Benzoxadinone compounds as KLK5 / 7 dual inhibitors

Abstract

The present invention provides compounds and pharmaceutical compositions comprising compounds for the treatment of skin disorders related to the proteolytic activity of one or more KLK proteases, wherein the compounds are represented by the following chemical formula (I): TIFF2022551583000068.tif41136, where R is as described herein.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Application No. 62 / 909,006, filed on 1 October 2019, which is incorporated herein by reference in its entirety for all purposes.

[0002] Description of the rights to inventions resulting from federally funded research and development. No results found.

[0003] Reference to the attached document for the "sequence listing," table, or computer program listing submitted on compact disc. No results found. [Background technology]

[0004] Background of the Invention Hereditary skin disorders are inherited diseases that manifest as skin conditions. These disorders are usually detectable unless immediately after birth. The type of disorder depends on how genes are affected, but they can have serious, rare, and significant impacts on the lives of patients, resulting in disability, shortened lifespan, and the development of other chronic diseases and cancers. There are approximately 400 different types of hereditary skin disorders, with incidence rates ranging from 1 in 6,000 to 1 in 500,000. The burden of the disease can be difficult for patients and their families, who may only have access to a very limited range of expensive medications that can help manage the condition. Hereditary skin disorders can be classified into monogenic disorders, where the skin disorder is caused by an abnormality in a single gene, and polygenic disorders, where several gene mutations (deletions) affect the type of disorder a patient experiences. Monogenic disorders include acute intermittent porphyria, epidermolysis bullosa, ichthyosis, Fabry disease, anhidrotic ectodermal dysplasia, and incontinence pigmenti. Genetic disorders associated with ichthyosis include Netherton syndrome.

[0005] Netherton syndrome (NS) is a rare autosomal recessive genetic skin disorder characterized by congenital ichthyosiform erythroderma, atopic predisposition, and a distinctive hair shaft abnormality known as trichorrhexis invaginata. Netherton syndrome is one of the most severe disorders of keratinization. Infants typically present with generalized scaling erythroderma at birth and are at high risk of life-threatening complications such as hypernatremic dehydration, stunted growth, and sepsis. Older children may present with widespread allergic symptoms, including severe atopic dermatitis, asthma, hay fever, and markedly elevated serum immunoglobulin E levels. In older children and adults, scaling may have a distinctive circular pattern (ichithyosis linearis circumflexa). However, in infants and young children, the skin is more typically red and scaly throughout the body, without the characteristic circular pattern. The hair shafts are short and sparse due to trichorrhexis invaginata, or "bamboo hair," which makes them brittle and easily broken. Another feature of Netherton syndrome is a predisposition to allergies, asthma, and eczema. Adults and children with Netherton syndrome are also predisposed to viral skin infections caused by herpes and human papillomavirus, in addition to bacterial skin infections and systemic infections. The incidence of Netherton syndrome is estimated at 1 in 200,000 births, and the prevalence is estimated at 1 to 9 in 1,000,000. Nevertheless, due to diagnostic challenges in infancy and early childhood, including overlapping features with atopic dermatitis and other recessive ichthyosis, regional studies suggest that the prevalence may be higher. Currently, there is no specific treatment for NS, and only palliative care is provided to manage skin infections and alleviate itching and pain.

[0006] NS is caused by truncated loss-of-function mutations in the serine protease inhibitor Cazal type 5 (SPINK5), the gene encoding LEKTI (lymphoepithelial Cazal type inhibitor). Loss of function in LEKTI results in uninhibited protease activity and overdigestion of epidermal structures and barrier proteins.

[0007] 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 an upstream initiator of KLK7. According to current hypotheses, pro-KLK is synthesized and activated in the granular layer, and the active KLK enzyme rapidly forms a complex with LEKTI, thereby preventing premature degradation of desmosomes at the stratum corneum / granular layer 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 to the outer layer of the stratum corneum, where the acidic microenvironment triggers the release of active KLK from LEKTI. Subsequently, the active KLK cleaves corneodesmosome proteins in the outermost layer of the stratum corneum. This ensures a sophisticated and balanced regulation of the desquamation process (Ovaere et al., Trends Biochem Sci 2009, 34:453-463).

[0008] Explosive ink5 - / - Mice reproduce NS disease (Descargues et al., Nat Genet 2005, 37:56-65; Hewett et al., Hum Mol Genet 2005, 14:335-346; Yang et al., Genes Dev 2004, 18:2354-2358). An important finding is that Spink5, as observed in NS subjects, is similar to that observed in NS subjects. - / -Mouse epidermis shows no inhibition of KLK5 and KLK7 protease activity (Descargues et al., Nat Genet 2005, 37:56-65).

[0009] In contrast, KLK5 deficiency is associated with Spink5 - / - Neonatal lethality in newborn mice is reversed, and skin characteristics of NS mice, including skin barrier defects, disordered epidermal structure, and skin inflammation, are reversed (Furio et al., PLoS Genet 2015, Sep; 11(9): e1005389). In particular, the loss of KLK5 leads to a decrease in epidermal proteolytic activity, especially KLK7 and KLK14. Subsequently, structural integrity and normal epidermal differentiation of desmosomes and cornodesmosomes are restored, as well as the normalized expression of IL-1β, IL-17A, and TSLP (thymic stromal lymphocyte neoplastic factor).

[0010] In addition to animal models, 3D organ cultures were generated in collagen gels containing normal human keratinocytes and fibroblasts transfected with SPINK5-targeted small interfering RNA (siRNA), and epidermal defects were observed in these culture models (Wang et al., Exp Dermatol 2014, Jul;23(7):524-6). Gene silencing of KLK5 or KLK7 significantly improved the impaired epidermal structure by reducing SPINK5 expression. In summary, these studies confirm the key roles of KLK5 and its upstream and downstream regulators in the NS.

[0011] KLK7 is expressed by keratinocytes in the granular layer and subsequently secreted into the extracellular space of the stratum corneum (i.e., the outermost layer of skin) as zymogen activated by KLK5. Once activated, KLK7 plays a central role in the skin's desquamation process. The proteolytic event of KLK7 is essential for desquamation and controlled skin regeneration. A tight balance between the generation of new keratinocytes and desquamation is essential for maintaining skin homeostasis. In contrast, an imbalance between these two processes leads to impaired skin function and ultimately causes skin diseases such as Netherton syndrome. Under normal conditions, this balance is maintained by the expression of an endogenous protein inhibitor of KLK7 (LEKTI), which counteracts its proteolytic activity. In skin disorders, overexpression and / or increased activity of KLK7 cause excessive desquamation. The involvement of KLK7 in the development of skin disorders is further supported by genetic associations in both animal models and humans. Transgenic mice that overexpress human KLK7 have been shown to express skin characteristics similar to those seen in patients with chronic atopic dermatitis, sharing certain features common to patients with Netherton syndrome.

[0012] In terms of the roles of KLK5 and KLK7 in regulating the desquamation process, and Spink5 - / - / Klk5 - / - / Klk7 - / - Combined with reports of complete phenotypic rescue from combinations of knockout model mice (Kasparek et al., PLoS Genet 2017, Jan; 13(1): e1006566), there is a need for potent and selective dual KLK5 / KLK7 inhibitors to treat hereditary skin disorders, such as Netherton syndrome, by regulating the proteolytic activity of these proteases. [Overview of the project]

[0013] Brief Overview of the Invention In one embodiment, the present invention is based on the following chemical formula (I): [ka] To provide a compound represented by the formula, or a pharmaceutically acceptable complex thereof, wherein R is H or -R 1 and -C(O)R 1a is a member selected from the group consisting of, where R 1 is C 1-12 alkyl, and R 1a is H or C 1-12 alkyl.

[0014] In a second aspect, the present invention provides a pharmaceutical composition comprising a compound of chemical formula (I) or a pharmaceutically acceptable complex thereof and a pharmaceutically acceptable carrier.

[0015] In a third aspect, the present invention provides a method of treating a skin disease related to the proteolytic activity of one or more KLK proteases in a subject that requires it. The method includes administering to the subject an effective amount of a compound of chemical formula (I) or a pharmaceutically acceptable complex thereof, or a pharmaceutical composition comprising a compound of chemical formula (I) or a pharmaceutically acceptable complex thereof.

[0016] In a fourth aspect, the present invention provides an in vitro assay for measuring the proteolytic activity of one or more KLK proteases in the skin. The method includes: 1) preparing a skin extract; 2) exposing a substrate and a compound of chemical formula (I) or a pharmaceutically acceptable complex thereof to the skin extract; and 3) measuring the protein cleavage rate of the substrate. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] [Figure 1] Figure 1 shows a scheme for preparing compound 1.001.

[0018] [Figure 2] Figure 2 shows a scheme for preparing compound 1.002.

[0019] [Figure 3] Figure 3 shows a scheme for preparing intermediate 100, which is used to prepare compounds 1.001 and 1.002.

[0020] [Figure 4A] Figures 4–4D show the protein cleavage rates of KLK5-selective substrates in skin extract assays with compounds 1.001, 1.002, or known compound 14. Figure 4A: Control; [Figure 4B] Figure 4B: Compound 14; [Figure 4C] Figure 4C: Compound 1.001; and [Figure 4D] Figure 4D: Compound 1.002. NKS refers to extracts from freshly prepared skin; and OSK refers to extracts from skin after freeze-thaw cycles. [Modes for carrying out the invention]

[0021] Detailed description of the invention I. Overview The present invention provides a compound of chemical formula (I) and a pharmaceutical composition comprising a compound of chemical formula (I) for the treatment of skin diseases associated with the proteolytic activity of one or more KLK proteases. In particular, the pharmaceutical composition may be a topical formulation comprising a compound of chemical formula (I) for topical use in the treatment of hereditary skin diseases. The present invention also provides a method for treating skin diseases associated with the proteolytic activity of one or more KLK proteases by administering a compound of chemical formula (I) or a pharmaceutical composition thereof. In particular, a compound of chemical formula (I) or a pharmaceutical composition thereof can be administered topically. Specifically, the skin disease is a hereditary skin disease such as Netherton syndrome.

[0022] II. Definition The abbreviations used herein have their conventional meanings among those skilled in the art of chemistry and biology.

[0023] "Alkyl" refers to a linear or branched, saturated, or aliphatic radical having the given number of carbon atoms (i.e., C1-12 ( means 1 to 12 carbon atoms). Alkyl means 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 5-6 , C 5-7 , C 5-8 , C 5-9 , 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 It can include any number of carbon atoms, such as C 1-12Alkyl 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, and n-dodecyl. Alkyl also refers to, but is not limited to, alkyl groups having up to 20 carbon atoms, such as tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, and octadecyl.

[0024] As used herein, “pharmaceutically acceptable complex” means a complex comprising a compound of chemical formula (I) and at least one complexing agent. The term “pharmaceutically acceptable” means that it is approved by a federal or state regulatory authority or is listed in the United States Pharmacopeia or other generally accepted pharmacopoeias for use in animals, and more specifically in humans. The complexing agent has the ability to form a complex with the compound of chemical formula (I) via non-covalent secondary interactions. Secondary interactions can be formed via electrostatic interactions such as ionic interactions, hydrogen bonds, dipole-dipole interactions, dipole-induced dipole interactions, London dispersion forces, π-π interactions, and hydrophobic interactions. A pharmaceutically acceptable complex of the compound of chemical formula (I) includes its pharmaceutically acceptable salt and / or solvate.

[0025] "Solvate" means a compound or salt thereof provided herein, further comprising a stoichiometric or non-stoichiometric amount of solvent bonded by non-covalent intermolecular forces. If the solvent is water, the solvate is a hydrate.

[0026] A "hydrate" refers to a compound that forms a complex with water molecules. The compounds of the present invention can form a complex with 1 / 2 water molecules or with 1 to 10 water molecules.

[0027] As used herein, "peptide-p-nitroanilide" or "peptide-pNA" refers to a peptide substrate having 4 to 6 amino acids linked by peptide bonds and a p-nitroanilide 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.

[0028] As used herein, “composition” is intended to include products containing a particular component in a particular amount, as well as any product obtained directly or indirectly from a particular combination of components in a particular amount. “Pharmacologically acceptable” means that the carrier, excipient, or additive must be compatible with the other components of the formulation and must not be harmful to its recipient.

[0029] A "pharmaceutically acceptable carrier or additive" refers to a substance that assists in the administration of an active agent to a target and its absorption by the target. Useful pharmaceutically acceptable additives in this invention include, but are not limited to, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavorings, and colorants. Useful pharmaceutically acceptable additives in this invention for transdermal / topical delivery include, but are not limited to, accelerators, solubilizers, antioxidants, plasticizers, thickeners, polymers, and pressure-sensitive adhesives. Those skilled in the art will recognize that other pharmaceutically acceptable additives are useful in this invention.

[0030] "I C 50 "In an assay measuring such a reaction, this refers to the amount, concentration, or dose of a specific test compound that achieves 50% inhibition of the maximum reaction.

[0031] "Inhibition," "to inhibit," and "inhibitor" refer to compounds or methods of inhibiting a particular action or function.

[0032] "Administering" refers to topical administration, such as lotions, sprays, ointments, creams, gels, pastes, or patches.

[0033] "Topical" means the application to the skin of a suitable compound (e.g., an active agent) or a composition containing a compound (e.g., an active agent) for treating a disease or condition, such as a hereditary skin disease. In some embodiments, "topical" means the application to the skin of a suitable compound (e.g., an active agent) or a composition containing a compound (e.g., an active agent) with sufficient penetration into the epidermis or dermis for treating a hereditary skin disease. In some embodiments of topical administration, the compound or composition penetrates into the epidermis or dermis without any intention of significant systemic exposure or the treatment or prevention of disease of other organ systems. In some embodiments of topical administration, the compound or composition is delivered transdermally through the skin for systemic distribution. Examples include transdermal patches used for drug delivery.

[0034] "Treatment," "to treat," and "treatment" mean any sign of success in treating or improving an injury, condition, or state, including any objective or subjective parameters such as: reduction of symptoms; remission; mitigation; or making the injury, condition, or state more tolerable to the patient; slowing the rate of degeneration or decline; preventing the terminal stage of degeneration from becoming debilitating; or improving the patient's physical and mental health. Treatment or improvement of symptoms may be based on objective or subjective parameters; including the results of a physical examination, neuropsychiatric examination, and / or psychiatric evaluation.

[0035] "Patient" or "Subject" means a living organism suffering from or prone to a disease or condition that can be treated by administration of the pharmaceutical compositions provided herein. Non-limiting examples include humans, other mammals, Bovidae, rats, mice, dogs, monkeys, goats, sheep, cattle, deer, and other non-mammals. In some embodiments, the subject is human.

[0036] "Therapeutic dose" refers to the amount of a useful compound or pharmaceutical composition that is effective in treating or improving an identified disease or condition, or that exhibits a detectable therapeutic or inhibitory effect. The amount extracted will depend on the therapeutic purpose and can be determined by those skilled in the art using known techniques (see, for example, Lieberman, Pharmaceutical Dosage Forms (vols. 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).

[0037] When "A," "an," or "a(n)" are used in reference to the group of substituents or "substituents" herein, they mean at least one. For example, if a compound is substituted with an "an" alkyl or aryl, the compound is optionally substituted with at least one alkyl and / or at least one aryl, where each alkyl and / or aryl is optionally different. In another example, if a compound is substituted with an "a" substituent, the compound is substituted with at least one substituent, where each substituent is optionally different.

[0038] III.Compound In one embodiment, the present invention is based on the following chemical formula (I): [ka] Provides a compound represented by or a pharmaceutically acceptable complex thereof; where R is H or -R 1 and -C(O)R 1a A member selected from the group consisting of R 1 is C 1-12 It is alkyl, and R 1ais H or C 1-12 It is alkyl.

[0039] In chemical formula (I), the methylsulfonyl (-SO2CH3) group can be in the ortho-, meta-, or para position relative to the 7,8-dihydro-4H-[1,4]dioxyno[2',3':4,5]benzo[1,2-d][1,3]oxazine-4-one moiety. In some embodiments, the -SO2CH3 group is in the ortho position relative to the 7,8-dihydro-4H-[1,4]dioxyno[2',3':4,5]benzo[1,2-d][1,3]oxazine-4-one moiety.

[0040] In chemical formula (I), the -CH2OR group can be in the ortho-, meta-, or para position relative to 7,8-dihydro-4H-[1,4]dioxyno[2',3':4,5]benzo[1,2-d][1,3]oxazine-4-one. In some embodiments, the -CH2OR group is in the meta position relative to 7,8-dihydro-4H-[1,4]dioxyno[2',3':4,5]benzo[1,2-d][1,3]oxazine-4-one. In some embodiments, the -CH2OR group is in the para position relative to 7,8-dihydro-4H-[1,4]dioxyno[2',3':4,5]benzo[1,2-d][1,3]oxazine-4-one.

[0041] In some embodiments, the compound is given by the following chemical formula (Ia): [ka] This is expressed as follows, where R is as defined and described in chemical formula (I).

[0042] In some embodiments of chemical formula (Ia), the -CH2OR group can be in the ortho-, meta-, or para position relative to the -SO2CH3 group. In some embodiments, the -CH2OR group is in the meta position relative to the -SO2CH3 group. In some embodiments, the -CH2OR group is in the para position relative to the -SO2CH3 group.

[0043] In some embodiments, the compound has the following chemical formula (Ib): [ka] This is expressed as follows, where R is as defined and described in chemical formula (I).

[0044] In some embodiments, the compound has the following chemical formula (Ic): [ka] This is expressed as follows, where R is as defined and described in chemical formula (I).

[0045] Referring to any one of the chemical formulas (I), (Ia), (Ib), and (Ic), in some embodiments, R is H.

[0046] In some embodiments, the compound has the following chemical formula: [ka] It is represented by [this].

[0047] In some embodiments, the compound has the following chemical formula: [ka] It is represented by [this].

[0048] Referring to any one of the chemical formulas (I), (Ia), (Ib), and (Ic), in some embodiments, R is -R 1 and -C(O)R 1a A member selected from the group consisting of R 1 is C 1-12 It is alkyl, and R 1a is H or C 1-12 It is alkyl.

[0049] In one embodiment of any one of Chemical Formulas (I), (Ia), (Ib), and (Ic), R is C 1-12 alkyl. 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 alkyl. 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 embodiments, R is methyl.

[0050] In some embodiments of any one of Chemical Formulas (I), (Ia), (Ib), and (Ic), R is -C(O)R 1a where R 1a is H or C 1-12 alkyl. In some embodiments, R 1a is H. In some embodiments, R 1a is C 1-12 alkyl. In 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. In some embodiments, R 1a is C<00​​​​​​R is methyl. In some embodiments, R is acetyl.

[0051] In some embodiments, the compound has the following chemical formula: [ka] It is represented by [this].

[0052] In some embodiments, the compound has the following chemical formula: [ka] It is represented by [this].

[0053] Exemplary compounds of chemical formula (I) are shown in Table 1.

[0054] [Table 1]

[0055] Compounds in other forms The compounds of the present invention may exist as complexes with at least one complexing agent. The complexing agent has the function of forming a complex structure with the compound of chemical formula (I) via non-covalent secondary interactions. Secondary interactions may be formed via electrostatic interactions such as ionic interactions, hydrogen bonds, dipole-dipole interactions, dipole-induced dipole interactions, London dispersion forces, π-π interactions, and hydrophobic interactions. A pharmaceutically acceptable complex of the compound of chemical formula (I) includes a pharmaceutically acceptable salt and / or solvate thereof. In some embodiments, a pharmaceutically acceptable complex of the compound of chemical formula (I) is its pharmaceutically acceptable salt and / or solvate. In some embodiments, a pharmaceutically acceptable complex of the compound of chemical formula (I) is its pharmaceutically acceptable salt. In some embodiments, a pharmaceutically acceptable complex of the compound of chemical formula (I) is its pharmaceutically acceptable solvate.

[0056] Certain compounds of the present invention can exist in solvated forms, including non-solvated and hydrated forms. Generally, the solvated forms are equivalent to the non-solvated forms and are included within the scope of the present invention. Certain compounds of the present invention can exist in multiple crystalline or amorphous forms. Generally, all physical forms are intended to be equivalent to the uses envisioned by the present invention and are within the scope of the present invention.

[0057] Unless otherwise stated, the compounds of the present invention may also contain unnatural proportions of atomic isotopes in one or more atoms constituting such compounds. For example, the compounds of the present invention may contain, for example, deuterium ( 2 H), tritium ( 3 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 They can be labeled with radioactive or stable isotopes such as C). All isotopic variations of the compounds of the present invention, whether radioactive or not, are included within the scope of the present invention.

[0058] The present invention provides compounds in the form of prodrugs. The prodrugs of the compounds described herein are compounds that readily undergo chemical changes under physiological conditions, thereby providing the compounds of the present invention. Furthermore, the prodrugs can be converted to the compounds of the present invention by chemical or biological methods in an ex vivo environment. For example, a prodrug can be slowly converted to the compounds of the present invention when placed in a transdermal patch reservoir with a suitable enzyme or chemical reagent.

[0059] IV. Composition In a second embodiment, the present invention provides a pharmaceutical composition comprising a compound of chemical formula (I) or a pharmaceutically acceptable complex thereof and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition comprises a compound of chemical formula (Ia) or a pharmaceutically acceptable complex thereof and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition comprises a compound of chemical formula (Ib) or a pharmaceutically acceptable complex thereof and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition comprises a compound of chemical formula (Ic) or a pharmaceutically acceptable complex thereof and a pharmaceutically acceptable carrier.

[0060] A pharmaceutically acceptable complex of any one compound of chemical formulas (Ia), (Ib), and (Ic) is defined and described herein. A pharmaceutically acceptable complex of any one compound of chemical formulas (I), (Ia), (Ib), and (Ic) may include a pharmaceutically acceptable salt and / or solvate thereof.

[0061] In some embodiments, one of the compounds of chemical formulas (I), (Ia), (Ib), and (Ic) is the following: [ka] Or it has a pharmaceutically acceptable complex.

[0062] In some embodiments, one of the compounds of chemical formulas (I), (Ia), (Ib), and (Ic) is the following: [ka] or having a pharmaceutically acceptable complex thereof.

[0063] The compounds provided herein can be formulated into pharmaceutical compositions using methods available to those skilled in the art and using those disclosed herein. Any compound disclosed herein can be provided in a suitable pharmaceutical composition and administered via a suitable route of administration.

[0064] The compounds described herein may be administered to a subject topically or non-systemically, for example, topically, intradermally, or intralesionally. In some embodiments, the compounds may be administered topically. In some embodiments, the compounds may be administered intradermally. In some embodiments, the compounds may be administered intralesionally, for example, by intralesional injection.

[0065] The methods provided herein involve administering a pharmaceutical composition comprising at least one compound described herein, preferably a compound of chemical formula (I), in the form of a salt or a complex, either alone or in combination with one or more compatible and pharmaceutically acceptable carriers, such as excipients or adjuvants, or in combination with other agents for the treatment of a genetic skin disease in which the subject requires it.

[0066] In certain embodiments, a second agent may be formulated or packaged together with the compound provided herein. Of course, the second agent may be formulated together with the compound provided herein only if, in the judgment of those skilled in the art, such a co-formulation should not interfere with the activity or method of administration of either agent. In certain embodiments, the compound provided herein and the second agent may be formulated separately. They may be packaged together or separately for the convenience of those skilled in the art.

[0067] In clinical practice, the active agents provided herein may be administered by any conventional route, particularly topically, intradermally, intralesionally, orally, parenterally, rectally, or by inhalation (e.g., in aerosol form). 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.

[0068] For oral administration, solid compositions such as tablets, pills, hard gelatin capsules, powders, or granules may be used. In these compositions, the active product is mixed with one or more inactive excipients or adjuvants, such as sucrose, lactose, or starch.

[0069] These compositions may include substances other than excipients, such as lubricants like magnesium stearate, or coating agents intended for release control.

[0070] For oral administration, pharmaceutically acceptable solutions, suspensions, emulsions, syrups, and elixirs containing water or an inert excipient such as liquid paraffin may be used as liquid compositions. These compositions may also contain substances other than excipients, in certain embodiments, wetting agents, sweeteners, or flavoring products.

[0071] The compositions may be used for topical administration as lotions, tinctures, creams, emulsions, gels, or ointments. In these compositions, the active product is mixed with one or more inert additives, including water, acetone, ethanol, ethylene glycol, propylene glycol, polyethylene glycol, butane 1,3-diol, isopropyl myristate, isopropyl palmitate, mineral oil, and mixtures thereof.

[0072] Compositions for parenteral, intrafocal, or intradermal administration may be emulsions or sterile solutions. Propylene glycol, polyethylene glycol, vegetable oils, particularly olive oil, or injectable organic esters, and in certain embodiments, ethyl oleate, may be used as solvents or vehicles. These compositions may also contain adjuvants, particularly wetting agents, isotonic agents, emulsions, dispersants, and stabilizers. Sterilization may be carried out by several methods, in certain embodiments by radiation or heating, using a bacteriological filter. They may also be prepared in the form of sterile solid compositions that can be dissolved in sterile water or any other injectable sterile medium at the time of use.

[0073] Compositions for rectal administration are suppositories or rectal capsules containing an active ingredient along with additives such as cocoa butter, semi-synthetic glycerides, or polyethylene glycol.

[0074] The composition may also be an aerosol. For use in liquid aerosol form, the composition may be a stable sterile solution or a solid composition dissolved in non-pyrogenic sterile water, saline solution or other pharmaceutically acceptable vehicle at the time of use. For use in dry aerosol form intended for direct inhalation, the active ingredient is finely divided and combined with a water-soluble solid diluent or vehicle, in certain embodiments, dextran, mannitol, or lactose.

[0075] In certain embodiments, the compounds provided herein are pharmaceutical compositions or single-unit dosage forms. Pharmaceutical compositions and single-unit dosage forms provided herein comprise one or more prophylactic or therapeutic agents (e.g., the compounds provided herein, or other prophylactic or therapeutic agents) in amounts effective for prevention or treatment, and typically one or more pharmaceutically acceptable carriers or excipients. In certain embodiments and in this context, the term “pharmaceutically acceptable” means approved by a federal or state government regulatory authority, or listed in the United States Pharmacopeia or other generally accepted pharmacopoeias for use in animals, more specifically in humans. The term “carrier” includes excipients, adjuvants (e.g., Freund’s adjuvants (complete and incomplete)), additives, or vehicles administered together with the therapeutic agent. Such pharmaceutical carriers can be sterile liquids such as water and oil, including those of petroleum, animal, plant, or synthetic origin, such as peanut oil, soybean oil, mineral oil, and sesame oil. When pharmaceutical compositions are administered intravenously, water can be used as a carrier. Saline solutions and aqueous glucose and glycerol 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).

[0076] Typical pharmaceutical compositions and dosage forms contain one or more additives. Suitable additives are well known to those skilled in the pharmaceutical art, and in certain embodiments, suitable additives include starch, glucose, lactose, sucrose, gelatin, malt, rice, wheat flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene, glycol, water, ethanol, and the like. Whether a particular additive is suitable for incorporation into a pharmaceutical composition or dosage form depends on various factors well known in the art, including, but not limited to, the method by which the dosage form will be administered to the target and the specific active ingredients in the dosage form. If desired, the composition or single-unit dosage form may also contain small amounts of wetting or emulsion agents or pH buffers.

[0077] The lactose-free compositions provided herein are well known to those skilled in the art and, in certain embodiments, may include additives listed in the United States Pharmacopeia (USP 36-NF 31 S2). Generally, lactose-free compositions contain pharmaceutically acceptable amounts of an active ingredient, a binder / filler, and a lubricant. An exemplary lactose-free dosage form contains an active ingredient, microcrystalline cellulose, pregelatinized starch, and magnesium stearate.

[0078] Since water can accelerate the decomposition of several compounds, this specification further encompasses anhydrous pharmaceutical compositions and dosage forms containing active ingredients. For example, the addition of water (e.g., 5%) is widely accepted in the pharmaceutical industry 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. Ed., Marcel Dekker, New York, 1995, pp. 379 80. In effect, water and heat accelerate the decomposition of several compounds. Therefore, the effect of water on a formulation can be very significant, as moisture and / or humidity are commonly encountered during the manufacture, handling, packaging, storage, shipping, and use of the formulation.

[0079] The anhydrous pharmaceutical compositions and dosage forms provided herein can be prepared using anhydrous or low-moisture-containing components and low-moisture or low-humidity conditions. Pharmaceutical compositions and dosage forms containing lactose and at least one active ingredient comprising a primary or secondary amine may be anhydrous if substantial contact with moisture and / or humidity is expected during manufacture, packaging, and / or storage.

[0080] Anhydrous pharmaceutical compositions should be prepared and stored in such a way that their anhydrous properties are maintained. Therefore, anhydrous compositions can be packaged using materials known to prevent exposure to water so that they can be included in appropriate formulation kits. In some embodiments, suitable packaging includes, but is not limited to, airtight-sealed foil, plastic, unit-dose containers (e.g., vials), blister packs, and strip packs.

[0081] Further provided are pharmaceutical compositions and dosage forms comprising one or more compounds that reduce the rate at which the active ingredient degrades. Such compounds, referred to herein as “stabilizers,” include, but are not limited to, antioxidants such as ascorbic acid, pH buffers, or salt buffers.

[0082] Pharmaceutical compositions and single-unit dosage forms can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, and the like. Oral formulations may include standard carriers such as mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, and magnesium carbonate. Such compositions and dosage forms will be contained together with an appropriate amount of carrier to provide a prophylactic or therapeutically effective amount, in particular embodiments, in a purified form, for appropriate administration to the subject. The formulation should be suitable for the mode of administration. In particular embodiments, the pharmaceutical composition or single-unit dosage form is sterile and suitable for administration to the subject, in particular embodiments for animal subjects such as mammalian subjects, and in particular embodiments for human subjects.

[0083] Pharmaceutical compositions are formulated to suit their intended route of administration. In certain embodiments, the route of administration includes, but is not limited to, parenteral, intradermal, subcutaneous, intramuscular, oral, buccal, sublingual, inhalation, intranasal, transdermal, topical, transmucosal, intratumoral, intra-synovial, and rectal administration. In certain embodiments, the route of administration is transdermal, topical, or intrafocal. In certain embodiments, the route of administration is non-systemic. In specific embodiments, the composition is formulated according to routine procedures as a pharmaceutical composition suitable for intravenous, subcutaneous, intramuscular, oral, intranasal, or topical administration to humans. In embodiments, the pharmaceutical composition is formulated according to routine procedures for subcutaneous administration to humans. Typically, a composition for intravenous administration is a solution in a sterile isotonic aqueous buffer. If necessary, the composition may also include a solubilizer and a local anesthetic such as a lignocaine to relieve pain at the injection site.

[0084] In certain embodiments, dosage forms include, but are not limited to, tablets; caplets; capsules such as soft-elastic gelatin capsules; cachets; lozenges; powders; suppositories; ointments; poultices; pastes; powders; bandages; creams; transdermal patches; solutions; patches; aerosols (e.g., nasal sprays or inhalants); gels; suspensions (e.g., aqueous or non-aqueous liquid suspensions, oil-in-water emulsions, or water-in-oil liquid emulsions), solutions, and liquid dosage forms suitable for oral or transmucosal administration to subjects; liquid dosage forms suitable for parenteral administration to subjects; and sterile solids (e.g., crystalline or amorphous solids) that can be redissolved to provide liquid dosage forms suitable for parenteral administration to subjects.

[0085] The composition, form, and type of dosage forms provided herein will typically vary depending on their intended use. In certain embodiments, a dosage form used for the initial treatment of a hereditary skin disease may contain a higher amount of one or more active ingredients than a dosage form used for the maintenance treatment of the same disorder or disease. Similarly, a parenteral dosage form may contain a lower amount of one or more active ingredients than an oral dosage form used for the treatment of the same disorder or disease. These and other ways in which specific dosage forms contained herein differ from one another will be readily apparent to those skilled in the art. See, for example, Remington: The Science and Practice of Pharmacy; Pharmaceutical Press; 22nd edition (September 15, 2012).

[0086] Generally, the components of a composition are provided in unit dosage forms, either separately or mixed together, and in certain embodiments, as dry lyophilized powder or water-free concentrate in sealed containers such as ampoules or pouches indicating the amount of active agent. When the composition is administered by infusion, it can be administered in an infusion bottle containing sterile pharmaceutical-grade water or saline. When the composition is administered by injection, ampoules of sterile water for injection or saline can be provided so that the components can be mixed before administration.

[0087] Typical dosage forms of the compounds provided herein, or pharmaceutically acceptable complexes, solvates, or hydrates thereof, are administered in a once-daily dose in the morning or in divided daily doses with meals, ranging from about 0.1 mg to about 1000 mg per day. Specific dosage forms may contain 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 the active compound.

[0088] Oral dosage form Pharmaceutical compositions suitable for oral administration may be presented in individual dosage forms such as tablets (e.g., chewable tablets), caplets, capsules, and liquids (e.g., flavored syrups), but are not limited to these. Such dosage forms may contain a predetermined amount of active ingredient and may be prepared by pharmaceutical methods well known to those skilled in the art. For general information, see Remington: The Science and Practice of Pharmacy; Pharmaceutical Press; 22nd edition (September 15, 2012).

[0089] In certain embodiments, the oral dosage form is solid and prepared with anhydrous components under anhydrous disease or disorder conditions, as described in detail herein. However, the range of compositions provided herein extends beyond anhydrous, solid oral dosage forms. Therefore, further forms are described herein.

[0090] Typical oral dosage forms are prepared by closely combining at least one excipient with the active ingredient(s) in the mixture, according to conventional pharmaceutical formulation techniques. Excipients can take a wide variety of forms depending on the desired formulation for administration. In certain embodiments, excipients suitable for use in oral liquid or aerosol dosage forms include, but are not limited to, water, glycols, oils, alcohols, fragrances, preservatives, and colorants. In certain embodiments, excipients suitable for use in solid oral dosage forms (e.g., powders, tablets, capsules, and caplets) include, but are not limited to, starch, sugars, microcrystalline cellulose, excipients, granulators, lubricants, binders, and disintegrants.

[0091] Due to their ease of administration, tablets and capsules present the most advantageous oral dosage forms, in which case solid excipients are used. If desired, tablets can be coated with standard aqueous or non-aqueous techniques. Such dosage forms can be prepared by any pharmaceutical method. Generally, pharmaceutical compositions and dosage forms are prepared by uniformly and closely mixing the active ingredient with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product into the desired presentation.

[0092] In certain embodiments, tablets can be prepared by compression or molding. Compressed tablets can be prepared by compressing an active ingredient, optionally mixed with additives in a free-flowing form such as powder or granules, using a suitable machine. Molded tablets can be produced by molding a mixture of powdered compounds moistened with an inert liquid excipient using a suitable machine.

[0093] In certain embodiments, additives that can be used in oral dosage forms include, but are not limited to, binders, fillers, disintegrants, and lubricants. Suitable binders for use in pharmaceutical compositions and dosage forms include, but are not limited to, corn starch, potato starch, or other starches, gelatin, natural and synthetic rubber such as acacia, sodium alginate, alginic acid, other alginates, tragacanth powder, guar gum, cellulose and its derivatives (e.g., ethylcellulose, cellulose acetate, carboxymethylcellulose calcium, carboxymethylcellulose sodium), polyvinylpyrrolidone, methylcellulose, pregelatinized starch, hydroxypropyl methylcellulose (e.g., Nos. 2208, 2906, 2910), microcrystalline cellulose, and mixtures thereof.

[0094] In some embodiments, fillers suitable for use in the 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, dextrate, kaolin, mannitol, silicic acid, sorbitol, starch, pregelatinized starch, and mixtures thereof. Binders or fillers in pharmaceutical compositions are typically present in about 50 to about 99 percent by weight of the pharmaceutical composition or dosage form.

[0095] In some embodiments, suitable forms of microcrystalline cellulose include, but are not limited to, materials marketed as AVIEL PH 101, AVIEL PH 103, AVIEL RC 581, AVIEL PH 105 (available from FMC Corporation, American Viscose Division, Avicel Sales, Marcus Hook, PA), and mixtures thereof. Specific binders include a mixture of microcrystalline cellulose and sodium carboxymethylcellulose marketed as AVIEL RC 581. Suitable anhydrous or low-moisture additives include AVIEL PH 103 (商標) and includes Starch 1500 LM.

[0096] Disintegrants are used in compositions to provide tablets that disintegrate when exposed to an aqueous environment. Tablets containing too much disintegrant may disintegrate during storage, while those containing too little may not disintegrate at the desired rate or under the desired conditions. Therefore, a sufficient amount of disintegrant, neither too much nor too little, should be used to form oral solid dosage forms, so as not to adversely alter the release of the active ingredient. The amount of disintegrant used varies depending on the type of formulation and is readily apparent to those skilled in the art. Typical pharmaceutical compositions contain about 0.5 to about 15 weight percent of disintegrant, specifically about 1 to about 5 weight percent.

[0097] Disintegrants that may be used in pharmaceutical compositions and dosage forms include, but are not limited to, agar, alginic acid, calcium carbonate, microcrystalline cellulose, croscarmellose sodium, crospovidone, polaritrin potassium, sodium starch glycolate, potato or tapioca starch, pregelatinized starch, other starches, clay, other algins, other celluloses, rubber, and mixtures thereof.

[0098] Lubricants that may be used in pharmaceutical compositions and dosage forms include, but are not limited to, calcium stearate, magnesium stearate, mineral oil, light mineral oil, glycerin, 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 laurylate, agar, and mixtures thereof. Additional lubricants, in certain embodiments, include syloid silica gel (MD, AEROSIL 200 manufactured by WRGrace Co. of Baltimore), synthetic silica solidification aerosol (TX, sold by Degussa Co. of Plano), CAB O SIL (MA, pyrogenic silicon dioxide product sold by Cabot Co. of Boston), and mixtures thereof. When not used at all, lubricants are typically used in amounts less than about 1 weight percent of the pharmaceutical composition or dosage form in which they are incorporated.

[0099] Delayed-release dosage form Active ingredients, such as compounds provided herein, can be administered by release control means or delivery devices well known to those skilled in the art. In certain embodiments, U.S. 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 are described, but are not limited thereto; each of them is incorporated herein by reference in whole. Such dosage forms can be used in certain embodiments to provide sustained release or controlled release of one or more active ingredients, using hydroxypropyl methylcellulose, other polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, liposomes, microspheres, or combinations thereof, to provide desired release profiles in various proportions. Suitable controlled-release formulations known to those skilled in the art, including those described herein, can be readily selected for use with the active ingredients provided herein. Therefore, what is included herein are, but are not limited to, single-unit dosage forms suitable for oral administration, such as tablets, capsules, gel capsules, and caplets, adapted for controlled release.

[0100] All controlled-release drugs share the common goal of improving upon the pharmacotherapy achieved by their non-controlled counterparts. Ideally, the use of optimally designed controlled-release formulations in medical treatment is characterized by the use of minimal drug substance to treat or control a disease or disorder in minimal time. Advantages of controlled-release formulations include extended drug activity, reduced administration frequency, and improved patient compliance. Furthermore, controlled-release formulations can be used to influence other characteristics such as onset time or drug blood levels, and thus may influence the occurrence of side effects (e.g., adverse effects).

[0101] Most controlled-release formulations are designed to release an initial, rapid dose of the drug (active ingredient) to produce the desired therapeutic effect, followed by a gradual and continuous release of other amounts of the drug to maintain this level of therapeutic or preventive effect over a long period. To maintain a constant level of this drug in the body, the drug must be released from the dosage form at a rate that is replaced by the amount of drug metabolized and excreted from the body. Controlled release of the active ingredient can be stimulated by various diseases or disorders, including but not limited to pH, temperature, enzymes, water, or other physiological disorders or compounds.

[0102] In certain embodiments, the drug may be administered by intravenous infusion, an implantable osmotic pump, a transdermal patch, liposomes, or other modes of administration. In certain embodiments, a pump may be used (see Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med. 321:574 (1989)). In another embodiment, a polymeric substance may be used. In yet another embodiment, a release control system may be placed at an appropriate site of the target as determined by those skilled in the art, i.e., requiring only a portion of the systemic dose (see, for example, Goodson, Medical Applications of Controlled Release, vol. 2, pp. 115-138 (1984)). Other release control systems are discussed in a review by Langer (Science 249:1527-1533 (1990)).The active ingredients are an outer polymer membrane insoluble in body fluids, such as polyethylene, polypropylene, ethylene / propylene copolymer, ethylene / ethyl acrylate copolymer, ethylene / vinyl acetate copolymer, silicone rubber, polydimethylsiloxane, neoprene rubber, chlorinated polyethylene, polyvinyl chloride, copolymer of vinyl chloride and vinyl acetate, vinylidene chloride, ethylene and propylene, ionomer polyethylene terephthalate, butyl rubber, epichlorohydrin rubber, ethylene / vinyl alcohol copolymer, ethylene / vinyl acetate / terpolymer, and ethylene / vinyl oxyethanol copolymer. The active ingredient can be dispersed in a matrix within a solid surrounded by polymers, such as hydrophilic polymers including polymethyl methacrylate, polybutyl methacrylate, plasticized or unplasticized polyvinyl chloride, plasticized nylon, plasticized polyethylene terephthalate, natural rubber, polyisoprene, polyisobutylene, polybutadiene, polyethylene, ethylene-vinyl acetate copolymer, silicone rubber, polydimethylsiloxane, silicone carbonate copolymer, hydrogels of acrylic and methacrylic acid esters, collagen, crosslinked polyvinyl alcohol, and crosslinked partially hydrolyzed polyvinyl acetate. Subsequently, the active ingredient diffuses through the outer polymer membrane, and the release rate is controlled. The proportion of the active ingredient in such a parenteral composition depends largely on its specific properties and the needs of the target.

[0103] Parenteral dosage form In some embodiments, parenteral dosage forms are provided herein. In some embodiments, parenteral dosage forms can be administered to a subject by various routes of administration, including, but not limited to, subcutaneous, intravenous (including bolus injection), intramuscular, and intra-arterial. In some embodiments, parenteral dosage forms can be administered to a subject by various routes of administration, including, but not limited to, topical, intradermal, or intrafocal. Because their administration typically circumvents the subject's natural defenses against contaminants, parenteral dosage forms are typically sterilized or in a sterilizable state before administration to the subject. In certain embodiments, parenteral dosage forms include, but are not limited to, solutions ready for injection, dried products ready to be dissolved or suspended in a pharmaceutically acceptable vehicle for injection, suspensions ready for injection, and emulsions.

[0104] Suitable vehicles that can be used to provide parenteral dosage forms are well known to those skilled in the art. In certain embodiments, suitable vehicles include, but are not limited to, water for injection (USP); aqueous vehicles such as sodium chloride injection, Ringer's injection, glucose injection, glucose and sodium chloride injection, and Ringer's lactate injection; water-miscible vehicles such as ethyl alcohol, polyethylene glycol, and polypropylene glycol; and non-aqueous vehicles such as corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate, among others.

[0105] Compounds that increase the solubility of one or more active ingredients disclosed herein may also be incorporated into parenteral dosage forms.

[0106] Transdermal, topical, and transmucosal dosage forms Transdermal, topical, and transmucosal dosage forms are also provided. These include, but are not limited to, eye drops, sprays, aerosols, creams, lotions, ointments, gels, solutions, emulsions, suspensions, or other dosage 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 ed., Lea & Febiger, Philadelphia (1985). Dosage forms suitable for oral mucosal tissue may be formulated as mouthwash or oral gel. Furthermore, transdermal dosage forms include "reservoir-type" or "matrix-type" patches, which, when applied to the skin and worn for a specific period, allow for the penetration of a desired amount of the active ingredient.

[0107] The term “pharmaceutically acceptable carrier” refers to a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, excipient, additive, solvent, or encapsulating material, that is involved in the transport or delivery of 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 is harmless to the patient. Suitable carriers (e.g., additives and excipients) and other materials that can be used to provide the transdermal, topical, and transmucosal dosage forms encompassed herein are well known to those skilled in the art of the pharmaceutical industry and depend on the specific tissue to which a given pharmaceutical composition or dosage form will be applied. With that in mind, typical carriers include, but are not limited to, water, acetone, ethanol, ethylene glycol, propylene glycol, polyethylene glycol, butane 1,3-diol, isopropyl myristate, isopropyl palmitate, mineral oil, and mixtures thereof, which form lotions, tinctures, creams, emulsions, gels, or ointments, and are non-toxic and pharmaceutically acceptable. In some embodiments, materials that can function 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 carboxymethylcellulose, ethylcellulose, and cellulose acetate; (4) tragacanth powder; (5) malt; (6) gelatin; (7) talc; (8) additives such as cocoa butter and suppository wax; (9) peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil, and (10) Oils such as soybean oil; glycols such as propylene glycol; (11) polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol; (12) esters such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) distilled water from which pyrogenic substances have been removed; (17) isotonic saline solution; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer; and (21) non-toxic, suitable substances used in other pharmaceutical formulations.Furthermore, humectants or moisturizers may be added to the pharmaceutical composition and dosage form as desired. Examples of such additional components are well known in the industry. See, for example, Remington: The Science and Practice of Pharmacy; Pharmaceutical Press; 22nd edition (September 15, 2012).

[0108] Depending on the specific tissue being treated, additional components may be used before, in combination with, or after treatment with the provided active ingredient. In certain embodiments, penetration enhancers may be used to aid in the delivery of the active ingredient to the tissue. Suitable penetration enhancers include, but are not limited to, acetone; various alcohols such as ethanol, oleyl, and tetrahydrofuryl; alkyl sulfoxides such as dimethyl sulfoxide; dimethylacetamide; dimethylformamide; polyethylene glycol; pyrrolidones such as polyvinylpyrrolidone; Kollidon grade (povidone, polyvidone); urea; and various water-soluble or insoluble sugar esters such as Tween® 80 (polysorbate 80) and Span 60 (sorbitan monostearate).

[0109] The pH of a pharmaceutical composition or dosage form, or the tissue to which the pharmaceutical composition or dosage form is applied, may be adjusted to improve the delivery of one or more active ingredients. Similarly, the polarity of a solvent carrier may be adjusted by modifying its ionic strength or tension to improve delivery. Compounds such as stearates may also be added to a pharmaceutical composition or dosage form to favorably alter the hydrophilicity or lipophilicity of one or more active ingredients and improve delivery. In this regard, stearates can function as a lipid vehicle for the formulation, as an emulsifier or surfactant, and as a delivery accelerator or penetration enhancer. Different complexes, salts, hydrates, or solvates of active ingredients can be used to further adjust the properties of the resulting composition.

[0110] Dosage and Unit Dosage Form In human treatment, physicians will determine the most appropriate pharmacokinetics based on whether the treatment is prophylactic or therapeutic, and on the age, weight, stage of disability or disease, and other specific factors of the person being treated. In certain embodiments, doses are approximately 1 to 1000 mg per day for adults, or approximately 5 to 250 mg per day or approximately 10 to 50 mg per day for adults. In certain embodiments, doses are approximately 5 to 400 mg per day or approximately 25 to 200 mg per day for adults. In certain embodiments, dose rates of approximately 50 to 500 mg per day are also intended.

[0111] In a further embodiment, a method is provided for treating a disease or disorder and / or a hereditary skin disorder in a subject in which the subject needs treatment, by administering a therapeutically or prophylactically effective amount of the compound or a pharmaceutically acceptable complex thereof provided herein to the subject in need. The amount of compound or composition that will be therapeutically or prophylactically effective in treating the disorder or one or more symptoms thereof will vary depending on the nature and severity of the disease or condition and the route through which the active ingredient is administered. The frequency and dose will also vary according to factors specific to each subject, depending on the particular therapy administered (e.g., therapeutic or prophylactic agent), the severity of the disorder, disease, or condition, the route of administration, and the subject's age, physical condition, weight, response, and past medical history. The effective dose can be estimated from dose-response curves obtained from in vitro or animal model test systems.

[0112] In certain embodiments, exemplary doses of a composition include milligrams or micrograms of the active compound per kilogram of the weight of the subject or sample (e.g., about 10 micrograms to about 50 milligrams per kilogram, about 100 micrograms to about 25 milligrams per kilogram, or about 100 micrograms to about 10 milligrams per kilogram). In the case of compositions provided herein, in certain embodiments, the dose administered to a subject is 0.140 mg / kg to 3 mg / kg of the subject's body weight, based on the weight of the active compound. In certain embodiments, the dose administered to a subject is (of) the weight of the subject. The dosages are approximately 5 mg / kg, 10 mg / kg, 15 mg / kg, 25 mg / kg, 30 mg / kg, 35 mg / kg, 40 mg / kg, 45 mg / kg, or 50 mg / kg.

[0113] In certain embodiments, the recommended daily dose range of the compositions provided herein for the diseases or disorders described herein is in the range of about 0.1 mg to about 1000 mg per day, given as a single dose once daily or as divided doses throughout the day. In certain embodiments, the daily dose is administered twice daily 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 between about 10 mg to about 150 mg per day, and in further embodiments between about 25 mg to about 100 mg per day. As will be apparent to those skilled in the art, in some examples it may be necessary to use dosages of the active ingredient outside the range disclosed herein. Furthermore, it should be noted that the clinician or treating physician will know how and when to interrupt, adjust, or discontinue treatment in relation to the response to the target.

[0114] As will be readily apparent to those skilled in the art, different therapeutically effective doses can be adapted to different diseases and conditions. Similarly, doses sufficient to prevent, manage, treat or improve such diseases, but not sufficient to cause, or reduce, the side effects associated with the compositions provided herein, are also included by the dosage and administration schedules described herein. Furthermore, if a subject is administered multiple doses of the compositions provided herein, all doses do not need to be the same. In certain embodiments, the dose administered to a subject may be increased to improve the prophylactic or therapeutic effect of the composition, or decreased to mitigate one or more side effects experienced by a particular subject.

[0115] In certain embodiments, based on the weight of the active compound, the dosage of the composition provided herein administered to prevent, treat, manage, or improve a disorder or one or more symptoms thereof in a subject 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 or more of the subject's body weight. In other embodiments, the dosage of the composition or composition provided herein administered to prevent, treat, manage, or improve a disorder or one or more symptoms thereof in a subject is 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, or 0.1 mg to 7.5 mg. The unit doses are g, 0.1mg-5mg, 0.1-2.5mg, 0.25mg-20mg, 0.25-15mg, 0.25-12mg, 0.25-10mg, 0.25mg-7.5mg, 0.25mg-5mg, 0.5mg-2.5mg, 1mg-20mg, 1mg-15mg, 1mg-12mg, 1mg-10mg, 1mg-7.5mg, 1mg-5mg, or 1mg-2.5mg.

[0116] In certain embodiments, treatment or prevention may be initiated with one or more loading doses of the compounds or compositions provided herein, followed by one or more maintenance doses. In such embodiments, the loading dose may be, for example, about 60 to about 400 mg per day, or about 100 to about 200 mg per day, for a period of 1 to 5 weeks. The loading dose may be followed by one or more maintenance doses. In certain embodiments, each maintenance dose may be independently between about 10 mg to about 200 mg per day, between about 25 mg to about 150 mg per day, or between about 25 mg to about 80 mg per day. The maintenance doses may be administered daily and may be administered as a single dose or as divided doses.

[0117] In certain embodiments, the dose of the compound or composition provided herein may be administered to achieve a steady-state concentration of the active ingredient in the blood or serum of the subject. The steady-state concentration may be determined by measurement by techniques available to those skilled in the art, or it may be based on the subject's physical characteristics such as height, weight, and age. In certain embodiments, a sufficient amount of the compound or composition provided herein is administered to achieve a steady-state concentration in the blood or serum of the subject of about 300 to about 4000 ng / mL, about 400 to about 1600 ng / mL, or about 600 to about 1200 ng / mL. In certain embodiments, a sufficient amount of the compound or composition provided herein is administered topically to achieve a steady-state concentration in the blood or serum of the subject 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. In some embodiments, the onboard dose may be administered to achieve a steady-state blood or serum concentration of approximately 1200–8000 ng / mL, or approximately 2000–4000 ng / mL, for 1–5 days. In some embodiments, the onboard dose may be administered topically to achieve a steady-state blood or serum concentration of approximately 0.05–1200 ng / mL, approximately 0.05–100 ng / mL, approximately 0.05–10 ng / mL, approximately 0.05–10 ng / mL, approximately 0.05–1 ng / mL, approximately 0.05–0.5 ng / mL, or approximately 0.05–0.1 ng / mL for 1–5 days. In certain embodiments, the maintenance dose may be administered to achieve a steady-state concentration in the target blood or serum of approximately 300–4000 ng / mL, approximately 400–1600 ng / mL, or approximately 600–1200 ng / mL. In certain embodiments, the maintenance dose may be administered to achieve a steady-state concentration in the subject's blood or serum of approximately 0.01 to 300 ng / mL, 0.01 to 100 ng / mL, 0.01 to 10 ng / mL, 0.01 to 1 ng / mL, 0.01 to 0.1 ng / mL, or 0.01 to 0.05 ng / mL.

[0118] In certain embodiments, the administration of the same composition may be repeated, and such administrations may be spaced 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 apart. In other embodiments, the administration of the same prophylactic or therapeutic agent may be repeated, and such administrations may be spaced 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 apart.

[0119] In certain embodiments, what is provided herein is a unit dosage form comprising a compound, or a pharmaceutically acceptable complex thereof, in a form suitable for administration. Such forms are described in detail herein. In certain embodiments, the unit dosage form contains 1 to 1000 mg, 5 to 250 mg, or 10 to 50 mg of the active ingredient. In certain embodiments, the unit dosage form contains about 1, 5, 10, 25, 50, 100, 125, 250, 500, or 1000 mg of the active ingredient. Such unit dosage forms can be prepared by techniques familiar to those skilled in the art.

[0120] Dosage may vary depending on the dosage form used and the route of administration. For any compound, the therapeutically effective dose can be initially estimated from cell culture assays. The dose is determined in cell culture systems in lesional skin (e.g., Netherton syndrome or KLK protease-mediated skin diseases). 50 In animal models, the drug may be formulated to achieve a level including (i.e., the concentration of the test compound that achieves median inhibition of symptoms). Such information can be used to more accurately determine an effective dose in humans. Furthermore, to confirm systemic exposure, the level in plasma can be measured, for example, by high-performance liquid chromatography.

[0121] It should also be understood that specific dosages and treatment regimens for any particular patient will depend on a variety of factors, including the activity of the specific compound used, age, size and number of lesions, general health, sex, diet, administration time, drug combinations, and the judgment of the treating physician and the severity of the specific disease being treated. The amount of the compound of chemical formula (I) described herein in a composition will also depend on the specific compound of chemical formula (I) in the composition.

[0122] In some embodiments, the local dose is approximately 0.01 μg / cm³. 2 , about 0.05μg / cm 2 , about 0.1μg / cm 2 , about 0.15μg / cm 2 , about 0.2μg / cm 2 , about 0.3μg / cm 2 , about 0.4μg / cm 2 , about 0.5μg / cm 2 , about 0.6μg / cm 2 , about 0.7μg / cm 2 , about 0.8μg / cm 2 , or approximately 0.9 μg / cm³ 2 It is; or approximately 0.01-0.03 μg / cm³ 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 90~100μg / cm 2, about 100~125μg / cm 2 , about 125~150μg / cm 2 , about 150~175μg / cm 2 , about 175~200μg / cm 2 , about 200~250μg / 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 approximately 950-1000 μg / cm³ 2 It is within.

[0123] In some embodiments, the local dose is approximately 0.5–1.0 mg / cm³. 2 , 1.0~1.5 mg / cm³ 2 , 1.5~2.0 mg / cm³ 2 , 2.5~2.5 mg / cm³ 2 , 3.0~3.5 mg / cm³ 2 3.5~5.0 mg / cm³ 2 5.0~7.5 mg / cm³ 2 7.5~10 mg / cm³ 2 , 1-10 mg / cm³ 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 / cm2 , 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 approximately 950-1000 mg / cm³ 2 It is within.

[0124] V. Method In a third embodiment, the Specified provides a method for treating a skin disease relating to the proteolytic activity of one or more KLK proteases, where the subject requires it. The method comprises administering to the subject a therapeutically effective amount of a compound of chemical formula (I) or a pharmaceutically acceptable complex thereof, or a pharmaceutical composition comprising a compound of chemical formula (I) or a pharmaceutically acceptable complex thereof.

[0125] In some embodiments, the compound of chemical formula (I) has the following chemical formula: [ka] or having a pharmaceutically acceptable complex thereof.

[0126] In some embodiments, the compound of chemical formula (I) has the following chemical formula: [ka] or having a pharmaceutically acceptable complex thereof.

[0127] A pharmaceutically acceptable complex of the compound of chemical formula (I) is defined or described herein. A pharmaceutically acceptable complex of the compound of chemical formula (I) may include a pharmaceutically acceptable salt and / or solvate thereof.

[0128] In some embodiments, the skin disease is a hereditary skin disease. In some embodiments, the hereditary skin disease is Netherton syndrome.

[0129] In some embodiments, the subject has truncated loss-of-function mutations in the serine protease inhibitor Cazal type 5 (SPINK5). In some embodiments, the subject has LEKTI (lymphoid epithelial cell Cazal type inhibitor) loss-of-function. In some embodiments, the subject has overexpression of one or more kallikrein (KLK) serine proteases. In some embodiments, the subject has overexpression of one or more KLK5, KLK7, and KLK14 proteases. In some embodiments, the subject has overexpression of KLK5 protease. In some embodiments, the subject has overexpression of KLK7 protease. In some embodiments, the subject has overexpression of KLK14 protease. In some embodiments, the subject has overexpression of KLK5 and KLK7 proteases. In some embodiments, the subject has overexpression of KLK5, KLK7, and KLK14 proteases. In some embodiments, the subject has increased proteolytic activity of one or more kallikrein (KLK) serine proteases. In some embodiments, the subject has increased proteolytic activity of one or more KLK5, KLK7, and KLK14 proteases. In some embodiments, the subject has increased proteolytic activity of KLK5 protease. In some embodiments, the subject has increased proteolytic activity of KLK7 protease. In some embodiments, the subject has increased proteolytic activity of KLK14 protease. In some embodiments, the subject has increased proteolytic activity of KLK5 and KLK7 proteases. In some embodiments, the subject has increased proteolytic activity of KLK5, KLK7, and KLK14 proteases.

[0130] In some embodiments, this specification provides a method for treating Netherton syndrome if the subject requires it. The method comprises administering to the subject a therapeutically effective amount of a compound of chemical formula (I) or a pharmaceutically acceptable complex thereof, or a pharmaceutical composition comprising a compound of chemical formula (I) or a pharmaceutically acceptable complex thereof.

[0131] In some embodiments, this specification provides a method for treating a hereditary skin disease in which a subject requires it and the hereditary skin disease is mediated by one or more KLK proteases. The method comprises administering to a subject a therapeutically effective amount of a compound of chemical formula (I) or a pharmaceutically acceptable complex thereof, or a pharmaceutical composition comprising chemical formula (I) or a pharmaceutically acceptable complex thereof. In some embodiments, the hereditary skin disease is mediated by KLK5 protease, KLK7 protease, KLK14 protease, or a combination thereof. In some embodiments, the hereditary skin disease is mediated by KLK5 protease. In some embodiments, the hereditary skin disease is mediated by KLK7 protease. In some embodiments, it is mediated by 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 disorder is Netherton syndrome.

[0132] In some embodiments, this specification provides a method for treating a hereditary skin disease 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 chemical formula (I) or a pharmaceutically acceptable complex thereof, or a pharmaceutical composition comprising a compound of chemical formula (I) or a pharmaceutically acceptable complex thereof. In some embodiments, the compound of chemical formula (I) inhibits the proteolytic activity of one or more KLK proteases. In some embodiments, the compound of chemical formula (I) inhibits the proteolytic activity of KLK5 protease, KLK7 protease, KLK14 protease, or a combination thereof. In some embodiments, the compound of chemical formula (I) inhibits the proteolytic activity of KLK5 protease. In some embodiments, the compound of chemical formula (I) inhibits the proteolytic activity of KLK7 protease. In some embodiments, the compound of chemical formula (I) inhibits the proteolytic activity of KLK14 protease. In some embodiments, the compound of chemical formula (I) inhibits the proteolytic activity of KLK5 and KLK7 proteases. In some embodiments, the compound of chemical formula (I) inhibits the proteolytic activity of KLK5, KLK7, and KLK14 proteases. In some embodiments, the compound of chemical formula (I) is a KLK5 inhibitor. In some embodiments, the compound of chemical formula (I) is a KLK7 inhibitor. In some embodiments, the compound of chemical formula (I) is a KLK14 inhibitor. In some embodiments, the compound of chemical formula (I) is a dual KLK5 / KLK7 inhibitor. In some embodiments, the hereditary skin disorder is Netherton syndrome.

[0133] In some embodiments, this specification provides 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 dose of a compound of chemical formula (I) or a pharmaceutically acceptable complex thereof, or a pharmaceutical composition comprising a compound of chemical formula (I) or a pharmaceutically acceptable complex thereof. In some embodiments, the compound of chemical formula (I) inhibits the proteolytic activity of one or more KLK proteases. In some embodiments, the compound of chemical formula (I) inhibits the proteolytic activity of KLK5 protease, KLK7 protease, KLK14 protease, or a combination thereof. In some embodiments, the compound of chemical formula (I) inhibits the proteolytic activity of KLK5 protease. In some embodiments, the compound of chemical formula (I) inhibits the proteolytic activity of KLK7 protease. In some embodiments, the compound of chemical formula (I) inhibits the proteolytic activity of KLK14 protease. In some embodiments, the compound of chemical formula (I) inhibits the proteolytic activity of KLK5 and KLK7 proteases. In some embodiments, the compound of chemical formula (I) inhibits the proteolytic activity of KLK5, KLK7, and KLK14 proteases. In some embodiments, the compound of chemical formula (I) is a KLK5 inhibitor. In some embodiments, the compound of chemical formula (I) is a KLK7 inhibitor. In some embodiments, the compound of chemical formula (I) is a KLK14 inhibitor. In some embodiments, the compound of chemical formula (I) is a dual KLK5 / KLK7 inhibitor.

[0134] The compounds of the present invention are useful for protecting the skin by inhibiting the proteolytic activity of one or more KLK proteases and promoting the development of healthy skin. Accordingly, in a further embodiment, the present invention provides a method for treating a skin disease in which a subject needs it and the skin disease is mediated by one or more KLK proteases. The method comprises administering to a subject a therapeutically effective amount of a compound of chemical formula (I) or a pharmaceutically acceptable complex thereof, or a pharmaceutical composition comprising a compound of chemical formula (I) or a pharmaceutically acceptable complex thereof. In some embodiments, the skin disease is mediated by KLK5 protease, KLK7 protease, KLK14 protease, or a combination thereof. In some embodiments, the skin disease is mediated by KLK5 protease. In some embodiments, the skin disease is mediated by KLK7 protease. In some embodiments, the skin disease is mediated by KLK14 protease. In some embodiments, the skin disease is mediated by both KLK5 and KLK7 proteases. In some embodiments, the disease is mediated by KLK5, KLK7, and KLK14 proteases. In some embodiments, the skin disease is a KLK-mediated disease selected from the group consisting of Netherton syndrome, peeling skin syndrome, rosacea, psoriasis, eczema, and atopic dermatitis. In some embodiments, the KLK-mediated disease is Netherton syndrome.

[0135] In some embodiments, this specification provides a method for treating a target skin disease by inhibiting the proteolytic activity of one or more KLK proteases. The method comprises administering an effective amount to a target a compound of chemical formula (I) or a pharmaceutically acceptable complex thereof, or a pharmaceutical composition containing a compound of chemical formula (I) or a pharmaceutically acceptable complex thereof. In some embodiments, the compound of chemical formula (I) inhibits the proteolytic activity of one or more KLK proteases. In some embodiments, the compound of chemical formula (I) inhibits the proteolytic activity of KLK5 protease, KLK7 protease, KLK14 protease, or a combination thereof. In some embodiments, the compound of chemical formula (I) inhibits the proteolytic activity of KLK5 protease. In some embodiments, the compound of chemical formula (I) inhibits the proteolytic activity of KLK7 protease. In some embodiments, the compound of chemical formula (I) inhibits the proteolytic activity of KLK14 protease. In some embodiments, the compound of chemical formula (I) inhibits the proteolytic activity of KLK5 and KLK7 proteases. In some embodiments, the compound of chemical formula (I) inhibits the proteolytic activity of KLK5, KLK7, and KLK14 proteases. In some embodiments, the compound of chemical formula (I) is a KLK5 inhibitor. In some embodiments, the compound of chemical formula (I) is a KLK7 inhibitor. In some embodiments, the compound of chemical formula (I) is a KLK14 inhibitor. In some embodiments, the compound of chemical formula (I) is a dual KLK5 / KLK7 inhibitor. In some embodiments, the skin disease is selected from the group consisting of Netherton syndrome, peeling skin syndrome, rosacea, psoriasis, eczema, and atopic dermatitis. In some embodiments, the skin disease is Netherton syndrome.

[0136] Compounds of chemical formula (I) or pharmaceutical compositions of the present invention, including a compound of chemical formula (I), can be used for both prophylactic and therapeutic treatment of a target skin disease or condition, or other undesirable skin condition. For example, compositions of the present invention can be used for the prevention of skin irritation, prevention of rashes, promotion of healing of skin tissue after a rash or irritation has occurred (i.e., building up the epidermis and / or dermis layers of the skin), prevention and / or delay of skin atrophy, prevention and / or delay of the appearance of spider veins and / or red spots on the skin, prevention and / or delay of skin peeling, prevention and / or relief of skin itching, modification of skin texture (e.g., improvement of peeling, roughness, swelling or pain), and improvement of skin color (e.g., reduction of redness).

[0137] Preventive or therapeutic treatment of hereditary skin diseases or skin conditions in a subject can be carried out by applying a compound of chemical formula (I) or a pharmaceutical composition thereof, preferably in the form of a skin lotion, cream, gel, foam, ointment, paste, emulsion, spray, conditioner, tonic, patch, etc., which is intended to remain on the skin. After application of the composition to the skin, it may remain 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.

[0138] The compounds of the present invention are useful for regulating desquamation in vitro by inhibiting the protease activity of one or more KLK proteases in cell / tissue culture procedures. Accordingly, in a further embodiment, the present invention provides a method for degrading cells or tissues in vitro, the method comprising the steps of exposing cells or tissues to a KLK protease (e.g., KLK5, KLK7 and / or KLK14), followed by exposure to a compound of the present invention's chemical formula (I) or a pharmaceutically acceptable complex thereof.

[0139] This method is understood to be generally applicable to cell culture procedures, and can be used to dissociate and detach cells for passaging. Furthermore, this method can be used in the culture of artificial skin, where selective cleavage of cell-cell adhesion proteins by KLK proteases (e.g., KLK5, KLK7, and / or KLK14) in binding, with regulation of such cleavage by compounds of chemical formula (I) and their pharmaceutically acceptable complexes, results in cells / tissues more suitable for subsequent therapeutic applications.

[0140] In another embodiment, this specification provides an in vitro assay for measuring the proteolytic activity of one or more KLK proteases in skin. The method comprises: 1) a method for preparing a skin extract; 2) a method for exposing the skin extract to a substrate and a compound of chemical formula (I) or a pharmaceutically acceptable complex thereof; and 3) a method for measuring the proteolytic rate of the substrate. In some embodiments, the substrate is a peptide-p-nitroanilide (also called peptide-pNA). In some embodiments, the substrate is a dipeptide-pNA, tripeptide-pNA, tetrapeptide-pNA, pentapeptide-pNA, or hexapeptide-pNA. In some embodiments, the substrate is a tetrapeptide-pNA. In some embodiments, the substrate is Tyr-Arg-Ser-Arg-pNA or Lys-His-Leu-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 may be a human skin extract or an animal skin extract (e.g., mouse). In some embodiments, the skin extract is a human skin extract. [Examples]

[0141] VI. Examples General synthesis methods Where used herein, the symbols and conventions used in these steps, schemes, and examples, whether or not specific abbreviations are specifically defined, are consistent with those used in modern scientific literature, such as the Journal of the American Chemical Society or the Journal of Biological Chemistry. Specifically, the following abbreviations may, but are not limited to, be used throughout the examples and specification: :eq. (equivalent); g (gram); mg (milligram); mL (milliliters); μL (microliters); mM (millimolar); μM (micromolar); Hz (hertz); MHz (megahertz); mmol (millimole); 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); Â (ethyl acetic acid); and MeOH (methanol).

[0142] All the following examples can utilize standard work-up and purification methods known to those skilled in the art. Unless otherwise stated, all temperatures are expressed in degrees Celsius (°C). Unless otherwise stated, all reactions are carried out at room temperature. The synthetic methodologies illustrated herein are intended to illustrate the applicable chemistry through the use of specific examples and do not imply the scope of this disclosure.

[0143] Example 1: 2-(4-(hydroxymethyl)-2-(methylsulfonyl)phenyl)-7,8-dihydro-4H-[1,4]dioxyno[2',3':4,5]benzo[1,2-d][1,3]oxazin-4-one (compound 1.001) [ka] The synthesis of compound 1.001 is shown in Figure 1. The synthesis of intermediate 100 is explained in Figure 3.

[0144] Step 1: 4-Bromo-2-mercaptobenzoic acid (intermediate 102) [ka] A mixture of concentrated HCl (280.0 mL) and ice-cold water (280.0 mL) was slowly added to a stirred solution of 2-amino-4-bromobenzoic acid (101) (200.0 g, 926.0 mmol, 1.0 equivalent), NaOH (37.0 g, 926.0 mmol, 1.0 equivalent), and sodium nitrite (63.90 g, 926.0 mmol, 1.0 equivalent) in DMF (4.0 L) at a rate that maintained the reaction temperature at 3-6°C. After the addition, the reaction mixture was stirred at 0°C for 30 minutes, and then neutralized with potassium acetate (454.0 g, 4.63 mol, 5.0 equivalent). This solution was added to a preheated solution of potassium O-ethylxanthate (445.0 g, 2.78 mol, 3.0 equivalent) in DMW (4.0 L) at 90°C. The reaction mixture was stirred at 90°C for 30 minutes, cooled to 0°C, and acidified with concentrated HCl (600.0 mL). The reaction mixture was basicized with 20% NaOH (1750 mL) and heated at 85°C for 2 hours. NaHSO3 (96.30 g, 926.0 mmol, 1.0 equivalent) was added little by little to this mixture, and the reaction mixture was heated at 85°C for 10 minutes. The reaction mixture was filtered, cooled to 0°C, and acidified with concentrated HCl (650.0 mL). The precipitated solid was recovered by filtration, washed with DMF (500 mL), then washed with n-hexane, and air-dried to obtain intermediate 102 (216.0 g, 100.0%) as a light gray 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 / z232.8(M-1).

[0145] Step 2: Methyl 4-bromo-2-(methylthio)benzoate (intermediate 103) [ka] Potassium carbonate (896.0 g, 6.49 mol, 7.0 equivalents) and methyl iodide (231.0 mL, 3.71 mol, 4.0 equivalents) were added to a stirred solution of 4-bromo-2-mercaptobenzoic acid (102) (216.0 g, 927.0 mol, 1.0 equivalent) in DMF (3.24 L) at room temperature. The reaction mixture was heated to 80°C and stirred at 80°C for 20 hours. The progress of the reaction was monitored by TLC (10% ethyl acetate in n-hexane) to confirm 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 ethyl acetate extract was washed with DMW (2 x 2.5 L) and brine (2.5 L), dried on sodium sulfate, filtered, and the filtrate was concentrated under vacuum to obtain 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).

[0146] Step 3: Methyl 4-bromo-2-(methylsulfonyl)benzoate (intermediate 104) [ka] A solution of oxone (1.17 kg, 3.79 mol, 5.50 equivalents) in DMW (4.50 L) was added at 0°C to a stirred solution of methyl 4-bromo-2-(methylthio)benzoate (103) (180.0 g, 589 mmol, 1.0 equivalent) in methanol (3.60 L). The reaction mixture was stirred at 60°C for 16 hours. The progress of the reaction was monitored by TLC (30% ethyl acetate in n-hexane). Approximately 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 obtain wet product 104. The wet product was dried in a vacuum oven below 50°C to obtain dried intermediate 104 (200 g, 99.0%) as an off-white solid. 1HNMR (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 / z311.5(M+18).

[0147] Step 4: 4-Bromo-2-(methylsulfonyl)benzoic acid (intermediate 105) [ka] A solution of NaOH (192.0 g, 4.80 mol, 7.0 equivalents) in water (2.01 L) was added to methyl 4-bromo-2-(methylsulfonyl)benzoate (104) (201.0 g, 0.68 mol, 1.0 equivalent) in methanol (2.01 L). The reaction mixture was stirred at room temperature for 3.0 hours. 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 obtain intermediate 105 (155.0 g, 81.0%) as an off-white solid. 1 HNMR(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 / z278.8(M-1)

[0148] Step 5: 4-Bromo-2-(methylsulfonyl)benzoyl chloride (intermediate 106) [ka] Thionyl chloride (68.4 mL, 940.0 mmol, 2.50 equivalents) was added to a stirred solution of 4-bromo-2-(methylsulfonyl)benzoic acid in THF (1.05 L), followed by the addition of DMF (1.37 mL, 18.80 mmol, 0.05 equivalents) at room temperature. The reaction mixture was heated to 80°C and stirred for 2 hours. The progress of the reaction was monitored by TLC (50% siRNA in n-hexane) to confirm completion of the reaction. The solvent was removed under vacuum to obtain intermediate 106 (112.0 g). The crude intermediate 106 was used directly in step 6.

[0149] Step 6: 7-(4-bromo-2-(methylsulfonyl)benzamide)-2,3-dihydrobenzo[b][1,4]dioxin-6-carboxylic acid (intermediate 107) [ka] Triethylamine (103.0 mL, 753.0 mmol, 2.0 equivalents) was added at 0°C to a stirred solution of 7-amino-2,3-dihydrobenzo[b][1,4]dioxin-6-carboxylic acid (100) (60.20 g, 309.0 mmol, 0.82 equivalents) in THF (1.68 L), and then 4-bromo-2-(methylsulfonyl)benzoyl chloride (106) (60.20 g, 309 mmol, 1.0 equivalent) in THF (1.68 L) was added at 0°C. The reaction mixture was stirred at 0°C for 10 minutes, and then at room temperature for 2.5 hours. The progress of the reaction was monitored by TLC (10% MeOH in DCM). The pH was adjusted to 7 by adding 1N HCl. Approximately 70% of the THF was removed by distillation, and the remaining residue was ground with diethyl ether (250 mL) to precipitate the solid product. The solid was recovered by filtration, washed with diethyl ether (50 mL), and dried to obtain intermediate 107 (120.0 g, 69.90%) as a light brown solid.

[0150] Intermediate 100, 7-amino-2,3-dihydrobenzo[b][1,4]dioxin-6-carboxylic acid, was synthesized via steps A to E as described herein.

[0151] Step A: Methyl 3,4-dihydroxybenzoate (intermediate 100B) [ka]

[0152] Sulfuric acid (138.0 mL, 2.60 mol, 2.0 equivalents) was added to a stirred solution of 3,4-dihydroxybenzoic acid (100A) (200.0 g, 1.30 mol, 1.0 equivalent) in methanol (2.0 L). The reaction mixture was stirred at 80°C for 15 hours. The progress of the reaction was monitored by TLC (50% ethyl acetate in n-hexane). The solvent was removed by distillation under vacuum, then DMW (1500 mL) was added, and the product was extracted with ethyl acetate (3 x 1.5 L). The combined organic extracts were washed with a saturated solution of sodium bicarbonate (2000 mL) and brine (1500 mL), dried on sodium sulfate, and concentrated under vacuum to obtain intermediate 100B (198.0 g, 90.70%) as an off-white solid. 1 HNMR (300MHz, DMSO): δ9.78ppm(s,1H), 9.37ppm(s,1H), 7.36-7.23ppm(m,2H), 6.81ppm(d,J=8.2Hz,1H), 3.76ppm(s,3H).

[0153] Step B: Methyl 2,3-dihydrobenzo[b][1,4]dioxin-6-carboxylate (intermediate 100C) [ka]

[0154] Potassium carbonate (405 g, 2.93 mol, 2.50 equivalents) and 1,2-dibromoethane (152.0 mL, 1.76 mol, 1.50 equivalents) were added to a stirred solution of methyl 3,4-dihydroxybenzoate (100B) (197.0 g, 1.17 mol, 1.0 equivalent) in DMF (2.96 L). The reaction mixture was heated to 90°C and stirred for 25 hours. The progress of the reaction was monitored by TLC (TLC analysis of 60% ethyl acetate in n-heptane). The reaction mixture was cooled to room temperature, then DMW (7.5 L) was added, followed by extraction with SiO (2 x 3.50 L). The combined organic extracts were washed with DMW (2 x 3.5 L) and brine (3.5 L), dried on sodium sulfate, and concentrated under vacuum to obtain intermediate 100C (214.0 g, 94.10%) as a light brown oil. 1 HNMR (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).

[0155] Step C: Methyl 7-nitro-2,3-dihydrobenzo[b][1,4]dioxin-6-carboxylate (intermediate 100D) [ka]

[0156] Concentrated HNO3 (832.0 mL, 14.50 mol, 17.0 equivalents) was slowly added at a temperature below 20°C to a stirred solution of methyl 2,3-dihydrobenzo[b][1,4]dioxin-6-carboxylate (100C) (214.0 g, 1.10 mol, 1.0 equivalent) in acetic acid (832.0 mL, 14.50 mol, 13.2 equivalents). The reaction mixture was stirred at room temperature for 2 hours while monitoring the progress of the reaction by TLC (eluent: 60% ethyl acetic acid in n-heptane). The reaction mixture was transferred to ice water (5.0 L) while vigorously stirring. The precipitated solid was collected by filtration, washed with DMW (1100 mL), and dried to obtain intermediate 100D (261 g, 99.0%) as a pale yellow solid. 1HNMR (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).

[0157] Step D: Methyl 7-amino-2,3-dihydrobenzo[b][1,4]dioxin-6-carboxylate (intermediate 100E) [ka]

[0158] A mixture of methyl-7-nitro-2,3-dihydrobenzo[b][1,4]dioxin-6-carboxylate (100D) (260 g, 1.09 mol, 1.0 equivalent), iron powder (217.0 g, 2.72 mol, 2.5 equivalents; mesh size 300-400) in ethanol (1.69 L, 6.50 volume equivalents), water (585 mL), and 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 acetic acid in n-heptane). The reaction mixture was cooled to room temperature, filtered through a celite bed, and the bed was washed with ethyl acetic acid (500 mL). Water (5.0 L) was added to the filtrate and mixed. The aqueous layer was separated and back-extracted with ethyl acetic acid (3 x 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 on sodium sulfate and filtered. The filtrate was concentrated under vacuum to obtain intermediate 100E (226 g, 99.40%) as a light brown solid. 1 HNMR (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).

[0159] Step E: 7-amino-2,3-dihydrobenzo[b][1,4]dioxin-6-carboxylic acid (intermediate 100) [ka]

[0160] A stirred solution of methyl-7-amino-2,3-dihydrobenzo[b][1,4]dioxin 6-carboxylate (100E) (225.0 g, 1.08 mol, 1.0 equivalent) in THF (1.58 mL) was added to a NaOH solution in water (1.58 mL) (237.0 g, 5.92 mol, 5.50 equivalents). The reaction mixture was heated to 80°C and stirred for 15 hours. The progress of the reaction was monitored by TLC (10% MeOH in DCM) to confirm completion of the reaction. The reaction mixture was cooled to room temperature, and the 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 obtain intermediate 100 (152.50 g, 72.5%) as a pale yellow solid. 1 HNMR (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).

[0161] Step 7: Synthesis of 2-(4-bromo-2-(methylsulfonyl)phenyl)-7,8-dihydro-4H-[1,4]dioxyno[2',3':4,5]benzo[1,2-d][1,3]oxazin-4-one (intermediate 108) [ka] 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 equivalent) in acetic anhydride (960 mL) was heated to 140 °C and stirred for 1.5 hours. The progress of the reaction was monitored by TLC (50% siRNA in n-hexane) to confirm completion of the reaction. Approximately 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 intermediate 108 (85.4 g, 74.1%) was dried as an off-white solid. 1 HNMR(300MHz,DMSO-d6):δ8.29-8.12ppm(m,2H),7.91ppm(d,J=8.2Hz,1H),7.59ppm(s, 1H),7.26ppm(s,1H),4.55-4.32ppm(m,4H),3.57ppm(s,3H).MS(ESI):m / z439.6(M+1).

[0162] Step 8: Synthesis of 2-(2-(methylsulfonyl)-4-vinylphenyl)-7,8-dihydro-4H-[1,4]dioxyno[2',3':4,5]benzo[1,2-d][1,3]oxazin-4-one (intermediate 109) [ka] A solution of 2-(4-bromo-2-(methylsulfonyl)phenyl)-7,8-dihydro-4H-[1,4]dioxyno[2',3':4,5]benzo[1,2-d][1,3]oxazine-4-one (108) (84.0 g, 192.0 mmol, 1.0 equivalent) in 1,4-dioxane (1.68 L) was degassed three times by alternately replenishing with vacuum and nitrogen to remove oxygen. Sodium bicarbonate (56.40 g, 671.0 mmol, 3.5 equivalents), potassium vinyl trifluoroborate (64.20 g, 479.0 mmol, 2.5 equivalents), tri(o-tolyl)-phosphine (14.60 g, 47.90 mmol, 0.25 equivalents), and Pd(OAc)2 (1.51 g, 6.71 mmol, 0.035 equivalents) were added under nitrogen, and the reaction flask was degassed twice more by alternately replenishing with vacuum and nitrogen. The reaction mixture was heated to 70°C and stirred for 3.5 hours. The progress of the reaction was monitored by TLC (40% siRNA in n-hexane) to confirm completion of the reaction. The reaction mixture was cooled to room temperature, quenched with 1N HCl (1000 mL), and filtered through a hyflo pad. The solvent was evaporated under reduced pressure, and DMW (2.5 L) was added to the residue. The product was extracted with ethyl acetic acid (3 x 2 L), the combined pharmaceutically acceptable 1 HNMR(300MHz,DMSO-d6):δ8.19-8.10ppm(m,1H),8.06-8.00ppm(m,1H),7.93ppm(dd,J=7.9,3.5Hz,1H),7.58ppm(s,1H),7.24ppm(s,1H) ,6.96ppm(dd,J=17.7,11.0Hz,1H),6.10ppm(t,J=17.7Hz,1H),5.56ppm(d,J=11.0Hz,1H),4.55-4.26ppm(m,4H),3.56-3.46ppm(m,3H).

[0163] Step 9: Synthesis 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 (Intermediate 110) [Chemical formula] Ruthenium(10%) on carbon (5.66 g, 5.60 mmol, 0.03 eq) was added 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 eq) in acetonitrile (1.44 L), EtOAc (1.44 L) and DMW (1.08 L) at room temperature. Sodium metaperiodate (130.0 g, 607.0 mmol, 3.25 eq) 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 n-hexane). The reaction mixture was filtered through a Celite bed. The solid was washed with ethyl acetate (4 x 1.5 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 under vacuum to give the crude product 110 (65.0, 95.30%) as a brown solid. The crude material of Intermediate 110 was used directly in the next Step 10.

[0164] Step 10: Synthesis of 2-(4-(hydroxymethyl)-2-(methylsulfonyl)phenyl)-7,8-dihydro-4H-[1,4]dioxino[2’,3’:4,5]benzo[1,2-d][1,3]oxazin-4-one (Compound 1.001) [Chemical formula] Sodium boro hydroxide (8.0 g, 1.0 equivalent) was added over 1 hour at 0°C to a stirred solution of 3-(methylsulfonyl)-4-(4-oxo-7,8-dihydro-4H-[1,4]dioxyno[2',3':4,5]benzo[1,2-d][1,3]oxazin-2-yl)benzaldehyde (110) (82.0 g, 212.0 mmol, 1.0 equivalent) in THF (1.23 mL) and ethanol (1.23 L). The reaction mixture was stirred at 0°C for 2 hours. The progress of the reaction was monitored by TLC (70% ethyl acetate in n-hexane) to confirm completion of the reaction. The reaction mixture was quenched with a saturated solution of ammonium chloride (100 mL), and the product was extracted with ethyl acetate (3 x 1500 mL). The combined organic extracts were washed with brine (500 mL), dried on sodium sulfate, and concentrated under vacuum to obtain the crude product. The crude product was purified by column chromatography on silica gel (230-400 mesh size) using a solvent gradient (20-40% n-hexane [(10% MeOH in toluene)]) to obtain compound 1.001 (15.50 g, 18.80%) as an off-white solid. 1 HNMR(300MHz,DMSO-d6):δ8.08ppm(d,J=1.5Hz,1H),7.91ppm(d,J=7.9Hz,1H),7.83ppm(dd,J=7.9,1.6Hz,1H),7.58pp m(s,1H),7.24ppm(s,1H),5.64ppm(t,J=5.7Hz,1H),4.70ppm(d,J=5.7Hz,2H),4.47-4.35ppm(m,4H),3.50ppm(s,3H).

[0165] Example 2: 2-(5-(hydroxymethyl)-2-(methylsulfonyl)phenyl)-7,8-dihydro-4H-[1,4]dioxyno[2',3':4,5]benzo[1,2-d][1,3]oxazin-4-one (compound 1.002) [ka] The synthesis of compound 1.002 is shown in Figure 2. The synthesis of intermediate 100 is explained in Figure 3.

[0166] Step 1: 5-Bromo-2-mercaptobenzoic acid (intermediate 202) [ka] A mixture of concentrated HCl (28.0 mL) and cold water (34 mL) was added dropwise to a stirred solution of 2-amino-5-bromobenzoic acid (201) (20.0 g, 92.6 mmol, 1.0 equivalent), NaOH (3.70 g, 92.6 mmol, 1.0 equivalent), and sodium nitrite (6.39 g, 92.6 mmol, 1.0 equivalent) in DMW (400.0 mL), while maintaining the internal temperature at 3-6°C. The reaction mixture was stirred at 0°C for 30 minutes, and then neutralized with potassium acetate (30.0 g, 306.0 mmol, 3.3 equivalents). This solution was added to a solution of potassium O-ethylxanthate (44.6 g, 278.0 mmol, 3.0 equivalent) in DMW (223 mL) preheated to 90°C. The mixture was stirred at the same temperature for 30 minutes, cooled to 0°C, and acidified with concentrated HCl (100 mL). The reaction mixture was basicized with 10% NaOH (200 mL) and heated to 85°C for 2 hours. NaHSO3 (9.81 g, 92.6 mmol) was added gradually to this mixture, and the mixture was heated to 85°C for 10 minutes. The mixture was filtered, cooled to 0°C, and acidified with concentrated HCl (100 mL). The precipitate was recovered by filtration, washed with H2O and then n-hexane, and intermediate 202 (20.2 g, 93.6%) was obtained as a light brown solid. MS(ESI): m / z 230.8 (M-1).

[0167] Step 2: Methyl 5-bromo-2-(methylthio)benzoate (intermediate 203) [ka] Potassium carbonate (83.0 g, 601 mmol, 7.0 equivalents) and ethyl iodide (21.4 mL, 343 mmol, 4.0 equivalents) were added at room temperature to a stirred solution of 5-bromo-2-mercaptobenzoic acid (202) (20 g, 85.8 mmol, 1 equivalent) in DMF (600 mL) at 0°C. The reaction mixture was stirred at 80°C for 4 hours. The progress of the reaction was monitored by TLC (10% ethyl ammonium in n-hexane) to confirm completion of the reaction. DMW (500 mL) was added, and the product was extracted with ethyl acetate (3 x 500 mL). The combined ethyl extract was washed with brine (300 mL), dried over sodium sulfate, filtered, and the filtrate was concentrated under vacuum to obtain the crude compound. The crude product was purified by column chromatography on silica gel (230-400 mesh size) and a solvent gradient (0-5% siRNA in n-hexane) to obtain intermediate 203 (20 g, 89.3%) as an off-white solid.

[0168] Step 3: Methyl 5-bromo-2-(methylsulfonyl)benzoate (intermediate 204) [ka] A solution of oxone (40.8 g, 268 mmol, 5.0 equivalents) in DMW (280 mL) was added to a solution of methyl 5-bromo-2-(methylthio)benzoate (203) (14.0 g, 53.6 mmol, 1.0 equivalent) in methanol (350 mL) with stirring at 0°C. The reaction mixture was stirred at 50°C for 16 hours. The progress of the reaction was monitored by TLC (30% ethyl acetate in n-hexane) to confirm completion of the reaction. The product was extracted with ethyl acetate (4 x 500 mL). The combined organic extracts were washed with brine (2 x 300 mL), dried on sodium sulfate, and concentrated under vacuum to obtain the crude product. The crude product was purified by column chromatography on silica gel (230-400 mesh size) and a solvent gradient (5-20% ethyl acetate in n-hexane) to obtain intermediate 204 (13.2 g, 84.0%) as an off-white solid. 1HNMR(300MHz,DMSO):δ8.08-7.98ppm(m,2H),7.93ppm(dd,J=7.9,0.9Hz,1H),3.87ppm(s,3H),3.44-3.35ppm(m,3H).MS(ESI):m / z292.8(M-1).

[0169] Step 4: Methyl 2-(methylsulfonyl)-5-vinylbenzoate (intermediate 205) [ka] A solution of methyl 5-bromo-2-(methylsulfonyl)benzoate (204) (5.0 g, 17.1 mmol, 1.0 equivalent) in DME (100.0 mL, 20 volume equivalents) was degassed three times by alternating vacuum and nitrogen replenishment, and then degassed twice by alternating vacuum and nitrogen replenishment. 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 under nitrogen, and the reaction flask was repeated two more times by alternating vacuum and nitrogen replenishment. The reaction mixture was heated to 80°C and stirred for 4 hours (N2 was removed when the temperature rose to 40-45°C). The progress of the reaction was monitored by TLC (30% SiO in n-hexane) to confirm completion of the reaction. The reaction mixture was cooled to room temperature and filtered through a hyflo pad. DMW (300 mL) was added to the filtrate, and the product was extracted with ethyl acetic acid (2 x 100 mL). The combined organic extracts were washed with brine (2 x 100 mL), dried on sodium sulfate, filtered, and concentrated to provide the crude product. The crude product was purified by column chromatography on silica gel (230-400 mesh size) using a solvent gradient (5-20% ethyl acetate in n-hexane) as the eluent to obtain intermediate 205 (3.36 g, 82.0%) as a pale yellow rubber.

[0170] Step 5: 2-(methylsulfonyl)-5-vinylbenzoic acid (intermediate 206) [ka] A solution of NaOH (3.92 g, 97.9 mmol, 7.0 equivalents) in water (67.2 mL) was added to a stirred solution of methyl 2-(methylsulfonyl)-5-vinylbenzoate (205) (3.36 g, 14.0 mmol, 1.0 equivalent) in methanol (67.2 mL). The reaction mixture was stirred at 60°C for 2 hours. The progress of the reaction was monitored by TLC (70% EtOH in n-hexane) to confirm 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 acetic acid (4 x 500 mL). The combined organic extracts were washed with brine, dried on sodium sulfate, filtered, and the filtrate was concentrated under vacuum to obtain the desired intermediate 206 (1.48 g, 46.8%) as an off-white solid. 1 HNMR(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).

[0171] Step 6: 2-(methylsulfonyl)-5-vinylbenzoyl chloride (intermediate 207) [ka] Oxalyl chloride (2.66 mL, 30.9 mmol, 5.0 equivalents) was added to a stirred solution of 2-(methylsulfonyl)-5-vinylbenzoic acid (206) (1.4 g, 6.19 mmol, 1.0 equivalent) in DCM (70 mL), and then 2 drops of DMF were added at room temperature. The reaction mixture was refluxed for 2 hours. The progress of the reaction was monitored by TLC (50% siRNA in n-hexane) to confirm completion of the reaction. The solvent was removed under vacuum, and the desired product was obtained as intermediate 207 (540 mg, 83.2%). The crude intermediate 207 was used directly in step 7, which is described below.

[0172] Step 7: 7-(2-(Methylsulfonyl)-5-vinylbenzamide)-2,3-dihydrobenzo[b][1,4]dioxine-6-carboxylic acid (Intermediate 208) [Chemical formula] A solution of 2-(Methylsulfonyl)-5-vinylbenzoyl chloride (207) (1.35 mg, 5.53 mmol, 1.2 eq) in triethylamine (1.93 mL, 13.8 mmol, 3.0 eq) and THF (36 mL) was added dropwise to a stirred solution of 7-amino-2,3-dihydrobenzo[b][1,4]dioxine-6-carboxylic acid (100) (900 mg, 4.61 mmol, 1.0 eq) in THF (36 mL) at 0 °C. The reaction mixture was stirred at the same temperature for 10 minutes and then at room temperature for 1.5 hours. The progress of the reaction was monitored by TLC (20% EtOAc in n-hexane), and the completion of the reaction was confirmed. The solvent was removed under vacuum. DMW was added to the residue, and the product was extracted with EtOAc (9 x 200 mL). The combined EtOAc extracts were washed with brine (200 mL), dried over sodium sulfate, and concentrated under vacuum to give 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).

[0173] Intermediate 100, 7-Amino-2,3-dihydrobenzo[b][1,4]dioxine 6-carboxylic acid was synthesized via Steps A - E as described herein.

[0174] Step A: Methyl 3,4-dihydroxybenzoate (Intermediate 100B) [Chemical formula]

[0175] A stirred solution of 3,4-dihydroxybenzoic acid (100A) (97%, 10.0 g, 62.9 mmol, 1.0 equivalent) in MeOH (500 mL) was mixed with sulfuric acid (96%, 7.0 mL, 126.0 mmol, 2.0 equivalents) at room temperature. The reaction mixture was refluxed overnight. TLC was checked to confirm the completion of the reaction. The solvent was removed under vacuum, and then water (500 mL) was added. The aqueous layer was extracted with RINKAN (3 x 100 mL). The combined organic extracts were washed with saturated NaHCO3 solution (250 mL) and brine (250 mL), dried on sodium sulfate, and concentrated under vacuum to obtain the desired intermediate 100B (8.72 g, 82.4%) as an off-white solid. 1 HNMR (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).

[0176] Step B: Methyl 2,3-dihydrobenzo[b][1,4]dioxin 6-carboxylate (intermediate 100C) [ka]

[0177] A stirred solution of methyl 3,4-dihydrobenzoate (100B) (10.0 g, 59.5 mmol, 1.0 equivalent) in acetone (200.0 mL) was added to potassium carbonate (20.5 g, 149 mmol, 2.50 equivalents), followed by the addition of 1,2-dibromoethane (7.72 mL, 89.2 mmol, 1.50 equivalents). The reaction mixture was refluxed overnight. TLC was checked to confirm completion of the reaction. The solvent was removed under vacuum, and then water was added. The aqueous layer was neutralized with 1N HCl and then extracted with siRNA (2 x 250 mL). The combined organic extracts were washed with brine (500 mL), dried on sodium sulfate, and concentrated under vacuum. The crude product was purified by combiflash using ethyl acetate in n-hexane (0-10%) to obtain the desired intermediate 100C (9.0 g, 77.9%) as a colorless oil. 1HNMR (300MHz, CDCl3): δ7.54-7.42ppm (m, 2H), 6.85-6.75ppm (m, 1H), 4.27-4.14ppm (m, 4H), 3.79ppm (s, 3H).

[0178] Step C: Methyl 7-nitro-2,3-dihydrobenzo[b][1,4]dioxin-6-carboxylate (intermediate 100D) [ka]

[0179] To a stirred solution of methyl 2,3-dihydrobenzo[b][1,4]dioxin-6-carboxylate 100C (9.0 g, 46.3 mmol, 1.0 equivalent) in acetic acid (35.0 mL, 612 mmol, 13.2 equivalents), concentrated HNO3 (47.0 mL, 788 mmol, 17.0 equivalents) was added dropwise at a temperature below 20°C, and the reaction mixture was stirred at room temperature for 2 hours. The completion of the reaction was confirmed by checking the TLC. The reaction mixture was poured into ice water with vigorous stirring, and the aqueous layer was extracted with RINKAN (3 x 500 mL). The combined organic extracts were washed with NaHCO3 and brine, dried on sodium sulfate, and concentrated under vacuum to obtain the desired product, intermediate 100D (9.7 g, 87.5%), as a pale 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).

[0180] Step D: Methyl 7-amino-2,3-dihydrobenzo[b][1,4]dioxin-6-carboxylate (intermediate 100E) [ka]

[0181] A mixture of methyl-7-nitro-2,3-dihydrobenzo[b][1,4]dioxin-6-carboxylate (100D) (25.0 g, 105.0 mmol, 1.0 equivalent), iron (20.4 g, 366.0 mmol, 3.5 equivalents), ethanol (625 mL), water (163 mL), and acetic acid (500 mL) was stirred at 70°C for 45 minutes. TLC was checked to confirm completion of the reaction. The reaction mixture was cooled to room temperature and filtered through a hyflo pad. The filtrate was diluted with water (1000 mL), and the aqueous layer was extracted with ethyl acetic acid (3 x 700 mL). The combined organic extracts were washed with saturated sodium bicarbonate (2 x 1000 mL) and brine (1000 mL), dried on sodium sulfate, and filtered. The filtrate was concentrated under vacuum to obtain the title compound, intermediate 100E (21.8 g, 99.7%), as a brown solid. 1 HNMR(300MHz,DMSO):δ7.14ppm(s,1H),6.25ppm(d,J=7.6Hz,3H),4.26-4.19ppm(m,2H),4.17-4.08ppm(m,2H),3.73ppm(s,3H).MS(ESI):m / z210.10(M+1).

[0182] Step E: 7-amino-2,3-dihydrobenzo[b][1,4]dioxin-6-carboxylic acid (intermediate 100) [ka]

[0183] A stirred solution of methyl-7-amino-2,3-dihydrobenzo[b][1,4]dioxin-6-carboxylate (100E) (22.0 g, 105 mmol, 1.0 equivalent) in THF (440.0 mL) was added to a NaOH solution (23.1 g, 578 mmol, 5.5 equivalents) in water (440.0 mL), and the reaction mixture was stirred at 80°C for 20 hours. The progress of the reaction was monitored by TLC (10% MeOH in DCM) to confirm 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 acetic acid (5 x 500 mL). The combined organic extracts were washed with brine, dried on sodium sulfate, filtered, and the filtrate was concentrated under vacuum to obtain a brown crude product. The crude product was purified with n-hexane to obtain the desired intermediate 100 (18.6 g, 90.6%) as a brown solid. 1 HNMR (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).

[0184] Step 8: 2-(2-(methylsulfonyl)-5-vinylphenyl)-7,8-dihydro-4H-[1,4]dioxyno[2',3':4,5]benzo[1,2-d][1,3]oxazine-4-one (intermediate 209) [ka] A solution of 7-(2-(methylsulfonyl)-5-vinylbenzamide)-2,3-dihydrobenzo[b][1,4]dioxin-6-carboxylic acid (208) (1.60 mg, 3.97 mmol, 1.0 equivalent) in acetic anhydride (40 mL) was stirred at 140 °C for 1.5 hours. The progress of the reaction was monitored by TLC (50% EtOH in n-hexane) to confirm completion of the reaction. The reaction mixture was cooled to room temperature, and then DMW (100 mL) was added. The product was extracted with siRNA (4 x 200 mL). The combined siRNA extracts were washed with brine (2 x 200 mL), dried on sodium sulfate, and concentrated under vacuum to obtain the crude product. The crude product was purified by column chromatography on silica gel (230-400 mesh size) and a solvent gradient (20-50% siRNA in n-hexane) to obtain intermediate 209 (700 mg, 45.8%) as a yellow solid. 1 HNMR (300MHz, CDC) l3 ):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=17.5,10.9Hz,1H),6.01p pm(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 / z385.80(M+1).

[0185] Step 9: 3-(methylsulfonyl)-4-(4-oxo-7,8-dihydro-4H-[1,4]dioxyno[2',3':4,5]benzo[1,2-d][1,3]oxazine-2-yl)benzaldehyde (intermediate 210) [ka] 10% ruthenium on carbon (50 mg, 0.049 mmol, 0.03 equivalents) was added at room temperature to a solution of 2-(2-(methylsulfonyl)-5-vinylphenyl)-7,8-dihydro-4H-[1,4]dioxyno[2',3':4,5]benzo[1,2-d][1,3]oxazin-4-one (209) (0.630 g, 1.63 mmol, 1.0 equivalent) in acetonitrile (12.6 mL), ethyl acetate (12.6 mL), and DMW (12.6 mL). Sodium metaperiodate (1.05 g, 4.9 mmol, 3.0 equivalents) 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% ethyl acetate in n-hexane) to confirm completion of the reaction. The reaction mixture was filtered through a Celite bed. The filtrate was diluted with water (100 mL), and the aqueous layer was extracted with ethyl acetic acid (3 x 200 mL). The combined organic extracts were washed with saturated sodium bicarbonate and brine, dried on sodium sulfate, and filtered. The filtrate was concentrated under vacuum to obtain crude intermediate 210. The crude product of intermediate 210 was used directly in step 10 shown below.

[0186] Step 10: 2-(4-(hydroxymethyl)-2-(methylsulfonyl)phenyl)-7,8-dihydro-4H-[1,4]dioxyno[2',3':4,5]benzo[1,2-d][1,3]oxazin-4-one (compound 1.002) [ka] Sodium boro hydroxide (9.77 mg, 0.258 mmol, 0.5 equivalents) was added over 1 hour at -10°C to a stirred solution of 4-(methylsulfonyl)-3-(4-oxo-7,8-dihydro-4H-[1,4]dioxyno[2',3':4,5]benzo[1,2-d][1,3]oxazin-2-yl)benzaldehyde (210) (0.20 g, 0.52 mmol, 1.0 equivalent) in THF (10.0 mL). The reaction mixture was stirred at the same temperature at 0°C for 30 minutes. The progress of the reaction was monitored by TLC (70% ethyl acetate in n-hexane) to confirm completion of the reaction. The reaction mixture was quenched with a saturated solution of ammonium chloride (50 mL), and the product was extracted with ethyl acetate (3 x 100 mL). The combined organic extracts were washed with brine (500 mL), dried on sodium sulfate, and concentrated under vacuum to obtain the crude product. The crude product was purified by column chromatography using silica gel (230-400 mesh size) with a solvent gradient (20-40% n-hexane [(10% MeOH in Â)]) to obtain compound 1.002 (70 mg, 34.80%) as an off-white solid. 1 HNMR (300MHz, DMSO): δ8.07ppm(d,J=8.2Hz,1H),7.88ppm(s,1H),7.79ppm(d,J=8.5Hz,1H),7.58ppm(s,1H),7.24ppm(s,1 H),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 / z390.0(M+1).

[0187] Example 3: In Vitro Activity Assay Recombinant human KLK5 expressed on human kallikrein protein:HEK293 was obtained from SpeedBio (Gaithersburg, MD). Both recombinant proenzymes KLK-7 and KLK-14 expressed in mouse myeloma cells were from R&D Biosystems (Minneapolis, MN). Plasmin and thrombin were from Haematologic Technologies (EssexJunction, VT). Trypsin and chymotrypsin were purchased from Worthington Biochemical Corporation (Lakewood, NJ), and plasma kallikrein from Prospec (East Brunswick, NJ). Neutrophil elastase was obtained from Innovative Research (PearyCourt, MI). Usiliconian enterokinase expressed in E. coli was purchased from Sigma (St. Louis, MI). Matryptase expressed in E. coli was obtained from EnzoLifeSciences (Farmingdale, NY). The control inhibitor nafamostat mesylate was 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 from Interbioscreen (Moscow, Russia).

[0188] 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-Ile-Pro-Arg-pNA) were purchased from Diapharma (West Chester, OH). MeOSuccAla-Ala-Pro-Val-pNA was purchased from Sigma (St Louis, MI). The substrates were dissolved in 10 mM water and frozen in aliquots at -20°C.

[0189] Activation of KLK-7 and KLK-14: 150 μL of 8 μM proenzyme KLK-7 or KLK-14 solution 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. KLK-7 was incubated for 2 hours and KLK-14 for 1 hour at 37°C. After incubation, 6 μL of 0.5 M EDTA pH 8.0 was added to stop the activation.

[0190] Assays: All assays were performed in 384-well polystyrene plates coated overnight with 0.1% Tween® 20 at 4°C. The plates were then washed twice with 100 μL of water and dried. IC 50 To measure the values, the inhibitor was diluted in pure DMSO at 50-fold the final concentration intended in the assay, in serial dilutions at 1:3 intervals. Typically, eight inhibitor dilutions and one DMSO control were prepared. The substrate was diluted in water to 2.2-fold the final concentration in the assay mixture. The plate wells were filled with 50 μL of enzyme diluted in 2x assay buffer (100 mM Tris, 200 mM NaCl, 0.2% PEG, 0.01% Tween®, 2 mM EDTA, pH 8.0) to 2-fold the desired concentration. Then, 5 μL of DMSO, 2.2 μL of inhibitor, and 45 μL of substrate were added to initiate the reaction. The assay was monitored for 1 hour at 37°C at a wavelength of 405 nm using a multi-well plate reader (Molecular Devices, San Jose, CA). Nafamostat, a tool inhibitor, was used at a maximum concentration of 10 μM against trypsin, thrombin, KLK-5, and KLK-14, while (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 at 5 μM against KLK-7.

[0191] [Table 2]

[0192] Data Analysis: The progress curve of substrate hydrolysis was analyzed by linear regression over the last 20 minutes of the reaction. The obtained rate dose-response was applied to the following equation: IC 50 Obtained: [ka] 1) c: Background rate at the highest inhibitory concentration, 2) d: Control response rate, 3) IC 50 : IC of inhibitors 50 4)x: inhibitor concentration; and 5)b: slope.

[0193] Example 4: Protease activity in human skin 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). Human epidermal samples were obtained from Biopredic (Saint-Gregoire, France). A BCA protein assay kit was purchased from VWR.

[0194] Preparation of human extracts: Epidermis was freeze-dried and lyophilized. A 15 mg sample of lyophilized epidermis was immersed in 1 mL of high-salt buffer (100 mM Tris, 5 mM EDTA, 2 M KCl, pH 8) and frozen and thawed twice on dry ice. After incubation overnight at 4°C and addition of 6 2.4 mm metal beads, the sample was homogenized three times at 5000 Hz for 20 seconds each time, with 30 seconds of cooling during the process. After centrifugation at maximum speed using a benchtop mini centrifuge, the protein concentration of the supernatant was measured by BCA assay (~0.5 mg / mL).

[0195] Inhibition of protease activity in skin extracts: 200, 66, 22, and 0 μM stock solutions of the test compounds were prepared in DMSO. 1100 μM PS-01 and PS-02 substrate stocks were prepared by dissolving them in water. Wells of a 384-well plate were filled with 50 μL of 2x assay buffer (see above), and then 45 μL of substrate stock and 5 μL of the test compound were added. The reaction was initiated by adding 10 μL of skin extract for substrate PS-02 and 5 μL for substrate PS-01. Substrate hydrolysis was monitored at 405 nm with a multi-well plate reader.

[0196] Example 5: Test compound assay results Compounds 1.001 and 1.002, as well as the known compound 14, were tested according to the assay protocols of Examples 3 and 4. The test results are listed in Table 2. Compound 14 is disclosed in WO2015 / 112081 and has the following chemical formula: [ka] It holds. [Table 3]

[0197] The progression of chromogenic substrate cleavage of compounds 1.001, 1.002, and 14 was tested by the human stratum corneum extract assay of Example 4 using preferred chromogenic peptide substrates (PS-01 and PS-02) for KLK5 and KLK7. A comparison of proteolytic cleavage of KLK5-selective substrates in skin extracts by compounds 1.001, 1.002, and 14 is shown in Figures 4A–4D. NSK refers to extracts from freshly prepared skin; and OSK refers to extracts from freeze-thawed skin.

[0198] As can be seen in Figures 4-4D(NSK), the initial linear portion of the curve for compound 1.001 shows a lower rate (slope) than the other two compounds, suggesting a higher inhibition of substrate cleavage. The observed low initial rate of proteolytic activity for compound 1.001 is also consistent with the observed low intercept of the curve. As can be seen in Figures 4-4D(NSK), the substrate cleavage rate slows exponentially as the substrate is depleted. The lower exponential rate constant for compound 1.001 suggests that compound 1.001 appears to have stronger inhibition of proteolytic activity.

[0199] In contrast to the results obtained with purified recombinant KLK5, compound 1.001 exhibits more potent inhibition of proteolytic activity in skin extracts compared to the known compound 14, based on three parameters (rate, substrate depletion, and initial rate of proteolytic activity).

[0200] Example 6: Chromogenic Assay Preparation of neonatal human epidermal keratinocyte lysate: Human epidermal keratinocyte pool cells (HEK) were purchased from Invitrogen (catalog no. A13401). Cells were cultured in the absence of serum in commercially available EpiLife medium (Thermo Fisher, catalog no. MEPI500CA) supplemented with commercially available supplement S7 (Thermo Fisher, catalog no. SO0175). Tissue culture was performed in a humidified chamber at 37°C with 5% carbon dioxide. HEK lysation buffer composition: 100 mM Tris-HCl, 150 mM NaCl, 1% Triton, 5 mM EDTA, final pH 7.6. After two passages, cells were lysed using HEK lysation buffer without protease and phosphatase inhibitors, and protein concentrations were measured using bicinchoninic acid assay (BCA) (Thermo Fisher, catalog no. 23225).

[0201] Preparation of healthy human keratin extract: Keratin sheets were purchased from Biopredic (catalog number STR0020). Extract preparation: Following the manufacturer's instructions, keratin lysates were homogenized and then prepared using the Minute Total Protein Extraction Kit for Adipose Tissues (InventBiotech, catalog number AT-022). The total protein concentration was measured by BCA assay (ThermoFisher, catalog number 23225). The extracts were rapidly frozen and stored at -80°C until use.

[0202] KLK5 concentration in stratum corneum extracts: The KLK5 concentration in stratum corneum extracts was determined by 1) ELISA from Abcam (catalog number ab131555); and 2) comparison of the hydrolysis rate of the selective chromogenic substrate PS-01 (YRSR-pNA Tyr-Arg-Ser-Arg-pNA) with the known concentration of purified KLK5 (Speed ​​Bio). Furthermore, the KLK5 concentration in unknown samples was measured from the hydrolysis rate of the selective substrate by interpolation from linear plots of hydrolysis rates of known samples.

[0203] To cleave high molecular weight substrates with endogenous and exogenous kallikrein, 5:10 ug of HEK lysis solution 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 adding loading buffer under reducing conditions (50 mM DTT) and boiling at 100°C for 5 minutes. Proteins were separated by electrophoresis on a bis-Tris gel with a 4–12% acrylamide gradient, run at 200 V for 22 minutes. After transferring the proteins to a PVDF membrane, nonspecific binding was blocked at room temperature for 1 hour using intercept blocking buffer (Licor). The signals on the blot were normalized with GAPDH.

[0204] The conditions for detecting specific proteins are summarized below.

[0205] Desmoglein 1: Desmoglein antibody sc-137164 (Santa Cruz Biochemicals) was diluted to 1 / 750 and incubated at 40°C for 18 hours. After washing, a secondary anti-mouse antibody (Licor) was added, diluted to 1 / 20000 of the stock solution. KLK5-absent controls were treated with a protease / phosphatase inhibitor mix (Cell Signalling, #5872).

[0206] Desmocolin 1: The anti-desmocolin 1 antibody (Abcam, 150382) was diluted 1:1000. The secondary antibody was diluted 1:15000. After washing, the secondary anti-rabbit antibody was added and diluted 1:15000.

[0207] Filaggrin: Anti-filaggrin (LS-Bio, 1561) was diluted to 1:400. The secondary antibody was diluted to 1:10000.

[0208] Results of the chromogenic assay:

[0209] Proteases: KLK5 (SpeedBioSystems); KLK7 (R&D Systems) were 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) were activated with enterokinase (1 hour, 37°C) prior to the assay; thrombin (Haematologic Technologies); neutrophil elastase (Athens Research & Technology); and KLK1 (Prospec).

[0210] Compound 408: See below for chemical formula [ka] This product is obtained by hydrolyzing and ring-opening the 4-quinazolinone moiety of compound 1.001, represented by [formula].

[0211] The assay results are summarized in Table 3.

[0212] [Table 4]

[0213] Conclusion: Compound 1.001 can be concluded to be an inhibitor of KLK5, KLK7, and chymotrypsin. In contrast, the deficiency of the 4-quinazolinone moiety (i.e., the pharmacophore of compound 1.001) makes protease inhibition impossible.

Claims

1. Chemical formula (I) below: 【Chemistry 1】 A compound represented by the formula; where R is H or -R 1 and -C(O)R 1a A member selected from the group consisting of R 1 is C 1-12 Alkyl, and R 1a is H or C 1-12 Alkyl compounds, or pharmaceutically acceptable salts thereof, and / or solvates thereof.

2. The following chemical formula (Ia): 【Chemistry 2】 The compound according to claim 1, represented by , or a pharmaceutically acceptable salt thereof, and / or a solvate thereof.

3. The following chemical formula (Ib): 【Transformation 3】 The compound according to claim 1, represented by , or a pharmaceutically acceptable salt thereof, and / or a solvate thereof.

4. The following chemical formula (Ic): 【Chemistry 4】 The compound according to claim 1, represented by , or a pharmaceutically acceptable salt thereof, and / or a solvate thereof.

5. The following chemical formula: 【Transformation 5】 The compound according to claim 1, represented by , or a pharmaceutically acceptable salt thereof, and / or a solvate thereof.

6. The following chemical formula: 【Transformation 6】 The compound according to claim 1, represented by , or a pharmaceutically acceptable salt thereof, and / or a solvate thereof.

7. A pharmaceutical composition comprising a compound according to any one of claims 1 to 6, a pharmaceutically acceptable salt thereof, and / or a solvate thereof, and a pharmaceutically acceptable carrier.

8. The aforementioned compound has the following chemical formula: 【Transformation 7】 The pharmaceutical composition according to claim 7, wherein the compound is represented by the compound.

9. The aforementioned compound has the following chemical formula: 【Transformation 8】 The pharmaceutical composition according to claim 7, wherein the compound is represented by the compound.

10. A pharmaceutical composition according to any one of claims 7 to 9 for local administration.

11. A pharmaceutical composition for treating skin diseases relating to the proteolytic activity of one or more KLK proteases in subjects requiring treatment of skin diseases, wherein an effective amount of the following chemical formula (I) 【Chemistry 9】 [wherein, R is H or -R 1 and -C(O)R 1a is a member selected from the group consisting of, R 1 is C 1-12 alkyl, and R 1a is H or C 1-12 alkyl]] A pharmaceutical composition comprising a compound having, or a pharmaceutically acceptable salt thereof, and / or a solvate thereof.

12. The aforementioned compound has the following chemical formula (Ia): 【Chemistry 10】 The pharmaceutical composition according to claim 11, wherein the compound is represented by [the formula shown].

13. The aforementioned compound has the following chemical formula: 【Chemistry 11】 The pharmaceutical composition according to claim 11, wherein the compound is represented by a chemical formula selected from the group consisting of the following.

14. The pharmaceutical composition according to any one of claims 11 to 13, wherein the skin disease is a hereditary skin disease.

15. The pharmaceutical composition according to claim 14, wherein the hereditary skin disease is Netherton syndrome.

16. The pharmaceutical composition according to any one of claims 11 to 15, wherein the one or more KLK proteases are KLK5 and / or KLK7.

17. The pharmaceutical composition according to any one of claims 11 to 16, wherein the pharmaceutical composition is administered topically.

18. An in vitro assay method for measuring the proteolytic activity of one or more KLK proteases, a) Preparing skin extracts; b) Substrate and the following chemical formula (I): 【Chemistry 12】 Exposing skin extracts to compounds thereof, or pharmaceutically acceptable salts thereof, and / or solvates thereof; and c) Measuring the rate of protein degradation and cleavage of the substrate, Includes, Here, the substrate is a peptide-p-nitroanilide; and R is H or -R 1 and -C(O)R 1a A member selected from the group consisting of R 1 C 1-12 It is alkyl, and R 1a is H or C 1-12 Alkyl assay method.

19. The assay method according to claim 18, wherein the substrate is Tyr-Arg-Ser-Arg-pNA or Lys-His-Leu-Tyr-pNA.

20. The aforementioned compound has the following chemical formula (Ia): 【Chemistry 13】 The assay method according to claim 18, as represented by [the specified expression].

21. The aforementioned compound has the following chemical formula: 【Chemistry 14】 The assay method according to claim 18, wherein the compound is represented by a chemical formula selected from the group consisting of the following.

22. The assay method according to claim 18, wherein the skin extract is a human skin extract.

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