 3.42ppm (s, 3H). MS (ESI): m / z 311.5 (M+18)

[0190] Step 4: 4-Bromo-2-(methylsulfonyl)benzoic acid (Intermediate 105)

[0191]

[0192] To a solution of methyl 4-bromo-2-(methylsulfonyl)benzoate (104) (201.0 g, 0.68 mol, 1.0 equiv) in methanol (2.01 L) was added a solution of NaOH (192.0 g, 4.80 mol, 7.0 equiv) in water (2.01 L). The reaction mixture was stirred at room temperature for 3.0 h. The progress of the reaction was monitored by TLC (20% MeOH in DCM). The solvent was removed under reduced pressure. Ice-cold DMW (1.5 L) was added to the residue and 5N HCl was added to adjust the pH to ~5. The precipitated solid was collected by filtration, washed with cold water (1000 mL) and dried to give intermediate 105 (155.0 g, 81.0%) as an off-white solid. 1 H NMR (300MHz, DMSO-d6): 8.09ppm (d, J=2.0Hz, 1H), 8.04ppm (dd, J=8.2, 2.0Hz, 1H), 7.70ppm (d, J=8.2Hz, 1H), 3.44ppm (s, 3H). MS (ESI): m / z 278.8 (M-1)

[0193] Step 5: 4-Bromo-2-(methylsulfonyl)benzoyl chloride (Intermediate 106)

[0194]

[0195] To a solution of 4-bromo-2-(methylsulfonyl)benzoic acid (105) (105.0 g, 376.0 mmol, 1.0 equiv) in THF (1.05 L) was added thionyl chloride (68.4 mL, 940.0 mmol, 2.50 equiv) at room temperature, followed by DMF (1.37 mL, 18.80 mmol, 0.05 equiv). The reaction mixture was heated to 80 °C and stirred for 2 hours. The progress of the reaction was monitored by TLC (50% EtOAc in hexane) to ensure that the reaction was complete. The solvent was removed under vacuum to obtain intermediate 106 (112.0 g). The crude product of intermediate 106 was used directly in step 6.

[0196] Step 6: 7-(4-bromo-2-(methylsulfonyl)benzamide)-2,3-dihydrobenzo[ b [1,4]Dioxin-6-carboxy Acid (Intermediate 107)

[0197]

[0198] To a solution of 7-amino-2,3-dihydrobenzo[b][1,4]dioxine-6-carboxylic acid (100) (60.20 g, 309.0 mmol, 0.82 equiv) in THF (1.68 L) at 0°C was added triethylamine (103.3 mL, 753.0 mmol, 2.0 equiv) followed by a solution of 4-bromo-2-(methylsulfonyl)benzoyl chloride (106) (60.20 g, 309.0 mmol, 1.0 equiv) in THF (1.68 L) at 0°C. The reaction mixture was stirred at 0°C for 10 min and then at room temperature for 2.5 h. The progress of the reaction was monitored by TLC (10% MeOH in DCM). 1 N HCl was added to adjust the pH to 7. About 70% of the THF was removed by distillation and the remaining residue was triturated with diethyl ether (250 mL) to precipitate the solid product. The solid was collected by filtration, washed with ether (50 mL) and dried to give Intermediate 107 (120.0 g, 69.90%) as a light brown solid.

[0199] Intermediate 100: 7-amino-2,3-dihydrobenzo[b][1,4]dioxin-6-carboxylic acid was synthesized by Procedures AE as described herein.

[0200] Step A: Methyl 3,4-dihydroxybenzoate (Intermediate 100B)

[0201]

[0202] To a solution of 3,4-dihydroxybenzoic acid (100A) (200.0 g, 1.30 mol, 1.0 equiv) in methanol (2.0 L) was added sulfuric acid (138.0 mL, 2.60 mol, 2.0 equiv). The reaction mixture was stirred at 80 °C for 15 h. The progress of the reaction was monitored by TLC (50% EtOAc in n-hexane). The solvent was removed by vacuum distillation, then DMW (1500 mL) was added and the product was extracted with ethyl acetate (3×1.5 L). The combined organic extracts were washed with saturated sodium bicarbonate solution (2000 mL), brine (1500 mL), dried over sodium sulfate and concentrated in vacuo to afford intermediate 100B (198.0 g, 90.70%) as an off-white solid. 1 H NMR (300MHz, DMSO): δ 9.78ppm (s, 1H), 9.37ppm (s, 1H), 7.36-7.23ppm (m, 2H), 6.81ppm (d, J = 8.2Hz, 1H), 3.76ppm (s, 3H).

[0203] Step B: 2,3-Dihydrobenzo[b][1,4]dioxin-6-carboxylic acid methyl ester (Intermediate 100C)

[0204]

[0205] To a solution of methyl 3,4-dihydroxybenzoate (100B) (197.0 g, 1.17 mol, 1.0 equiv) in DMF (2.96 L) was added potassium carbonate (405 g, 2.93 mol, 2.50 equiv) and 1,2-dibromoethane (152.0 mL, 1.76 mol, 1.50 equiv). The reaction mixture was heated to 90 °C and stirred for 25 h. The progress of the reaction was monitored by TLC (TLC analysis of 60% ethyl acetate in n-heptane). The reaction was cooled to room temperature and then DMW (7.50 L) was added, followed by extraction with EtOAc (2 x 3.50 L). The combined organic extracts were washed with DMW (2 x 3.5 L), brine (3.5 L), dried over sodium sulfate and concentrated in vacuo to afford intermediate 100C (214.0 g, 94.10%) as a light brown oil. 1 H NMR (300MHz, CDCl3): δ7.63-7.51ppm (m, 2H), 6.89ppm (d, J = 9.0Hz, 1H), 4.35-4.24ppm (m, 4H), 3.88ppm (s, 3H).

[0206] Step C: 7-nitro-2,3-dihydrobenzo[b][1,4]dioxin-6-carboxylic acid methyl ester (Intermediate 100D)

[0207]

[0208] Concentrated nitric acid (832.0 mL, 14.50 mol, 17.0 equiv) was added to a solution of methyl 2,3-dihydrobenzo[b][1,4]dioxin-6-carboxylate (100C) (214.0 g, 1.10 mol, 1.0 equiv) in acetic acid (832.0 mL, 14.50 mol, 13.2 equiv) at below 20°C. The reaction mixture was stirred at room temperature for 2 h, during which the progress of the reaction was monitored by TLC (eluent: 60% ethyl acetate in n-heptane). The reaction mixture was poured into ice water (5.0 L) under vigorous stirring. The precipitated solid was collected by filtration, washed with DMW (1100 mL), and dried to give intermediate 100D (261 g, 99.0%) as a light yellow solid. 1 HNMR (300MHz, DMSO-d6): δ7.65ppm(s,1H),7.30ppm(s,1H),4.47-4.34ppm(m,1H),4.39ppm(s,4H),3.80ppm(s,3H).

[0209] Step D: 7-amino-2,3-dihydrobenzo[b][1,4]dioxin-6-carboxylic acid methyl ester (Intermediate 100E)

[0210]

[0211] A mixture of methyl 7-nitro-2,3-dihydrobenzo[b][1,4]dioxin-6-carboxylate (100D) (260 g, 1.09 mol, 1.0 equiv), iron powder (217.0 g, 2.72 mol, 2.5 equiv; mesh size 300-400) and ethanol (1.69 L, 6.50 volume equiv), water (585 mL), acetic acid (1.69 L) was heated to 80°C and stirred at this temperature for 45 minutes. The progress of the reaction was monitored by TLC (eluent: 60% ethyl acetate in n-heptane). The reaction mixture was cooled to room temperature and filtered through a celite bed, which was washed with ethyl acetate (500 mL). Water (5.0 L) was added to the filtrate and mixed. The aqueous layer was separated and back extracted with ethyl acetate (3×2.0 L). The combined organic extracts were washed with saturated sodium bicarbonate (3 x 3.0 L) and brine (5.0 L), then dried over sodium sulfate and filtered. The filtrate was concentrated under vacuum to afford Intermediate 100E (226 g, 99.40%) as a light brown solid. 1 H NMR (300MHz, DMSO-d6): δ7.14ppm(s,1H),6.27ppm(s,2H),6.24ppm(s,1H),4.28-4.19ppm(m,2H),4.16-4.06ppm(m,2H),3.73ppm(s,3H).

[0212] Step E: 7-amino-2,3-dihydrobenzo[b][1,4]dioxin-6-carboxylic acid (Intermediate 100)

[0213]

[0214] To a solution of 7-amino-2,3-dihydrobenzo[b][1,4]dioxin-6-carboxylate (100E) (225.0 g, 1.08 mol, 1.0 equiv) in THF (1.58 mL) was added NaOH (237.0 g, 5.92 mol, 5.5 equiv) in water (1.58 mL) and the reaction mixture was heated to 80 °C and stirred for 15 h. The progress of the reaction was monitored by TLC (10% MeOH in DCM) to ensure the completion of the reaction. The reaction mixture was cooled to room temperature and THF was evaporated under reduced pressure. Subsequently, cooled DMW (1500 mL) was added to the residue and the pH was adjusted to ~6.0 using 5N HCl. The precipitated solid was collected by filtration, washed with DMW (300 mL) and dried to give intermediate 100 (152.50 g, 72.5%) as a light yellow solid. 1H NMR (300MHz, DMSO): δ8.17ppm(s,2H),7.13ppm(s,1H),6.20ppm(s,1H),4.27-4.17ppm(m,2H),4.17-4.02ppm(m,2H).

[0215] Step 7: 2-(4-bromo-2-(methylsulfonyl)phenyl)-7,8-dihydro-4H-[1,4]dioxin[2',3':4,5] Synthesis of benzo[1,2-d][l,3]oxazin-4-one (Intermediate 108)

[0216]

[0217] A solution of 7-(4-bromo-2-(methylsulfonyl)benzamide)-2,3-dihydrobenzo[b][1,4]dioxin-6-carboxylic acid (107) (120.0 g, 263.0 mmol, 1.0 equiv) in acetic anhydride (960 mL) was heated to 140 °C and stirred for 1.5 h. The progress of the reaction was monitored by TLC (50% EtOAc in hexane) to ensure the completion of the reaction. About 70% of the solvent was removed by vacuum distillation. The remaining residue was cooled to room temperature, then DMW (1000 mL) was added and the mixture was stirred for 40 minutes. The precipitated solid was collected by filtration, washed with DMW (1000 mL) and dried to intermediate 108 (85.4 g, 74.1%) as an off-white solid. 1 HNMR (300MHz, DMSO-d6): δ8.29-8.12ppm (m, 2H), 7.91ppm (d, J = 8.2Hz, IH), 7.59 ppm(s,IH),7.26ppm(s,IH),4.55-4.32ppm(m,4H),3.57ppm(s,3H).MS(ESI):m / z 439.6(M+1).

[0218] Step 8: 2-(2-(methylsulfonyl)-4-vinylphenyl)-7,8-dihydro-4H-[1,4]dioxin [2',3': Synthesis of 4,5]benzo[1,2-d][l,3]oxazin-4-one (Intermediate 109)

[0219]

[0220] A solution of 2-(4-bromo-2-(methylsulfonyl)phenyl)-7,8-dihydro-4H[1,4]dioxino[2',3':4,5]benzo[1,2-d][l,3]oxazin-4-one (108) (84.0 g, 192.0 mmol, 1.0 equiv) in 1,4-dioxane (1.68 L) was degassed three times by alternating application of vacuum and nitrogen filling to remove oxygen. Under nitrogen, sodium bicarbonate (56.40g, 671.0mmol, 3.5 equivalents), potassium vinyl trifluoroborate (64.20g, 479.0mmol, 2.5 equivalents), tri(o-tolyl)-phosphine (14.60g, 47.90mmol, 0.25 equivalents) and Pd(OAc)2(1.51g, 6.71mmol, 0.035 equivalents), and by filling with alternating vacuum and nitrogen, the reaction flask is degassed twice again. The reaction mixture is heated and stirred for 3.5h at 70 ℃. The reaction process is monitored by TLC (n-hexane solution of 40%EtOAc) to ensure that the reaction is complete. The reaction mixture is cooled to room temperature, quenched with 1N HCl (1000mL) and filtered through a diatomaceous earth pad. The solvent is evaporated under reduced pressure and DMW (2.5L) is added to the residue. The product was extracted with ethyl acetate (3x2 L), the combined EtOAc extracts were washed with brine (2.5 L), dried over sodium sulfate, filtered and concentrated to give the crude product. The crude product was stirred in ether (150 mL) and filtered to give Intermediate 109 (72.0 g, 97.50%) as a light yellow solid. 1 H NMR (300MHz, DMSO-d6): δ7.14ppm(s,1H),6.27ppm(s,2H),6.24ppm(s,1H),4.28-4.19ppm(m,2H),4.16-4.06ppm(m,2H),3.73ppm(s,3H).

[0221] Step 9: 3-(Methylsulfonyl)-4-(4-oxo-7,8-dihydro-4H-[1,4]dioxin[2',3':4,5]benzo Synthesis of [1,2-d][1,3]oxazin-2-yl)benzaldehyde (Intermediate 110)

[0222]

[0223] To a solution of 2-(2-(methylsulfonyl)-4-vinylphenyl)-7,8-dihydro-4H-[1,4]dioxino[2',3':4,5]benzo[1,2-d][1,3]oxazin-4-one (109) (72.0 g, 187.0 mmol, 1.0 equiv) in acetonitrile (1.44 L), EtOAc (1.44 L) and DMW (1.08 L) was added ruthenium on carbon (5.66 g, 5.60 mmol, 0.03 equiv) at room temperature. Sodium periodate (130.0 g, 607.0 mmol, 3.25 equiv) was added at 0°C. The reaction mixture was stirred at 0°C for 3.0 h. The progress of the reaction was monitored by TLC (50% EtOAc in hexane). The reaction mixture was filtered through a bed of celite. The solid was washed with ethyl acetate (4x1.5 L). The filtrate was washed with water (3.0 L) and the aqueous layer was back extracted with ethyl acetate (800 mL). The combined organic extracts were washed with brine (2.0 L), dried over sodium sulfate and filtered. The filtrate was concentrated in vacuo to give crude 110 (65.0, 95.30%) as a brown solid. The crude intermediate 110 was used directly in the subsequent step 10.

[0224] Step 10: 2-(4-(Hydroxymethyl)-2-(methylsulfonyl)phenyl)-7,8-dihydro-4H-[1,4]dioxin[2', Synthesis of 3':4,5]phenylpropano[l,2-d][l,3]oxazin-4-one (Compound 1.001)

[0225]

[0226] To a solution of 3-(methylsulfonyl)-4-(4-oxo-7,8-dihydro-4H-[1,4]dioxino[2',3':4,5]benzo[1,2-d][1,3]oxazin-2-yl)benzaldehyde (110) (82.0 g, 212.0 mmol, 1.0 equiv) in THF (1.23 mL) and ethanol (1.23 L) was added sodium borohydride (8.0 g, 1.0 equiv) slowly over 1.0 h at 0 °C. The reaction mixture was stirred at 0 °C for 2 h. The progress of the reaction was monitored by TLC (70% EtOAc in hexane) to ensure the completion of the reaction. The reaction mixture was quenched with saturated ammonium chloride solution (100 mL) and the product was extracted with ethyl acetate (3x1500 mL). The combined organic extracts were washed with brine (500 mL), dried over sodium sulfate and concentrated in vacuo to give the crude product. The crude product was purified by silica gel column chromatography (230-400 mesh) using a gradient solvent (20-40% [(10% MeOH in EtOAc)] in hexanes) to afford compound 1.001 (15.50 g, 18.80%)) as an off-white solid. 1H NMR(300 MHz, DMSO-d6): δ8.08ppm(d,J=1.5Hz,1H),7.91 ppm(d,J=7.9 Hz,1H),7.83 ppm(dd,J=7.9,1.6 Hz,1H),7.58 ppm(s,1H),7.24 ppm(s,1H),5.64 ppm(t,J=5.7 Hz,1H),4.70 ppm(d,J=5.7 Hz,2H),4.47-4.35ppm(m,4H),3.50 ppm(s,3H).

[0227] Example 2: 2-(5-(Hydroxymethyl)-2-(methylsulfonyl)phenyl)-7,8-dihydro-4H[1,4]dioxin[2',3':4,5]benzo[1,2-d][1,3]oxazin-4-one (Compound 1.002)

[0228]

[0229] The synthesis of compound 1.002 is as follows Figure 2 The synthesis of intermediate 100 is as shown. Figure 3 shown.

[0230] Step 1: 5-Bromo-2-mercaptobenzoic acid (Intermediate 202)

[0231]

[0232] To a solution of 2-amino-5-bromobenzoic acid (201) (20.0 g, 92.6 mmol, 1.0 eq), NaOH (3.70 g, 92.6 mmol, 1.0 eq) and sodium nitrite (6.39 g, 92.6 mmol, 1.0 eq) in DMW (400 mL) was added a mixture of concentrated HCl (28.0 mL) and cold water (34 mL) slowly, dropwise, such that the reaction temperature was maintained at 3-6 °C. After the addition, the reaction mixture was stirred at 0 °C for 30 min and then neutralized with potassium acetate (30 g, 306.0 mmol, 3.3 eq). This solution was added to a solution of potassium O-ethylxanthate (44.6 g, 278 mmol, 3.0 eq) in DMW (223 mL) preheated to 90 °C. The reaction mixture was stirred at 90 °C for 30 min, cooled to 0 °C, and acidified with concentrated HCl (100 mL). The reaction mixture was basified with 10% NaOH (200 ml) and heated to 85°C for 2 hours. NaHSO3 (9.81 g, 92.6 mmol) was added to the mixture in small portions and the reaction mixture was heated to 85°C for 10 minutes. The reaction mixture was filtered, cooled to 0°C, and acidified with concentrated HCl (100 mL). The precipitated solid was collected by filtration, washed with H2O and then with n-hexane to give intermediate 202 (20.2 g, 93.6%) as a light grey solid. MS (ESI): m / z 230.8 (M-1).

[0233] Step 2: Methyl 5-bromo-2-(methylthio)benzoate (Intermediate 203)

[0234]

[0235] To a solution of 4-bromo-2-mercaptobenzoic acid (202) (20 g, 85.8 mmol, 1 eq) in DMF (600 mL) was added potassium carbonate (83.0 g, 601 mmol, 7.0 eq) and iodoethane (21.4 mL, 343 mmol, 4.0 eq) at 0°C. The reaction mixture was stirred at 80°C for 20 h. The progress of the reaction was monitored by TLC (10% EtOAc in hexane) to determine the completion of the reaction. DMW (500 mL) was added and the product was extracted with ethyl acetate (3x500 mL). The combined EtOAc extracts were washed with brine (300 mL), dried over sodium sulfate, filtered, and the filtrate was concentrated in vacuo to give the crude product. The crude product was purified by silica gel (230-400 mesh) column chromatography (solvent gradient 0-5% EtOAc in n-hexane) to afford intermediate 203 (20 g, 89.30%) as an off-white solid.

[0236] Step 3: Methyl 5-bromo-2-(methylsulfonyl)benzoate (Intermediate 104)

[0237]

[0238] To a solution of methyl 5-bromo-2-(methylthio)benzoate (203) (14.0 g, 53.6 mmol, 1.0 equiv) in methanol (350 mL) was added a solution of hydrogen peroxide (40.8 g, 268 mmol, 5.0 equiv) in DMW (280 mL) at 0°C. The reaction mixture was stirred at 50°C for 16 h. The progress of the reaction was monitored by TLC (30% EtOAc in hexane) to ensure the completion of the reaction. The product was extracted with EtOAc (4x500 mL). The combined organic phases were washed with brine (2x300 mL), dried over sodium sulfate, and concentrated in vacuo to give the crude product. The crude product was purified by column chromatography on silica gel (230-400 mesh) (solvent gradient 5-20% EtOAc) in hexane to give intermediate 204 (13.2 g, 84.0%) as an off-white solid. 1 H NMR (300MHz, DMSO): δ8.08-7.98ppm (m, 2H), 7.93ppm (dd, J=7.9, 0.9Hz, IH), 3.87ppm (s, 3H), 3.44-3.35ppm (m, 3H). MS (ESI): m / z292.8 (M-1).

[0239] Step 4: Methyl 2-(methylsulfonyl)-5-vinylbenzoate (Intermediate 205)

[0240]

[0241] A solution of 5-bromo-2-(methylsulfonyl)benzoic acid methyl ester (204) (5.0 g, 17.1 mmol, 1.0 equivalent) in DME (100.0 mL, 20 volume equivalents) was degassed three times in a vacuum and nitrogen alternate filling mode. Potassium carbonate (4.71 g, 34.1 mmol, 2.0 equivalents), potassium vinyl trifluoroborate (4.57 g, 34.1 mmol, 2.0 equivalents) and Pd(PPh3)4 (591 mg, 512 μmol, 0.03 equivalents) were added in nitrogen and degassed twice in a vacuum and nitrogen alternate filling mode. The reaction mixture was heated to 80 ° C and stirred for 4 h (nitrogen was removed when the temperature rose to 40-45 ° C). The reaction progress was monitored by TLC (30% EtOAc in n-hexane) to ensure that the reaction was complete. The reaction mixture was cooled to room temperature and filtered through diatomaceous earth. In the filtrate, DMW (300mL) is added and the product is extracted with ethyl acetate (2x100mL). The organic extracts merged are washed with brine (2x100mL), dried over sodium sulfate, filtered and concentrated to provide a crude product. The crude product is purified by silica gel column chromatography (230-400 mesh) using a gradient solvent (5-20% EtOAc in n-hexane) as an eluent to obtain intermediate 205 (3.36g, 82.0%), which is a light yellow jelly.

[0242] Step 5: 2-(Methylsulfonyl)-5-vinylbenzoic acid (Intermediate 206)

[0243]

[0244] To a solution of methyl 2-(methylsulfonyl)-5-vinylbenzoate (205) (3.36 g, 14.0 mmol, 1.0 equiv) in methanol (67.2 mL) was added a solution of NaOH (3.92 g, 97.9 mmol, 7.0 equiv) in water (67.2 mL).

[0245] The reaction mixture was stirred at 60 ℃ for 2 hours. The reaction process was monitored by TLC (70% EtOH in normal hexane) to ensure that the reaction was complete. The reaction mixture was cooled to room temperature, 3N HCl was used to adjust the pH to ~6, and aqueous layer was extracted with ethyl acetate (4x500mL). The organic extracts merged were washed with salt water, dried over sodium sulfate, filtered and concentrated in vacuo to obtain the required intermediate 206 (1.48 g, 46.8%), which was a pale solid. 1H NMR (300MHz, DMSO): δ7.93ppm (t, J=7.6Hz, 1H), 7.88-7.78ppm (m, 2H), 6.87ppm (dd, J=17.7, 11.0Hz, 1 H), 6.13ppm (d, J = 17.6Hz, 1H), 5.53ppm (d, J = 10.9Hz, 1H), 3.37ppm (s, 3H). MS (ESI): m / z224.95 (M-1).

[0246] Step 6: 2-(Methylsulfonyl)-5-vinylbenzoyl chloride (Intermediate 207)

[0247]

[0248] To a solution of 2-(methylsulfonyl)-5-vinylbenzoic acid (206) (1.4 g, 6.19 mmol, 1.0 equiv) in DCM (70 mL) was added oxalyl chloride (2.66 mL, 30.9 mmol, 5.0 equiv) at room temperature, followed by 2 drops of DMF. The reaction mixture was refluxed for 2 hours. The progress of the reaction was monitored by TLC (50% EtOAc in hexane) to ensure completion of the reaction. The solvent was removed in vacuo to give the desired product, intermediate 207 (540 mg, 83.2%). The crude product of intermediate 207 was used directly in subsequent step 7.

[0249] Step 7: 7-(2-(methylsulfonyl)-5-vinylbenzamido)-2,3-dihydrobenzo[b][1,4]dioxadioxane Indole-6-carboxylic acid (Intermediate 208)

[0250]

[0251] At 0 ° C, triethylamine (1.93 mL, 13.8 mmol, 3.0 eq) and 2-(methylsulfonyl)-5-vinylbenzoyl chloride (207) (1.35 mg, 5.53 mmol, 1.2 eq) THF (36 mL) solution were added to 7-amino-2,3-dihydrobenzo [b] [1,4] dioxin-6-carboxylic acid (100) (900 mg, 4.61 mmol, 1.0 eq) THF (36 mL) solution. The reaction mixture was stirred at the same temperature for 10 minutes, then stirred at room temperature for 1.5 hours. The reaction progress was monitored by TLC (20% EtOAc in n-hexane) to ensure that the reaction was complete. The solvent was removed in vacuo. DMW was added to the residue and the product was extracted with EtOAc (9x200 mL). The combined EtOAc extracts were washed with brine (200 mL), dried over sodium sulfate and concentrated in vacuo to afford Intermediate 208 (1.6 g, 86.0%) as a brown solid. MS (ESI): m / z 404.10 (M+1), m / z 401.90 (M-1).

[0252] Intermediate 100: 7-amino-2,3-dihydrobenzo[b][1,4]dioxin-6-carboxylic acid was synthesized by Procedures AE as described herein.

[0253] Step A: Methyl 3,4-dihydroxybenzoate (Intermediate 100B)

[0254]

[0255] To a solution of 3,4-dihydroxybenzoic acid (100A) (97%, 10.0 g, 62.9 mmol, 1.0 equiv) in methanol (500 mL) was added sulfuric acid (96%, 7.0 mL, 126.0 mmol, 2.0 equiv) at room temperature. The reaction mixture was stirred overnight. TLC ensured that the reaction was complete. The solvent was removed in vacuo and water (500 mL) was added. The aqueous layer was extracted with EtOAc (3×100 mL). The combined organic layers were washed with saturated NaHCO3 solution (250 mL), brine (250 mL), dried over sodium sulfate and concentrated in vacuo to give the desired intermediate 100B (8.72 g, 82.4%) as an off-white solid. 1 H NMR (300MHz, DMSO-d6): δ9.81ppm (d,J=131.1Hz,2H), 7.41-7.19ppm (m,2H), 6.81ppm (d,J=8.2Hz,1H), 3.77ppm (s,3H).

[0256] Step B: 2,3-Dihydrobenzo[b][1,4]dioxin-6-carboxylic acid methyl ester (Intermediate 100C)

[0257]

[0258] To a solution of methyl 3,4-dihydroxybenzoate (100B) (10.0 g, 59.5 mmol, 1.0 equiv) in acetone (200 mL) was added potassium carbonate (20.5 g, 149 mmol, 2.50 equiv) followed by 1,2-dibromoethane (7.72 mL, 89.2 mmol, 1.50 equiv) and the reaction mixture was refluxed overnight. Completion of the reaction was ensured by TLC. The solvent was removed in vacuo and water was then added. The aqueous layer was neutralized with 1N HCl and then extracted with EtOAc (2x250 mL). The combined organic extracts were washed with brine (500 mL), dried over sodium sulfate and concentrated in vacuo. The crude product was purified by combiflash chromatography using ethyl acetate / hexane (0 to 10%) to give the desired intermediate 100C (9.0 g, 77.9%) as a colorless oil. 1H NMR (300MHz, CDCl3): δ7.54-7.42ppm (m, 2H), 6.85-6.75ppm (m, IH), 4.27-4.14ppm (m, 4H), 3.79ppm (s, 3H).

[0259] Step C: 7-nitro-2,3-dihydrobenzo[b][1,4]dioxin-6-carboxylic acid methyl ester (Intermediate 100D)

[0260]

[0261] To a solution of methyl 2,3-dihydrobenzo[b][1,4]dioxin-6-carboxylate (100C) (9.0 g, 46.3 mmol, 1.0 equiv) in acetic acid (35.0 mL, 612 mmol, 13.2 equiv) was added concentrated nitric acid (47.0 mL, 788 mmol, 17.0 equiv) dropwise below 20°C, and the reaction mixture was allowed to stir at room temperature for 2 h. The reaction was determined to be complete by TLC. The reaction mixture was poured into ice water with vigorous stirring, and the aqueous phase was washed with EtOAc (3 x 500 mL). The combined organic extracts were washed with NaHCO3, brine, dried over anhydrous sodium sulfate, and concentrated in vacuo to give the product intermediate 100D (9.7 g, 87.5%) as a light yellow solid. 1 HNMR (300MHz, DMSO-d6): δ7.65ppm(s,IH),7.30ppm(s,IH),4.47-4.34ppm(m,IH),4.39ppm(s,4H),3.80ppm(s,3H).

[0262] Step D: 7-amino-2,3-dihydrobenzo[b][1,4]dioxin-6-carboxylic acid methyl ester (Intermediate 100E)

[0263]

[0264] A mixture of 7-nitro-2,3-dihydrobenzo[b][1,4]dioxin-6-carboxylic acid methyl ester (100D) (25.0 g, 105.0 mmol, 1.0 equiv), iron powder (20.4 g, 366.0 mmol, 3.5 equiv) and ethanol (625 mL), water (163 mL) and acetic acid (500 mL) was heated to 70 ° C and stirred at this temperature for 45 minutes. The reaction was completed by TLC. The reaction mixture was cooled to room temperature and filtered through diatomaceous earth. The filtrate was diluted with water (1000 mL) and the aqueous layer was extracted with ethyl acetate (3x700 mL). The combined organic extracts were washed with a saturated solution. Sodium bicarbonate (2x1000 mL), brine (1000 mL), dried over sodium sulfate and filtered. The filtrate was concentrated in vacuo to give the title compound intermediate 100E (21.8 g, 99.7%) as a brown solid. 1 H NMR (300MHz, DMSO): δ7.14ppm (s, 1H), 6.25ppm (d, J = 7.6Hz, 3H), 4.26-10 4.19ppm (m, 2H), 4.17-4.08ppm (m, 2H), 3.73ppm (s, 3H). MS (ESI): m / z 210.10(M+1).

[0265] Step E: 7-amino-2,3-dihydrobenzo[b][1,4]dioxin-6-carboxylic acid (Intermediate 100)

[0266]

[0267] To a solution of 7-amino-2,3-dihydrobenzo[b][1,4]dioxin-6-carboxylate (100E) (22.0 g, 105 mmol, 1.0 equiv) in THF (440.0 mL) was added NaOH (23.1 g, 578 mmol, 5.5 equiv) in water (440.0 mL) and the reaction mixture was stirred at 80 °C for 20 h. The progress of the reaction was monitored by TLC (10% MeOH in DCM) to ensure the completion of the reaction. The reaction mixture was cooled to room temperature, the pH was adjusted to 6 using 3N HCl, and the aqueous layer was extracted with ethyl acetate (5x500 mL). The combined organic layers were washed with brine, dried over sodium sulfate, filtered and the filtrate was concentrated in vacuo to give a solid brown crude product. The crude product was purified with n-hexane to give the desired intermediate 100 (18.6 g, 90.6%) as a brown solid. 1 H NMR (300MHz, DMSO): δ8.27ppm(s,2H),7.13ppm(s,1H),6.20ppm(s,1H),4.27-4.19ppm(m,2H),4.18-4.08ppm(m,2H).

[0268] Step 8: 2-(2-(methylsulfonyl)-5-vinylphenyl)-7,8-dihydro-4H-[1,4]dioxin [2',3': Synthesis of 4,5]benzo[1,2-d][l,3]oxazin-4-one (Intermediate 209)

[0269]

[0270] A solution of 7-(2-(methylsulfonyl)-5-vinylbenzamido)-2,3-dihydrobenzo[b][1,4]dioxine-6-carboxylic acid (208) (1.60 g, 3.97 mmol, 1.0 equiv) in acetic anhydride (40 mL) was heated to 140 °C and stirred for 1.5 h. The progress of the reaction was monitored by TLC (50% EtOH in hexane) to ensure the completion of the reaction. The reaction mixture was cooled to room temperature and DMW (100 mL) was added. The product was extracted with EtOAc (4x200 mL). The combined EtOAc extracts were washed with brine (2x200 mL), dried over sodium sulfate and concentrated in vacuo to give the crude product. The crude product was purified by column chromatography on silica gel (230-400 mesh) and gradient solvent (20-50% EtOAc / hexane) to give intermediate 209 (700 mg, 45.8%) as a yellow solid. 1 HNMR (300MHz, CDCl3): δ8.15ppm (d,J=8.2Hz,1H), 7.90ppm (d,J=1.7Hz,1H), 7.77-7.68ppm (m,2H), 7.16ppm (s,1H), 6.82ppm (dd,J=17.5,1 0.9Hz,1H),6.01ppm(d,J=17.5Hz,1H),5.57ppm(d,J=10.9Hz,1H),4.40ppm(dd,J=11.8,5.3Hz,5H),3.52ppm(d,J=3.0Hz,3H).MS(ESI):m / z 385.80(M+1).

[0271] Step 9: 3-(Methylsulfonyl)-4-(4-oxo-7,8-dihydro-4H-[1,4]dioxin[2',3':4,5]benzo Synthesis of [1,2-d][1,3]oxazin-2-yl)benzaldehyde (Intermediate 210)

[0272]

[0273] To a solution of 2-(2-(methylsulfonyl)-5-vinylphenyl)-7,8-dihydro-4H-[1,4]dioxin[2',3':4,5]benzo[1,2-d][1,3]oxazin-4-one (209) (0.630 g, 1.63 mmol, 1.0 equiv) in acetonitrile (12.6 mL), EtOAc (12.6 mL) and DMW (12.6 L) at room temperature was added ruthenium on carbon (10%, 50 mg, 0.049 mmol, 0.03 equiv). Sodium periodate (1.05 g, 4.9 mmol, 3.0 equiv) was added at 0°C. The reaction mixture was stirred at room temperature for 1 hour. The progress of the reaction was monitored by TLC (30% EtOAc in hexane) to ensure the completion of the reaction. The reaction mixture was filtered through a bed of celite. The filtrate was diluted with water (100 mL) and the aqueous layer was extracted with ethyl acetate (3x200 mL). The combined organic extracts were washed with saturated sodium bicarbonate solution, brine, dried over sodium sulfate and filtered. The filtrate was concentrated in vacuo to give crude intermediate 210. The crude product of intermediate 210 was used directly in the subsequent step 10.

[0274] Step 10: 2-(4-(Hydroxymethyl)-2-(methylsulfonyl)phenyl)-7,8-dihydro-4H-[1,4]dioxin[2', Synthesis of 3':4,5]phenylpropano[l,2-d][l,3]oxazin-4-one (Compound 1.002)

[0275]

[0276] To a solution of 3-(methylsulfonyl)-4-(4-oxo-7,8-dihydro-4H-[1,4]dioxino[2',3':4,5]benzo[1,2-d][1,3]oxazin-2-yl)benzaldehyde (210) (0.20 g, 0.52 mmol, 1.0 equiv) in THF (10.0 mL) was added sodium borohydride (9.77 mg, 0.258 mmol, 0.5 equiv) slowly over 1 h at -10 °C. The reaction mixture was stirred at the same temperature of 0 °C for 30 min. The progress of the reaction was monitored by TLC (70% EtOAc in n-hexane) to ensure the completion of the reaction. The reaction mixture was quenched with saturated ammonium chloride solution (50 mL) and the product was extracted with ethyl acetate (3×100 mL). The combined organic extracts were washed with brine (500 mL), dried over sodium sulfate and concentrated in vacuo to obtain the crude product. The crude product was purified by silica gel column chromatography (230-400) using a gradient solvent (20-40% [(10% MeOH in EtOAc)] in hexanes) to afford compound 1.002 (70 mg, 34.80%) as an off-white solid. 1H NMR (300MHz, DMSO): δ8.07ppm(d,J=8.2Hz,1H),7.88ppm(s,1H),7.79ppm(d,J=8.5Hz,1H),7.58ppm(s,1H),7.24pp m(s,1H),5.63ppm(t,J=5.7Hz,1H),4.69ppm(d,J=5.6Hz,2H),4.52-4.31ppm(m,4H),3.48ppm(s,3H).MS(ESI):m / z 390.0(M+1).

[0277] Example 3: In vitro activity assay

[0278] Human kallikrein proteins: Recombinant human KLK5 expressed in HEK293 was obtained from SpeedBio (Gaithersburg, MD). Recombinant proenzymes KLK-7 and KLK-14 expressed in mouse myeloma cells were purchased from R&D Biosystems (Minneapolis, MN). Plasmin and thrombin were purchased from Haematologic Technologies (Essex Junction, VT). Trypsin and chymotrypsin were purchased from Worthington Biochemicals (Lakewood, NJ), and plasma kallikrein was purchased from Prospec (East Brunswick, NJ). Neutrophil elastase was purchased from Innovative Research (Peary Court, MI). Bovine recombinant enterokinase expressed in Escherichia coli was purchased from Sigma (St. Louis, MI). Matrixase expressed in Escherichia coli was purchased from Enzo Life Sciences (Farmingdale, NY). The control inhibitor nafamostat mesylate was purchased from Sigma (St. Louis, MI), and (2-(2-fluorophenyl)-5,6,7,8-tetrahydro-4H-benzo[4,5]thieno[2,3-d][1,3]oxazin-4-one was purchased from Interbioscreen (Moscow, Russia).

[0279] Substrates: S-2288 (D-Ile-Pro-Arg-pNA), CS-PSA (MeO-Suc-Arg-Pro-Tyr-pNA), S-2266 (HD-Val-Leu-Arg-pNA) and S-2288 (HD-lle-Pro-Arg-pNA) were purchased from Diapharma (Westchester, Ohio). MeOSuccAla-Ala-Pro-Val-pNA was purchased from Sigma (St. Louis, Michigan). The substrates were dissolved in water at 10 mM and frozen in aliquots at -20°C.

[0280] KLK-7 and KLK-14 stimulation: 150 μL of 8 μM KLK-7 or KLK-14 zymogen in 50 mM Tris, 10 mM CaCl2, 150 mM NaCl, 0.05% (w / v) Brij-35, pH 7.5 was mixed with 150 μL of 10 μg / mL bovine enterokinase in the same buffer system at 37 degrees C and incubated for 2 hours for KLK-7 and 1 hour for KLK. After the incubation period, stimulation was stopped by adding 6 μL of 0.5 M, pH 8.0 EDTA solution.

[0281] Assays: All assays were performed in 384-well polystyrene plates coated with 0.1% Tween 20 overnight at 4 °C. The plates were then washed twice with 100 μL of water and dried. To determine the IC 50 The inhibitor was serially diluted into pure DMSO at intervals of 1:3, with a concentration of 50 times the expected final concentration in the test. Eight inhibitor dilutions and a DMSO control were usually prepared. The substrate was diluted into water to 2.2 times the final concentration in the test mixture. 50 μL of enzyme was filled into the wells of the plate, and the enzyme was diluted in 2x assay buffer (100mM Tris, 200mM NaCl, 0.2% PEG, 0.01% Tween, 2mM EDTA, pH 8.0) at twice the expected concentration. Then 5uL DMSO, 2.2uL inhibitor and 45uL substrate were added to start the reaction. At 37°C, the assay was monitored for 1 hour at a wavelength of 405nm (Molecular Devices, San Jose, CA) in a multiwell plate reader. The instrumental inhibitor nafamostat was used as a control for trypsin, thrombin, KLK-5, and KLK-14 at a maximum concentration of 10 μM, and (2-(2-fluorophenyl)-5,6,7,8-tetrahydro-4H-benzo[4,5]thieno[2,3-d][1,3]oxazin-4-one was used as a control for KLK-7 at a maximum concentration of 5 μM.

[0282] The following table summarizes the assay conditions for each enzyme:

[0283] Enzymes Enzyme concentration (nM) Substrate Substrate concentration (μM) KLK-7 20 CS-PSA 500 KLK-5 10 S-2288 500 KLK-14 5 S-2288 500 Plasma kallikrein 5 S-2266 200 Trypsin 0.1 S-2288 500 Chymotrypsin 0.2 CS-PSA 500 Plasmin 1 S-2288 500 Thrombin 0.5 S-2288 200 Matrix protease 0.015 U / μL S-2288 200

[0284] Data Analysis: Progress curves for substrate hydrolysis were analyzed by linear regression over the last 20 min of the reaction. The resulting rate dose responses were fitted to the following equation to obtain the IC 50 :

[0285]

[0286] Where 1) c: background rate at the highest inhibitor concentration; 2) d: rate of the control reaction; 3) IC50 :IC of inhibitor 50 ; 4) x: inhibitor concentration; 5) b: slope.

[0287] Example 4: Protease activity in human skin

[0288] Materials: Peptide substrates PS-01 (YRSR-pNA Tyr-Arg-Ser-Arg-pNA) and PS-02 (Lys-His-Leu-Tyr-pNA) were synthesized by Zamboni Chemical Solutions (Montreal, Canada).

[0289] Preparation of skin extracts: The epidermis was frozen and lyophilized. A 15 mg freeze-dried epidermis sample was immersed in 1 mL of high salt buffer (100 mM Tris, 5 mM EDTA, 2 M KCl, pH 8), frozen twice in dry ice and thawed. After incubation at 4 ° C overnight and the addition of six 2.4 mm metal beads, the sample was homogenized 3 times at 5000 Hz in a Bertin Precellys homogenizer (Atkinson, New Hampshire) for 20 seconds each time with 30 seconds of intermediate cooling. After centrifugation at maximum speed in a tabletop microcentrifuge, the protein concentration of the supernatant was determined by the BCA assay (~0.5 mg / mL).

[0290] Inhibition of protease activity in skin extracts: Prepare stock solutions of test compounds in DMSO at 200, 66, 22, and 0 μM. Prepare 1100 μM substrate stock solutions of PS-01 and PS-02 by dissolving in water. Add 50 μL of 2x assay buffer (see above) to the wells of a 384-well plate, followed by 45 μL of substrate stock solution and 5 μL of test compound. Start the reaction by adding 10 μL of skin extract to substrate PS-02 and 5 μL of skin extract to substrate PS-01. Monitor substrate hydrolysis at 405 nm in a multiwell plate reader.

[0291] Example 5: Test results of the tested compounds

[0292] Compounds 1.001 and 1.002 and known compound 14 were tested according to the assay protocol of Examples 3 and 4. The test results are listed in Table 2. Compound 14 is disclosed in WO 2015 / 112081 and has the following formula:

[0293]

[0294] Table 2: Assay results of test compounds

[0295]

[0296] *Absorbance 405 / min

[0297] Compounds 1.001, 1.002 and 14 were tested for chromogenic substrate cleavage progress using KLK5 and KLK7 preferred chromogenic peptide substrates (PS-01 and PS-02) according to the human stratum corneum extract assay of Example 4. Figures 4A-4D Proteolytic cleavage of KLK5 selective substrates in skin extracts by compounds 1.001, 1.002, and 14 is compared and shown in NSK refers to freshly prepared skin extracts; OSK refers to frozen-thawed skin extracts.

[0298] from Figures 4A-4D (NSK) It can be seen that for compound 1.001, the rate (slope) of the initial linear portion of the curve appears to be lower than for the other two compounds, indicating a higher inhibition of substrate cleavage. The observed lower initial rate of proteolytic activity for compound 1.001 is also consistent with the observed lower intercept of the curve. Figures 4A-4D (NSK) As can be seen, the rate of substrate cleavage slows down exponentially as substrate is depleted. The lower exponential rate constant of compound 1.001 indicates that compound 1.001 appears to have a stronger inhibitory effect on proteolytic activity.

[0299] Compared with the results of purified recombinant KLK5, compound 1.001 appears to be a more potent inhibitor of the proteolytic activity of skin extracts compared with known compound 14 based on three parameters (rate, substrate consumption and initial rate of proteolytic activity).

[0300] Example 6: Chromogenic method

[0301] Preparation of neonatal human epidermal keratinocyte lysate: Pooled human epidermal keratinocytes (HEK) were purchased from Invitrogen (Catalog No. Al3401). In the absence of serum, cells were grown in commercial medium EpiLife (Thermo Fisher, Catalog No. MEPI500CA) supplemented with commercial supplement S7 (Thermo Fisher, Catalog No. S00175). Tissue culture was carried out in a humidified chamber at 37°C and 5% carbon dioxide. HEK lysis buffer composition: 100mM Tris-HCl, 150mM NaCl, 1% triton, 5mM EDTA, final pH 7.6. After two passages, cells were lysed using HEK lysis buffer without the addition of protease and phosphatase inhibitors, and protein concentration was determined using bicinchoninic acid assay (BCA) (ThermoFisher, Catalog No. 23225).

[0302] Preparation of stratum corneum extracts from healthy human volunteers: Stratum corneum sheets were purchased from Biopredic (Cat. No. STR0020). Extract preparation: Stratum corneum lysates were prepared using the Minute Adipose Tissue Total Protein Extraction Kit (InventBiotech, Cat. No. AT-022) after homogenization according to the manufacturer's instructions. Total protein concentration was determined using the BCA assay (ThermoFisher, Cat. No. 23225). The extracts were snap frozen and stored at -80°C until use.

[0303] Concentration of KLK5 in stratum corneum extracts: The concentration of KLK5 in stratum corneum extracts was determined by 1) ELISA purchased from Abcam (Catalog No. ab131555); 2) comparison of the hydrolysis rate of the selective chromogenic substrate PS-01 (YRSR-pNATyr-Arg-Ser-Arg-pNA) with known concentrations of purified KLK5 (SpeedBio). In addition, the concentration of KLK5 in unknown samples was determined by interpolating the rate of hydrolysis of the selective substrate from a linear plot of the rates of the known samples.

[0304] Endogenous and exogenous kallikrein 5 cleavage of macromolecular substrates: 10ug HEK lysate was incubated with an equal volume of stratum corneum extract or protease in buffer at 37°C for 1.5 hours. The reaction was quenched by boiling with loading buffer at 100°C for 5 minutes under reducing conditions (50mM DTT). Proteins were separated by electrophoresis on bis-tris gels with a 4-12% acrylamide gradient, and the gel was electrophoresed at 200V for 22 minutes. After transferring the protein to a PVDF membrane, nonspecific binding was blocked for 1 hour at room temperature using Intercept blocking buffer (Licor). Signals on the blot were normalized by GAPDH.

[0305] The conditions for detecting specific proteins are summarized below:

[0306] Desmoglein 1 : Desmoglein antibody sc-137164 (Santa Cruz Biochemicals) was diluted 1 / 750 and incubated at 40°C for 18 hours. After washing, the stock solution was diluted 1 / 20000 and anti-mouse secondary antibody (Licor) was added. A control group without KLK5 was treated with a protease / phosphatase inhibitor cocktail (Cell Signalling, #5872).

[0307] Desmoglein 1 : Anti-desmoglein 1 antibody (Abcam, 150382) was diluted 1:1000. Secondary antibody was diluted 1:15000. After washing, secondary rabbit antibody was added at a dilution of 1:15000.

[0308] Filaggrin : Anti-filagrin (LS-Bio, 1561) was diluted 1:400. Secondary antibody was diluted 1:10000.

[0309] Colorimetric results:

[0310] Protease : KLK5 (Speed ​​BioSystems); KLK7 (R&D systems) activated with enterokinase (2 hours, 37°C) prior to the assay; trypsin (Sigma Aldrich); chymotrypsin (Sigma Aldrich); Factor Xa (Haematologic Technologies); plasmin (Haematologic Technologies); KLK14 (R&D systems) activated with enterokinase (1 hour, 37°C) prior to the assay; thrombin (Haematologic Technologies); neutrophil elastase (Athens Research Technologies); and KLK1 (Prospec).

[0311] Compound 408 : The product of the 4-quinazolinone partial hydrolysis ring opening of compound 1.001 is represented by the following formula:

[0312]

[0313] The measurement results are shown in Table 3.

[0314] Table 3: Chromogenic assay results of compound 1.001 and compound 408.

[0315]

[0316]

[0317] S-2288, S-2266 and CS-PSA were from Diapharma and dissolved in water at 10 mM solution; Elastase substrate 1 was from Sigma and dissolved in water at 0.8 mM solution. a: IC50 concentration of KLK14 was 5 nM; b: IC50 concentration of KLK14 was 1 nM.

[0318] Conclusion: It can be concluded that compound 1.001 is an inhibitor of KLK5, KLK7 and chymotrypsin. In contrast, compound 408, which lacks the 4-quinazolinone moiety (i.e., the pharmacophore of compound 1.001), cannot inhibit proteases.

Claims

1. A compound represented by formula (I): , or a pharmaceutically acceptable salt thereof.

2. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that It is a compound represented by the following formula: .

3. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises a compound as described in the following formula, or a pharmaceutically acceptable salt thereof: and a pharmaceutically acceptable carrier.

4. The pharmaceutical composition according to claim 3, characterized in that The pharmaceutical composition comprises a compound described in the following formula: .

5. The pharmaceutical composition according to claim 3, characterized in that It is prepared for topical administration.

6. A use of a compound as shown in the following formula, or a pharmaceutically acceptable salt thereof, characterized in that: For the preparation of a pharmaceutical composition for treating a skin disease associated with the proteolytic activity of one or more KLK proteases: .

7. The use according to claim 6, characterized in that The pharmaceutical composition comprises a compound shown in the following formula: .

8. The use according to claim 6, characterized in that The skin disease is a hereditary skin disease.

9. The use according to claim 8, characterized in that The genetic skin disease is Netherton Syndrome.

10. The use according to claim 6, characterized in that The one or more KLK proteases are KLK5 and / or KLK7.

11. An in vitro non-therapeutic assay for determining the proteolytic activity of one or more KLK proteases, comprising the steps of: a) preparing a skin extract; b) reacting a substrate and a compound represented by the following formula, or a pharmaceutically acceptable salt thereof: exposing to said skin extract; and c) determining the rate of proteolytic cleavage of said substrate, in, The substrate is a peptide-p-nitroaniline selected from Tyr-Arg-Ser-Arg-pNA or Lys-His-Leu-Tyr-pNA.

12. The measuring method according to claim 11, characterized in that In step (b), a compound shown in the following formula is used: .

13. The measuring method according to claim 11, characterized in that The skin extract is a human skin extract.

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