RU2831430C1 - Benzoxazinone compounds as double inhibitors klk5/7 - Google Patents

Abstract

FIELD: pharmaceutics.

SUBSTANCE: invention relates to a compound of formula (I), where R is hydrogen, and a pharmaceutical composition thereof for treating a skin disease associated with proteolytic activity of one or more KLK proteases.

EFFECT: disclosed are benzoxazinone compounds as double inhibitors of KLK5/7.

22 cl, 4 dwg, 3 tbl, 6 ex

Description

Cross-references to related applications

This application claims priority to U.S. Provisional Application No. 62/909,006, filed October 1, 2019, which is incorporated herein in its entirety for all purposes.

Statement of Rights to Inventions Made During Federally Supported Research

Not applicable

Link to a "sequence listing", table or appendix with a computer program provided on the CD

Not applicable

Background of the invention

Genodermatoses are genetic disorders that manifest as skin conditions. These diseases are usually detected at or shortly after birth. The type of disease depends on how the genes are affected, but the resulting conditions can be serious, rare, and have a significant impact on the patient’s life, leading to disability, shortened life expectancy, and the development of other chronic diseases and cancers. There are approximately 400 different types of genodermatoses, with an incidence that can range from 1 in 6,000 people to 1 in 500,000 people. The burden of the disease can be severe for the patient and their family, as they potentially have access to only a very limited number of expensive drugs to help treat the disease. Genodermatoses can be divided into single-gene (monogenic) diseases, where the skin condition is caused by an abnormality in a single gene, and polygenic diseases, where mutations (defects) in several genes influence the type of disease that the patient suffers from. Monogenic disorders include acute intermittent porphyrin, epidermolysis bullosa, ichthyosis, Fabry disease, anhidrotic ectodermal dysplasia, and incontinentia pigmenti. Genetic disorders with ichthyosis include Netherton syndrome.

Netherton syndrome (NS) is a rare autosomal recessive genodermatosis characterized by congenital ichthyosiform erythroderma, atopic diathesis, and a characteristic hair shaft abnormality known as trichorrhexis invaginata. Netherton syndrome is one of the most severe keratinization disorders. Infants typically present with generalized scaly erythroderma at birth and are at high risk for life-threatening complications such as hypernatremic dehydration, failure to thrive, and sepsis. Older children may present with a wide range of allergic manifestations, including severe atopic dermatitis, asthma, hay fever, and markedly elevated serum immunoglobulin E levels. In older children and adults, the scaling may have a characteristic circular pattern (ichthyosis linearis circumflexa). However, in infants and young children, the skin is more often red and scaly, without the characteristic circular pattern. The hair shafts are brittle and easily broken due to trichorrhexis invaginata or "bamboo hair", resulting in short, sparse hair. Another characteristic of Netherton syndrome is a predisposition to allergies, asthma, and eczema. Adults and children with Netherton syndrome are also predisposed to cutaneous viral infections caused by herpes and human papillomavirus, in addition to bacterial skin and systemic infections. The incidence of Netherton syndrome is estimated at 1/200,000 births, and the prevalence is estimated at 1-9/1,000,000. However, regional studies suggest that the prevalence may be higher due to diagnostic problems in infancy and early childhood, including overlap with atopic dermatitis and other recessive ichthyoses. There is currently no specific therapy for NS, only palliative treatment of skin infections, relief of itching and pain.

NS is caused by loss-of-function truncation mutations in the serine protease inhibitor Kazal-type 5 (SPINK5), the gene that encodes LEKTI (lymphoepithelial inhibitor Kazal-type). Loss of LEKTI function results in unimpeded protease activity and excessive hydrolysis of epidermal structural and barrier proteins.

LETKI is an inhibitor specific for members of the kallikrein (KLK) family of serine proteases (KLK5, KLK7, and KLK14) (Chavanas et al., Nat Genet 2000, 25: 141-142). KLK5 is characterized as an upstream initiator of KLK7. According to the current hypothesis, pro-KLKs are synthesized and activated in the granular layer, and active KLK enzymes rapidly form complexes with LEKTI, thereby preventing premature degradation of desmosomes at the SC/Granulosa 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). KLK–LEKTI complexes diffuse into the outer SC, where the acidic SC microenvironment triggers the release of active KLKs from LEKTIs. Active KLKs subsequently cleave corneodesmosomal proteins in the most superficial layers of the SC. This ensures a finely balanced regulation of the desquamation process (Ovaere et al., Trends Biochem Sci 2009, 34:453-463).

Spink5-/- mice recapitulate 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). A key finding is that, similar to what has been observed in NS subjects, the epidermis of Spink5-/- mice exhibits decompensated KLK5 and KLK7 protease activity (Descargues et al., Nat Genet 2005, 37:56-65).

Conversely, deletion of KLK5 rescues neonatal lethality in Spink5-/- mice and abolishes cutaneous hallmarks of NS, including skin barrier defect, disrupted epidermal architecture, and skin inflammation (Furio et al., PLoS Genet 2015, Sep; 11(9): e1005389). Notably, loss of KLK5 results in decreased epidermal proteolytic activity, particularly KLK7 and KLK14. Subsequently, it restores the structural integrity of desmosomes and corneodesmosomes and normal epidermal differentiation, and normalizes IL-1β, IL-17A, and TSLP (thymic stromal lymphopoietin) expression.

In addition to animal models, organotypic 3D cultures with normal human keratinocytes transfected with small interfering RNA (siRNA) targeting SPINK5 and collagen gels populated with fibroblasts have been generated, and epidermal defects are observed in these culture models (Wang et al., Exp Dermatol 2014, Jul;23(7):524-6). Silencing of KLK5 or KLK7 genes markedly improves the epidermal architecture impaired by decreased SPINK5 expression. Collectively, these studies support an important role for KLK5 and its upstream and downstream regulators in NS.

KLK7 is expressed by keratinocytes in the granular layer and secreted into the extracellular space of the stratum corneum (i.e., the outermost layer of skin) as a zymogen, which is subsequently activated by KLK5. Once activated, KLK7 plays a central role in the skin desquamation process. The proteolytic actions of KLK7 are essential for the desquamation process and controlled skin regeneration. A tight balance between the formation of new corneocytes and desquamation is essential for maintaining skin homeostasis. In contrast, an imbalance between these two processes leads to impaired skin function and ultimately to dermatological diseases such as Netherton syndrome. Under normal conditions, this balance is maintained by the expression of endogenous protein inhibitors (LEKTIs) of KLK7, which counteract its proteolytic activity. In skin diseases, overexpression and/or increased activity of KLK7 lead to excessive desquamation. The involvement of KLK7 in skin disease is further supported by genetic associations in both animal models and humans. Transgenic mice overexpressing human KLK7 have been shown to develop skin features similar to those of patients with chronic atopic dermatitis, which share some features with patients with Netherton syndrome.

Given the role of KLK5 and KLK7 in regulating the desquamation process, as well as the results of complete phenotypic rescue in the Spink5-/-/Klk5-/-/Klk7-/- combined knockout mouse model (Kasparek et al., PLoS Genet 2017, Jan; 13(1): e1006566), potent and selective dual KLK5/KLK7 inhibitors modulating the proteolytic activity of these proteases are needed for the treatment of genodermatoses such as Netherton syndrome.

Brief description of the invention

In one aspect, the present invention provides a compound represented by formula (I):

… (I),

or a pharmaceutically acceptable complex thereof; wherein R is H or a member selected from the group consisting of R ¹ and –C(O)R ^1a , wherein R ¹ is C _1-12 alkyl, and R ^1a is H or C _1-12 alkyl.

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

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

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

Brief description of the drawings

Fig. 1 shows a scheme for obtaining Compound 1.001.

Fig. 2 shows a diagram for obtaining compound 1.002.

Fig. 3 shows a scheme for the preparation of intermediate compound 100, which is used to prepare compounds 1.001 and 1.002.

Fig. 4A-4D show the rates of proteolytic cleavage of the selective substrate KLK5 in a skin extract assay by compounds 1.001, 1.002, or known compound 14. Fig. 4A is a control; Fig. 4B is compound 14; Fig. 4C is compound 1.001; and Fig. 4D is compound 1.002. NSK refers to freshly prepared skin extract; and OSK refers to freeze-thawed skin extract.

Detailed description of the invention

I. General

The present invention provides compounds of formula (I) and pharmaceutical compositions comprising compounds of formula (I) for the treatment of a skin disease associated with the proteolytic activity of one or more KLK proteases. In particular, the pharmaceutical compositions can be topical formulations comprising compounds of formula (I) intended for topical use in the treatment of genodermatoses. The present invention also relates to methods for the treatment of a skin disease associated with the proteolytic activity of one or more KLK proteases by administering compounds of formula (I) or pharmaceutical compositions thereof. In particular, compounds of formula (I) or pharmaceutical compositions thereof can be administered topically. In particular, the skin disease is a genodermatosis, such as Netherton syndrome.

II. Definitions

The abbreviations used in this document have their usual meanings in the fields of chemistry and biology.

"Alkyl" refers to a straight or branched, saturated aliphatic radical having the stated number of carbon atoms (i.e. C _1-12 means one to twelve carbon atoms). An alkyl may include any number of carbon atoms such as _C1-2 , _C1-3 , _C1-4 , _C1-5 , _C1-6 , _C1-7 , _C1-8 , _C1-9 , _C1-10 , C1-11, C1-12, _{C2-3, C2-4, C2-5 , C2-6 , C2-7, C2-8 , C2-9} , _C2-10 , _C2-11 , _C2-12 , _{C3-4 , C3-5} , _C3-6 , C3-7, _{C3-8 , C3-9} , _C3-10 , _C3-11 , _{C3-12 , C4-5} , 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 . For example, C _1-12 alkyl includes, but is not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, n-hexyl, n-heptyl, n-octyl, n-nonyl, n-decyl, n-undecyl, n-dodecyl, etc. Alkyl can also refer to alkyl groups having up to 20 carbon atoms, such as, but not limited to, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl and octadecyl, etc.

"Pharmaceutically acceptable complex" as used herein refers to a complex comprising a compound of formula (I) and at least one complexing agent. The term "pharmaceutically acceptable" and the like means approved by a regulatory agency of the federal or state government or listed in the United States Pharmacopeia or other generally recognized pharmacopeias for use in animals, and more particularly in humans. Complexing agents have the function of forming a complex structure with the compound of formula (I) through non-covalent secondary interactions. The secondary interactions can be formed through electrostatic interactions such as ionic interactions, H-bonding, dipole-dipole interactions, dipole-induced dipole interactions, London dispersion forces, π-π interactions and hydrophobic interactions. A pharmaceutically acceptable complex of a compound of formula (I) includes a pharmaceutically acceptable salt and/or solvate thereof.

"Solvate" refers to a compound provided herein or a salt thereof, which further comprises a stoichiometric or non-stoichiometric amount of a solvent bound by non-covalent intermolecular forces. If the solvent is water, the solvate is a hydrate.

"Hydrate" refers to a compound that is in a complex with a water molecule. The compounds of the present invention can form complexes with ½ a water molecule or with 1-10 water molecules.

"Peptide-p-nitroanilide" or "peptide-pNA" as used herein 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 according to the present invention include Tyr-Arg-Ser-Arg-pNA and Lys-His-Leu-Tyr-pNA.

"Composition" as used herein includes a product containing the stated ingredients in the stated amounts, as well as any product that directly or indirectly results from a combination of the stated ingredients in the stated amounts. By "pharmaceutically acceptable" it is meant that the carrier, diluent or excipient must be compatible with the other ingredients of the formulation and not deleterious to the recipient.

"A pharmaceutically acceptable carrier or excipient" refers to a substance that facilitates the administration of an active agent and absorption by a subject. Pharmaceutical excipients useful in the present invention include, but are not limited to, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavors, and colorants. Pharmaceutical excipients useful in the present invention for transdermal/topical delivery include, but are not limited to, enhancers, solubilizers, antioxidants, plasticizers, thickeners, polymers, and pressure-sensitive adhesives. One skilled in the art will appreciate that other pharmaceutical excipients may be used in the present invention.

"IC ₅₀ " refers to the amount, concentration, or dosage of a particular test compound that produces 50% inhibition of the maximal response in an assay that measures such response.

"Inhibition," "inhibits," and "inhibitor" refer to a compound that interferes with, or a method of interfering with, a particular action or function.

"Administer" refers to topical application, such as a lotion, spray, ointment, cream, gel, paste, or patch.

"Topical" means applying a suitable compound (e.g., an active agent) or a composition comprising a compound (e.g., an active agent) to the skin to treat diseases or conditions, such as genodermatoses. In some embodiments, "topical" means applying a suitable compound (e.g., an active agent) or a composition comprising a compound (e.g., an active agent) to the skin with adequate penetration into the epidermis or dermis to treat a genodermatosis. In some embodiments of topical administration, the compound or composition penetrates the epidermis or dermis without significant systemic exposure and without the intent to treat or prevent disease in another organ system. In some embodiments of topical administration, the compound or composition is delivered transdermally through the skin for systemic distribution. Examples include transdermal patches used to deliver drugs.

“Treat,” “treat,” and “treatment” refer to any measure of success in treating or alleviating an injury, pathology, or condition, including any objective or subjective parameters such as reduction; remission; reduction in symptoms or improvement in the patient’s tolerance of the injury, pathology, or condition; slowing the rate of deterioration or decline; making the end point of deterioration less debilitating; and improvement in the patient’s physical or mental well-being. Treatment or symptom relief may be based on objective or subjective parameters; including the results of a physical examination, neuropsychiatric examination, and/or psychiatric evaluation.

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

"Therapeutically effective amount" refers to the amount of a compound or pharmaceutical composition useful in treating or ameliorating an identified disease or condition or in exhibiting a detectable therapeutic or inhibitory effect. Precise amounts will depend on the purpose of treatment and can be determined by one of skill in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols. 13, 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).

The singular terms (in the English text, preceded by the articles "a", "an" or "a(n)"), when used in reference to a substituent group or "substituent group" herein, mean at least one. For example, when a compound is substituted with an alkyl or aryl, the compound is optionally substituted with at least one such alkyl and/or at least one such aryl, wherein each alkyl and/or aryl may optionally be different. In another example, when a compound is substituted with a substituent group, the compound is substituted with at least one such substituent group, wherein each substituent group may optionally be different.

III. Connections

In one aspect, the present invention provides a compound represented by formula (I):

… (I),

or a pharmaceutically acceptable complex thereof; wherein R is H or a member selected from the group consisting of R ¹ and –C(O)R ^1a , wherein R ¹ is C _1-12 alkyl, and R ^1a is H or C _1-12 alkyl.

The methylsulfonyl (-SO ₂ CH ₃ ) group in formula (I) may be in the ortho-, meta-, or para-position relative to the 7,8-dihydro-4H-[1,4]dioxino[2',3':4,5]benzo[1,2-d][1,3]oxazin-4-one moiety. In some embodiments, the -SO ₂ CH ₃ group is in the ortho-position relative to the 7,8-dihydro-4H-[1,4]dioxino[2',3':4,5]benzo[1,2-d][1,3]oxazin-4-one moiety.

The _–CH2OR group in formula (I) may be in the ortho, meta, or para position relative to the 7,8-dihydro-4H-[1,4]dioxino[2',3':4,5]benzo[1,2-d][1,3]oxazin-4-one moiety. In some embodiments, the _–CH2OR group is in the meta position relative to the 7,8-dihydro-4H-[1,4]dioxino[2',3':4,5]benzo[1,2-d][1,3]oxazin-4-one moiety. In some embodiments, the _–CH2OR group is in the para position relative to the 7,8-dihydro-4H-[1,4]dioxino[2',3':4,5]benzo[1,2-d][1,3]oxazin-4-one moiety.

In some embodiments, the compound is represented by formula (Ia):

… (Ia),

where R has the meaning defined and described for formula (I).

In some embodiments of formula (Ia), the _-CH2OR group may 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.

In some embodiments, the compound is represented by formula (Ib):

… (Ib),

where R has the meaning defined and described for formula (I).

In some embodiments, the compound is represented by formula (Ic):

… (Ic),

where R has the meaning defined and described for formula (I).

With respect to any of formulas (I), (Ia), (Ib) and (Ic), in some embodiments R is H.

In some embodiments, the compound is represented by the formula:

.

In some embodiments, the compound is represented by the formula:

.

With respect to any of formulas (I), (Ia), (Ib), and (Ic), in some embodiments R is a member of the group consisting of R ¹ and –C(O)R ^1a , wherein R ¹ is C _1-12 alkyl and R ^1a is H or C _1-12 alkyl.

In some embodiments, any of Formulae (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 selected embodiments, R is methyl.

In some embodiments, any of formulae (I), (Ia), (Ib), and (Ic), R is –C(O)R ^1a , wherein 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 _1-6 alkyl. In some embodiments, R ^1a is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, or n-hexyl. In some embodiments, R ^1a is methyl or ethyl. In some selected embodiments, R ^1a is methyl. In some embodiments, R is acetyl.

In some embodiments, the compound is represented by the formula:

.

In some embodiments, the compound is represented by the formula:

.

Examples of compounds of formula (I) are listed in Table 1.

Table 1. Compounds of formula (I)

No. Structure No. Structure 1.001 1.002

Compounds in other forms

The compounds of the present invention can be formed as complexes with at least one complexing agent. The complexing agents have the function of forming a complex structure with the compound of formula (I) through non-covalent secondary interactions. The secondary interactions can be formed through electrostatic interactions such as ionic interactions, H-bonding, dipole-dipole interactions, dipole-induced dipole interactions, London dispersion forces, π-π interactions and hydrophobic interactions. A pharmaceutically acceptable complex of a compound of formula (I) includes a pharmaceutically acceptable salt and/or solvate thereof. In some embodiments, a pharmaceutically acceptable complex of a compound of formula (I) is a pharmaceutically acceptable salt and/or solvate thereof. In some embodiments, a pharmaceutically acceptable complex of a compound of formula (I) is a pharmaceutically acceptable salt thereof. In some embodiments, a pharmaceutically acceptable complex of a compound of formula (I) is a pharmaceutically acceptable solvate thereof.

Some compounds of the present invention may exist in unsolvated forms as well as solvated forms, including hydrogenated forms. In general, solvated forms are equivalent to unsolvated forms and are within the scope of the present invention. Some compounds of the present invention may exist in a variety of crystalline or amorphous forms. In general, all physical forms are equivalent for their intended use in the present invention and are within the scope of the present invention.

Unless otherwise indicated, the compounds of the present invention may also contain unnatural ratios of atomic isotopes with respect to one or more of the atoms comprising such compounds. For example, the compounds of the present invention may be labeled with radioactive or stable isotopes such as, for example, deuterium ( ^2H ), tritium ( ^3H ), iodine-125 ( ^125I ), fluorine- ¹⁸ (18F), nitrogen-15 ( ^15N ), oxygen-17 ( ^17O ), oxygen-18 ( ^18O ), carbon-13 ( ^13C ), or carbon-14 ( ^14C ). All isotopic variations of the compounds of the present invention, whether radioactive or not, are within the scope of the present invention.

The present invention provides compounds that are in the form of a prodrug. Prodrugs of the compounds described herein are those compounds that readily undergo chemical changes under physiological conditions to release the compounds of the present invention. In addition, prodrugs can be converted into the compounds of the present invention by chemical or biochemical methods under ex vivo conditions. For example, prodrugs can be slowly converted into the compounds of the present invention when they are placed in a transdermal patch reservoir with a suitable enzyme or chemical agent.

IV. Composition

In a second aspect, the present invention provides a pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable complex thereof and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition comprises a compound of formula (Ia) or a pharmaceutically acceptable complex thereof and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition comprises a compound of formula (Ib) or a pharmaceutically acceptable complex thereof and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition comprises a compound of formula (Ic) or a pharmaceutically acceptable complex thereof and a pharmaceutically acceptable carrier.

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

In some embodiments, the compound of any of formulas (I), (Ia), (Ib), and (Ic) has the formula:

,

or a pharmaceutically acceptable complex thereof.

In some embodiments, the compound of any of formulas (I), (Ia), (Ib), and (Ic) has the formula:

,

or a pharmaceutically acceptable complex thereof.

The compounds provided herein can be formulated into pharmaceutical compositions using methods available in the art and the methods disclosed herein. Any of the compounds disclosed herein can be in the form of an appropriate pharmaceutical composition and can be administered by a suitable route of administration.

Administration of a compound described herein to a subject may be local or non-systemic, such as local, intradermal, or intralesional. In some embodiments, the compound may be administered by topical administration. In some embodiments, the compound may be administered intradermally. In some embodiments, the compound may be administered by intralesional administration, such as by intralesional injection.

The methods provided herein include the administration of pharmaceutical compositions comprising at least one compound described herein, including a compound of formula (I), if appropriate, in salt or complex form, used either alone or in combination with one or more compatible and pharmaceutically acceptable carriers, such as diluents or adjuvants, or with another agent for the treatment of genodermatosis, if the subject is in need thereof.

In some embodiments, the second agent may be formulated or packaged with the compound provided herein. It goes without saying that the second agent will be formulated with the compound provided herein only if, in the opinion of those skilled in the art, such co-formulation should not interfere with the activity of either agent or the route of administration. In some embodiments, the compound provided herein and the second agent are included in separate formulations. They may be packaged together or packaged separately for the convenience of one skilled in the art.

In clinical practice, the active agents provided herein may be administered by any conventional route, including topically, intradermally, intralesionally, orally, parenterally, rectally, or by inhalation (e.g., in the form of aerosols). In some embodiments, the compound provided herein is administered topically, intradermally, or intralesionally. In some embodiments, the compound provided herein is administered topically. In some embodiments, the compound provided herein is administered intradermally. In some embodiments, the compound provided herein is administered intralesionally.

Solid compositions for oral administration may be tablets, pills, hard gelatin capsules, powders or granules. In these compositions, the active product is mixed with one or more inert diluents or adjuvants, such as sucrose, lactose or starch.

These compositions may contain substances other than diluents, for example a lubricant such as magnesium stearate, or a coating designed to provide controlled release.

Liquid compositions for oral administration may include pharmaceutically acceptable solutions, suspensions, emulsions, syrups and elixirs containing inert diluents such as water or liquid paraffin. These compositions may also contain substances other than diluents, in some embodiments wetting, sweetening or flavoring products.

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

Compositions for parenteral, intralesional or intradermal administration may be emulsions or sterile solutions. Propylene glycol, polyethylene glycol, vegetable oils, in particular olive oil, or organic esters for injection, in some embodiments ethyl oleate, can be used as a solvent or carrier. These compositions can also contain adjuvants, in particular wetting, isotonizing, emulsifying, dispersing and stabilizing agents. Sterilization can be carried out in several ways, in some embodiments using a bacteriological filter, irradiation or heating. They can also be prepared in the form of sterile solid compositions that can be dissolved during use in sterile water or any other sterile injection medium.

Compositions for rectal administration are suppositories or rectal capsules which contain, in addition to the active principle, excipients such as cocoa butter, semi-synthetic glycerides or polyethylene glycols.

The compositions may also be aerosols. For use in the form of liquid aerosols, the compositions may be stable sterile solutions or solid compositions dissolved at the time of use in apyrogenic sterile water, physiological solution or any other pharmaceutically acceptable carrier. For use in the form of dry aerosols intended for direct inhalation, the active principle is finely divided and mixed with a water-soluble solid diluent or carrier, in some embodiments dextran, mannitol or lactose.

In some embodiments, the composition provided herein is a pharmaceutical composition or a unit dosage form. The pharmaceutical compositions and unit dosage forms provided herein comprise a prophylactically or therapeutically effective amount of one or more prophylactic or therapeutic agents (e.g., a compound provided herein or another prophylactic or therapeutic agent), and typically one or more pharmaceutically acceptable carriers or excipients. In one particular embodiment and in this context, the term "pharmaceutically acceptable" means approved by a regulatory agency of the federal or state government or registered in the United States Pharmacopeia or other generally recognized pharmacopeias for use in animals, and more particularly in humans. The term "carrier" includes a diluent, an adjuvant (e.g., Freund's adjuvant (complete and incomplete)), an excipient, or a vehicle with which the therapeutic agent is administered. Such physiological carriers may be sterile liquids such as water or oils including oils of petroleum, animal or synthetic origin such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water may be used as a carrier when administering the pharmaceutical composition intravenously. Saline solutions and aqueous dextrose and glycerol solutions may 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).

Typical pharmaceutical compositions and dosage forms contain one or more excipients. Suitable excipients are well known to those skilled in the pharmaceutical arts, and in some embodiments, suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dry skim milk, glycerol, propylene, glycol, water, ethanol, and the like. Whether a particular excipient is suitable for inclusion in a pharmaceutical composition or dosage form depends on a variety of factors well known in the art, including, but not limited to, the route of administration of the dosage form to the subject and the specific active ingredients in the dosage form. The composition or unit dosage form, if desired, may also contain minor amounts of wetting or emulsifying agents or buffering agents.

The lactose-free compositions provided herein may contain excipients that are well known in the art and are listed, in certain embodiments, in the United States Pharmacopeia (USP 36-NF 31 S2). Generally, lactose-free compositions contain the active ingredient, a binder/filler, and a lubricant in pharmaceutically compatible and pharmaceutically acceptable amounts. Typical lactose-free dosage forms contain the active ingredient, microcrystalline cellulose, pregelatinized starch, and magnesium stearate.

In addition, anhydrous pharmaceutical compositions and dosage forms containing active ingredients are covered in this document since water can promote the degradation of some compounds. For example, the addition of water (e.g., 5%) is widely used in the pharmaceutical industry as a means of simulating long-term storage to determine characteristics such as shelf life or the stability of formulations over time. See, e.g., Jens T. Carstensen, Drug Stability: Principles & Practice, 2d. Ed., Marcel Dekker, New York, 1995, pp. 379-80. In fact, water and heat accelerate the degradation of some compounds. Thus, the effect of water on a formulation can be significant since moisture and/or humidity are often encountered during the manufacture, processing, packaging, storage, transportation, and use of formulations.

The anhydrous pharmaceutical compositions and dosage forms provided herein may be prepared using anhydrous or low moisture ingredients and under low moisture or low humidity conditions. Pharmaceutical compositions and dosage forms containing lactose and at least one active ingredient containing a primary or secondary amine may be anhydrous if significant contact with moisture and/or humidity is expected during manufacturing, packaging and/or storage.

An anhydrous pharmaceutical composition should be prepared and stored in a manner that maintains its anhydrous nature. Accordingly, anhydrous compositions can be packaged using materials that are known to prevent exposure to water, so that they can be included in suitable pharmacological kits. In some embodiments, suitable packaging includes, but is not limited to, hermetically sealed foil, plastics, single-dose containers (e.g., vials), blister packs, and strip packs.

Also provided are pharmaceutical compositions and dosage forms containing one or more compounds that reduce the rate of degradation of the active ingredient. Such compounds, which are referred to herein as "stabilizers," include, but are not limited to, antioxidants such as ascorbic acid, pH buffers, or salt buffers.

The pharmaceutical compositions and unit dosage forms may be in the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations, etc. The formulation for oral use may include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. Such compositions and dosage forms will contain a prophylactically or therapeutically effective amount of a prophylactic or therapeutic agent, in some embodiments, in purified form, together with a suitable amount of a carrier to provide a form for proper administration to a subject. The composition must be appropriate for the route of administration. In some embodiments, the pharmaceutical compositions or unit dosage forms are sterile and are in a form suitable for administration to a subject, in some embodiments an animal such as a mammal, in some embodiments a human.

The pharmaceutical composition is formulated to be compatible with its intended route of administration. In some embodiments, routes of administration include, but are not limited to, parenteral, such as intravenous, intradermal, subcutaneous, intramuscular, subcutaneous, oral, buccal, sublingual, inhalation, intranasal, transdermal, topical, transmucosal, intratumoral, intrasynovial and rectal administration. In some embodiments, the route of administration is intradermal, topical or intralesional administration. In some embodiments, the route of administration is non-systemic administration. In a particular embodiment, the composition is formulated according to conventional procedures as a pharmaceutical composition adapted for intravenous, subcutaneous, intramuscular, oral, intranasal or topical administration to humans. In one embodiment, the pharmaceutical composition is formulated according to conventional procedures for subcutaneous administration to humans. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. If necessary, the composition may also include a solubilizing agent and a local anesthetic such as lidocaine to relieve pain at the injection site.

In some embodiments, the dosage forms include, but are not limited to: tablets; capsules; capsules such as soft elastic gelatin capsules; wafers; lozenges; pastilles; dispersions; suppositories; ointments; hot compresses (poultices); pastes; powders; dressings; creams; plasters; solutions; patches; aerosols (e.g., nasal sprays or inhalers); gels; liquid dosage forms suitable for oral or transmucosal administration to a subject, including suspensions (e.g., aqueous or non-aqueous liquid suspensions, oil-in-water emulsions, or water-in-oil liquid emulsions), solutions, and elixirs; liquid dosage forms suitable for parenteral administration to a subject; and sterile solids (e.g., crystalline or amorphous solids) that can be reconstituted to produce liquid dosage forms suitable for parenteral administration to a subject.

The composition, form, and type of dosage forms provided herein will generally vary depending on their intended use. In some embodiments, a dosage form used in the initial treatment of a genodermatosis may contain a greater amount of one or more active ingredients it contains than a dosage form used in the maintenance treatment of the same disorder or condition. Similarly, a parenteral dosage form may contain lesser amounts of one or more active ingredients than an oral dosage form used to treat the same disorder or condition. These and other ways in which specific dosage forms covered herein will differ from one another will be apparent to those skilled in the art. See, e.g., Remington: The Science and Practice of Pharmacy; Pharmaceutical Press; 22nd edition (September 15, 2012).

Typically, the ingredients of the compositions are supplied either separately or mixed together in a unit dosage form, in some embodiments as a dry lyophilized powder or anhydrous concentrate in a hermetically sealed container such as an ampoule or sachet indicating the amount of active agent. If the composition is administered by infusion, it can be dispensed by an infusion bottle containing sterile pharmaceutical grade water or saline. When the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed before administration.

Exemplary dosage forms include the compound provided herein, or a pharmaceutically acceptable complex, solvate, or hydrate thereof, in a range of from about 0.1 mg to about 1000 mg per day, taken as a single dose once daily in the morning or as divided doses throughout the day taken with meals. 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.

Oral dosage forms

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

In some embodiments, the oral dosage forms are solid and are prepared for anhydrous diseases or disorders using anhydrous ingredients, as described in detail herein. However, the scope of the compositions provided herein extends beyond anhydrous solid oral dosage forms. Thus, additional forms are described herein.

Typical oral dosage forms are prepared by mixing the active ingredient(s) in intimate admixture with at least one excipient according to conventional pharmaceutical compounding techniques. Excipients may take a wide variety of forms depending on the form of the composition desired for administration. In some embodiments, excipients suitable for use in oral liquid or aerosol dosage forms include, but are not limited to, water, glycols, oils, alcohols, flavoring agents, preservatives, and coloring agents. In some embodiments, excipients suitable for use in solid oral dosage forms (e.g., powders, tablets, capsules, and caplets) include, but are not limited to, starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, and disintegrating agents.

Because of their ease of administration, tablets and capsules are the most preferred oral dosage unit forms, in which case solid excipients are used. If desired, tablets may be coated by standard aqueous or non-aqueous processes. Such dosage forms may be prepared by any of the methods known in the art of pharmacy. In general, pharmaceutical compositions and dosage forms are prepared by uniformly and intimately admixing the active ingredients with liquid carriers, finely divided solid carriers, or both, and then, if necessary, shaping the product into the desired presentation.

In some embodiments, the tablet may be prepared by compression or molding. Compressed tablets may be prepared by compressing in a suitable machine the active ingredients in a free-flowing form such as powder or granules, optionally mixed with an excipient. Molded tablets may be made by molding in a suitable machine a mixture of a powdered compound moistened with an inert liquid diluent.

In some embodiments, excipients that may be used in oral dosage forms include, but are not limited to, binders, fillers, disintegrants, and lubricants. Binders suitable 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 gums such as acacia, sodium alginate, alginic acid, other alginates, powdered tragacanth, guar gum, cellulose and its derivatives (e.g., ethyl cellulose, cellulose acetate, calcium carboxymethyl cellulose, sodium carboxymethyl cellulose), polyvinylpyrrolidone, methyl cellulose, pregelatinized starch, hydroxypropyl methyl cellulose (e.g., No. 2208, 2906, 2910), microcrystalline cellulose, and mixtures thereof.

In some embodiments, excipients 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, dextrates, kaolin, mannitol, silicic acid, sorbitol, starch, pregelatinized starch, and mixtures thereof. The binder or filler in the pharmaceutical compositions is typically present in an amount of from about 50 to about 99% by weight of the pharmaceutical composition or dosage form.

In some embodiments, suitable forms of microcrystalline cellulose include, but are not limited to, those sold as AVICEL PH 101, AVICEL PH 103, AVICEL RC 581, AVICEL PH 105 (available from FMC Corporation, American Viscose Division, Avicel Sales, Marcus Hook, Pa.) and mixtures thereof. A specific binder is a mixture of microcrystalline cellulose and sodium carboxymethylcellulose sold as AVICEL RC 581. Suitable anhydrous excipients or low-moisture fillers or additives include AVICEL PH 103™ and Starch 1500 LM.

Disintegrants are used in compositions to produce tablets that disintegrate upon exposure to an aqueous medium. Tablets containing too much disintegrant may disintegrate upon storage, while tablets containing too little may not disintegrate at the desired rate or under the desired conditions. Thus, to form solid oral dosage forms, a sufficient amount of disintegrant should be used, which is neither too much nor too little so as not to adversely alter the release characteristics of the active ingredients. The amount of disintegrant used varies depending on the type of formulation and is readily determined by those skilled in the art. Typical pharmaceutical compositions contain from about 0.5 to about 15% by weight of disintegrant, in particular from about 1 to about 5% by weight of disintegrant.

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

Lubricants that can 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 oil (e.g., peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil, and soybean oil), zinc stearate, ethyl oleate, ethyl laureate, agar, and mixtures thereof. Additional lubricants include, in some embodiments, SYLOID silica gel (AEROSIL 200, manufactured by W.R. Grace Co. Baltimore, Md.), coagulated aerosol synthetic silicon dioxide (sold by Degussa Co. Plano, Tex.), CAB O SIL (a pyrogenic silicon dioxide product sold by Cabot Co. Boston, Mass.), and mixtures thereof. If lubricants are used at all, they are typically used in an amount of less than about 1% by weight of the pharmaceutical compositions or dosage forms in which they are included.

Delayed release dosage forms

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

All controlled-release pharmaceutical products share the common goal of improving drug therapy compared to their uncontrolled counterparts. Ideally, the use of an optimally designed controlled-release formulation in medical treatment is characterized by the use of a minimum amount of drug substance to treat or control a disease or disorder in a minimum amount of time. The benefits of controlled-release formulations include prolonged drug activity, reduced dosing frequency, and increased patient compliance. In addition, controlled-release dosage forms can be used to influence the time of onset of action or other characteristics such as blood levels of the drug, and thus may influence the occurrence of side (e.g., adverse) effects.

Most controlled release formulations are designed to initially release an amount of drug (active ingredient) that rapidly produces the desired therapeutic effect, and gradually and continuously release other amounts of drug to maintain this level of therapeutic or prophylactic effect over an extended period of time. To maintain this constant level of drug in the body, the drug must be released from the dosage form at a rate that will replace the amount of drug metabolized and excreted from the body. Controlled release of the active ingredient can be stimulated by a variety of diseases or disorders, including, but not limited to, pH, temperature, enzymes, water, or other physiological diseases, disorders, or compounds.

In some embodiments, the drug may be administered by intravenous infusion, an implantable osmotic pump, a transdermal patch, liposomes, or other routes of administration. In some 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, polymeric materials may be used. In yet another embodiment, the controlled release system may be placed in the subject at a suitable location determined by the practitioner, i.e., only a fraction of the systemic dose is required (see, e.g., Goodson, Medical Applications of Controlled Release, vol. 2, pp. 115-138 (1984)). Other controlled release systems are discussed in the review by Langer (Science 249:1527–1533 (1990)). The active ingredient can be dispersed in a solid inner matrix such as polymethyl methacrylate, polybutyl methacrylate, plasticized or unplasticized polyvinyl chloride, plasticized nylon, plasticized polyethylene terephthalate, natural rubber, polyisoprene, polyisobutylene, polybutadiene, polyethylene, ethylene-vinyl acetate copolymers, silicone rubbers, polydimethyl siloxanes, silicon carbonate copolymers, hydrophilic polymers such as acrylic and methacrylic acid ester hydrogels, collagen, cross-linked polyvinyl alcohol and cross-linked partially hydrolyzed polyvinyl acetate, surrounded by an outer polymer membrane such as polyethylene, polypropylene, ethylene/propylene copolymers, ethylene/ethyl acrylate copolymers, ethylene/vinyl acetate copolymers, silicone rubbers, polydimethyl siloxanes, neoprene rubber, chlorinated polyethylene, polyvinyl chloride, copolymers of vinyl chloride with vinyl acetate, vinylidene chloride, ethylene and propylene, polyethylene terephthalate ionomer, butyl rubber, epichlorohydrin rubbers, ethylene/vinyl alcohol copolymer, ethylene/vinyl acetate/vinyl alcohol terpolymer and ethylene/vinyloxyethanol copolymer, insoluble in body fluids. The active ingredient then diffuses through the outer polymer membrane in a rate-controlled release step. The percentage of active ingredient in such parenteral compositions varies greatly depending on their specific nature as well as the needs of the target.

Parenteral dosage forms

In some embodiments, parenteral dosage forms are provided herein. In some embodiments, parenteral dosage forms can be administered to subjects via a variety of routes, including, but not limited to, subcutaneous, intravenous (including bolus injection), intramuscular, and intraarterial. In some embodiments, parenteral dosage forms can be administered to subjects via a variety of routes, including, but not limited to, topical, intradermal, or intralesional. Because their administration typically bypasses the subject's natural defenses against contaminants, parenteral dosage forms are typically sterile or can be sterilized prior to administration to the subject. In some embodiments, parenteral dosage forms include, but are not limited to, solutions ready for injection, dry products ready for solution or suspension in a pharmaceutically acceptable vehicle for injection, suspensions ready for injection, and emulsions.

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

Compounds that enhance the solubility of one or more of the active ingredients disclosed herein may also be included in parenteral dosage forms.

Transdermal, topical and transmucosal dosage forms

Transdermal, topical, and transmucosal dosage forms are also provided. Transdermal, topical, and transmucosal dosage forms include, but are not limited to, ophthalmic solutions, sprays, aerosols, creams, lotions, ointments, gels, solutions, emulsions, suspensions, or other forms known to one of skill in the art. See, e.g., 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 treatment of oral mucosal tissues can be prepared as mouth rinses or oral gels. Additionally, transdermal dosage forms include "reservoir type" or "matrix type" patches that can be applied to the skin and worn for a specified period of time to ensure penetration of the desired amount of active ingredients.

The term "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, involved in the carriage or transport of any subject composition or component thereof. Each carrier must be "acceptable" in the sense of being compatible with the subject composition and its components and not deleterious to the patient. Suitable carriers (e.g., excipients and diluents) and other materials that can be used to prepare the transdermal, topical, and mucosal dosage forms encompassed herein are well known to those skilled in the pharmaceutical arts and will depend on the particular tissue to which the pharmaceutical composition or dosage form is to be administered. In view of this fact, 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 to form lotions, tinctures, creams, emulsions, gels, or ointments that are nontoxic and pharmaceutically acceptable. In some embodiments, materials that can serve 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) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients such as cocoa butter and suppository wax; (9) oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; (10) glycols such as propylene glycol; (11) polyols such as glycerol, 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) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances used in pharmaceutical formulations. If desired, wetting agents or humectants may also be added to the pharmaceutical compositions and dosage forms. Examples of such additional ingredients are well known in the art. See, for example, Remington: The Science and Practice of Pharmacy; Pharmaceutical Press; 22nd edition (September 15, 2012).

Depending on the particular tissue to be treated, additional components may be used prior to, in combination with, or subsequent to treatment with the provided active ingredients. In some embodiments, penetration enhancers may be used to facilitate delivery of the active ingredients into 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; polyethyleneglycol; pyrrolidones such as polyvinylpyrrolidone; Kollidon grades (povidone, polyvidone); urea; and various water-soluble or insoluble sugar esters such as Tween 80 (polysorbate 80) and Span 60 (sorbitan monostearate).

The pH of the pharmaceutical composition or dosage form or the tissue to which the pharmaceutical composition or dosage form is applied can also be adjusted to improve the delivery of one or more active ingredients. Likewise, the polarity of the carrier solvent, its ionic strength or tonicity can be adjusted to improve delivery. Compounds such as stearates can also be added to pharmaceutical compositions or dosage forms to advantageously change the hydrophilicity or lipophilicity of one or more active ingredients to improve delivery. In this regard, stearates can serve as a lipid carrier for the formulation, as an emulsifying agent or surfactant, as well as a delivery enhancing or penetration enhancing agent. Various complexes, salts, hydrates or solvates of the active ingredients can be used to further adjust the properties of the resulting composition.

Dosage and single dosage forms

In human therapy, the physician determines the dosage he considers most appropriate, in accordance with the prophylactic or curative treatment, as well as in accordance with the age, body weight, stage of the disorder or disease and other factors characteristic of the subject to be treated. In some embodiments, the doses are from about 1 to about 1000 mg per day for an adult, or from about 5 to about 250 mg per day, or from about 10 to 50 mg per day for an adult. In some embodiments, the doses are from about 5 to about 400 mg per day or from 25 to 200 mg per day per adult. In some embodiments, doses from about 50 to about 500 mg per day are also contemplated.

In additional aspects, methods are provided for treating a disease or disorder in need thereof and/or a genodermatosis in a subject by administering to a subject in need thereof a therapeutically or prophylactically effective amount of a compound as provided herein, or a pharmaceutically acceptable complex thereof. The amount of a compound or composition that will be therapeutically or prophylactically effective in treating a disorder or one or more symptoms thereof will vary depending on the nature and severity of the disease or condition and the route of administration of the active ingredient. The frequency and dosage will also vary depending on factors specific to each subject, depending on the particular therapy (e.g., therapeutic or prophylactic agents) administered, the severity of the disorder, disease or condition, the route of administration, and the age, body weight, response and medical history of the subject. Effective doses can be extrapolated from dose-response curves obtained in in vitro test systems or in animal models.

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

In some embodiments, the recommended daily dosage range of the composition provided herein for the diseases or disorders described herein is in the range of about 0.1 mg to about 1000 mg per day, administered as a single dose once a day or in divided doses throughout the day. In some embodiments, the daily dose is administered twice a day in equal doses. In some embodiments, the daily dosage range should be from about 10 to about 200 mg per day, in other embodiments from about 10 to about 150 mg per day, in further embodiments from about 25 to about 100 mg per day. In some cases, it may be necessary to use dosages of the active ingredient outside the ranges disclosed herein, as will be apparent to those skilled in the art. Furthermore, it should be noted that the clinician or physician will know how and when to interrupt, adjust, or discontinue therapy depending on the response of the subject.

Various therapeutically effective amounts may be useful for various diseases and conditions, as will be readily known to those skilled in the art. Likewise, amounts sufficient to prevent, control, treat, or alleviate such disorders, but insufficient to cause or sufficient to reduce side effects associated with the composition provided herein, are also encompassed by the dosage amounts and dosing frequency regimens described herein. Furthermore, when multiple doses of the composition provided herein are administered to a subject, it is not necessary that all doses be the same. In some embodiments, the dose administered to a subject may be increased to improve the prophylactic or therapeutic effect of the composition, or may be decreased to reduce one or more side effects experienced by a particular subject.

In some embodiments, the dosage of the composition provided herein, based on the weight of the active compound administered to prevent, treat, control, or alleviate 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 another embodiment, the dosage of the composition or the composition provided herein administered to prevent, treat, control or ameliorate the disorder or one or more symptoms thereof in a subject is a unit dose of from 0.1 mg to 200 mg, from 0.1 mg to 100 mg, from 0.1 mg to 50 mg, from 0.1 mg to 25 mg, from 0.1 mg to 20 mg, from 0.1 mg to 15 mg, from 0.1 mg to 10 mg, from 0.1 mg to 7.5 mg, from 0.1 mg to 5 mg, from 0.1 to 2.5 mg, from 0.25 mg to 20 mg, from 0.25 to 15 mg, from 0.25 to 12 mg, from 0.25 to 10 mg, from 0.25 mg to 7.5 mg, from 0.25 mg to 5 mg, from 0.5 mg to 2.5 mg, 1 mg to 20 mg, 1 mg to 15 mg, 1 mg to 12 mg, 1 mg to 10 mg, 1 mg to 7.5 mg, 1 mg to 5 mg, or 1 mg to 2.5 mg.

In some embodiments, treatment or prophylaxis may be initiated with one or more loading doses of a compound or composition provided herein, followed by one or more maintenance doses. In such embodiments, the loading dose may be, for example, from about 60 to about 400 mg per day, or from about 100 to about 200 mg per day for from one day to five weeks. The loading dose may be followed by one or more maintenance doses. In some embodiments, each maintenance dose is independently from about 10 mg to about 200 mg per day, from about 25 mg to about 150 mg per day, or from about 25 to about 80 mg per day. The maintenance doses may be administered daily and in single doses or in divided doses.

In some embodiments, a dose of a compound or composition provided herein can be administered to achieve a steady state concentration of the active ingredient in the blood or serum of the subject. The steady state concentration can be determined by measurement according to methods available to those skilled in the art, or can be based on physical characteristics of the subject, such as height, weight, and age. In some embodiments, a sufficient amount of a compound or composition provided herein is administered to achieve a steady state concentration in the blood or serum of the subject of from about 300 to about 4000 ng/mL, from about 400 to about 1600 ng/mL, from about 600 to about 1200 ng/mL. In some embodiments, a sufficient amount of a compound or composition provided herein is administered locally to achieve a steady state concentration in the blood or serum of the subject of from about 0.01 to about 300 ng/mL, from about 0.01 to about 100 ng/mL. ml, from about 0.01 to about 10 ng/ml, from about 0.01 to about 1 ng/ml, from about 0.01 to about 0.1 ng/ml, or from about 0.01 to about 0.05 ng/ml. In some embodiments, loading doses can be administered to achieve steady state blood or serum concentrations of from about 1200 to about 8000 ng/ml or from about 2000 to about 4000 ng/ml over one to five days. In some embodiments, loading doses can be administered locally to achieve steady-state concentrations in the blood or serum of from about 0.05 to about 1200 ng/mL, from about 0.05 to about 100 ng/mL, from about 0.05 to about 10 ng/mL, from about 0.05 to about 1 ng/mL, from about 0.05 to about 0.5 ng/mL, or from about 0.05 to about 0.1 ng/mL for from one to five days. In some embodiments, maintenance doses can be administered to achieve a steady-state concentration in the blood or serum of the subject of from about 300 to about 4000 ng/mL, from about 400 to about 1600 ng/mL, or from about 600 to about 1200 ng/mL. In some embodiments, maintenance doses can be administered locally to achieve a steady state concentration in the blood or serum of a subject of from about 0.01 to about 300 ng/mL, from about 0.01 to about 100 ng/mL, from about 0.01 to about 10 ng/mL, from about 0.01 to about 1 ng/mL, from about 0.01 to about 0.1 ng/mL, or from about 0.01 to about 0.05 ng/mL.

In some embodiments, the administration of the same composition may be repeated, and the administrations may be separated by at least 1 day, 2 days, 3 days, 5 days, 10 days, 15 days, 30 days, 45 days, 2 months, 75 days, 3 months or 6 months. In other embodiments, the administration of the same prophylactic or therapeutic agent may be repeated, and the administration may be separated by at least 1 day, 2 days, 3 days, 5 days, 10 days, 15 days, 30 days, 45 days, 2 months, 75 days, 3 months or 6 months.

In some aspects, provided herein are unit doses comprising a compound or a pharmaceutically acceptable complex thereof in a form suitable for administration. Such forms are described in detail herein. In some embodiments, a unit dose comprises 1 to 1000 mg, 5 to 250 mg, or 10 to 50 mg of the active ingredient. In particular embodiments, unit doses comprise about 1, 5, 10, 25, 50, 100, 125, 250, 500, or 1000 mg of the active ingredient. Such unit doses can be prepared according to methods known to those skilled in the art.

The dosage may vary over a range depending on the dosage form used and the route of administration employed. For any compound, the therapeutically effective dose can be estimated initially from cell culture assays. The dose can be formulated in animal models to achieve a level in the skin of the lesion (e.g. Netherton syndrome or KLK protease-mediated skin disease) that includes the IC ₅₀ (i.e., the concentration of the test compound that achieves half the maximal suppression of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses for humans. In addition, plasma levels can be measured, for example, by high-performance liquid chromatography, to determine systemic exposure.

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

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

In some embodiments, the local dose is about 0.5-1.0 mg/ ^cm2 , 1.0-1.5 mg/ ^cm2 , 1.5-2.0 mg/ ^cm2 , 2.5-2.5 mg/ ^cm2 , 3.0-3.5 mg/ ^cm2 , 3.5-5.0 mg/ ^cm2 , 5.0-7.5 mg/ ^cm2 , 7.5-10 mg/ ^cm2 , 1-10 mg/ ^cm2 , about 10-20 mg/ ^cm2 , about 20-30 mg/ ^cm2 , about 30-40 mg/ ^cm2 , about 40-50 mg/ ^cm2 , about 50-60 mg/ ^cm2 , about 60-70 mg/ ^cm2 , about 70-80 mg/ ^cm2 , about 80-90 mg/ ^cm2 , about 90-100 mg/ ^cm2 , about 100-125 mg/ ^cm2 , about 125-150 mg/ ^cm2 , about 150-175 mg/ ^cm2 , about 175-200 mg/ ^cm2 , about 200-250 mg/ ^cm2 , about 250-300 mg/ ^cm2 , about 300-350 mg/ ^cm2 , about 350-400 mg/ ^cm2 , about 400-450 mg/ ^cm2 , about 450-500 mg/ ^cm2 , about 500-550 mg/ ^cm2 , about 550-600 mg/ ^cm2 , about 600-650 mg/ ^cm2 , about 650-700 mg/ ^cm2 , about 700-750 mg/ ^cm2 , about 750-800 mg/ ^cm2 , about 800-850 mg/ ^cm2 , about 850-900 mg/ ^cm2 , about 900-950 mg/ ^cm2 or about 950-1000 mg/ ^cm2 .

V. Methods

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

In some embodiments, the compound of formula (I) has the formula:

,

or a pharmaceutically acceptable complex thereof.

In some embodiments, the compound of formula (I) has the formula:

,

or a pharmaceutically acceptable complex thereof.

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

In some embodiments, the skin disorder is a genodermatosis. In some embodiments, the genodermatosis is Netherton syndrome.

In some embodiments, the subject has loss-of-function truncating mutations in Kazal-type serine protease inhibitor 5 (SPINK5). In some embodiments, the subject has a loss of function of LEKTI (lymphoepithelial inhibitor Kazal-type). In some embodiments, the subject overexpresses one or more kallikrein (KLK) serine proteases. In some embodiments, the subject overexpresses one or more of KLK5, KLK7, and KLK14 proteases. In some embodiments, the subject overexpresses KLK5 protease. In some embodiments, the subject overexpresses KLK7 protease. In some embodiments, the subject overexpresses KLK14 protease. In some embodiments, the subject overexpresses KLK5 and KLK7 proteases. In some embodiments, the subject overexpresses 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 of the KLK5, KLK7, and KLK14 proteases. In some embodiments, the subject has increased proteolytic activity of the KLK5 protease. In some embodiments, the subject has increased proteolytic activity of the KLK7 protease. In some embodiments, the subject has increased proteolytic activity of the KLK14 protease. In some embodiments, the subject has increased proteolytic activity of the KLK5 and KLK7 proteases. In some embodiments, the subject has increased proteolytic activity of the KLK5, KLK7, and KLK14 proteases.

In some embodiments, the present document provides a method of treating Netherton syndrome when a subject is in need thereof. The method comprises administering to the subject an effective amount of a compound of formula (I) or a pharmaceutically acceptable complex thereof, or a pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable complex thereof.

In some embodiments, provided herein is a method of treating a genodermatosis wherein a subject is in need thereof and the genodermatosis is mediated by one or more KLK proteases. The method comprises administering to the subject an effective amount of a compound of formula (I) or a pharmaceutically acceptable complex thereof, or a pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable complex thereof. In some embodiments, the genodermatosis is mediated by a KLK5 protease, a KLK7 protease, a KLK14 protease, or combinations thereof. In some embodiments, the genodermatosis is mediated by a KLK5 protease. In some embodiments, the genodermatosis is mediated by a KLK7 protease. In some embodiments, the genodermatosis is mediated by a KLK14 protease. In some embodiments, the genodermatosis is mediated by a KLK5 and KLK7 proteases. In some embodiments, the genodermatosis is mediated by the proteases KLK5, KLK7, and KLK14. In some embodiments, the genodermatosis is Netherton syndrome.

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

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

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. Thus, in a further aspect, the present document provides a method of treating a skin disease in need thereof and the skin disease is mediated by one or more KLK proteases. The method comprises administering to the subject an effective amount of a compound of formula (I) or a pharmaceutically acceptable complex thereof, or a pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable complex thereof. In some embodiments, the skin disease is mediated by KLK5 protease, KLK7 protease, KLK14 protease, or combinations 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 the proteases KLK5 and KLK7. In some embodiments, the skin disease is mediated by the proteases KLK5, KLK7, and KLK14. In some embodiments, the skin disease is a KLK-mediated disease selected from the group consisting of Netherton syndrome, scaly skin syndrome, acne rosacea, psoriasis, eczema, and atopic dermatitis. In some embodiments, the KLK-mediated disease is Netherton syndrome.

In some embodiments, provided herein is a method of treating a skin disease in a subject by inhibiting the proteolytic activity of one or more KLK proteases. The method comprises administering to the subject an effective amount of a compound of Formula (I), or a pharmaceutically acceptable complex thereof, or a pharmaceutical composition comprising a compound of Formula (I), or a pharmaceutically acceptable complex thereof. In some embodiments, the compound of Formula (I) inhibits the proteolytic activity of one or more KLK proteases. In some embodiments, the compound of Formula (I) inhibits the proteolytic activity of KLK5 protease, KLK7 protease, KLK14 protease, or combinations thereof. In some embodiments, the compound of Formula (I) inhibits the proteolytic activity of KLK5 protease. In some embodiments, the compound of Formula (I) inhibits the proteolytic activity of KLK7 protease. In some embodiments, the compound of Formula (I) inhibits the proteolytic activity of KLK14 protease. In some embodiments, the compound of formula (I) inhibits the proteolytic activity of the KLK5 and KLK7 proteases. In some embodiments, the compound of formula (I) inhibits the proteolytic activity of the KLK5, KLK7 and KLK14 proteases. In some embodiments, the compound of formula (I) is a KLK5 inhibitor. In some embodiments, the compound of formula (I) is a KLK7 inhibitor. In some embodiments, the compound of formula (I) is a KLK14 inhibitor. In some embodiments, the compound of formula (I) is a dual KLK5/KLK7 inhibitor. In some embodiments, the skin disease is selected from the group consisting of Netherton syndrome, scaly skin syndrome, acne rosacea, psoriasis, eczema and atopic dermatitis. In some embodiments, the skin disease is Netherton syndrome.

A compound of formula (I) or a pharmaceutical composition comprising a compound of formula (I) according to the invention can be used for both prophylactic and therapeutic treatment of a skin disease or pathology or other undesirable skin condition in a subject. For example, the compositions of the present invention can be used to prevent skin irritation, prevent rashes, accelerate the healing of skin tissue after the occurrence of a rash or irritation (i.e. the formation of epidermal and/or dermal layers of the skin), prevent and/or slow down skin atrophy, prevent and/or slow down the appearance of spider veins and/or red spots on the skin, prevent and/or slow down skin flaking, prevent and/or relieve skin itching, regulate skin texture (e.g., reduce flaking, roughness, swelling and soreness) and improve skin color (e.g., reduce redness).

Prophylactic or therapeutic treatment of a genodermatosis or skin disease in a subject can be carried out by applying a compound of formula (I) or a pharmaceutical composition thereof in the form of a skin lotion, cream, gel, foam, ointment, paste, emulsion, spray, conditioner, tonic, patch, etc., which are preferably intended to be left on the skin. After applying the composition to the skin, it can be left on the skin for at least about 15 minutes, or at least about 30 minutes, or at least about 1 hour, or at least several hours, such as at least about 2 hours, 3 hours, 6 hours, 12 hours, or 24 hours.

The compounds of the present invention are also useful for regulating desquamation in vitro by inhibiting the protease activity of one or more KLK proteases in cell/tissue culture procedures. Thus, in a further aspect, the invention relates to a method for disaggregating cells or tissue in vitro, the method comprising the steps of exposing the cells or tissues to a KLK protease (e.g., KLK5, KLK7 and/or KLK14) followed by exposure to a compound of formula (I) or a pharmaceutically acceptable complex thereof according to the present invention.

It should be understood that this method is generally applicable to cell culture procedures, where this method can be used to dissociate and separate cells for passage. In addition, this method can be used in the culture of artificial skin, where selective cleavage of intercellular adhesion proteins by the KLK protease (e.g., KLK5, KLK7 and/or KLK14) in combination with the regulation of such cleavage by the compound of formula (I), a pharmaceutically acceptable complex thereof, leads to the fact that cells/tissues are better suited for subsequent therapeutic use.

In another aspect, provided herein is an in vitro assay for determining the proteolytic activity of one or more KLK proteases in skin. The method comprises 1) preparing a skin extract; 2) exposing a substrate and a compound of Formula (I), or a pharmaceutically acceptable complex thereof, to the skin extract; and 3) determining the rate of proteolytic cleavage of the substrate. In some embodiments, the substrate is a peptide-p-nitroanilide (also referred to as peptide-pNA). In some embodiments, the substrate is a dipeptide-pNA, a tripeptide-pNA, a tetrapeptide-pNA, a pentapeptide-pNA, or a 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 (e.g., mouse) skin extract. In some embodiments, the skin extract is a human skin extract.

VI. Examples

General synthetic methods

The symbols and conventions used in these processes, diagrams, and examples, whether or not a specific abbreviation is specifically defined, as used herein are consistent with those used in contemporary scientific literature, such as the American Chemical Society or the Journal of Biological Chemistry. In particular, but not limited to, the following abbreviations may be used in the examples and throughout the specification: eq (equivalent); g (grams); mg (milligrams); ml (milliliters); µl (microliter); mM (millimolar); µM (micromolar); Hz (Hertz); MHz (megahertz); mmol (millimoles); hr or hr (hours); min (minutes); RT (room temperature); MS (mass spectrometry); ESI (electrospray ionization); TLC (thin layer chromatography); HPLC (high pressure liquid chromatography); THF (tetrahydrofuran); _CDCl3 (deuterated chloroform); AcOH (acetic acid); DCM (dichloromethane); DMW (demineralized water); DME (dimethoxyethane); DMF (N,N-dimethylformamide); DMSO (dimethyl sulfoxide); DMSO-d6 (deuterated dimethyl sulfoxide); EtOAc (ethyl acetate); and MeOH (methanol).

For all of the following examples, standard work-up and purification methods known to those skilled in the art can be used. Unless otherwise noted, all temperatures are in °C (degrees Celsius). All reactions are conducted at room temperature unless otherwise noted. The synthetic methodologies illustrated herein are intended to illustrate the applicable chemistry through the use of specific examples and do not define the scope of the invention.

Example 1. 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)

The synthesis of compound 1.001 is shown in Fig. 1. The synthesis of intermediate compound 100 is illustrated in Fig. 3.

Step 1: 4-bromo-2-mercaptobenzoic acid (intermediate 102).

A mixture of conc. HCl (280.0 mL) and ice 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 equiv), NaOH (37.0 g, 926.0 mmol, 1.0 equiv), and sodium nitrite (63.90 g, 926.0 mmol, 1.0 equiv) in DMW (4.0 L) at a rate such that the reaction temperature was maintained at 3-6 °C. After the addition, the reaction mixture was stirred at 0 °C for 30 min and then neutralized with potassium acetate (454.0 g, 4.63 mol, 5.0 equiv). This solution was added to a preheated solution of potassium O-ethylxanthane (445.0 g, 2.78 mol, 3.0 equiv.) in DMW (4.0 L) to 90 °C. The reaction mixture was stirred at 90 °C for 30 min, cooled to 0 °C, and acidified with conc. HCl (600.0 mL). The reaction mixture was basified with 20% NaOH (1750 mL) and heated to 85 °C for 2 h. To this mixture was added NaHSO ₃ (96.30 g, 926.0 mmol, 1.0 equiv.) in small portions, and the reaction mixture was heated to 85 °C for 10 min. The reaction mixture was filtered, cooled to 0 °C, and acidified with conc. HCl (600.0 mL). The precipitated solid was collected by filtration, washed with DMW (500 mL) and then with n-hexane, and air-dried to afford intermediate 102 (216.0 g, 100.0%) as a light gray solid. ¹ H NMR (300 MHz, DMSO-d6): δ 7.88 ppm (d, J = 1.9 Hz, 1H), 7.81 ppm (t, J = 7.4 Hz, 1H), 7.38 ppm (dd, J = 8.4, 2.0 Hz, 1H). MS (ESI): m/z 232.8 (M-1).

Step 2: Methyl 4-bromo-2-(methylthio)benzoate (intermediate 103)

Potassium carbonate (896.0 g, 6.49 mol, 7.0 equiv) and methyl iodide (231.0 mL, 3.71 mol, 4.0 equiv) were added to a stirred solution of 4-bromo-2-mercaptobenzoic acid (102) (216.0 g, 927.0 mol, 1.0 equiv) in DMF (3.24 L) at RT. The reaction mixture was heated to 80 °C and stirred at 80 °C for 20 h. The reaction progress was monitored by TLC (10% EtOAc in n-hexane) to ensure completion of the reaction. DMW (2.5 L) was added and the product was extracted with ethyl acetate (2×2.5 L). The combined EtOAc extract was washed with DMW (2×2.5 L), brine (2.5 L), dried over sodium sulfate, filtered and the filtrate was concentrated in vacuo to give intermediate 103 (180 g, 74.40%) as a brown oil. ¹ H NMR (300 MHz, DMSO-d6): δ 7.83 ppm (d, J = 8.3 Hz, 1H), 7.51-7.39 ppm (m, 2H), 3.83 ppm (s, 3H), 2.46 ppm (s, 3H).

Step 3: Methyl 4-bromo-2-(methylsulfonyl)benzoate (intermediate 104)

A solution of oxone (1.17 kg, 3.79 mol, 5.50 equiv) in DMW (4.50 L) was added to a stirred solution of methyl 4-bromo-2-(methylthio)benzoate (103) (180.0 g, 589 mmol, 1.0 equiv) in methanol (3.60 L) at 0 °C. The reaction mixture was stirred at 60 °C for 16 h. The reaction progress was monitored by TLC (50% EtOAc in n-hexane). About 40% of the solvent was removed by distillation, then cooled DMW (4.50 L) was added to the reaction mixture and stirred for 15 min. The precipitated solid was collected by filtration to give the wet product 104. The wet product was dried in a vacuum oven below 50 °C to give dried intermediate 104 (200 g, 99.0%) as an off-white solid. ¹ H NMR (300 MHz, DMSO-d6): δ 8.19–8.02 ppm (m, 2H), 7.73 ppm (d, J = 8.1 Hz, 1H), 3.87 ppm (s, 3H), 3.42 ppm (s, 3H). MS (ESI): m/z 311.5 (M+18).

Step 4: 4-bromo-2-(methylsulfonyl)benzoic acid (intermediate 105)

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

Step 5: 4-bromo-2-(methylsulfonyl)benzoyl chloride (intermediate 106)

Thionyl chloride (68.4 mL, 940.0 mmol, 2.50 equiv) was added to a stirred solution of 4-bromo-2-(methylsulfonyl)benzoic acid (105) (105.0 g, 376.0 mmol, 1.0 equiv) in THF (1.05 L), followed by the addition of DMF (1.37 mL, 18.80 mmol, 0.05 equiv) at RT. The reaction mixture was heated to 80 °C and stirred for 2 h. The reaction progress was monitored by TLC (50% EtOAc in n-hexane) to ensure completion of the reaction. The solvent was removed in vacuo to yield intermediate 106 (112.0 g). The crude material of intermediate 106 was used directly in step 6.

Step 6: 7-(4-bromo-2-(methylsulfonyl)benzamido)-2,3-dihydrobenzo[b][1,4]dioxin-6-carboxylic acid (intermediate 107)

Triethylamine (103.0 mL, 753.0 mmol, 2.0 equiv) was added 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 equiv) in THF (1.68 L) at 0 °C, followed by a solution of 4-bromo-2-(methylsulfonyl)benzoyl chloride (106) (60.20 g, 309 mmol, 1.0 equiv) in THF (1.68 L) at 0 °C. The reaction mixture was stirred at 0 °C for 10 min and then at RT for 2.5 h. The progress of the reaction was monitored by TLC (10% MeOH in DCM). 1 N HCl was added to adjust the pH to 7. About 70% of the THF was removed by distillation and the remaining residue was triturated with diethyl ether (250 mL) to precipitate a solid. The solid was collected by filtration, washed with diethyl ether (50 mL), and dried to give intermediate 107 (120.0 g, 69.90%) as a light brown solid.

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

Step A: Methyl 3,4-dihydroxybenzoate (intermediate 100B).

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

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

Potassium carbonate (405 g, 2.93 mol, 2.50 equiv) and 1,2-dibromoethane (152.0 mL, 1.76 mol, 1.50 equiv) were added to a stirred solution of methyl 3,4-dihydroxybenzoate (100B) (197.0 g, 1.17 mol, 1.0 equiv) in DMF (2.96 L). The reaction mixture was heated at 90 °C and stirred for 25 h. The reaction progress was monitored by TLC (TLC analysis of 60% ethyl acetate in n-heptane). The reaction mixture was cooled to RT, then DMW (7.50 L) was added, then extracted with EtOAc (2 x 3.50 L). The combined organic extract was washed with DMW (2×3.5 L) and brine (3.5 L), dried over sodium sulfate and concentrated in vacuo to give intermediate 100C (214.0 g, 94.10%) as a light brown oil. ¹ H NMR (300 MHz, CDCl3): δ 7.63 – 7.51 ppm (m, 2H), 6.89 ppm (d, J = 9.0 Hz, 1H), 4.35 – 4.24 ppm (m, 4H), 3.88 ppm (s, 3H).

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

Conc. _HNO3 (832.0 mL, 14.50 mol, 17.0 equiv) was slowly added to a stirred solution of methyl 2,3-dihydrobenzo[b][1,4]dioxin-6-carboxylate (100°C) (214.0 g, 1.10 mol, 1.0 equiv) in acetic acid (832.0 mL, 14.50 mol, 13.2 equiv) at a temperature below 20°C. The reaction mixture was stirred at room temperature for 2 h, monitoring the reaction progress by TLC (eluent: 60% ethyl acetate in n-heptane). The reaction mixture was poured into ice water (5.0 L) with vigorous stirring. The precipitated solid was collected by filtration, washed with DMW (1100 mL) and dried to give intermediate 100D (261 g, 99.0%) as a light yellow solid. ¹ H NMR (300 MHz, DMSO-d6): δ 7.65 ppm (s, 1H), 7.30 ppm (s, 1H), 4.47 – 4.34 ppm (m, 1H), 4.39 ppm (s, 4H), 3.80 ppm (s, 3H).

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

A mixture of methyl 7-nitro-2,3-dihydrobenzo[b][1,4]dioxin-6-carboxylate (100D) (260 g, 1.09 mol, 1.0 equiv), iron dust (217.0 g, 2.72 mol, 2.5 equiv, 300-400 mesh size sieve) in ethanol (1.69 L, 6.50 vol equiv), water (585 mL), and acetic acid (1.69 L) was heated to 80 °C and stirred at this temperature for 45 min. The progress of the reaction was monitored by TLC (eluent: 60% ethyl acetate in n-heptane). The reaction mixture was cooled to room temperature, filtered through a pad of celite, and the pad was washed with ethyl acetate (500 mL). Water (5.0 L) was added to the filtrate and stirred. The aqueous layer was separated and re-extracted with ethyl acetate (3 x 2.0 L). The combined organic extract was washed with saturated sodium bicarbonate (3 x 3.0 L) and brine (5.0 L), then dried over sodium sulfate and filtered. The filtrate was concentrated in vacuo to give intermediate 100E (226 g, 99.40%) as a light brown solid. ¹ H NMR (300 MHz, DMSO-d6): δ 7.14 ppm (s, 1H), 6.27 ppm (s, 2H), 6.24 ppm (s, 1H), 4.28 – 4.19 ppm (m, 2H), 4.16 – 4.06 ppm. (m, 2H), 3.73 ppm (s, 3H).

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

To 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 equiv) in THF (1.58 mL) was added a solution of NaOH (237.0 g, 5.92 mol, 5.50 equiv) in water (1.58 mL), the reaction mixture was heated to 80 °C and stirred for 15 h. The reaction progress was monitored by TLC (10% MeOH in DCM) to ensure completion of the reaction. The reaction mixture was cooled to room temperature and THF was evaporated under reduced pressure. Then cold DMW (1500 mL) was added to the residue and the pH was adjusted to ~6.0 with 5 N HCl. The precipitated solid was collected by filtration, washed with DMW (300 mL) and dried to give intermediate 100 (152.50 g, 72.5%) as a light yellow solid. ¹ H NMR (300 MHz, DMSO): δ 8.17 ppm (s, 2H), 7.13 ppm (s, 1H), 6.20 ppm (s, 1H), 4.27 – 4.17 ppm (m, 2H), 4.17 – 4.02 ppm (m, 2H).

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

A solution of 7-(4-bromo-2-(methylsulfonyl)benzamido)-2,3-dihydrobenzo[b][1,4]dioxin-6-carboxylic acid (107) (120.0 g, 263.0 mmol, 1.0 equiv.) in acetic anhydride (960 mL) was heated to 140 °C and stirred for 1.5 h. The reaction progress was monitored by TLC (50% EtOAc in n-hexane) to ensure completion of the reaction. About 70% of the solvent was removed by vacuum distillation. The remaining residue was cooled to room temperature, then DMW (1000 mL) was added and the mixture was stirred for 40 min. The precipitated solid was collected by filtration, washed with DMW (1000 mL) and dried to afford intermediate 108 (85.4 g, 74.1%) as an off-white solid. ¹ H NMR (300 MHz, DMSO-d6): δ 8.29 – 8.12 ppm (m, 2H), 7.91 ppm (d, J = 8.2 Hz, 1H), 7.59 ppm (s, 1H), 7.26 ppm (s, 1H), 4.55-4.32 ppm (m, 4H), 3.57 ppm (s, 3H). MS (ESI): m/z 439.6 (M+1).

Step 8: Synthesis 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 (intermediate 109)

A solution of 2-(4-bromo-2-(methylsulfonyl)phenyl)-7,8-dihydro-4H-[1,4]dioxino[2',3':4,5]benzo[1,2-d ][1,3]oxazin-4-one (108) (84.0 g, 192.0 mmol, 1.0 equiv) in 1,4-dioxane (1.68 L) was degassed three times using alternating vacuum and nitrogen refill to remove oxygen. Sodium bicarbonate (56.40 g, 671.0 mmol, 3.5 equiv), potassium vinyl trifluoroborate (64.20 g, 479.0 mmol, 2.5 equiv), tri(o-tolyl)phosphine (14.60 g, 47.90 mmol, 0.25 equiv), and Pd(OAc)2 (1.51 g, 6.71 mmol, 0.035 equiv) were added under nitrogen atmosphere and the reaction flask was degassed two more times by alternating vacuum and nitrogen fill. The reaction mixture was heated at 70 °C and stirred for 3.5 h. The reaction progress was monitored by TLC (40% EtOAc in n-hexane) to ensure completion of the reaction. The reaction mixture was cooled to room temperature, quenched with 1 N. The mixture was diluted with 100 ml of HCl solution (1000 ml) and filtered through a Hiflo filter. The solvent was evaporated under reduced pressure and DMW (2.5 L) was added to the residue. The product was extracted with ethyl acetate (3×2 L) and the combined EtOAc extract was washed with brine (2.5 L), dried over sodium sulfate, filtered and concentrated to give the crude product. The crude product was stirred in diethyl ether (150 ml) and filtered to give intermediate 109 (72.0 g, 97.50%) as a light yellow solid. ¹ H NMR (300 MHz, DMSO-d6): δ 8.19–8.10 ppm (m, 1H), 8.06–8.00 ppm (m, 1H), 7.93 ppm (m, 1H). (dd, J = 7.9, 3.5 Hz, 1H), 7.58 ppm (s, 1H), 7.24 ppm (s, 1H), 6.96 ppm (dd, J = 17.7, 11.0 Hz, 1H), 6.10 ppm (t, J = 17.7 Hz, 1H), 5.56 ppm. (d, J = 11.0 Hz), 1H), 4.55–4.26 ppm. (m, 4H), 3.56–3.46 ppm (m, 3H).

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)

Ruthenium on carbon (10%) (5.66 g, 5.60 mmol, 0.03 equiv) 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 equiv) 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 equiv) was added at 0 °C. The reaction mixture was stirred at 0 °C for 3.0 h. The progress of the reaction was monitored by TLC (50% EtOAc in n-hexane). The reaction mixture was filtered through a pad of Celite. 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 re-extracted with ethyl acetate (800 mL). The combined organic extract was washed with brine (2.0 L), dried over sodium sulfate and filtered. The filtrate was concentrated in vacuo to give 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.

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)

Sodium borohydride (8.0 g, 1.0 equiv) was slowly added to a stirred solution of 3-(methylsulfonyl)-4-(4-oxo-7,8-dihydro-4H-[1,4]dioxino[2',3':4,5]benzo[1,2-d][1,3]oxazin-2-yl)benzaldehyde (110) (82.0 g, 212.0 mmol, 1.0 equiv) in THF (1.23 mL) and ethanol (1.23 L) at 0 °C over 1.0 h. The reaction mixture was stirred at 0 °C for 2 h. The reaction progress was monitored by TLC (70% EtOAc in n-hexane) to ensure completion of the reaction. The reaction mixture was quenched with saturated ammonium chloride solution (100 mL) and the product was extracted with ethyl acetate (3 x 1500 mL). The combined organic extract was washed with brine (500 mL), dried over sodium sulfate and concentrated in vacuo to give the crude product. The crude product was purified by column chromatography on silica gel (230-400 mesh) using a gradient solvent (20-40% [(10% MeOH in EtOAc)] in n-hexane) to give compound 1.001 (15.50 g, 18.80%) as an off-white solid. ¹ H NMR (300 MHz, DMSO-d6): δ 8.08 ppm (d, J = 1.5 Hz, 1H), 7.91 ppm (d, J = 1.5 Hz, 1H). (d, J = 7.9 Hz, 1H), 7.83 ppm (dd, J = 7.9, 1.6 Hz, 1H), 7.58 ppm (s, 1H), 7.24 ppm (s, 1H), 5.64 ppm (t, J = 5.7 Hz, 1H), 4.70 ppm. (d, J = 5.7 Hz, 2H), 4.47 – 4.35 ppm. (m, 4H), 3.50 ppm (c, 3H).

Example 2: 2-(5-(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 1.002)

The synthesis of compound 1.002 is shown in Fig. 2. The synthesis of intermediate 100 is illustrated in Fig. 3.

Step 1: 5-bromo-2-mercaptobenzoic acid (intermediate 202)

A mixture of concentrated HCl (28.0 mL) and chilled 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 equiv), NaOH (3.70 g, 92.6 mmol, 1.0 equiv), and sodium nitrite (6.39 g, 92.6 mmol, 1.0 equiv) in DMW (400.0 mL) while maintaining the internal temperature at 3-6 °C. The reaction mixture was stirred at 0 °C for 30 min and then neutralized with potassium acetate (30.0 g, 306.0 mmol, 3.3 equiv). This solution was added to a solution of potassium O-ethyl xanthate (44.6 g, 278.0 mmol, 3.0 equiv.) in DMW (223 mL) preheated to 90 °C. The mixture was stirred at the same temperature for 30 min, cooled to 0 °C and acidified with conc. HCl (100 mL). The reaction mixture was basified with 10% NaOH (200 mL) and heated to 85 °C for 2 h. To this mixture, NaHSO3 (9.81 g, 92.6 mmol) was added portionwise and the mixture was heated to 85 °C for 10 min. The mixture was filtered, cooled to 0 °C and acidified with conc. HCl (100 mL). The precipitate was collected by filtration and washed with water and then n-hexane to give intermediate 202 (20.2 g, 93.6%) as a pale brown solid. MS (ESI): m/z 230.8 (M-1).

Step 2: Methyl 5-bromo-2-(methylthio)benzoate (intermediate 203)

Potassium carbonate (83.0 g, 601 mmol, 7.0 equiv) and ethyl iodide (21.4 mL, 343 mmol, 4.0 equiv) were added at 0 °C to a stirred solution of 5-bromo-2-mercaptobenzoic acid (202) (20 g, 85.8 mmol, 1 equiv) in DMF (600 mL) at room temperature. The reaction mixture was stirred at 80 °C for 4 h. The reaction progress was monitored by TLC (10% EtOAc in n-hexane) to ensure completion of the reaction. DMW (500 mL) was added and the product was extracted with ethyl acetate (3×500 mL). The combined EtOAc extract was washed with brine (300 mL), dried over sodium sulfate, filtered, and the filtrate was concentrated in vacuo to give the crude compound. The crude product was purified by column chromatography on silica gel (230-400 mesh) and a gradient solvent (0-5% EtOAc in n-hexane) to give intermediate 203 (20 g, 89.3%) as an off-white solid.

Step 3: Methyl 5-bromo-2-(methylsulfonyl)benzoate (intermediate 204)

A solution of oxone (40.8 g, 268 mmol, 5.0 equiv) in DMW (280 mL) was added to a stirred solution of methyl 5-bromo-2-(methylthio)benzoate (203) (14.0 g, 53.6 mmol, 1.0 equiv) in methanol (350 mL) at 0 °C. The reaction mixture was stirred at 50 °C for 16 h. The reaction progress was monitored by TLC (70% EtOAc in n-hexane) to ensure completion. The product was extracted with EtOAc (4 x 500 mL). The combined organic extract was washed with brine (2 x 300 mL), dried over sodium sulfate, and concentrated in vacuo to give the crude product. The crude product was purified by column chromatography on silica gel (230-400 mesh) and a gradient solvent (5-20% EtOAc in n-hexane) to afford intermediate 204 (13.2 g, 84.0%) as an off-white solid. ¹ H NMR (300 MHz, DMSO): δ 8.08 – 7.98 ppm (m, 2H), 7.93 ppm (dd, J = 7.9, 0.9 Hz, 1H), 3.87 ppm (s, 3H), 3.44 – 3.35 ppm (m, 3H). MS (ESI): m/z 292.8 (M-1).

Step 4: Methyl 2-(methylsulfonyl)-5-vinyl benzoate (intermediate 205)

A solution of methyl 5-bromo-2-(methylsulfonyl)benzoate (204) (5.0 g, 17.1 mmol, 1.0 equiv.) in DME (100.0 mL, 20 vol equiv.) was degassed three times with alternating vacuum and refilled with nitrogen twice with alternating vacuum and nitrogen. Potassium carbonate (4.71 g, 34.1 mmol, 2.0 equiv.), potassium vinyl trifluoroborate (4.57 g, 34.1 mmol, 2.0 equiv.), and Pd( _PPh3 ) ₄ (591 mg, 512 μmol, 0.03 equiv.) were added under nitrogen and the reaction flask was degassed two additional times with alternating vacuum and refill with nitrogen. The reaction mixture was heated at 80 °C and stirred for 4 h (N ₂ was removed by raising the temperature to 40-45 °C). The reaction progress was monitored by TLC (70% EtOAc in n-hexane) to ensure completion. The reaction mixture was cooled to room temperature and filtered through a Hyflo filter. DMW (300 mL) was added to the filtrate and the product was extracted with ethyl acetate (2 x 100 mL). The combined organic extract was washed with brine (2 x 100 mL), dried over sodium sulfate, filtered and concentrated to give the crude product. The crude product was purified by column chromatography on silica gel (230-400 mesh) using a gradient solvent (5-20% EtOAc in n-hexane) as eluent to give intermediate 205 (3.36 g, 82.0%) as a light yellow gum.

Step 5: 2-(methylsulfonyl)-5-vinylbenzoic acid (intermediate 206).

To a stirred solution of methyl 2-(methylsulfonyl)-5-vinylbenzoate (205) (3.36 g, 14.0 mmol, 1.0 equiv) in methanol (67.2 mL) was added a solution of NaOH (3.92 g, 97.9 mmol, 7.0 equiv) in water (67.2 mL). The reaction mixture was stirred at 60 °C for 2 h. The reaction progress was monitored by TLC (70% EtOH in n-hexane) to ensure completion of the reaction. The reaction mixture was cooled to room temperature, the pH was adjusted to ~6 with 3 N HCl, and the aqueous layer was extracted with ethyl acetate (4×500 mL). The combined organic extract was washed with brine, dried over sodium sulfate, filtered and the filtrate was concentrated in vacuo to give the desired intermediate 206 (1.48 g, 46.8%) as an off-white solid. ¹ H NMR (300 MHz, DMSO): δ 7.93 ppm (t, J = 7.6 Hz, 1H), 7.88 – 7.78 ppm (m, 2H), 6.87 ppm (dd, J = 17.7, 11.0 Hz, 1H), 6.13 ppm (g, J = 17.6 Hz, 1H), 5.53 ppm (d, J = 10.9 Hz, 1H), 3.37 ppm (s, 3H). MS (ESI): m/z 224.95 (M-1).

Step 6: 2-(methylsulfonyl)-5-vinylbenzoyl chloride (intermediate 207).

Oxalyl chloride (2.66 mL, 30.9 mmol, 5.0 equiv) was added to a stirred solution of 2-(methylsulfonyl)-5-vinylbenzoic acid (206) (1.4 g, 6.19 mmol, 1.0 equiv) in DCM (70 mL), followed by the addition of 2 drops of DMF at RT. The reaction mixture was heated under reflux for 2 h. The reaction progress was monitored by TLC (50% EtOAc in n-hexane) to ensure completion of the reaction. The solvent was removed in vacuo to yield the desired product as intermediate 207 (540 mg, 83.2%). The crude material of intermediate 207 was used directly in the next step 7.

Step 7: 7-(2-(methylsulfonyl)-5-vinylbenzamido)-2,3-dihydrobenzo[b][1,4]dioxin-6-carboxylic acid (intermediate 208)

Triethylamine (1.93 mL, 13.8 mmol, 3.0 equiv) and a solution of 2-(methylsulfonyl)-5-vinylbenzoyl chloride (207) (1.35 mg, 5.53 mmol, 1.2 equiv) in THF (36 mL) were added to a stirred solution of 7-amino-2,3-dihydrobenzo[b][1,4]dioxin-6-carboxylic acid (100) (900 mg, 4.61 mmol, 1.0 equiv) in THF (36.0 mL) at 0 °C. The reaction mixture was stirred at the same temperature for 10 min and then at room temperature for 1.5 h. The reaction progress was monitored by TLC (20% EtOAc in n-hexane) to ensure completion of the reaction. The solvent was removed in vacuo. DMW was added to the residue and the product was extracted with EtOAc (9×200 mL). The combined EtOAc extract was washed with brine (200 mL), dried over sodium sulfate and concentrated in vacuo 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).

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

Step A: Methyl 3,4-dihydroxybenzoate (intermediate 100B).

To a stirred solution of 3,4-dihydroxybenzoic acid (100A) (97%, 10.0 g, 62.9 mmol, 1.0 equiv.) in MeOH (500 mL) was added sulfuric acid (96%, 7.0 mL, 126.0 mmol, 2.0 equiv.) at RT. The reaction mixture was heated at reflux overnight. The reaction was checked for completion by TLC. The solvent was removed in vacuo and then water (500 mL) was added. The aqueous layer was extracted with EtOAc (3 x 100 mL). The combined organic layers were washed with sat. _NaHCO3 solution (250 mL), brine (250 mL), dried over sodium sulfate and concentrated in vacuo to give the desired intermediate 100B (8.72 g, 82.4%) as an off-white solid. ¹ H NMR (300 MHz, DMSO-d6): δ 9.81 ppm (d, J = 131.1 Hz, 2H), 7.41 – 7.19 ppm (m, 2H), 6.81 ppm (d, J = 8.2 Hz, 1H), 3.77 ppm (s, 3H).

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

To a stirred solution of methyl 3,4-dihydroxybenzoate (100B) (10.0 g, 59.5 mmol, 1.0 equiv) in acetone (200.0 mL) was added potassium carbonate (20.5 g, 149 mmol, 2.50 equiv), followed by the addition of 1,2-dibromoethane (7.72 mL, 89.2 mmol, 1.50 equiv) and the reaction mixture was heated at reflux overnight. The reaction was checked for completion by TLC. The solvent was removed in vacuo and then water was added. The aqueous layer was neutralized with 1 N HCl and then extracted with EtOAc (2 x 250 mL). The combined organic extract was washed with brine (500 mL), dried over sodium sulfate and concentrated in vacuo. The crude product was purified by combiflash using ethyl acetate in n-hexane (0 to 10%) to give the desired intermediate 100C (9.0 g, 77.9%) as a colorless oil. ¹ H NMR (300 MHz, CDCl3): δ 7.54 – 7.42 ppm (m, 2H), 6.85 – 6.75 ppm (m, 1H), 4.27 – 4.14 ppm (m, 4H), 3.79 ppm (s, 3H).

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

To a stirred solution of methyl 2,3-dihydrobenzo[b][1,4]dioxin-6-carboxylate 100C (9.0 g, 46.3 mmol, 1.0 equiv.) in acetic acid (35.0 mL, 612 mmol, 13.2 equiv.) was added conc. HNO3 (47.0 mL, 788 mmol, 17.0 equiv.) dropwise below 20 °C, and the reaction mixture was stirred at room temperature for 2 h. The completion of the reaction was checked by TLC. The reaction mixture was poured into ice water with vigorous stirring, and the aqueous layer was extracted with EtOAc (3×500 mL). The combined organic extract was washed with NaHCO3, brine, dried over sodium sulfate and concentrated in vacuo to give the desired product as intermediate 100D (9.7 g, 87.5%) as a pale yellow solid. ¹ H NMR (300 MHz, DMSO-d6): δ 7.65 ppm (s, 1H), 7.30 ppm (s, 1H), 4.47 – 4.34 ppm (m, 1H), 4.39 ppm (s, 4H), 3.80 ppm (s, 3H).

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

A mixture of methyl 7-nitro-2,3-dihydrobenzo[b][1,4]dioxin-6-carboxylate (100D) (25.0 g, 105.0 mmol, 1.0 equiv), iron (20.4 g, 366.0 mmol, 3.5 equiv), ethanol (625 mL), water (163 mL), and acetic acid (500 mL) was stirred at 70 °C for 45 min. TLC was checked to ensure the reaction was complete. The reaction mixture was cooled to room temperature and filtered through a Hiflo filter. The filtrate was diluted with water (1000 mL), and the aqueous layer was extracted with ethyl acetate (3 x 700 mL). The combined organic extract was washed with saturated sodium bicarbonate (2 x 1000 mL), brine (1000 mL), dried over sodium sulfate, and filtered. The filtrate was concentrated in vacuo to give the title intermediate 100E (21.8 g, 99.7%) as a brown solid. ¹ H NMR (300 MHz, DMSO): δ 7.14 ppm (s, 1H), 6.25 ppm (d, J = 7.6 Hz, 3H), 4.26 – 4.19 ppm (m, 2H), 4.17 – 4.08 ppm (m, 2H), 3.73 ppm (s, 3H). MS (ESI): m/z 210.10 (M+1).

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

To a stirred solution of methyl 7-amino-2,3-dihydrobenzo[b][1,4]dioxin-6-carboxylate (100E) (22.0 g, 105 mmol, 1.0 equiv) in THF (440.0 mL) was added a solution of NaOH (23.1 g, 578 mmol, 5.5 equiv) in water (440.0 mL) and the reaction mixture was stirred at 80 °C for 20 h. The reaction progress was monitored by TLC (10% MeOH in DCM) to ensure completion of the reaction. The reaction mixture was cooled to room temperature, the pH was adjusted to 6 with 3 N HCl and the aqueous layer was extracted with ethyl acetate (5×500 mL). The combined organic layers were washed with brine, dried over sodium sulfate, filtered and the filtrate was concentrated in vacuo to give a brown crude solid. The crude product was purified with n-hexane to give the desired intermediate 100 (18.6 g, 90.6%) as a brown solid. ¹ H NMR (300 MHz, DMSO): δ 8.27 ppm (s, 2H), 7.13 ppm (s, 1H), 6.20 ppm (s, 1H), 4.27 – 4.19 ppm (m, 2H), 4.18 – 4.08 ppm (m, 2H).

Step 8: 2-(2-(methylsulfonyl)-5-vinylphenyl)-7,8-dihydro-4H-[1,4]dioxino[2',3':4,5]benzo[1,2-d][1,3]oxazin-4-one (intermediate 209)

A solution of 7-(2-(methylsulfonyl)-5-vinylbenzamido)-2,3-dihydrobenzo[b][1,4]dioxin-6-carboxylic acid (208) (1.60 mg, 3.97 mmol, 1.0 equiv) in acetic anhydride (40 mL) was stirred at 140 °C for 1.5 h. The reaction progress was monitored by TLC (50% EtOH in n-hexane) to ensure completion of the reaction. The reaction mixture was cooled to room temperature, then DMW (100 mL) was added. The product was extracted with EtOAc (4 x 200 mL). The combined EtOAc extract was washed with brine (2 x 200 mL), dried over sodium sulfate, and concentrated in vacuo to give the crude product. The crude product was purified by column chromatography on silica gel (230-400 mesh) and a gradient solvent (20-50% EtOAc in n-hexane) to give intermediate 209 (700 mg, 45.8%) as a yellow solid. ¹ H NMR (300 MHz, CDCl3): δ 8.15 ppm (d, J = 8.2 Hz, 1H), 7.90 ppm (d, J = 1.7 Hz, 1H), 7.77 – 7.68 ppm (m, 2H), 7.16 ppm (s, 1H), 6.82 ppm (dd, J = 17.5, 10.9 Hz, 1H), 6.01 ppm. (d, J = 17.5 Hz, 1H), 5.57 ppm. (d, J = 10.9 Hz, 1H), 4.40 ppm (dd, J = 11.8, 5.3 Hz, 5H), 3.52 ppm (d, J = 3.0 Hz, 3H). MS(ESI): m/z 385.80 (M+1).

Step 9: 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 210)

Ruthenium on carbon (10%) (50 mg, 0.049 mmol, 0.03 equiv) was added to a solution of 2-(2-(methylsulfonyl)-5-vinylphenyl)-7,8-dihydro-4H-[1,4]dioxino[2',3':4,5]benzo[1,2-d][1,3]oxazin-4-one (209) (0.630 g, 1.63 mmol, 1.0 equiv) in acetonitrile (12.6 mL), EtOAc (12.6 mL), and DMW (12.6 mL) at RT. Sodium metaperiodate (1.05 g, 4.9 mmol, 3.0 equiv) was added at 0 °C. The reaction mixture was stirred at room temperature for 1 h. The reaction progress was monitored by TLC (70% EtOAc in n-hexane) to ensure completion of the reaction. The reaction mixture was filtered through a pad of Celite. The filtrate was diluted with water (100 mL) and the aqueous layer was extracted with ethyl acetate (3 x 200 mL). The combined organic extract was washed with saturated sodium bicarbonate, brine, dried over sodium sulfate and filtered. The filtrate was concentrated in vacuo to give crude intermediate 210. The crude intermediate 210 was used directly in the next step 10.

Step 10: 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 1.002)

Sodium borohydride (9.77 mg, 0.258 mmol, 0.5 equiv) was added to a stirred solution of 4-(methylsulfonyl)-3-(4-oxo-7,8-dihydro-4H-[1, 4]dioxino[2',3':4,5]benzo[1,2-d][1,3]oxazin-2-yl)benzaldehyde (210) (0.20 g, 0.52 mmol, 1.0 equiv) in THF (10.0 mL) at -10 °C over 1 h. The reaction mixture was stirred at the same temperature of 0 °C for 30 min. The reaction progress was monitored by TLC (70% EtOAc in n-hexane) to ensure the completion of the reaction. The reaction mixture was quenched with saturated ammonium chloride solution (50 mL) and the product was extracted with ethyl acetate (3 x 100 mL). The combined organic extract was washed with brine (500 mL), dried over sodium sulfate and concentrated in vacuo to give the crude product. The crude product was purified by column chromatography on silica gel (230-400 mesh) using a gradient solvent (20-40% [(10% MeOH in EtOAc)] in n-hexane) to give compound 1.002 (70 mg, 34.80%) as an off-white solid. ¹ H NMR (300 MHz, DMSO): δ 8.07 ppm (d, J = 8.2 Hz, 1H), 7.88 ppm (d, J = 8.5 Hz, 1H). (s, 1H), 7.79 ppm (d, J = 8.5 Hz, 1H), 7.58 ppm (s, 1H), 7.24 ppm (s, 1H), 5.63 ppm (t, J = 5.7 Hz, 1H), 4.69 ppm. (d, J = 5.6 Hz, 2H), 4.52 – 4.31 ppm. (m, 4H), 3.48 ppm (c, 3H). MS(ESI): m/z 390.0 (M+1).

Example 3. In vitro activity assays

Human kallikrein proteins: Recombinant human KLK5 expressed in HEK293 was obtained from SpeedBio (Gaithersburg, MD). Recombinant proenzyme KLK-7 and recombinant proenzyme KLK-14, both expressed in murine myeloma cells, were obtained from R&D Biosystems (Minneapolis, MN). Plasmin and thrombin were obtained from Haematologic Technologies (Essex Junction, VT). Trypsin and chymotrypsin were purchased from Worthington Biochemical Corporation (Lakewood, NJ), and plasma kallikrein was from Prospec (East Brunswick, NJ). Neutrophil elastase was obtained from Innovative Research (Piri Court, MI). Bovine recombinant enterokinase expressed in E. coli was purchased from Sigma (St. Louis, MI). Matriptase expressed in E. coli was obtained from Enzo Life Sciences (Farmingdale, NY). As control inhibitors, nafamostat mesylate was obtained 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 obtained from Interbioscreen (Moscow, Russia).

Substrates: S-2288 (D-Ile-Pro-Arg-pNA), CS-PSA (MeO-Suc-Arg-Pro-Tyr-pNA), S-2266 (HD-Val-Leu-Arg-pNA), and S-2288 (HD-lle-Pro-Arg-pNA) were purchased from Diapharma (West Chester, OH). MeOSuccAla-Ala-Pro-Val-pNA was purchased from Sigma (St. Louis, MI). Substrates were dissolved in water at a concentration of 10 mM and frozen in aliquots at -20°C.

Activation of KLK-7 and KLK-14: 150 μl of 8 μM KLK-7 or KLK-14 proenzyme solution in 50 mM Tris, 10 mM CaCl ₂ , 150 mM NaCl, 0.05% (w/v) Brij-35, pH 7.5 were mixed with 150 μl of 10 μg/ml bovine enterokinase in the same buffer and incubated for 2 h for KLK-7 and 1 h for KLK-14 at 37°C. After the incubation period, activation was stopped by adding 6 μl of 0.5 M EDTA, pH 8.0.

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. For IC50 determinations, inhibitors were diluted in serial 1:3 intervals in neat DMSO to 50-fold the intended final assay concentrations. Typically, eight inhibitor dilutions and one DMSO control were prepared. Substrates were diluted with water to 2.2-fold the final assay concentration. Wells of the plate were filled with 50 μl of enzyme diluted in 2× assay buffer (100 mM Tris, 200 mM NaCl, 0.2% PEG, 0.01% Tween, 2 mM EDTA, pH 8.0) at 2× the intended concentration. Then, 5 μl of DMSO, 2.2 μl of inhibitor, and 45 μl of substrate were added to start the reaction. Assays were monitored at 37°C in a multiwell plate reader at 405 nm (Molecular Devices, San Jose, CA) for 1 h. The instrument inhibitor, nafamostat, was used as a control for trypsin, thrombin, KLK-5 and KLK-14 at a maximum concentration of 10 μM and (2-(2-fluorophenyl)-5,6,7,8-tetrahydro-4H-benzo[4,5]thieno[2,3-d][1,3]oxazin-4-one for KLK-7 at 5 μM.

The table shows the analysis conditions for individual enzymes.

Enzyme Enzyme concentration (nM) Substrate Substrate concentration
(μM) KLK-7 20 CS-PSA 500 KLK-5 10 S-2288 500 KLK-14 5 S-2288 500 Plasma kallikrein 5 S-2266 200 trypsin 0.1 S-2288 500 chymotrypsin 0.2 CS-PSA 500 plasmin 1 S-2288 500 thrombin 0.5 S-2288 200 matriptase 0.015 U/µl S-2288 200

Data analysis: The substrate hydrolysis progress curves were analyzed by linear regression of the last 20 minutes of reaction. Fitting the dose-response rates obtained to the following equation yields the IC50:

,

where 1) c: background velocity at the highest inhibitor concentration; 2) d: control reaction velocity; 3) IC ₅₀ : IC ₅₀ of inhibitor; 4) x: inhibitor concentration; and 5) b: slope.

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). BCA protein assay kit was purchased from VWR.

Preparation of skin extract. Epidermis was frozen 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), frozen twice on dry ice, and thawed. After overnight incubation at 4°C and the addition of six 2.4 mm metal beads, the sample was homogenized in a Bertin Precellys homogenizer (Atkinson, NH) three times at 5000 Hz for 20 sec each time and cooled for 30 sec in between. After centrifugation at maximum speed in a tabletop minicentrifuge, the protein concentration of the supernatant was determined by BCA assay (~0.5 mg/ml).

Inhibition of protease activity in skin extract: Stock solutions of 200, 66, 22, and 0 μM test compound were prepared in DMSO. Stock substrates of 1100 μM PS-01 and PS-02 were prepared by dissolution in water. Wells of a 384-well plate were filled with 50 μL of 2× assay buffer (see above), and then 45 μL of stock substrate and 5 μL of test compound were added. The reaction was started by adding 10 μL of skin extract to the PS-02 substrate and 5 μL to the PS-01 substrate. Substrate hydrolysis was monitored in a multiwell plate reader at 405 nm.

Example 5. Results of analysis of test compounds

Compounds 1.001 and 1.002, as well as the known compound 14, were tested according to the analysis protocols of Examples 3 and 4. The test results are given in Table 2. Compound 14 is disclosed in WO 2015/112081 and has the formula:

.

Table 2. Results of the analysis of the tested compounds

Conc. Conn. 14 Conn. 1.001 Comp. 1.002 Recombinant enzyme KLK5 (IC ₅₀ ) 0.24 µM 0.21 µM 1.57 µM Recombinant enzyme KLK7 (IC ₅₀ ) 0.13 µM 0.34 µM 0.22 µM Speed in skin analysis 0.14 ^* 0.004 ^* 0.009 ^* Substrate depletion rate constant 1 µM 0.1 ^* 0.02 ^* 0.06 ^* 3.3 µM 0.08 ^* 0.04 ^* 0.05 ^* Initial rate of proteolytic activity 1 µM 0.13 ^* 0.04 ^* 0.1 ^* 3.3 µM 0.11 ^* 0.07 ^* 0.09 ^*

* Absorption 405/min.

The chromogenic substrate cleavage progressions of compounds 1.001, 1.002, and 14 were tested in accordance with the human stratum corneum extract assay of Example 4 using the preferred chromogenic peptide substrates KLK5 and KLK7 (PS-01 and PS-02). The proteolytic cleavage of the selective substrate KLK5 in the skin extract by compounds 1.001, 1.002, and 14 is compared and shown in Figs. 4A-4D. NSK refers to freshly prepared skin extract; and OSK refers to freeze-thawed skin extract.

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

In contrast to the results of purified recombinant KLK 5 based on three parameters (rate, substrate depletion and initial rate of proteolytic activity), compound 1.001 was found to be a more potent inhibitor of the proteolytic activity of the skin extract compared to the known compound 14.

Example 6. Chromogenic analysis

Preparation of neonatal human epidermal keratinocyte lysate: Pooled human epidermal keratinocytes (HEK) were purchased from Invitrogen (Cat.# A13401). Cells were grown in commercial EpiLife medium (ThermoFisher, Cat.# MEPI500CA) supplemented with commercial S7 supplement (ThermoFisher, Cat.# SO0175) in the absence of serum. Tissue culture was performed at 37°C in a humidified chamber with 5% carbon dioxide. The composition of the HEK lysis buffer was: 100 mM Tris-HCl, 150 mM NaCl, 1% Triton, 5 mM EDTA, final pH 7.6. After two passages, cells were lysed using HEK lysis buffer without added protease and phosphatase inhibitors and protein concentration was determined using the bicinchoninic acid (BCA) assay (ThermoFisher, cat. #23225).

Preparation of Stratum Corneum Extract from Healthy Human Volunteers: Stratum corneum sheets were purchased from Biopredic (Cat.# STR0020). Extract Preparation: Stratum corneum lysate was prepared using the Minute Total Protein Extraction Kit for Adipose Tissue (InventBiotech, Cat.# AT-022) after homogenization according to the manufacturer’s instructions. Total protein concentration was determined using the BCA assay (ThermoFisher, Cat.# 23225). Extracts were snap frozen and stored at -80°C until use.

KLK5 Concentration in Stretch Marker Extract: KLK5 concentration in stratum corneum extract was determined by 1) ELISA from Abcam (Cat# ab131555); and 2) comparing the hydrolysis rate of the selective chromogenic substrate PS-01 (YRSR-pNA Tyr-Arg-Ser-Arg-pNA) to the rate of known concentrations of purified KLK5 (Speed Bio). The KLK5 concentration in the unknown sample was then determined from the hydrolysis rate of the selective substrate by interpolation from a linear plot of the rates of the known samples.

Cleavage of macromolecular substrates by endogenous and exogenous kallikrein 5: 10 μg of HEK lysate was incubated with an equal volume of stratum corneum extract or protease in buffer at 37°C for 1.5 h. Reactions were quenched by boiling for 5 min at 100°C with loading buffer under reducing conditions (50 mM DTT). Proteins were separated by electrophoresis on bis-tris gels with a 4-12% acrylamide gradient run for 22 min at 200 V. After transfer of proteins to PVDF membranes, nonspecific binding was blocked using Intercept blocking buffer (Licor) for one hour at room temperature. Blot signals were normalized with GAPDH.

The conditions for detection of specific proteins are given below:

Desmoglein 1: Anti-desmoglein antibody sc-137164 (Santa Cruz Biochemicals) was diluted 1/750 and incubated for 18 h at 40°C. After washing, anti-mouse secondary antibody (Licor) was added after dilution of 1/20,000 of stock solution. Controls lacking KLK5 were treated with protease/phosphatase inhibitor cocktail (Cell Signaling, #5872).

Desmocollin 1: Anti-desmocollin 1 antibody (Abcam, 150382) was diluted 1:1000. Secondary antibody was diluted 1:15000. After washing, secondary anti-rabbit antibody was added after dilution 1:15000.

Filaggrin: Antifilaggrin (LS-Bio, 1561) was diluted 1:400. Secondary antibody was diluted 1:10000.

Results of chromogenic analysis:

Proteases: KLK5 (SpeedBioSystems); KLK7 (R&Dsystems) activated with enterokinase (2 h, 37°C) before the assay; trypsin (Sigma Aldrich); chymotrypsin (Sigma Aldrich); Factor Xa (Haematologic Technologies); Plasmin (Haematologic Technologies); KLK14 (R&Dsystems) activated with enterokinase (1 h, 37°C) before the assay; Thrombin (Haematologic Technologies); neutrophil elastase (Athens Research&Technology); and KLK1 (Prospec).

Compound 408: a product obtained by hydrolytic opening of the 4-quinazolinone group of Compound 1.001, represented by the formula:

.

The results of the analysis are presented in Table 3.

Table 3. Results of chromogenic analysis of compound 1.001 and compound 408.

Protease Substrate Conn. 1.001 Conn. 408 Name under test
conc. Name under test
conc. Highest
conc. IC ₅₀ (µM) IC ₅₀ KLK5 10 nM S-2288 500 µM 10 µM 0.176 µM (n=2); 0.188 µM (n=2) Without
inhibition KLK7 20 nM CS-PSA 500 µM 20 µM 0.693 µM (n=2); 0.647 µM (n=2) Without
inhibition Trypsin 0.1 nM S-2288 500 µM 50 µM 1.664 µM (n=2); 1.363 µM n=2); 2.204 µM (n=2) Without
inhibition Chymotrypsin 0.2 nM CS-PSA 500 µM 50 µM 0.249 µM (n=2); 0.317 µM (n=2) Without
inhibition Factor Xa 0.5 nM S-2288 500 µM 20 µM 3.217 µM (n=2); 10.40 µM (n=2); 3.935 µM (n=2) Without
inhibition Plasmin 1.0 nM S-2288 500 µM 50 µM 3.357 µM (n=2); 4.196 µM (n=2) Without
inhibition KLK14 5 nM, 1 nM S-2288 500 µM 50 µM 0.633 µM (5 nM) ^a ; 0.442 µM (1 nM) ^b Without
inhibition Thrombin 0.5 nM S-2288 500 µM 100 µM >10 µM (n=2); >10 µM (n=2);
>10 µM (n=2) Without
inhibition neutrophil elastase 0.5 nM Elastase substrate 1 200 µM 100 µM >50 μM (n=2);
>50 μM (n=2) > 500 µM KLK1 10 nM S-2266 200 µM 50 µM Without
inhibition Without inhibition

S-2288, S-2266 and CS-PSA were obtained from Diapharma and dissolved in water as a 10 mM solution; and

Elastase substrate 1 was from Sigma and dissolved in water as a 0.8 mM solution.

a: _IC50 at 5 nM KLK14 concentration; and b: _IC50 at 1 nM KLK14 concentration.

Conclusions: It can be concluded that compound 1.001 is an inhibitor of KLK5, KLK7 and chymotrypsin. In contrast, compound 408, which lacks the 4-quinazolinone moiety (i.e., the pharmacophore of compound 1.001), is unable to inhibit proteases.

Claims (55)

1. The compound represented by formula (I) (I), or a pharmaceutically acceptable complex thereof, wherein R is H. 2. The compound according to claim 1, represented by formula (Ia) (Ia), or a pharmaceutically acceptable complex thereof. 3. The compound according to claim 1, represented by formula (Ib) (Ib), or a pharmaceutically acceptable complex thereof. 4. The compound according to claim 1, represented by formula (Ic) (Ic), or a pharmaceutically acceptable complex thereof. 5. The compound according to claim 1, represented by the formula , or a pharmaceutically acceptable complex thereof. 6. The compound according to claim 1, represented by the formula , or a pharmaceutically acceptable complex thereof. 7. A pharmaceutical composition for treating skin diseases associated with the proteolytic activity of one or more KLK proteases, containing a therapeutically effective amount of a compound according to any one of claims 1-6 and a pharmaceutically acceptable carrier. 8. A pharmaceutical composition according to claim 7, characterized in that said compound has the formula , or a pharmaceutically acceptable complex thereof. 9. The pharmaceutical composition according to item 7, characterized in that the said compound has the formula , or a pharmaceutically acceptable complex thereof. 10. A pharmaceutical composition according to any one of claims 7-9, obtained for topical use. 11. A method for treating a skin disease associated with the proteolytic activity of one or more KLK proteases in a subject in need thereof, comprising administering to said subject an effective amount of a compound having formula (I) (I), or a pharmaceutically acceptable complex thereof, wherein R is H. 12. The method according to claim 11, characterized in that said compound has the formula (Ia) (Ia), or a pharmaceutically acceptable complex thereof. 13. The method according to claim 11, characterized in that said compound has a formula selected from the group consisting of: And , or a pharmaceutically acceptable complex thereof. 14. The method according to any one of paragraphs 11-13, characterized in that the skin disease is genodermatosis. 15. The method according to claim 14, characterized in that the genodermatosis is Netherton syndrome. 16. The method according to any one of claims 11-15, characterized in that one or more KLK proteases are KLK5 and/or KLK7. 17. The method according to any of paragraphs 11-16, characterized in that the said compound is administered locally. 18. An in vitro method for determining the proteolytic activity of one or more KLK proteases, comprising: a) preparation of skin extract; b) exposing the substrate and the compound of formula (I) (I) or a pharmaceutically acceptable complex thereof, a skin extract; and c) determination of the rate of proteolytic cleavage of the substrate, wherein the substrate is a peptide p-nitroanilide; and R represents H. 19. The method according to claim 18, characterized in that the substrate is Tyr-Arg-Ser-Arg-pNA or Lys-His-Leu-Tyr-pNA. 20. The method according to claim 18, characterized in that said compound has the formula (Ia): (Ia), or a pharmaceutically acceptable complex thereof. 21. The method according to claim 18, characterized in that said compound has a formula selected from the group consisting of: And , or a pharmaceutically acceptable complex thereof. 22. The method according to claim 18, characterized in that the skin extract is an extract of human skin.